Cardiovascular progenitor cells (CPCs) expressing the ISL1-LIM–homeodomain transcription factor contribute developmentally to cardiomyocytes in all 4 chambers of the heart. Here, we show that ISL1-CPCs can be applied to myocardial regeneration following injury. We used a rapid 3D methylcellulose approach to form murine and human ISL1-CPC spheroids that engrafted after myocardial infarction in murine hearts, where they differentiated into cardiomyocytes and endothelial cells, integrating into the myocardium and forming new blood vessels. ISL1-CPC spheroid–treated mice exhibited reduced infarct area and increased blood vessel formation compared with control animals. Moreover, left ventricular (LV) contractile function was significantly better in mice transplanted with ISL1-CPCs 4 weeks after injury than that in control animals. These results provide proof-of-concept of a cardiac repair strategy employing ISL1-CPCs that, based on our previous lineage-tracing studies, are committed to forming heart tissue, in combination with a robust methylcellulose spheroid–based delivery approach.
Oscar Bartulos, Zhen Wu Zhuang, Yan Huang, Nicole Mikush, Carol Suh, Alda Bregasi, Lin Wang, William Chang, Diane S. Krause, Lawrence H. Young, Jordan S. Pober, Yibing Qyang
The prevention of organ damage and early death in young adults is a major clinical concern in sickle cell disease (SCD). However, mechanisms that control adult progression of SCD during the transition from adolescence are poorly defined with no cognate prophylaxis. Here, we demonstrate in a longitudinal cohort of homozygous SCD (SS) mice a link between intravascular hemolysis, vascular inflammation, lung injury, and early death. Prophylactic Nrf2 activation in young SS mice stabilized intravascular hemolysis, reversed vascular inflammation, and attenuated lung edema in adulthood. Enhanced Nrf2 activation in endothelial cells in vitro concurred with the dramatic effect on vascular inflammation in the mice. BM chimeric SS mice lacking Nrf2 expression in nonhematopoietic tissues were created to dissect the role of nonerythroid Nrf2 in SCD progression. The SS chimeras developed severe intravascular hemolysis despite having erythroid Nrf2. In addition, they developed premature vascular inflammation and pulmonary edema and died younger than donor littermates with intact nonhematopoietic Nrf2. Our results reveal a dominant protective role for nonhematopoietic Nrf2 against tissue damage in both erythroid and nonerythroid tissues in SCD. Furthermore, we show that prophylactic augmentation of Nrf2-coordinated cytoprotection effectively impedes onset of the severe adult phenotype of SCD in mice.
Samit Ghosh, Chibueze A. Ihunnah, Rimi Hazra, Aisha L. Walker, Jason M. Hansen, David R. Archer, Amma T. Owusu-Ansah, Solomon F. Ofori-Acquah
We have previously reported that obesity attenuates pulmonary inflammation in both patients with acute respiratory distress syndrome (ARDS) and in mouse models of the disease. We hypothesized that obesity-associated hyperleptinemia, and not body mass per se, drives attenuation of the pulmonary inflammatory response and that this effect could also impair the host response to pneumonia. We examined the correlation between circulating leptin levels and risk, severity, and outcome of pneumonia in 2 patient cohorts (NHANES III and ARDSNet-ALVEOLI) and in mouse models of diet-induced obesity and lean hyperleptinemia. Plasma leptin levels in ambulatory subjects (NHANES) correlated positively with annual risk of respiratory infection independent of BMI. In patients with severe pneumonia resulting in ARDS (ARDSNet-ALVEOLI), plasma leptin levels were found to correlate positively with subsequent mortality. In obese mice with pneumonia, plasma leptin levels were associated with pneumonia severity, and in obese mice with sterile lung injury, leptin levels were inversely related to bronchoalveolar lavage neutrophilia, as well as to plasma IL-6 and G-CSF levels. These results were recapitulated in lean mice with experimentally induced hyperleptinemia. Our findings suggest that the association between obesity and elevated risk of pulmonary infection may be driven by hyperleptinemia.
Niki D.J. Ubags, Renee D. Stapleton, Juanita H.J. Vernooy, Elianne Burg, Jenna Bement, Catherine M. Hayes, Sebastian Ventrone, Lennart Zabeau, Jan Tavernier, Matthew E. Poynter, Polly E. Parsons, Anne E. Dixon, Matthew J. Wargo, Benjamin Littenberg, Emiel F.M. Wouters, Benjamin T. Suratt
Idiopathic pulmonary fibrosis (IPF) is a fatal disease without any cure. Both human disease and animal models demonstrate dysregulated wound healing and unregulated fibrogenesis in a background of low-grade chronic T lymphocyte infiltration. Tissue-resident memory T cells (Trm) are emerging as important regulators of the immune microenvironment in response to pathogens, and we hypothesized that they might play a role in regulating the unremitting inflammation that promotes lung fibrosis. Herein, we demonstrate that lung-directed immunotherapy, in the form of i.n. vaccination, induces an antifibrotic T cell response capable of arresting and reversing lung fibrosis. In mice with established lung fibrosis, lung-specific T cell responses were able to reverse established pathology — as measured by decreased lung collagen, fibrocytes, and histologic injury — and improve physiologic function. Mechanistically, we demonstrate that this effect is mediated by vaccine-induced lung Trm. These data not only have implications for the development of immunotherapeutic regimens to treat IPF, but also suggest a role for targeting tissue-resident memory T cells to treat other tissue-specific inflammatory/autoimmune disorders.
Samuel L. Collins, Yee Chan-Li, MinHee Oh, Christine L. Vigeland, Nathachit Limjunyawong, Wayne Mitzner, Jonathan D. Powell, Maureen R. Horton
To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.
Nobuyuki Tanaka, Takeo Kosaka, Yasumasa Miyazaki, Shuji Mikami, Naoya Niwa, Yutaro Otsuka, Yoji Andrew Minamishima, Ryuichi Mizuno, Eiji Kikuchi, Akira Miyajima, Hisataka Sabe, Yasunori Okada, Per Uhlén, Makoto Suematsu, Mototsugu Oya
Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia–derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/β-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.
Giuseppina Federico, Michael Meister, Daniel Mathow, Gunnar H. Heine, Gerhard Moldenhauer, Zoran V. Popovic, Viola Nordström, Annette Kopp-Schneider, Thomas Hielscher, Peter J. Nelson, Franz Schaefer, Stefan Porubsky, Danilo Fliser, Bernd Arnold, Hermann-Josef Gröne
Stimulation of β1-adrenergic receptor (β1AR), a GPCR, and the receptor for advanced glycation end-products (RAGE), a pattern recognition receptor (PRR), have been independently implicated in the pathogenesis of cardiomyopathy caused by various etiologies, including myocardial infarction, ischemia/reperfusion injury, and metabolic stress. Here, we show that the two distinctly different receptors, β1AR and RAGE, are mutually dependent in mediating myocardial injury and the sequelae of cardiomyopathy. Deficiency or inhibition of RAGE blocks β1AR- and RAGE-mediated myocardial cell death and maladaptive remodeling. Ablation or blockade of β1AR fully abolishes RAGE-induced detrimental effects. Mechanistically, RAGE and β1AR form a complex, which in turn activates Ca2+/calmodulin-dependent kinase II (CaMKII), resulting in loss of cardiomyocytes and myocardial remodeling. These results indicate that RAGE and β1AR not only physically crosstalk at the receptor level, but also functionally converge at the common mediator, CaMKII, highlighting a combined inhibition of RAGE and β1AR as a more effective therapy to treat diverse cardiovascular diseases, such as myocardial infarction, ischemia/reperfusion injury, and diabetic cardiovascular complications.
Weizhong Zhu, Sharon Tsang, David M. Browe, Anthony Y.H. Woo, Ying Huang, Chanjuan Xu, Jian-Feng Liu, Fengxiang Lv, Yan Zhang, Rui-ping Xiao
Acute allograft rejection is mediated by host CD8+ cytotoxic T lymphocytes (CTL) targeting graft class I major histocompatibility complex (MHC) molecules. In experimental rodent models, rejection requires differentiation of naive CD8+ T cells into alloreactive CTL within secondary lymphoid organs, whereas in humans, CTL may alternatively develop within the graft from circulating CD8+ effector memory T cells (TEM) that recognize class I MHC molecules on graft endothelial cells (EC). This latter pathway is poorly understood. Here, we show that host CD4+ TEM, activated by EC class II MHC molecules, provide critical help for this process. First, blocking HLA-DR on EC lining human artery grafts in immunodeficient mice reduces CD8+ CTL development within and acute rejection of the artery by adoptively transferred allogeneic human lymphocytes. Second, siRNA knockdown or CRISPR/Cas9 ablation of class II MHC molecules on EC prevents CD4+ TEM from helping CD8+ TEM to develop into CTL in vitro. Finally, implanted synthetic microvessels, formed from CRISPR/Cas9-modified EC lacking class II MHC molecules, are significantly protected from CD8+ T cell–mediated destruction in vivo. We conclude that human CD8+ TEM–mediated rejection targeting graft EC class I MHC molecules requires help from CD4+ TEM cells activated by recognition of class II MHC molecules.
Parwiz Abrahimi, Lingfeng Qin, William G. Chang, Alfred L.M. Bothwell, George Tellides, W. Mark Saltzman, Jordan S. Pober
Homeostasis of the gastrointestinal (GI) tract is controlled by complex interactions between epithelial and immune cells and the resident microbiota. Here, we studied the role of Wnt signaling in GI homeostasis using
Haim Belinson, Adam K. Savage, Douglas Fadrosh, Yien-Ming Kuo, Din Lin, Ricardo Valladares, Ysbrand Nusse, Anthony Wynshaw-Boris, Susan V. Lynch, Richard M. Locksley, Ophir D. Klein
Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation.
Shijun Hu, Ming-Tao Zhao, Fereshteh Jahanbani, Ning-Yi Shao, Won Hee Lee, Haodong Chen, Michael P. Snyder, Joseph C. Wu
Limited transendothelial permeability across tumor microvessels represents a significant bottleneck in the development of tumor-specific diagnostic agents and theranostic drugs. Here, we show an approach to increase transendothelial permeability of macromolecular and nanoparticle-based contrast agents via inhibition of the type I TGF-β receptor, activin-like kinase 5 (Alk5), in tumors. Alk5 inhibition significantly increased tumor contrast agent delivery and enhancement on imaging studies, while healthy organs remained relatively unaffected. Imaging data correlated with significantly decreased tumor interstitial fluid pressure, while tumor vascular density remained unchanged. This immediately clinically translatable concept involving Alk5 inhibitor pretreatment prior to an imaging study could be leveraged for improved tumor delivery of macromolecular and nanoparticle-based imaging probes and, thereby, facilitate development of more sensitive imaging tests for cancer diagnosis, enhanced tumor characterization, and personalized, image-guided therapies.
Heike E. Daldrup-Link, Suchismita Mohanty, Celina Ansari, Olga Lenkov, Aubie Shaw, Ken Ito, Su Hyun Hong, Matthias Hoffmann, Laura Pisani, Nancy Boudreau, Sanjiv Sam Gambhir, Lisa M. Coussens
CD4+ T cells predominate in salivary gland (SG) inflammatory lesions in Sjögren’s syndrome (SS). However, their antigen specificity, degree of clonal expansion, and relationship to clinical disease features remain unknown. We used multiplex reverse-transcriptase PCR to amplify paired T cell receptor α (TCRα) and β transcripts of single CD4+CD45RA– T cells from SG and peripheral blood (PB) of 10 individuals with primary SS, 9 of whom shared the HLA DR3/DQ2 risk haplotype. TCRα and β sequences were obtained from a median of 91 SG and 107 PB cells per subject. The degree of clonal expansion and frequency of cells expressing two productively rearranged α genes were increased in SG versus PB. Expanded clones from SG exhibited complementary-determining region 3 (CDR3) sequence similarity both within and among subjects, suggesting antigenic selection and shared antigen recognition. CDR3 similarities were shared among expanded clones from individuals discordant for canonical Ro and La autoantibodies, suggesting recognition of alternative SG antigen(s). The extent of SG clonal expansion correlated with reduced saliva production and increased SG fibrosis, linking expanded SG T cells with glandular dysfunction. Knowledge of paired TCRα and β sequences enables further work toward identification of target antigens and development of novel therapies.
Michelle L. Joachims, Kerry M. Leehan, Christina Lawrence, Richard C. Pelikan, Jacen S. Moore, Zijian Pan, Astrid Rasmussen, Lida Radfar, David M. Lewis, Kiely M. Grundahl, Jennifer A. Kelly, Graham B. Wiley, Mikhail Shugay, Dmitriy M. Chudakov, Christopher J. Lessard, Donald U. Stone, R. Hal Scofield, Courtney G. Montgomery, Kathy L. Sivils, Linda F. Thompson, A. Darise Farris
Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-
David H. McMillan, Jos L.J. van der Velden, Karolyn G. Lahue, Xi Qian, Robert W. Schneider, Martina S. Iberg, James D. Nolin, Sarah Abdalla, Dylan T. Casey, Kenneth D. Tew, Danyelle M. Townsend, Colin J. Henderson, C. Roland Wolf, Kelly J. Butnor, Douglas J. Taatjes, Ralph C. Budd, Charles G. Irvin, Albert van der Vliet, Stevenson Flemer, Vikas Anathy, Yvonne M.W. Janssen-Heininger
David Carrick, Caroline Haig, Jaclyn Carberry, Vannesa Teng Yue May, Peter McCartney, Paul Welsh, Nadeem Ahmed, Margaret McEntegart, Mark C. Petrie, Hany Eteiba, Mitchell Lindsay, Stuart Hood, Stuart Watkins, Ahmed Mahrous, Samuli M.O. Rauhalammi, Ify Mordi, Ian Ford, Aleksandra Radjenovic, Naveed Sattar, Keith G. Oldroyd, Colin Berry
Tissue-resident memory T cells (TRM) are a recently defined, noncirculating subset with the potential for rapid in situ protective responses, although their generation and role in vaccine-mediated immune responses is unclear. Here, we assessed TRM generation and lung-localized protection following administration of currently licensed influenza vaccines, including injectable inactivated influenza virus (IIV, Fluzone) and i.n. administered live-attenuated influenza virus (LAIV, FluMist) vaccines. We found that, while IIV preferentially induced strain-specific neutralizing antibodies, LAIV generated lung-localized, virus-specific T cell responses. Moreover, LAIV but not IIV generated lung CD4+ TRM and virus-specific CD8+ TRM, similar in phenotype to those generated by influenza virus infection. Importantly, these vaccine-generated TRM mediated cross-strain protection, independent of circulating T cells and neutralizing antibodies, which persisted long-term after vaccination. Interestingly, intranasal administration of IIV or injection of LAIV failed to elicit T cell responses or provide protection against viral infection, demonstrating dual requirements for respiratory targeting and a live-attenuated strain to establish TRM. The ability of LAIV to generate lung TRM capable of providing long-term protection against nonvaccine viral strains, as demonstrated here, has important implications for protecting the population against emergent influenza pandemics by direct fortification of lung-specific immunity.
Kyra D. Zens, Jun Kui Chen, Donna L. Farber
Dopamine D2 receptor (DRD2) deficiency increases renal inflammation and blood pressure in mice. We show here that long-term renal-selective silencing of
Prasad R. Konkalmatt, Laureano D. Asico, Yanrong Zhang, Yu Yang, Cinthia Drachenberg, Xiaoxu Zheng, Fei Han, Pedro A. Jose, Ines Armando
Immune checkpoint therapy with PD-1 blockade has emerged as an effective therapy for many advanced cancers; however, only a small fraction of patients achieve durable responses. To date, there is no validated blood-based means of predicting the response to PD-1 blockade. We report that Bim is a downstream signaling molecule of the PD-1 pathway, and its detection in T cells is significantly associated with expression of PD-1 and effector T cell markers. High levels of Bim in circulating tumor-reactive (PD-1+CD11ahiCD8+) T cells were prognostic of poor survival in patients with metastatic melanoma who did not receive anti–PD-1 therapy and were also predictive of clinical benefit in patients with metastatic melanoma who were treated with anti–PD-1 therapy. Moreover, this circulating tumor-reactive T cell population significantly decreased after successful anti–PD-1 therapy. Our study supports a crucial role of Bim in both T cell activation and apoptosis as regulated by PD-1 and PD-L1 interactions in effector CD8+ T cells. Measurement of Bim levels in circulating T cells of patients with cancer may provide a less invasive strategy to predict and monitor responses to anti–PD-1 therapy, although future prospective analyses are needed to validate its utility.
Roxana S. Dronca, Xin Liu, Susan M. Harrington, Lingling Chen, Siyu Cao, Lisa A. Kottschade, Robert R. McWilliams, Matthew S. Block, Wendy K. Nevala, Michael A. Thompson, Aaron S. Mansfield, Sean S. Park, Svetomir N. Markovic, Haidong Dong
Pirfenidone is a recently approved antifibrotic drug for the treatment of idiopathic pulmonary fibrosis (IPF). Because tuberculosis (TB) is characterized by granulomatous inflammation in conjunction with parenchymal destruction and replacement fibrosis, we sought to determine whether the addition of pirfenidone as an adjunctive, host-directed therapy provides a beneficial effect during antimicrobial treatment of TB. We hypothesized that pirfenidone’s antiinflammatory and antifibrotic properties would reduce inflammatory lung damage and increase antimicrobial drug penetration in granulomas to accelerate treatment response. The effectiveness of adjunctive pirfenidone during TB drug therapy was evaluated using a murine model of chronic TB. Mice treated with standard therapy 2HRZ/4HR (H, isoniazid; R, rifampin; and Z, pyrazinamide) were compared with 2 alternative regimens containing pirfenidone (Pf) (2HRZPf/4HRPf and 2HRZPf/4HR). Contrary to our hypothesis, adjunctive pirfenidone use leads to reduced bacterial clearance and increased relapse rates. This treatment failure is closely associated with the emergence of isoniazid monoresistant bacilli, increased cavitation, and significant lung pathology. While antifibrotic agents may eventually be used as part of adjunctive host-directed therapy of TB, this study clearly demonstrates that caution must be exercised. Moreover, as pirfenidone becomes more widely used in clinical practice, increased patient monitoring would be required in endemic TB settings.
Bintou A. Ahidjo, Mariama C. Maiga, Elizabeth A. Ihms, Mamoudou Maiga, Alvaro A. Ordonez, Laurene S. Cheung, Sarah Beck, Bruno B. Andrade, Sanjay Jain, William R. Bishai
To elucidate the mechanisms for reverse LV remodeling, we generated a conditional (doxycycline [dox] off) transgenic mouse tetracycline transactivating factor–TRAF2 (tTA-TRAF2) that develops a dilated heart failure (HF) phenotype upon expression of a proinflammatory transgene, TNF receptor–associated factor 2 (TRAF2), and complete normalization of LV structure and function when the transgene is suppressed. tTA-TRAF2 mice developed a significant increase in LV dimension with decreased contractile function, which was completely normalized in the tTA-TRAF2 mice fed dox for 4 weeks (tTA-TRAF2dox4W). Normalization of LV structure and function was accompanied by partial normalization (~60%) of gene expression associated with incident HF. Similar findings were observed in patients with dilated cardiomyopathy who underwent reverse LV remodeling following mechanical circulatory support. Persistence of the HF gene program was associated with an exaggerated hypertrophic response and increased mortality in tTA-TRAF2dox4W mice following transaortic constriction (TAC). These effects were no longer observed following TAC in tTA-TRAF2dox8W, wherein there was a more complete (88%) reversal of the incident HF genes. These results demonstrate that reverse LV remodeling is associated with improvements in cardiac myocyte biology; however, the persistence of the abnormal HF gene program may be maladaptive following perturbations in hemodynamic loading conditions.
Veli K. Topkara, Kari T. Chambers, Kai-Chien Yang, Huei-Ping Tzeng, Sarah Evans, Carla Weinheimer, Attila Kovacs, Jeffrey Robbins, Philip Barger, Douglas L. Mann
The majority of metastatic renal cell carcinoma (RCC) patients are treated with tyrosine kinase inhibitors (TKI) in first-line treatment; however, a fraction are refractory to these antiangiogenic drugs. MicroRNAs (miRNAs) are regulatory molecules proven to be accurate biomarkers in cancer. Here, we identified miRNAs predictive of progressive disease under TKI treatment through deep sequencing of 74 metastatic clear cell RCC cases uniformly treated with these drugs. Twenty-nine miRNAs were differentially expressed in the tumors of patients who progressed under TKI therapy (
Jesús García-Donas, Benoit Beuselinck, Lucía Inglada-Pérez, Osvaldo Graña, Patrick Schöffski, Agnieszka Wozniak, Oliver Bechter, Maria Apellániz-Ruiz, Luis Javier Leandro-García, Emilio Esteban, Daniel E. Castellano, Aranzazu González del Alba, Miguel Angel Climent, Susana Hernando, José Angel Arranz, Manuel Morente, David G. Pisano, Mercedes Robledo, Cristina Rodriguez-Antona
IL-1β is a well-established inducer of both insulin resistance and impaired pancreatic islet function. Despite this, findings examining IL-1 receptor deficiency or antagonism in in vivo animal models, as well as in clinical studies of type 2 diabetic (T2D) patients, have led to conflicting results, suggesting that the actions of IL-1β on glycemic control may be pleiotropic in nature. In the present work, we find that the ability of IL-1β to amplify glucose-stimulated insulin secretion from human islets correlates with donor BMI. Islets from obese donors are sensitized to the insulinotropic effects of this cytokine, whereas the stimulatory effects of IL-1β are lost in islets from obese T2D patients, suggesting a role for IL-1 signaling in islet compensation. Indeed, mice deficient in IL-1 receptor type I become glucose intolerant more rapidly than their WT littermates and have impaired secretory responses during the acute stages of inflammatory and metabolic stress induced by LPS and high-fat diet, respectively. IL-1β directly enhances β cell insulin secretion by increasing granule docking and soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex formation at the plasma membrane. Together, our study highlights the importance of IL-1β signaling in islet compensation to metabolic and inflammatory stress.
Catherine Hajmrle, Nancy Smith, Aliya F. Spigelman, Xiaoqing Dai, Laura Senior, Austin Bautista, Mourad Ferdaoussi, Patrick E. MacDonald
High-affinity self-reactive thymocytes are purged in the thymus, and residual self-reactive T cells, which are detectable in healthy subjects, are controlled by peripheral tolerance mechanisms. Breakdown in these mechanisms results in autoimmune disease, but antigen-specific therapy to augment natural mechanisms can prevent this. We aimed to determine when antigen-specific therapy is most effective. Islet autoantigens, proinsulin (PI), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) were expressed in the antigen-presenting cells (APCs) of autoimmune diabetes-prone nonobese diabetic (NOD) mice in a temporally controlled manner. PI expression from gestation until weaning was sufficient to completely protect NOD mice from diabetes, insulitis, and development of insulin autoantibodies. Insulin-specific T cells were significantly diminished, were naive, and did not express IFN-γ when challenged. This long-lasting effect from a brief period of treatment suggests that autoreactive T cells are not produced subsequently. We tracked IGRP206–214-specific CD8+ T cells in NOD mice expressing IGRP in APCs. When IGRP was expressed only until weaning, IGRP206–214-specific CD8+ T cells were not detected later in life. Thus, anti-islet autoimmunity is determined during early life, and autoreactive T cells are not generated in later life. Bolstering tolerance to islet antigens in the perinatal period is sufficient to impart lasting protection from diabetes.
Gaurang Jhala, Jonathan Chee, Prerak M. Trivedi, Claudia Selck, Esteban N. Gurzov, Kate L. Graham, Helen E. Thomas, Thomas W.H. Kay, Balasubramanian Krishnamurthy
BCR-ABL1+ B progenitor acute lymphoblastic leukemia (Ph+ B-ALL) is an aggressive disease that frequently responds poorly to currently available therapies. Alterations in
Michelle L. Churchman, Kathryn Evans, Jennifer Richmond, Alissa Robbins, Luke Jones, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Peter J. Houghton, Malcolm A. Smith, Richard B. Lock, Charles G. Mullighan
To date, the major target of biologic therapeutics in systemic lupus erythematosus (SLE) has been the B cell, which produces pathogenic autoantibodies. Recently, targeting type I IFN, which is elaborated by plasmacytoid dendritic cells (pDCs) in response to endosomal TLR7 and TLR9 stimulation by SLE immune complexes, has shown promising results. pDCs express high levels of the IL-3Rα chain (CD123), suggesting an alternative potential targeting strategy. We have developed an anti-CD123 monoclonal antibody, CSL362, and show here that it affects key cell types and cytokines that contribute to SLE. CSL362 potently depletes pDCs via antibody-dependent cell-mediated cytotoxicity, markedly reducing TLR7, TLR9, and SLE serum-induced IFN-α production and IFN-α-upregulated gene expression. The antibody also inhibits TLR7- and TLR9-induced plasmablast expansion by reducing IFN-α and IL-6 production. These effects are more pronounced than with IFN-α blockade alone, possibly because pDC depletion reduces production of other IFN subtypes, such as type III, as well as non-IFN proinflammatory cytokines, such as IL-6. In addition, CSL362 depletes basophils and inhibits IL-3 signaling. These effects were confirmed in cells derived from a heterogeneous population of SLE donors, various IFN-dependent autoimmune diseases, and healthy controls. We also demonstrate in vivo activity of CSL362 following its s.c. administration to cynomolgus monkeys. This spectrum of effects provides a preclinical rationale for the therapeutic evaluation of CSL362 in SLE.
Shereen Oon, Huy Huynh, Tsin Yee Tai, Milica Ng, Katherine Monaghan, Mark Biondo, Gino Vairo, Eugene Maraskovsky, Andrew D. Nash, Ian P. Wicks, Nicholas J. Wilson
Ryan J. Adam, Katherine B. Hisert, Jonathan D. Dodd, Brenda Grogan, Janice L. Launspach, Janel K. Barnes, Charles G. Gallagher, Jered P. Sieren, Thomas J. Gross, Anthony J. Fischer, Joseph E. Cavanaugh, Eric A. Hoffman, Pradeep K. Singh, Michael J. Welsh, Edward F. McKone, David A. Stoltz
Osteochondromas are common benign osteocartilaginous tumors in children and adolescents characterized by cartilage-capped bony projections on the surface of bones. These tumors often cause pain, deformity, fracture, and musculoskeletal dysfunction, and they occasionally undergo malignant transformation. The pathogenesis of osteochondromas remains poorly understood. Here, we demonstrate that nuclear factor of activated T cells c1 and c2 (NFATc1 and NFATc2) suppress osteochondromagenesis through individual and combinatorial mechanisms. In mice, conditional deletion of NFATc1 in mesenchymal limb progenitors, Scleraxis-expressing (Scx-expressing) tendoligamentous cells, or postnatally in
Xianpeng Ge, Kelly Tsang, Lizhi He, Roberto A. Garcia, Joerg Ermann, Fumitaka Mizoguchi, Minjie Zhang, Bin Zhou, Bin Zhou, Antonios O. Aliprantis
Xenografting primary tumor cells allows modeling of the heterogeneous natures of malignant diseases and the influences of the tissue microenvironment. Here, we demonstrate that xenografting primary chronic lymphocytic leukemia (CLL) B lymphocytes with activated autologous T cells into alymphoid mice results in considerable CLL B cell division and sizable T cell expansion. Nevertheless, most/all CD5+CD19+ cells are eventually lost, due in part to differentiation into antibody-secreting plasmablasts/plasma cells. CLL B cell differentiation is associated with isotype class switching and development of new
Piers E.M. Patten, Gerardo Ferrer, Shih-Shih Chen, Rita Simone, Sonia Marsilio, Xiao-Jie Yan, Zachary Gitto, Chaohui Yuan, Jonathan E. Kolitz, Jacqueline Barrientos, Steven L. Allen, Kanti R. Rai, Thomas MacCarthy, Charles C. Chu, Nicholas Chiorazzi
Conventional memory CD8+ T cells and mucosal-associated invariant T cells (MAIT cells) are found in blood, liver, and mucosal tissues and have similar effector potential following activation, specifically expression of IFN-γ and granzyme B. To better understand each subset’s unique contributions to immunity and pathology, we interrogated inflammation- and TCR-driven activation requirements using human memory CD8+ T and MAIT cells isolated from blood and mucosal tissue biopsies in ex vivo functional assays and single cell gene expression experiments. We found that MAIT cells had a robust IFN-γ and granzyme B response to inflammatory signals but limited responsiveness when stimulated directly via their TCR. Importantly, this is not due to an overall hyporesponsiveness to TCR signals. When delivered together, TCR and inflammatory signals synergize to elicit potent effector function in MAIT cells. This unique control of effector function allows MAIT cells to respond to the same TCR signal in a dichotomous and situation-specific manner. We propose that this could serve to prevent responses to antigen in noninflamed healthy mucosal tissue, while maintaining responsiveness and great sensitivity to inflammation-eliciting infections. We discuss the implications of these findings in context of inflammation-inducing damage to tissues such as BM transplant conditioning or HIV infection.
Chloe K. Slichter, Andrew McDavid, Hannah W. Miller, Greg Finak, Brenda J. Seymour, John P. McNevin, Gabriela Diaz, Julie L. Czartoski, M. Juliana McElrath, Raphael Gottardo, Martin Prlic
Loss of functional pancreatic β cells is a hallmark of both type 1 and 2 diabetes. Identifying the pathways that promote β cell proliferation and/or block β cell apoptosis is a potential strategy for diabetes therapy. The transcriptional coactivator Yes-associated protein (YAP), a major downstream effector of the Hippo signaling pathway, is a key regulator of organ size and tissue homeostasis by modulating cell proliferation and apoptosis. YAP is not expressed in mature primary human and mouse β cells. We aimed to identify whether reexpression of a constitutively active form of YAP promotes β cell proliferation/survival. Overexpression of YAP remarkably induced β cell proliferation in isolated human islets, while β cell function and functional identity genes were fully preserved. The transcription factor forkhead box M1 (FOXM1) was upregulated upon YAP overexpression and necessary for YAP-dependent β cell proliferation. YAP overexpression protected β cells from apoptosis triggered by multiple diabetic conditions. The small redox proteins thioredoxin-1 and thioredoxin-2 (Trx1/2) were upregulated by YAP; disruption of the Trx system revealed that Trx1/2 was required for the antiapoptotic action of YAP in insulin-producing β cells. Our data show the robust proproliferative and antiapoptotic function of YAP in pancreatic β cells. YAP reconstitution may represent a disease-modifying approach to restore a functional β cell mass in diabetes.
Ting Yuan, Sahar Rafizadeh, Zahra Azizi, Blaz Lupse, Kanaka Durga Devi Gorrepati, Sushil Awal, Jose Oberholzer, Kathrin Maedler, Amin Ardestani
The efficacy of allogeneic hematopoietic stem cell transplantation for hematologic malignancies is limited by the difficulty in suppressing graft-versus-host disease (GVHD) without compromising graft-versus-tumor (GVT) effects. We previously showed that RAS/MEK/ERK signaling depends on memory differentiation in human T cells, which confers susceptibility to selective inhibition of naive T cells. Actually, antineoplastic MEK inhibitors selectively suppress alloreactive T cells, sparing virus-specific T cells in vitro. Here, we show that trametinib, a MEK inhibitor clinically approved for melanoma, suppresses GVHD safely without affecting GVT effects in vivo. Trametinib prolonged survival of GVHD mice and attenuated GVHD symptoms and pathology in the gut and skin. It inhibited ERK1/2 phosphorylation and expansion of donor T cells, sparing Tregs and B cells. Although high-dose trametinib inhibited myeloid cell engraftment, low-dose trametinib suppressed GVHD without severe adverse events. Notably, trametinib facilitated the survival of mice transplanted with allogeneic T cells and P815 tumor cells with no residual P815 cells observed in the livers and spleens, whereas tacrolimus resulted in P815 expansion. These results confirm that trametinib selectively suppresses GVHD-inducing T cells while sparing antitumor T cells in vivo, which makes it a promising candidate for translational studies aimed at preventing or treating GVHD.
Hidekazu Itamura, Takero Shindo, Isao Tawara, Yasushi Kubota, Ryusho Kariya, Seiji Okada, Krishna V. Komanduri, Shinya Kimura
BACKGROUND. Some adult patients presenting with unexplained pyrexia, serositis, skin rashes, arthralgia, myalgia, and other symptoms commonly found in autoinflammatory disorders may not fit a specific diagnosis, either because their clinical phenotype is nondiagnostic or genetic tests are negative. We used the term undifferentiated systemic autoinflammatory disorder (uSAID) to describe such cases. Given that well-defined autoinflammatory diseases show responses to IL-1 blockade, we evaluated whether anakinra was useful for both diagnosing and treating uSAID patients.
METHODS. We performed a retrospective analysis of consecutive patients presenting with uSAID between 2012–2015 who were treated with the recombinant IL-1 receptor antagonist anakinra. uSAID was diagnosed after excluding malignancy, infection, and pathogenic mutations in known hereditary fever syndromes (HFS) genes and where clinical criteria for adult onset Still’s disease (AOSD) were not met.
RESULTS. A total of 11 patients presented with uSAID (5 males and 6 females), with a mean time to diagnosis of 3.5 years (1–8 years). Patients were unresponsive or only partially controlled on disease-modifying antirheumatic drug (DMARD)/steroid treatment. Anakinra controlled symptoms within 4–6 weeks of starting treatment in 9 of 11 cases. Two patients discontinued therapy — one due to incomplete response and another due to severe injection-site reactions.
CONCLUSION. This retrospective case series demonstrates that the spectrum of poorly defined autoinflammatory disorders that show responsiveness to anakinra is considerable. Anakinra seems a viable treatment option for these patients, who are unresponsive to standard steroid/DMARD treatments. Moreover, given the mechanisms of action, response to anakinra implicates underlying IL-1 dysregulation in the disease pathogenesis of responding uSAIDs patients.
Stephanie R. Harrison, Dennis McGonagle, Sharmin Nizam, Stephen Jarrett, Jeroen van der Hilst, Michael F. McDermott, Sinisa Savic
We have previously identified prohibitin (PHB) and annexin A2 (ANX2) as proteins interacting on the surface of vascular endothelial cells in white adipose tissue (WAT) of humans and mice. Here, we demonstrate that ANX2 and PHB also interact in adipocytes. Mice lacking ANX2 have normal WAT vascularization, adipogenesis, and glucose metabolism but display WAT hypotrophy due to reduced fatty acid uptake by WAT endothelium and adipocytes. By using cell culture systems in which ANX2/PHB binding is disrupted either genetically or through treatment with a blocking peptide, we show that fatty acid transport efficiency relies on this protein complex. We also provide evidence that the interaction between ANX2 and PHB mediates fatty acid transport from the endothelium into adipocytes. Moreover, we demonstrate that ANX2 and PHB form a complex with the fatty acid transporter CD36. Finally, we show that the colocalization of PHB and CD36 on adipocyte surface is induced by extracellular fatty acids. Together, our results suggest that an unrecognized biochemical interaction between ANX2 and PHB regulates CD36-mediated fatty acid transport in WAT, thus revealing a new potential pathway for intervention in metabolic diseases.
Ahmad Salameh, Alexes C. Daquinag, Daniela I. Staquicini, Zhiqiang An, Katherine A. Hajjar, Renata Pasqualini, Wadih Arap, Mikhail G. Kolonin
We recently identified a genome-wide genetic association of eosinophilic esophagitis (EoE) at 2p23 spanning the calpain 14 (
Benjamin P. Davis, Emily M. Stucke, M. Eyad Khorki, Vladislav A. Litosh, Jeffrey K. Rymer, Mark Rochman, Jared Travers, Leah C. Kottyan, Marc E. Rothenberg
Victor Gura, Matthew B. Rivara, Scott Bieber, Raj Munshi, Nancy Colobong Smith, Lori Linke, John Kundzins, Masoud Beizai, Carlos Ezon, Larry Kessler, Jonathan Himmelfarb
Successful induction of allograft tolerance has been achieved in nonhuman primates (NHPs) and humans via induction of transient hematopoietic chimerism. Since allograft tolerance was achieved in these recipients without durable chimerism, peripheral mechanisms are postulated to play a major role. Here, we report our studies of T cell immunity in NHP recipients that achieved long-term tolerance versus those that rejected the allograft (AR). All kidney, heart, and lung transplant recipients underwent simultaneous or delayed donor bone marrow transplantation (DBMT) following conditioning with a nonmyeloablative regimen. After DBMT, mixed lymphocyte culture with CFSE consistently revealed donor-specific loss of CD8+ T cell responses in tolerant (TOL) recipients, while marked CD4+ T cell proliferation in response to donor antigens was found to persist. Interestingly, a significant proportion of the proliferated CD4+ cells were FOXP3+ in TOL recipients, but not in AR or naive NHPs. In TOL recipients, CD4+FOXP3+ cell proliferation against donor antigens was greater than that observed against third-party antigens. Finally, the expanded Tregs appeared to be induced Tregs (iTregs) that were converted from non-Tregs. These data provide support for the hypothesis that specific induction of iTregs by donor antigens is key to long-term allograft tolerance induced by transient mixed chimerism.
Kiyohiko Hotta, Akihiro Aoyama, Tetsu Oura, Yohei Yamada, Makoto Tonsho, Kyu Ha Huh, Kento Kawai, David Schoenfeld, James S. Allan, Joren C. Madsen, Gilles Benichou, Rex-Neal Smith, Robert B. Colvin, David H. Sachs, A. Benedict Cosimi, Tatsuo Kawai
Immune-suppressed organ transplant recipients (OTRs) can develop catastrophic squamous cell carcinoma (SCC), characterized by multiple primary tumors, extensive body surface area involvement, or metastases. There are currently no curative systemic therapies available. We previously showed that IL-22 enhances SCC proliferation. Herein, we examined links between cyclosporine (CSA), IL-22, and SCC in patients, cell lines, and mice with UV light–induced SCC. Eighteen of 114 OTRs developed catastrophic SCC, which was strongly associated with CSA treatment. We found that CSA drives T cell polarization toward IL-22–producing T22 cells, and CSA treatment increased IL-22 receptor in SCC cells. SCC tissue from OTRs showed increased expression of IL-22RA1. CSA potentiated rescue by IL-22 of serum-starved SCC cells; treatment of SCC cells with IL-22 and CSA increased both their migratory and invasive capacity. In a UV-induced model of SCC in SKH-1 immunocompetent mice, treatment with anti–IL-22 antibody reduced tumor number and tumor burden. We found that catastrophic SCC in OTRs is associated with CSA use, which may be acting by favoring T22 polarization. Since anti–IL-22 antibody administration decreased tumor number and tumor burden in vivo, blockade of the IL-22 axis may be developed as a viable therapeutic option for catastrophic SCC.
Melody Abikhair, Hiroshi Mitsui, Valerie Yanofsky, Nazanin Roudiani, Channa Ovits, Teddy Bryan, Tatiana M. Oberyszyn, Kathleen L. Tober, Juana Gonzalez, James G. Krueger, Diane Felsen, John A. Carucci
We are delighted to publish the first set of articles in
Sarah Jackson
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a pregnancy-related condition caused by maternal antibodies binding an alloantigen on fetal platelets. In most cases the alloantigen is formed by a single amino acid, integrin β3 Leu33, referred to as human platelet antigen–1a (HPA-1a). Production of anti–HPA-1a antibodies likely depends on CD4+ T cells that recognize the same alloantigen in complex with the HLA-DRA/DRB3*01:01 molecule. While this complex is well characterized, T cell recognition of it is not. Here, to examine the nature of antigen recognition by HPA-1a–specific T cells, we assayed native and synthetic variants of the integrin β3 peptide antigen for binding to DRA/DRB3*01:01-positive antigen-presenting cells and for T cell activation. We found that HPA-1a–specific T cells recognize non-allogeneic integrin β3 residues anchored to DRA/DRB3*01:01 by the allogeneic Leu33, which itself is not directly recognized by these T cells. Furthermore, these T cell responses are diverse, with different T cells depending on different residues for recognition. This represents a unique form of indirect allorecognition in which a non-allogeneic peptide sequence becomes immunogenic by stable anchoring to MHC by an allogeneic residue.
Maria Therese Ahlen, Anne Husebekk, Ida Løken Killie, Bjørn Skogen, Tor Brynjar Stuge
Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of
Kyoungmin Park, Akira Mima, Qian Li, Christian Rask-Madsen, Pingnian He, Koji Mizutani, Sayaka Katagiri, Yasutaka Maeda, I-Hsien Wu, Mogher Khamaisi, Simone Rordam Preil, Ernesto Maddaloni, Ditte Sørensen, Lars Melholt Rasmussen, Paul L. Huang, George L. King
Emerging knowledge indicates the difficulty in categorizing unusual cystic fibrosis (CF) mutations, with regard to both pathogenic mechanism and theratype. As case in point, we present data concerning P67L mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a defect carried by a small number of individuals with CF and sometimes attributed to a channel conductance abnormality. Findings from our laboratory and others establish that P67L causes protein misfolding, disrupts maturation, confers gating defects, is thermally stable, and exhibits near normal conductance. These results provide one framework by which rare CF alleles such as P67L can be more comprehensively profiled vis-à-vis molecular pathogenesis. We also demonstrate that emerging CF treatments — ivacaftor and lumacaftor — can mediate pronounced pharmacologic activation of P67L CFTR. Infrequent CF alleles are often improperly characterized, in part, due to the small numbers of patients involved. Moreover, access to new personalized treatments among patients with ultra-orphan genotypes has been limited by difficulty arranging phase III clinical trials, and off-label prescribing has been impaired by high drug cost and difficulty arranging third party reimbursement. Rare CFTR mutations such as P67L are emblematic of the challenges to “precision” medicine, including use of the best available mechanistic knowledge to treat patients with unusual forms of disease.
Carleen M. Sabusap, Wei Wang, Carmel M. McNicholas, W. Joon Chung, Lianwu Fu, Hui Wen, Marina Mazur, Kevin L. Kirk, James F. Collawn, Jeong S. Hong, Eric J. Sorscher
Maternal obesity is proposed to alter the programming of metabolic systems in the offspring, increasing the risk for developing metabolic diseases; however, the cellular mechanisms remain poorly understood. Here, we used a nonhuman primate model to examine the impact of a maternal Western-style diet (WSD) alone, or in combination with obesity (Ob/WSD), on fetal skeletal muscle metabolism studied in the early third trimester. We find that fetal muscle responds to Ob/WSD by upregulating fatty acid metabolism, mitochondrial complex activity, and metabolic switches (CPT-1, PDK4) that promote lipid utilization over glucose oxidation. Ob/WSD fetuses also had reduced mitochondrial content, diminished oxidative capacity, and lower mitochondrial efficiency in muscle. The decrease in oxidative capacity and glucose metabolism was persistent in primary myotubes from Ob/WSD fetuses despite no additional lipid-induced stress. Switching obese mothers to a healthy diet prior to pregnancy did not improve fetal muscle mitochondrial function. Lastly, while maternal WSD alone led only to intermediary changes in fetal muscle metabolism, it was sufficient to increase oxidative damage and cellular stress. Our findings suggest that maternal obesity or WSD, alone or in combination, leads to programmed decreases in oxidative metabolism in offspring muscle. These alterations may have important implications for future health.
Carrie E. McCurdy, Simon Schenk, Byron Hetrick, Julie Houck, Brian G. Drew, Spencer Kaye, Melanie Lashbrook, Bryan C. Bergman, Diana L. Takahashi, Tyler A. Dean, Travis Nemkov, Ilya Gertsman, Kirk C. Hansen, Andrew Philp, Andrea L. Hevener, Adam J. Chicco, Kjersti M. Aagaard, Kevin L. Grove, Jacob E. Friedman
Pancreatic ductal adenocarcinoma (Pdac) is a malignancy with a poor prognosis due to difficulties in early detection. Although promising biomarkers are increasingly reported, such methods are not yet easy to apply clinically, mainly due to their low reproducibility or technical difficulties. In this study, we developed a convenient and sensitive method for quantifying aberrantly expressed satellite repeat RNAs in sera, which can be used to efficiently detect patients with Pdac. Here, we introduce a Tandem Repeat Amplification by nuclease Protection (TRAP) method combined with droplet digital PCR (ddPCR) to detect human satellite II (HSATII) RNAs, which are specifically expressed in human Pdacs at greater levels than normal tissues but are difficult to measure due to their repetitive sequences and irregularities. HSATII RNA core sequence levels in sera were significantly higher in Pdac patients compared with noncancer patients (median copy number: 14.75 and 3.17 per μl in the training set and 17.35 and 2.9 in the validation set, respectively). In addition, patients with intraductal papillary mucinous neoplasm (IPMN), a precancerous lesion of Pdac, could also be efficiently detected. This method can be routinely applied to screen patients with Pdac and high-risk patients, facilitating the development of preventive medicine for this disease.
Takahiro Kishikawa, Motoyuki Otsuka, Takeshi Yoshikawa, Motoko Ohno, Keisuke Yamamoto, Natsuyo Yamamoto, Ai Kotani, Kazuhiko Koike
Gastrointestinal graft-versus-host-disease (GI-GVHD) is a life-threatening complication occurring after allogeneic hematopoietic cell transplantation (HCT), and a blood biomarker that permits stratification of HCT patients according to their risk of developing GI-GVHD would greatly aid treatment planning. Through in-depth, large-scale proteomic profiling of presymptomatic samples, we identified a T cell population expressing both CD146, a cell adhesion molecule, and CCR5, a chemokine receptor that is upregulated as early as 14 days after transplantation in patients who develop GI-GVHD. The CD4+CD146+CCR5+ T cell population is Th17 prone and increased by ICOS stimulation. shRNA knockdown of CD146 in T cells reduced their transmigration through endothelial cells, and maraviroc, a CCR5 inhibitor, reduced chemotaxis of the CD4+CD146+CCR5+ T cell population toward CCL14. Mice that received CD146 shRNA–transduced human T cells did not lose weight, showed better survival, and had fewer CD4+CD146+CCR5+ T cells and less pathogenic Th17 infiltration in the intestine, even compared with mice receiving maraviroc with control shRNA–transduced human T cells. Furthermore, the frequency of CD4+CD146+CCR5+ Tregs was increased in GI-GVHD patients, and these cells showed increased plasticity toward Th17 upon ICOS stimulation. Our findings can be applied to early risk stratification, as well as specific preventative therapeutic strategies following HCT.
Wei Li, Liangyi Liu, Aurelie Gomez, Jilu Zhang, Abdulraouf Ramadan, Qing Zhang, Sung W. Choi, Peng Zhang, Joel K. Greenson, Chen Liu, Di Jiang, Elizabeth Virts, Stephanie L. Kelich, Hong Wei Chu, Ryan Flynn, Bruce R. Blazar, Helmut Hanenberg, Samir Hanash, Sophie Paczesny
Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s.
Sandhya Bangaru, Travis Nieusma, Nurgun Kose, Natalie J. Thornburg, Jessica A. Finn, Bryan S. Kaplan, Hannah G. King, Vidisha Singh, Rebecca M. Lampley, Gopal Sapparapu, Alberto Cisneros III, Kathryn M. Edwards, James C. Slaughter, Srilatha Edupuganti, Lilin Lai, Juergen A. Richt, Richard J. Webby, Andrew B. Ward, James E. Crowe Jr.
The tumor suppressor p53 blocks tumor progression in multiple tumor types. Radiation-induced cancer following exposure to radiation therapy or space travel may also be regulated by p53 because p53 has been proposed to respond to DNA damage to suppress tumorigenesis. Here, we investigate the role of p53 in lung carcinogenesis and lymphomagenesis in
Everett J. Moding, Hooney D. Min, Katherine D. Castle, Moiez Ali, Loretta Woodlief, Nerissa Williams, Yan Ma, Yongbaek Kim, Chang-Lung Lee, David G. Kirsch
Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (
Ram P. Naikawadi, Supparerk Disayabutr, Benat Mallavia, Matthew L. Donne, Gary Green, Janet L. La, Jason R. Rock, Mark R. Looney, Paul J. Wolters
Osteoarthritis (OA) of spine (facet joints [FJs]) is one of the major causes of severe low back pain and disability worldwide. The degeneration of facet cartilage is a hallmark of FJ OA. However, endogenous mechanisms that initiate degeneration of facet cartilage are unknown, and there are no disease-modifying therapies to stop FJ OA. In this study, we have identified microRNAs (small noncoding RNAs) as mediators of FJ cartilage degeneration. We first established a cohort of patients with varying degrees of facet cartilage degeneration (control group: normal or mild facet cartilage degeneration; FJ OA group: moderate to severe facet cartilage degeneration) and then screened 2,100 miRNAs and identified 2 miRNAs (miR-181a-5p and miR-4454) that were significantly elevated in FJ OA cartilage compared with control facet cartilage. We further explored their role, function, and signaling mechanisms using computational, in vitro functional, and in vivo studies. We specifically indicate that miR-181a-5p and miR-4454 are involved in promoting inflammatory, catabolic, and cell death activity in FJ chondrocytes. This is the first report to our knowledge that identifies miR-181a-5p and miR-4454 as mediators of cartilage degeneration in FJs and potential therapeutic targets for stopping cartilage degeneration.
Akihiro Nakamura, Y. Raja Rampersaud, Anirudh Sharma, Stephen J. Lewis, Brian Wu, Poulami Datta, Kala Sundararajan, Helal Endisha, Evgeny Rossomacha, Jason S. Rockel, Igor Jurisica, Mohit Kapoor
High-grade serous ovarian carcinoma (HGS-OvCa) harbors p53 mutations and can originate from the epithelial cell compartment of the fallopian tube fimbriae. From this site, neoplastic cells detach, survive in the peritoneal cavity, and form cellular clusters that intercalate into the mesothelium to form ovarian and peritoneal masses. To examine the contribution of mutant p53 to phenotypic alterations associated with HGS-OvCA, we developed live-cell microscopy assays that recapitulate these early events in cultured fallopian tube nonciliated epithelial (FNE) cells. Expression of stabilizing mutant variants of p53, but not depletion of endogenous wild-type p53, in FNE cells promoted survival and cell-cell aggregation under conditions of cell detachment, leading to the formation of cell clusters with mesothelium-intercalation capacity. Mutant p53R175H-induced phenotypes were dependent on fibronectin production, α5β1 fibronectin receptor engagement, and
Marcin P. Iwanicki, Hsing-Yu Chen, Claudia Iavarone, Ioannis K. Zervantonakis, Taru Muranen, Marián Novak, Tan A. Ince, Ronny Drapkin, Joan S. Brugge
Cancer testis antigens (CTAs) are of clinical interest as biomarkers and present valuable targets for immunotherapy. To comprehensively characterize the CTA landscape of non–small-cell lung cancer (NSCLC), we compared RNAseq data from 199 NSCLC tissues to the normal transcriptome of 142 samples from 32 different normal organs. Of 232 CTAs currently annotated in the Caner Testis Database (CTdatabase), 96 were confirmed in NSCLC. To obtain an unbiased CTA profile of NSCLC, we applied stringent criteria on our RNAseq data set and defined 90 genes as CTAs, of which 55 genes were not annotated in the CTdatabase, thus representing potential new CTAs. Cluster analysis revealed that CTA expression is histology dependent and concurrent expression is common. IHC confirmed tissue-specific protein expression of selected new CTAs (TKTL1, TGIF2LX, VCX, and CXORF67). Furthermore, methylation was identified as a regulatory mechanism of CTA expression based on independent data from The Cancer Genome Atlas. The proposed prognostic impact of CTAs in lung cancer was not confirmed, neither in our RNAseq cohort nor in an independent meta-analysis of 1,117 NSCLC cases. In summary, we defined a set of 90 reliable CTAs, including information on protein expression, methylation, and survival association. The detailed RNAseq catalog can guide biomarker studies and efforts to identify targets for immunotherapeutic strategies.
Dijana Djureinovic, Björn M. Hallström, Masafumi Horie, Johanna Sofia Margareta Mattsson, Linnea La Fleur, Linn Fagerberg, Hans Brunnström, Cecilia Lindskog, Katrin Madjar, Jörg Rahnenführer, Simon Ekman, Elisabeth Ståhle, Hirsh Koyi, Eva Brandén, Karolina Edlund, Jan G. Hengstler, Mats Lambe, Akira Saito, Johan Botling, Fredrik Pontén, Mathias Uhlén, Patrick Micke
Dilated cardiomyopathy (DCM) is defined by progressive functional and structural changes. We performed RNA-seq at different stages of disease to define molecular signaling in the progression from pre-DCM hearts to DCM and overt heart failure (HF) using a genetic model of DCM (phospholamban missense mutation, PLNR9C/+). Pre-DCM hearts were phenotypically normal yet displayed proliferation of nonmyocytes (59% relative increase vs. WT,
Michael A. Burke, Stephen Chang, Hiroko Wakimoto, Joshua M. Gorham, David A. Conner, Danos C. Christodoulou, Michael G. Parfenov, Steve R. DePalma, Seda Eminaga, Tetsuo Konno, Jonathan G. Seidman, Christine E. Seidman
BACKGROUND. Paneth cell dysfunction has been implicated in a subset of Crohn’s disease (CD) patients. We previously stratified clinical outcomes of CD patients by using Paneth cell phenotypes, which we defined by the intracellular distribution of antimicrobial proteins. Animal studies suggest that Paneth cells shape the intestinal microbiome. However, it is unclear whether Paneth cell phenotypes alter the microbiome complexity in CD subjects. Therefore, we analyzed the correlation of Paneth cell phenotypes with mucosal microbiome composition and ileal RNA expression in pediatric CD and noninflammatory bowel disease (non-IBD) patients.
METHODS. Pediatric CD (
RESULTS. The prevalence of abnormal Paneth cells was higher in pediatric versus adult CD cohorts. For pediatric CD patients, those with abnormal Paneth cells showed significant changes in their ileal mucosal microbiome, highlighted by reduced protective microbes and enriched proinflammatory microbes. Ileal transcriptome profiles showed reduced transcripts for genes that control oxidative phosphorylation in CD patients with abnormal Paneth cells. These transcriptional changes in turn were correlated with specific microbiome alterations. In non-IBD patients, a subset contained abnormal Paneth cells. However, this subset was not associated with alterations in the microbiome or host transcriptome.
CONCLUSION. Paneth cell abnormalities in human subjects are associated with mucosal dysbiosis in the context of CD, and these changes are associated with alterations in oxidative phosphorylation, potentially in a feedback loop.
FUNDING. The research was funded by Helmsley Charitable Trust (to T.S. Stappenbeck, R.J. Xavier, and D.P.B. McGovern), Crohn’s and Colitis Foundation of America (to N.H. Salzman, T.S. Stappenbeck, R.J. Xavier, and C. Huttenhower), and Doris Duke Charitable Foundation grant 2014103 (to T.C. Liu).
Ta-Chiang Liu, Bhaskar Gurram, Megan T. Baldridge, Richard Head, Vy Lam, Chengwei Luo, Yumei Cao, Pippa Simpson, Michael Hayward, Mary L. Holtz, Pavlos Bousounis, Joshua Noe, Diana Lerner, Jose Cabrera, Vincent Biank, Michael Stephens, Curtis Huttenhower, Dermot P.B. McGovern, Ramnik J. Xavier, Thaddeus S. Stappenbeck, Nita H. Salzman
Infections are an important cause of morbidity and mortality in patients with decompensated cirrhosis and ascites. Hypothesizing that innate immune dysfunction contributes to susceptibility to infection, we assessed ascitic fluid macrophage phenotype and function. The expression of complement receptor of the immunoglobulin superfamily (CRIg) and CCR2 defined two phenotypically and functionally distinct peritoneal macrophage subpopulations. The proportion of CRIghi macrophages differed between patients and in the same patient over time, and a high proportion of CRIghi macrophages was associated with reduced disease severity (model for end-stage liver disease) score. As compared with CRIglo macrophages, CRIghi macrophages were highly phagocytic and displayed enhanced antimicrobial effector activity. Transcriptional profiling by RNA sequencing and comparison with human macrophage and murine peritoneal macrophage expression signatures highlighted similarities among CRIghi cells, human macrophages, and mouse F4/80hi resident peritoneal macrophages and among CRIglo macrophages, human monocytes, and mouse F4/80lo monocyte-derived peritoneal macrophages. These data suggest that CRIghi and CRIglo macrophages may represent a tissue-resident population and a monocyte-derived population, respectively. In conclusion, ascites fluid macrophage subset distribution and phagocytic capacity is highly variable among patients with chronic liver disease. Regulating the numbers and/or functions of these macrophage populations could provide therapeutic opportunities in cirrhotic patients.
Katharine M. Irvine, Xuan Banh, Victoria L. Gadd, Kyle K. Wojcik, Juliana K. Ariffin, Sara Jose, Samuel Lukowski, Gregory J. Baillie, Matthew J. Sweet, Elizabeth E. Powell
Atopic dermatitis (AD) is characterized by reduced barrier function, reduced innate immune activation, and susceptibility to
Ian A. Myles, Kelli W. Williams, Jensen D. Reckhow, Momodou L. Jammeh, Nathan B. Pincus, Inka Sastalla, Danial Saleem, Kelly D. Stone, Sandip K. Datta
Pulmonary arterial (PA) stiffness is associated with increased mortality in patients with pulmonary hypertension (PH); however, the role of PA stiffening in the pathogenesis of PH remains elusive. Here, we show that distal vascular matrix stiffening is an early mechanobiological regulator of experimental PH. We identify cyclooxygenase-2 (COX-2) suppression and corresponding reduction in prostaglandin production as pivotal regulators of stiffness-dependent vascular cell activation. Atomic force microscopy microindentation demonstrated early PA stiffening in experimental PH and human lung tissue. Pulmonary artery smooth muscle cells (PASMC) grown on substrates with the stiffness of remodeled PAs showed increased proliferation, decreased apoptosis, exaggerated contraction, enhanced matrix deposition, and reduced COX-2–derived prostanoid production compared with cells grown on substrates approximating normal PA stiffness. Treatment with a prostaglandin I2 analog abrogated monocrotaline-induced PA stiffening and attenuated stiffness-dependent increases in proliferation, matrix deposition, and contraction in PASMC. Our results suggest a pivotal role for early PA stiffening in PH and demonstrate the therapeutic potential of interrupting mechanobiological feedback amplification of vascular remodeling in experimental PH.
Fei Liu, Christina Mallarino Haeger, Paul B. Dieffenbach, Delphine Sicard, Izabela Chrobak, Anna Maria F. Coronata, Margarita M. Suárez Velandia, Sally Vitali, Romain A. Colas, Paul C. Norris, Aleksandar Marinković, Xiaoli Liu, Jun Ma, Chase D. Rose, Seon-Jin Lee, Suzy A.A. Comhair, Serpil C. Erzurum, Jacob D. McDonald, Charles N. Serhan, Stephen R. Walsh, Daniel J. Tschumperlin, Laura E. Fredenburgh
Mucous membrane pemphigoid (MMP) is a systemic mucosal scarring disease, commonly causing blindness, for which there is no antifibrotic therapy. Aldehyde dehydrogenase family 1 (ALDH1) is upregulated in both ocular MMP (OMMP) conjunctiva and cultured fibroblasts. Application of the ALDH metabolite, retinoic acid (RA), to normal human conjunctival fibroblasts in vitro induced a diseased phenotype. Conversely, application of ALDH inhibitors, including disulfiram, to OMMP fibroblasts in vitro restored their functionality to that of normal controls. ALDH1 is also upregulated in the mucosa of the mouse model of scarring allergic eye disease (AED), used here as a surrogate for OMMP, in which topical application of disulfiram decreased fibrosis in vivo. These data suggest that progressive scarring in OMMP results from ALDH/RA fibroblast autoregulation, that the ALDH1 subfamily has a central role in immune-mediated ocular mucosal scarring, and that ALDH inhibition with disulfiram is a potential and readily translatable antifibrotic therapy.
Sarah D. Ahadome, David J. Abraham, Suryanarayana Rayapureddi, Valerie P. Saw, Daniel R. Saban, Virginia L. Calder, Jill T. Norman, Markella Ponticos, Julie T. Daniels, John K. Dart
Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC–derived ALDH was associated with 9-
Sarah D. Ahadome, Rose Mathew, Nancy J. Reyes, Priyatham S. Mettu, Scott W. Cousins, Virginia L. Calder, Daniel R. Saban
Center for Transplantation Sciences, Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts, USA.
Maria Lucia L. Madariaga, Philip J. Spencer, Kumaran Shanmugarajah, Kerry A. Crisalli, David C. Chang, James F. Markmann, Nahel Elias, A. Benedict Cosimi, David H. Sachs, Tatsuo Kawai
The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss,
Anup Sood, Alexandra M. Miller, Edi Brogi, Yunxia Sui, Joshua Armenia, Elizabeth McDonough, Alberto Santamaria-Pang, Sean Carlin, Aleksandra Stamper, Carl Campos, Zhengyu Pang, Qing Li, Elisa Port, Thomas G. Graeber, Nikolaus Schultz, Fiona Ginty, Steven M. Larson, Ingo K. Mellinghoff
Current methods of drug screening in human blood focus on the immediate products of the affected pathway and mostly rely on approaches that lack sensitivity and the capacity for multiplex analysis. We have developed a sensitive and selective method based on ultra-performance liquid chromatography–tandem mass spectrometry to scan the effect of drugs on the bioactive eicosanoid lipidome in vitro and ex vivo. Using small sample sizes, we can reproducibly measure a broad spectrum of eicosanoids in human blood and capture drug-induced substrate rediversion and unexpected shifts in product formation. Microsomal prostaglandin E synthase-1 (mPGES-1) is an antiinflammatory drug target alternative to COX-1/-2. Contrasting effects of targeting mPGES-1 versus COX-1/-2, due to differential substrate shifts across the lipidome, were observed and can be used to rationalize and evaluate drug combinations. Finally, the in vitro results were extrapolated to ex vivo studies by administration of the COX-2 inhibitor, celecoxib, to volunteers, illustrating how this approach can be used to integrate preclinical and clinical studies during drug development.
Liudmila L. Mazaleuskaya, John A. Lawson, Xuanwen Li, Gregory Grant, Clementina Mesaros, Tilo Grosser, Ian A. Blair, Emanuela Ricciotti, Garret A. FitzGerald
DC vaccination with autologous tumor lysate has demonstrated promising results for the treatment of glioblastoma (GBM) in preclinical and clinical studies. While the vaccine appears capable of inducing T cell infiltration into tumors, the effectiveness of active vaccination in progressively growing tumors is less profound. In parallel, a number of studies have identified negative costimulatory pathways, such as programmed death 1/programmed death ligand 1 (PD-1/PD-L1), as relevant mediators of the intratumoral immune responses. Clinical responses to PD-1 pathway inhibition, however, have also been varied. To evaluate the relevance to established glioma, the effects of PD-1 blockade following DC vaccination were tested in intracranial (i.c.) glioma tumor–bearing mice. Treatment with both DC vaccination and PD-1 mAb blockade resulted in long-term survival, while neither agent alone induced a survival benefit in animals with larger, established tumors. This survival benefit was completely dependent on CD8+ T cells. Additionally, DC vaccine plus PD-1 mAb blockade resulted in the upregulation of integrin homing and immunologic memory markers on tumor-infiltrating lymphocytes (TILs). In clinical samples, DC vaccination in GBM patients was associated with upregulation of PD-1 expression in vivo, while ex vivo blockade of PD-1 on freshly isolated TILs dramatically enhanced autologous tumor cell cytolysis. These findings strongly suggest that the PD-1/PD-L1 pathway plays an important role in the adaptive immune resistance of established GBM in response to antitumor active vaccination and provide us with a rationale for the clinical translation of this combination therapy.
Joseph P. Antonios, Horacio Soto, Richard G. Everson, Joey Orpilla, Diana Moughon, Namjo Shin, Shaina Sedighim, William H. Yong, Gang Li, Timothy F. Cloughesy, Linda M. Liau, Robert M. Prins
Early after HIV infection there is substantial depletion of CD4+ T cells in the gastrointestinal (GI) tract lamina propria (LP), with associated epithelial barrier damage, leading to microbial translocation and systemic inflammation and immune activation. In this study, we analyzed these early events in the GI tract in a cohort of Thai acute HIV-infected patients and determined the effect of early combination antiretroviral treatment (cART). HIV-uninfected and chronically and acutely HIV-infected patients at different Fiebig stages (I–V) underwent colonic biopsies and then received cART. Immunohistochemistry and quantitative image analysis were performed on cross-sectional and longitudinal colon biopsy specimens (day 0 to week 96) to measure GI tract damage (infiltration of polymorphonuclear cells), inflammation (Mx1, TNF-α), immune activation (Ki-67), and the CD4+ T cell population in the LP. The magnitude of GI tract damage, immune activation, and inflammation was significantly increased, with significantly depleted CD4+ T cells in the LP in all acutely infected groups prior to cART compared with HIV-uninfected control participants. While most patients treated during acute infection resolved GI tract inflammation and immune activation back to baseline levels after 24 weeks of cART, most acutely infected participants did not restore their CD4+ T cells after 96 weeks of cART.
Claire Deleage, Alexandra Schuetz, W. Gregory Alvord, Leslie Johnston, Xing-Pei Hao, David R. Morcock, Rungsun Rerknimitr, James L.K. Fletcher, Suwanna Puttamaswin, Nittaya Phanuphak, Robin Dewar, Joseph M. McCune, Irini Sereti, Merlin Robb, Jerome H. Kim, Timothy W. Schacker, Peter Hunt, Jeffrey D. Lifson, Jintanat Ananworanich, Jacob D. Estes, on behalf of the RV254/SEARCH 010 and RV304/SEARCH 013 Study Groups
Little is known about the role of IL-3 in multiple sclerosis (MS) in humans and in experimental autoimmune encephalomyelitis (EAE). Using myelin oligodendrocyte glycoprotein (MOG) peptide–induced EAE, we show that CD4+ T cells are the main source of IL-3 and that cerebral IL-3 expression correlates with the influx of T cells into the brain. Blockade of IL-3 with monoclonal antibodies, analysis of IL-3 deficient mice, and adoptive transfer of leukocytes demonstrate that IL-3 plays an important role for development of clinical symptoms of EAE, for migration of leukocytes into the brain, and for cerebral expression of adhesion molecules and chemokines. In contrast, injection of recombinant IL-3 exacerbates EAE symptoms and cerebral inflammation. In patients with relapsing-remitting MS (RRMS), IL-3 expression by T cells is markedly upregulated during episodes of relapse. Our data indicate that IL-3 plays an important role in EAE and may represent a new target for treatment of MS.
Kerstin Renner, Sonja Hellerbrand, Fabian Hermann, Christine Riedhammer, Yvonne Talke, Gabriela Schiechl, Manuel Rodriguez Gomez, Simone Kutzi, Dagmar Halbritter, Nicole Goebel, Hilke Brühl, Robert Weissert, Matthias Mack
Ectopic lymphoid tissues (ELT) can be found in multiple sclerosis (MS) and other organ-specific inflammatory conditions. Whether ELT in the meninges of central nervous system (CNS) autoimmune disease exhibit local germinal center (GC) activity remains unknown. In an experimental autoimmune encephalomyelitis model of CNS autoimmunity, we found activation-induced cytidine deaminase, a GC-defining enzyme, in meningeal ELT (mELT) densely populated by B and T cells. To determine GC activity in mELT, we excised meningeal lymphoid aggregates using laser capture microscopy and evaluated B cell repertoires in mELT and secondary lymphoid organs by next-generation immune repertoire sequencing. We found immunoglobulin heavy chain variable region sequences that were unique to mELT and had accumulated functionally relevant somatic mutations, together indicating localized antigen-driven affinity maturation. Our results suggest that B cells in mELT actively participate in CNS autoimmunity, which may be relevant to mELT in MS and ELT in other chronic inflammatory conditions.
Klaus Lehmann-Horn, Sheng-zhi Wang, Sharon A. Sagan, Scott S. Zamvil, H.-Christian von Büdingen
Jennifer K Roe, Niclas Thomas, Eliza Gil, Katharine Best, Evdokia Tsaliki, Stephen Morris‑Jones, Sian Stafford, Nandi Simpson, Karolina D Witt, Benjamin Chain, Robert F Miller, Adrian Martineau, Mahdad Noursadeghi
Lymphangioleiomyomatosis (LAM) is a rare lung disease of women that leads to progressive cyst formation and accelerated loss of pulmonary function. Neoplastic smooth muscle cells from an unknown source metastasize to the lung and drive destructive remodeling. Given the role of NK cells in immune surveillance, we postulated that NK cell activating receptors and their cognate ligands are involved in LAM pathogenesis. We found that ligands for the NKG2D activating receptor UL-16 binding protein 2 (ULBP2) and ULBP3 are localized in cystic LAM lesions and pulmonary nodules. We found elevated soluble serum ULBP2 (mean = 575 pg/ml ± 142) in 50 of 100 subjects and ULBP3 in 30 of 100 (mean = 8,300 pg/ml ± 1,515) subjects. LAM patients had fewer circulating NKG2D+ NK cells and decreased NKG2D surface expression. Lung function decline was associated with soluble NKG2D ligand (sNKG2DL) detection. The greatest rate of decline forced expiratory volume in 1 second (FEV1, –124 ± 30 ml/year) in the 48 months after enrollment (NHLBI LAM Registry) occurred in patients expressing both ULBP2 and ULBP3, whereas patients with undetectable sNKG2DL levels had the lowest rate of FEV1 decline (–32.7 ± 10 ml/year). These data suggest a role for NK cells, sNKG2DL, and the innate immune system in LAM pathogenesis.
Andrew R. Osterburg, Rebecca L. Nelson, Benyamin Z. Yaniv, Rachel Foot, Walter R.F. Donica, Madison A. Nashu, Huan Liu, Kathryn A. Wikenheiser-Brokamp, Joel Moss, Nishant Gupta, Francis X. McCormack, Michael T. Borchers
It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant–mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2+ monocyte–derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury.
Wenjun Li, Hsi-Min Hsiao, Ryuji Higashikubo, Brian T. Saunders, Ankit Bharat, Daniel R. Goldstein, Alexander S. Krupnick, Andrew E. Gelman, Kory J. Lavine, Daniel Kreisel
The small intestine has an underappreciated role as a lipid storage organ. Under conditions of high dietary fat intake, enterocytes can minimize the extent of postprandial lipemia by storing newly absorbed dietary fat in cytoplasmic lipid droplets. Lipid droplets can be subsequently mobilized for the production of chylomicrons. The mechanisms that regulate this process are poorly understood. We report here that the milk protein Mfge8 regulates hydrolysis of cytoplasmic lipid droplets in enterocytes after interacting with the αvβ3 and αvβ5 integrins. Mice deficient in Mfge8 or the αvβ3 and αvβ5 integrins accumulate excess cytoplasmic lipid droplets after a fat challenge. Mechanistically, interruption of the Mfge8-integrin axis leads to impaired enterocyte intracellular triglyceride hydrolase activity in vitro and in vivo. Furthermore, Mfge8 increases triglyceride hydrolase activity through a PI3 kinase/mTORC2–dependent signaling pathway. These data identify a key role for Mfge8 and the αvβ3 and αvβ5 integrins in regulating enterocyte lipid processing.
Amin Khalifeh-Soltani, Deepti Gupta, Arnold Ha, Jahangir Iqbal, Mahmood Hussain, Michael J. Podolsky, Kamran Atabai
Pentraxin-2 (PTX-2), also known as serum amyloid P component (SAP/APCS), is a constitutive, antiinflammatory, innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in
Naoki Nakagawa, Luke Barron, Ivan G. Gomez, Bryce G. Johnson, Allie M. Roach, Sei Kameoka, Richard M. Jack, Mark L. Lupher Jr., Sina A. Gharib, Jeremy S. Duffield
In cystic fibrosis (CF), loss of CF transmembrane conductance regulator (CFTR) anion channel activity causes airway surface liquid (ASL) pH to become acidic, which impairs airway host defenses. One potential therapeutic approach is to correct the acidic pH in CF airways by aerosolizing HCO3– and/or nonbicarbonate pH buffers. Here, we show that raising ASL pH with inhaled HCO3– increased pH. However, the effect was transient, and pH returned to baseline values within 30 minutes. Tromethamine (Tham) is a buffer with a long serum half-life used as an i.v. formulation to treat metabolic acidosis. We found that Tham aerosols increased ASL pH in vivo for at least 2 hours and enhanced bacterial killing. Inhaled hypertonic saline (7% NaCl) is delivered to people with CF in an attempt to promote mucus clearance. Because an increased ionic strength inhibits ASL antimicrobial factors, we added Tham to hypertonic saline and applied it to CF sputum. We found that Tham alone and in combination with hypertonic saline increased pH and enhanced bacterial killing. These findings suggest that aerosolizing the HCO3–-independent buffer Tham, either alone or in combination with hypertonic saline, might be of therapeutic benefit in CF airway disease.
Mahmoud H. Abou Alaiwa, Janice L. Launspach, Kelsey A. Sheets, Jade A. Rivera, Nicholas D. Gansemer, Peter J. Taft, Peter S. Thorne, Michael J. Welsh, David A. Stoltz, Joseph Zabner
Although a close connection between uterine regeneration and successful pregnancy in both humans and mice has been consistently observed, its molecular basis remains unclear. We here established a mouse model of decellularized uterine matrix (DUM) transplantation. Resected mouse uteri were processed with SDS to make DUMs without any intact cells. DUMs were transplanted into the mouse uteri with artificially induced defects, and all the uterine layers were recovered at the DUM transplantation sites within a month. In the regenerated uteri, normal hormone responsiveness in early pregnancy was observed, suggesting the regeneration of functional uteri. Uterine epithelial cells rapidly migrated and formed a normal uterine epithelial layer within a week, indicating a robust epithelial-regenerating capacity. Stromal and myometrial regeneration occurred following epithelial regeneration. In ovariectomized mice, uterine regeneration of the DUM transplantation was similarly observed, suggesting that ovarian hormones are not essential for this regeneration process. Importantly, the regenerating epithelium around the DUM demonstrated heightened STAT3 phosphorylation and cell proliferation, which was suppressed in uteri of
Takehiro Hiraoka, Yasushi Hirota, Tomoko Saito-Fujita, Mitsunori Matsuo, Mahiro Egashira, Leona Matsumoto, Hirofumi Haraguchi, Sudhansu K. Dey, Katsuko S. Furukawa, Tomoyuki Fujii, Yutaka Osuga
Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the
Michihiro Hashimoto, Azusa Asai, Hiroyuki Kawagishi, Ryuta Mikawa, Yuji Iwashita, Kazuki Kanayama, Kazushi Sugimoto, Tadashi Sato, Mitsuo Maruyama, Masataka Sugimoto
Genome-wide association studies of asthma have identified genetic variants in the
Erin D. Gordon, Joe Palandra, Agata Wesolowska-Andersen, Lando Ringel, Cydney L. Rios, Marrah E. Lachowicz-Scroggins, Louis Z. Sharp, Jamie L. Everman, Hannah J. MacLeod, Jae W. Lee, Robert J. Mason, Michael A. Matthay, Richard T. Sheldon, Michael C. Peters, Karl H. Nocka, John V. Fahy, Max A. Seibold
Necrotizing fasciitis caused by group A streptococcus (GAS) is a life-threatening, rapidly progressing infection. At present, biofilm is not recognized as a potential problem in GAS necrotizing soft tissue infections (NSTI), as it is typically linked to chronic infections or associated with foreign devices. Here, we present a case of a previously healthy male presenting with NSTI caused by GAS. The infection persisted over 24 days, and the surgeon documented the presence of a “thick layer biofilm” in the fascia. Subsequent analysis of NSTI patient tissue biopsies prospectively included in a multicenter study revealed multiple areas of biofilm in 32% of the patients studied. Biopsies associated with biofilm formation were characterized by massive bacterial load, a pronounced inflammatory response, and clinical signs of more severe tissue involvement. In vitro infections of a human skin tissue model with GAS NSTI isolates also revealed multilayered fibrous biofilm structures, which were found to be under the control of the global Nra gene regulator. The finding of GAS biofilm formation in NSTIs emphasizes the urgent need for biofilm to be considered as a potential complicating microbiological feature of GAS NSTI and, consequently, emphasizes reconsideration of antibiotic treatment protocols.
Nikolai Siemens, Bhavya Chakrakodi, Srikanth Mairpady Shambat, Marina Morgan, Helena Bergsten, Ole Hyldegaard, Steinar Skrede, Per Arnell, Martin B. Madsen, Linda Johansson, INFECT Study Group, Julius Juarez, Lidija Bosnjak, Matthias Mörgelin, Mattias Svensson, Anna Norrby-Teglund
BACKGROUND. Children treated with cerebrospinal fluid (CSF) shunts to manage hydrocephalus frequently develop shunt failure and/or infections, conditions that present with overlapping symptoms. The potential life-threatening nature of shunt infections requires rapid diagnosis; however, traditional microbiology is time consuming, expensive, and potentially unreliable. We set out to identify a biomarker that would identify shunt infection.
METHODS. CSF was assayed for the soluble membrane attack complex (sMAC) by ELISA in patients with suspected shunt failure or infection. CSF was obtained at the time of initial surgical intervention. Statistical analysis was performed to assess the diagnostic potential of sMAC in pyogenic-infected versus noninfected patients.
RESULTS. Children with pyogenic shunt infection had significantly increased sMAC levels compared with noninfected patients (3,211 ± 1,111 ng/ml vs. 26 ± 3.8 ng/ml,
CONCLUSION. Elevated CSF sMAC levels are both sensitive and specific for diagnosing pyogenic shunt infection and may serve as a useful prognostic biomarker during recovery from infection.
FUNDING. This work was supported in part by the Impact Fund of Children’s of Alabama.
Theresa N. Ramos, Anastasia A. Arynchyna, Tessa E. Blackburn, Scott R. Barnum, James M. Johnston
Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design.
Giridhar Murlidharan, Andrew Crowther, Rebecca A. Reardon, Juan Song, Aravind Asokan
Systemic sclerosis (SSc) is a potentially fatal autoimmune disorder with limited therapeutic options. Sclerodermatous graft versus host disease (sclGvHD), induced by transfer of B10.D2 splenocytes into BALB/c
Katia Urso, David Alvarez, Viviana Cremasco, Kelly Tsang, Angelo Grauel, Robert Lafyatis, Ulrich H. von Andrian, Joerg Ermann, Antonios O. Aliprantis
Ashutosh Lal, Esteban Gomez, Cassandra Calloway
The stasis of mucus secretions in the lungs of cystic fibrosis (CF) patients leads to recurrent infections and pulmonary exacerbations, resulting in decreased survival. Prior studies have assessed the biochemical and biophysical features of airway mucus in individuals with CF. However, these measurements are unable to probe mucus structure on microscopic length scales relevant to key players in the progression of CF-related lung disease, namely, viruses, bacteria, and neutrophils. In this study, we quantitatively determined sputum microstructure based on the diffusion of muco-inert nanoparticle probes in CF sputum and found that a reduction in sputum mesh pore size is characteristic of CF patients with reduced lung function, as indicated by measured FEV1. We also discovered that the effect of ex vivo treatment of CF sputum with rhDNase I (Pulmozyme) on microstructure is dependent upon the time interval between the most recent inhaled rhDNase I treatment and the sample collection. Microstructure of mucus may serve as a marker for the extent of CF lung disease and as a parameter for assessing the effectiveness of mucus-altering agents.
Gregg A. Duncan, James Jung, Andrea Joseph, Abigail L. Thaxton, Natalie E. West, Michael P. Boyle, Justin Hanes, Jung Soo Suk
Systemic mitochondrial energy deficiency is implicated in the pathophysiology of many age-related human diseases. Currently available tools to estimate mitochondrial oxidative phosphorylation (OXPHOS) capacity in skeletal muscle in vivo lack high anatomic resolution. Muscle groups vary with respect to their contractile and metabolic properties. Therefore, muscle group–specific estimates of OXPHOS would be advantageous. To address this need, a noninvasive creatine chemical exchange saturation transfer (CrCEST) MRI technique has recently been developed, which provides a measure of free creatine. After exercise, skeletal muscle can be imaged with CrCEST in order to make muscle group–specific measurements of OXPHOS capacity, reflected in the recovery rate (τCr) of free Cr. In this study, we found that individuals with genetic mitochondrial diseases had significantly (
Catherine DeBrosse, Ravi Prakash Reddy Nanga, Neil Wilson, Kevin D’Aquilla, Mark Elliott, Hari Hariharan, Felicia Yan, Kristin Wade, Sara Nguyen, Diana Worsley, Chevonne Parris-Skeete, Elizabeth McCormick, Rui Xiao, Zuela Zolkipli Cunningham, Lauren Fishbein, Katherine L. Nathanson, David R. Lynch, Virginia A. Stallings, Marc Yudkoff, Marni J. Falk, Ravinder Reddy, Shana E. McCormack
The adaptive immune repertoire plays a critical role in type 1 diabetes (T1D) pathogenesis. However, efforts to characterize B cell and T cell receptor (TCR) profiles in T1D subjects have been largely limited to peripheral blood sampling and restricted to known antigens. To address this, we collected pancreatic draining lymph nodes (pLN), “irrelevant” nonpancreatic draining lymph nodes, peripheral blood mononuclear cells (PBMC), and splenocytes from T1D subjects (
Howard R. Seay, Erik Yusko, Stephanie J. Rothweiler, Lin Zhang, Amanda L. Posgai, Martha Campbell-Thompson, Marissa Vignali, Ryan O. Emerson, John S. Kaddis, Dave Ko, Maki Nakayama, Mia J. Smith, John C. Cambier, Alberto Pugliese, Mark A. Atkinson, Harlan S. Robins, Todd M. Brusko
Metastatic dissemination of cancer cells, which accounts for 90% of cancer mortality, is the ultimate hallmark of malignancy. Growing evidence suggests that blood platelets have a predominant role in tumor metastasis; however, the molecular mechanisms involved remain elusive. Here, we demonstrate that genetic deficiency of integrin α6β1 on platelets markedly decreases experimental and spontaneous lung metastasis. In vitro and in vivo assays reveal that human and mouse platelet α6β1 supports platelet adhesion to various types of cancer cells. Using a knockdown approach, we identified ADAM9 as the major counter receptor of α6β1 on both human and mouse tumor cells. Static and flow-based adhesion assays of platelets binding to DC-9, a recombinant protein covering the disintegrin-cysteine domain of ADAM9, demonstrated that this receptor directly binds to platelet α6β1. In vivo studies showed that the interplay between platelet α6β1 and tumor cell–expressed ADAM9 promotes efficient lung metastasis. The integrin α6β1–dependent platelet-tumor cell interaction induces platelet activation and favors the extravasation process of tumor cells. Finally, we demonstrate that a pharmacological approach targeting α6β1 efficiently impairs tumor metastasis through a platelet-dependent mechanism. Our study reveals a mechanism by which platelets promote tumor metastasis and suggests that integrin α6β1 represents a promising target for antimetastatic therapies.
Elmina Mammadova-Bach, Paola Zigrino, Camille Brucker, Catherine Bourdon, Monique Freund, Adèle De Arcangelis, Scott I. Abrams, Gertaud Orend, Christian Gachet, Pierre Henri Mangin
IFN-ε is a unique type I IFN that is not induced by pattern recognition response elements. IFN-ε is constitutively expressed in mucosal tissues, including the female genital mucosa. Although the direct antiviral activity of IFN-ε was thought to be weak compared with IFN-α, IFN-ε controls
Carley Tasker, Selvakumar Subbian, Pan Gao, Jennifer Couret, Carly Levine, Saleena Ghanny, Patricia Soteropoulos, Xilin Zhao, Nathaniel Landau, Wuyuan Lu, Theresa L. Chang
In carcinogen-driven cancers, a high mutational burden results in neoepitopes that can be recognized immunologically. Such carcinogen-induced tumors may evade this immune response through “immunoediting,” whereby tumors adapt to immune pressure and escape T cell–mediated killing. Many tumors lack a high neoepitope burden, and it remains unclear whether immunoediting occurs in such cases. Here, we evaluated T cell immunity in an autochthonous mouse model of pancreatic cancer and found a low mutational burden, absence of predicted neoepitopes derived from tumor mutations, and resistance to checkpoint immunotherapy. Spontaneous tumor progression was identical in the presence or absence of T cells. Moreover, tumors arising in T cell–depleted mice grew unchecked in immune-competent hosts. However, introduction of the neoantigen ovalbumin (OVA) led to tumor rejection and T cell memory, but this did not occur in OVA immune-tolerant mice. Thus, immunoediting does not occur in this mouse model — a likely consequence, not a cause, of absent neoepitopes. Because many human tumors also have a low missense mutational load and minimal neoepitope burden, our findings have clinical implications for the design of immunotherapy for patients with such tumors.
Rebecca A. Evans, Mark S. Diamond, Andrew J. Rech, Timothy Chao, Max W. Richardson, Jeffrey H. Lin, David L. Bajor, Katelyn T. Byrne, Ben Z. Stanger, James L. Riley, Nune Markosyan, Rafael Winograd, Robert H. Vonderheide
A large portion of the global population carries latent herpes simplex virus (HSV), which can periodically reactivate, resulting in asymptomatic shedding or formation of ulcerative lesions. Current anti-HSV drugs do not eliminate latent virus from sensory neurons where HSV resides, and therefore do not eliminate the risk of transmission or recurrent disease. Here, we report the ability of HSV-specific endonucleases to induce mutations of essential HSV genes both in cultured neurons and in latently infected mice. In neurons, viral genomes are susceptible to endonuclease-mediated mutagenesis, regardless of the time of treatment after HSV infection, suggesting that both HSV lytic and latent forms can be targeted. Mutagenesis frequency after endonuclease exposure can be increased nearly 2-fold by treatment with a histone deacetylase (HDAC) inhibitor. Using a mouse model of latent HSV infection, we demonstrate that a targeted endonuclease can be delivered to viral latency sites via an adeno-associated virus (AAV) vector, where it is able to induce mutation of latent HSV genomes. These data provide the first proof-of-principle to our knowledge for the use of a targeted endonuclease as an antiviral agent to treat an established latent viral infection in vivo.
Martine Aubert, Emily A. Madden, Michelle Loprieno, Harshana S. DeSilva Feelixge, Laurence Stensland, Meei-Li Huang, Alexander L. Greninger, Pavitra Roychoudhury, Nixon Niyonzima, Thuy Nguyen, Amalia Magaret, Roman Galleto, Daniel Stone, Keith R. Jerome
The ALVAC prime/ALVAC + AIDSVAX B/E boost RV144 vaccine trial induced an estimated 31% efficacy in a low-risk cohort where HIV‑1 exposures were likely at mucosal surfaces. An immune correlates study demonstrated that antibodies targeting the V2 region and in a secondary analysis antibody-dependent cellular cytotoxicity (ADCC), in the presence of low envelope-specific (Env-specific) IgA, correlated with decreased risk of infection. Thus, understanding the B cell repertoires induced by this vaccine in systemic and mucosal compartments are key to understanding the potential protective mechanisms of this vaccine regimen. We immunized rhesus macaques with the ALVAC/AIDSVAX B/E gp120 vaccine regimen given in RV144, and then gave a boost 6 months later, after which the animals were necropsied. We isolated systemic and intestinal vaccine Env-specific memory B cells. Whereas Env-specific B cell clonal lineages were shared between spleen, draining inguinal, anterior pelvic, posterior pelvic, and periaortic lymph nodes, members of Env‑specific B cell clonal lineages were absent in the terminal ileum. Env‑specific antibodies were detectable in rectal fluids, suggesting that IgG antibodies present at mucosal sites were likely systemically produced and transported to intestinal mucosal sites.
Kan Luo, Hua-Xin Liao, Ruijun Zhang, David Easterhoff, Kevin Wiehe, Thaddeus C. Gurley, Lawrence C. Armand, Ashley A. Allen, Tarra A. Von Holle, Dawn J. Marshall, John F. Whitesides, Jamie Pritchett, Andrew Foulger, Giovanna Hernandez, Robert Parks, Krissey E. Lloyd, Christina Stolarchuk, Sheetal Sawant, Jessica Peel, Nicole L. Yates, Erika Dunford, Sabrina Arora, Amy Wang, Cindy M. Bowman, Laura L. Sutherland, Richard M. Scearce, Shi-Mao Xia, Mattia Bonsignori, Justin Pollara, R. Whitney Edwards, Sampa Santra, Norman L. Letvin, James Tartaglia, Donald Francis, Faruk Sinangil, Carter Lee, Jaranit Kaewkungwal, Sorachai Nitayaphan, Punnee Pitisuttithum, Supachai Rerks-ngarm, Nelson L. Michael, Jerome H. Kim, S. Munir Alam, Nathan A. Vandergrift, Guido Ferrari, David C. Montefiori, Georgia D. Tomaras, Barton F. Haynes, M. Anthony Moody
A single-cycle herpes simplex virus (HSV) deleted in glycoprotein D (ΔgD-2) elicited high titer HSV-specific antibodies (Abs) that (i) were rapidly transported into the vaginal mucosa; (ii) elicited antibody-dependent cell-mediated cytotoxicity but little neutralization; (iii) provided complete protection against lethal intravaginal challenge; and (iv) prevented establishment of latency in mice. However, clinical isolates may differ antigenically and impact vaccine efficacy. To determine the breadth and further define mechanisms of protection of this vaccine candidate, we tested ΔgD-2 against a panel of clinical isolates in a murine skin challenge model. The isolates were genetically diverse, as evidenced by genomic sequencing and in vivo virulence. Prime and boost immunization (s.c.) with live but not heat- or UV-inactivated ΔgD-2 completely protected mice from challenge with the most virulent HSV-1 and HSV-2 isolates. Furthermore, mice were completely protected against 100 times the lethal dose that typically kills 90% of animals (LD90) of a South African isolate (SD90), and no latent virus was detected in dorsal root ganglia. Immunization was associated with rapid recruitment of HSV-specific FcγRIII- and FcγRIV-activating IgG2 Abs into the skin, resolution of local cytokine and cellular inflammatory responses, and viral clearance by day 5 after challenge. Rapid clearance and the absence of latent virus suggest that ΔgD-2 elicits sterilizing immunity.
Christopher D. Petro, Brian Weinrick, Nazanin Khajoueinejad, Clare Burn, Rani Sellers, William R. Jacobs Jr, Betsy C. Herold
Aberrant wnt/β-catenin signaling and amplification/overexpression of Myc are associated with hepatoblastoma (HB), the most prevalent type of childhood liver cancer. To address their roles in the pathogenesis of HB, we generated mice in which Myc and mutant β-catenin were targeted to immature cells of the developing mouse liver. Perinatal coexpression of both genes promoted the preferential development of HBs over other tumor types in neonatal mice, all of which bore striking resemblance to their human counterparts. Integrated analysis indicated that tumors emerged as a consequence of Myc-driven alterations in hepatoblast fate in a background of pan-hepatic injury, inflammation, and nuclear factor (erythroid-derived 2)-like 2/Nrf2-dependent antioxidant signaling, which was specifically associated with expression of mutant β-catenin but not Myc. Immunoprofiling of human HBs confirmed that approximately 50% of tumors demonstrated aberrant activation of either Myc or Nfe2l2/Nrf2, while knockdown of Nrf2 in a cell line–derived from a human HB with
Sarah A. Comerford, Elizabeth A. Hinnant, Yidong Chen, Hima Bansal, Shawn Klapproth, Dinesh Rakheja, Milton J. Finegold, Dolores Lopez-Terrada, Kathryn A. O’Donnell, Gail E. Tomlinson, Robert E. Hammer
Recent gene-profiling analyses showed significant upregulation of the folate hydrolase (
Rana Rais, Weiwei Jiang, Huihong Zhai, Krystyna M. Wozniak, Marigo Stathis, Kristen R. Hollinger, Ajit G. Thomas, Camilo Rojas, James J. Vornov, Michael Marohn, Xuhang Li, Barbara S. Slusher
von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein’s multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine
Alexandre Kauskot, Sonia Poirault-Chassac, Frédéric Adam, Vincent Muczynski, Gabriel Aymé, Caterina Casari, Jean-Claude Bordet, Christelle Soukaseum, Chantal Rothschild, Valérie Proulle, Audrey Pietrzyk-Nivau, Eliane Berrou, Olivier D. Christophe, Jean-Philippe Rosa, Peter J. Lenting, Marijke Bryckaert, Cécile V. Denis, Dominique Baruch
Emerging evidence indicates that upregulation of the ER stress–induced pro-osteogenic transcription factor ATF4 plays an important role in vascular calcification, a common complication in patients with aging, diabetes, and chronic kidney disease (CKD). In this study, we demonstrated the pathophysiological role of ATF4 in vascular calcification using global
Masashi Masuda, Shinobu Miyazaki-Anzai, Audrey L. Keenan, Yuji Shiozaki, Kayo Okamura, Wallace S. Chick, Kristina Williams, Xiaoyun Zhao, Shaikh Mizanoor Rahman, Yin Tintut, Christopher M. Adams, Makoto Miyazaki
Hepatic fibrosis arises from inflammation in the liver initiated by resident macrophage activation and massive leukocyte accumulation. Hepatic macrophages hold a central position in maintaining homeostasis in the liver and in the pathogenesis of acute and chronic liver injury linked to fibrogenesis. Interferon regulatory factor 5 (IRF5) has recently emerged as an important proinflammatory transcription factor involved in macrophage activation under acute and chronic inflammation. Here, we revealed that IRF5 is significantly induced in liver macrophages from human subjects developing liver fibrosis from nonalcoholic fatty liver disease or hepatitis C virus infection. Furthermore, IRF5 expression positively correlated with clinical markers of liver damage, such as plasma transaminase and bilirubin levels. Interestingly, mice lacking IRF5 in myeloid cells (MKO) were protected from hepatic fibrosis induced by metabolic or toxic stresses. Transcriptional reprogramming of macrophages lacking IRF5 was characterized by immunosuppressive and antiapoptotic properties. Consequently, IRF5 MKO mice respond to hepatocellular stress by promoting hepatocyte survival, leading to complete protection from hepatic fibrogenesis. Our findings reveal a regulatory network, governed by IRF5, that mediates hepatocyte death and liver fibrosis in mice and humans. Therefore, modulating IRF5 function may be an attractive approach to experimental therapeutics in fibroinflammatory liver disease.
Fawaz Alzaid, Floriane Lagadec, Miguel Albuquerque, Raphaëlle Ballaire, Lucie Orliaguet, Isabelle Hainault, Corinne Blugeon, Sophie Lemoine, Agnès Lehuen, David G. Saliba, Irina A. Udalova, Valérie Paradis, Fabienne Foufelle, Nicolas Venteclef
The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects.
Benjamin Steines, David D. Dickey, Jamie Bergen, Katherine J.D.A. Excoffon, John R. Weinstein, Xiaopeng Li, Ziying Yan, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Linda S. Powers, Nicholas D. Gansemer, Lynda S. Ostedgaard, John F. Engelhardt, David A. Stoltz, Michael J. Welsh, Patrick L. Sinn, David V. Schaffer, Joseph Zabner
Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in CF transmembrane conductance regulator (
Ashley L. Cooney, Mahmoud H. Abou Alaiwa, Viral S. Shah, Drake C. Bouzek, Mallory R. Stroik, Linda S. Powers, Nick D. Gansemer, David K. Meyerholz, Michael J. Welsh, David A. Stoltz, Patrick L. Sinn, Paul B. McCray Jr.
Esophageal squamous cell carcinoma (ESCC) is endemic in regions of sub-Saharan Africa (SSA), where it is the third most common cancer. Here, we describe whole-exome tumor/normal sequencing and RNA transcriptomic analysis of 59 patients with ESCC in Malawi. We observed similar genetic aberrations as reported in Asian and North American cohorts, including mutations of
Wenjin Liu, Jeff M. Snell, William R. Jeck, Katherine A. Hoadley, Matthew D. Wilkerson, Joel S. Parker, Nirali Patel, Yohannie B. Mlombe, Gift Mulima, N. George Liomba, Lindsey L. Wolf, Carol G. Shores, Satish Gopal, Norman E. Sharpless
Following the advent of molecular assays that measure T cell receptor excision circles (TRECs) present in recent thymic emigrants, it has been conclusively shown that thymopoiesis persists in most adults, but that functional output decreases with age, influencing the maintenance of a diverse and functional T cell receptor (TCR) repertoire. Space flight has been shown to result in a variety of phenotypic and functional changes in human T cells and in the reactivation of latent viruses. While space flight has been shown to influence thymic architecture in rodents, thymopoiesis has not previously been assessed in astronauts. Here, we assessed thymopoiesis longitudinally over a 1-year period prior to and after long-term space flight (median duration, 184 days) in 16 astronauts. While preflight assessments of thymopoiesis remained quite stable in individual astronauts, we detected significant suppression of thymopoiesis in all subjects upon return from space flight. We also found significant increases in urine and plasma levels of endogenous glucocorticoids coincident with the suppression of thymopoiesis. The glucocorticoid induction and thymopoiesis suppression were transient, and they normalized shortly after return to Earth. This is the first report to our knowledge to prospectively demonstrate a significant change in thymopoiesis in healthy individuals in association with a defined physiologic emotional and physical stress event. These results suggest that suppression of thymopoiesis has the potential to influence the maintenance of the TCR repertoire during extended space travel. Further studies of thymopoiesis and endogenous glucocorticoids in other stress states, including illness, are warranted.
Cara L. Benjamin, Raymond P. Stowe, Lisa St. John, Clarence F. Sams, Satish K. Mehta, Brian E. Crucian, Duane L. Pierson, Krishna V. Komanduri
Mutagenesis screening is a powerful forward genetic approach that has been successfully applied in lower-model organisms to discover genetic factors for biological processes. This phenotype-based approach has yet to be established in vertebrates for probing major human diseases, largely because of the complexity of colony management. Herein, we report a rapid strategy for identifying genetic modifiers of cardiomyopathy (CM). Based on the application of doxorubicin stress to zebrafish insertional cardiac (ZIC) mutants, we identified 4 candidate CM-modifying genes, of which 3 have been linked previously to CM. The long isoform of DnaJ (Hsp40) homolog, subfamily B, member 6b (
Yonghe Ding, Pamela A. Long, J. Martijn Bos, Yu-Huan Shih, Xiao Ma, Rhianna S. Sundsbak, Jianhua Chen, Yiwen Jiang, Liqun Zhao, Xinyang Hu, Jianan Wang, Yongyong Shi, Michael J. Ackerman, Xueying Lin, Stephen C. Ekker, Margaret M. Redfield, Timothy M. Olson, Xiaolei Xu
Chronic inflammation with mucous metaplasia and airway remodeling are hallmarks of allergic asthma, and these outcomes have been associated with enhanced expression and activation of EGFR signaling. Here, we demonstrate enhanced expression of EGFR ligands such as amphiregulin as well as constitutive EGFR activation in cultured nasal epithelial cells from asthmatic subjects compared with nonasthmatic controls and in lung tissues of mice during house dust mite–induced (HDM-induced) allergic inflammation. EGFR activation was associated with cysteine oxidation within EGFR and the nonreceptor tyrosine kinase Src, and both amphiregulin production and oxidative EGFR activation were diminished by pharmacologic or genetic inhibition of the epithelial NADPH oxidase dual oxidase 1 (DUOX1). DUOX1 deficiency also attenuated several EGFR-dependent features of HDM-induced allergic airway inflammation, including neutrophilic inflammation, type 2 cytokine production (IL-33, IL-13), mucous metaplasia, subepithelial fibrosis, and central airway resistance. Moreover, targeted inhibition of airway DUOX1 in mice with previously established HDM-induced allergic inflammation, by intratracheal administration of DUOX1-targeted siRNA or pharmacological NADPH oxidase inhibitors, reversed most of these outcomes. Our findings indicate an important function for DUOX1 in allergic inflammation related to persistent EGFR activation and suggest that DUOX1 targeting may represent an attractive strategy in asthma management.
Aida Habibovic, Milena Hristova, David E. Heppner, Karamatullah Danyal, Jennifer L. Ather, Yvonne M.W. Janssen-Heininger, Charles G. Irvin, Matthew E. Poynter, Lennart K. Lundblad, Anne E. Dixon, Miklos Geiszt, Albert van der Vliet
Infantile hemangioma (IH) is the most common vascular tumor of infancy, and it uniquely regresses in response to oral propranolol. MicroRNAs (miRNAs) have emerged as key regulators of vascular development and are dysregulated in many disease processes, but the role of miRNAs in IH growth has not been investigated. We report expression of C19MC, a primate-specific megacluster of miRNAs expressed in placenta with rare expression in postnatal tissues, in glucose transporter 1–expressing (GLUT-1–expressing) IH endothelial cells and in the plasma of children with IH. Tissue or circulating C19MC miRNAs were not detectable in patients having 9 other types of vascular anomalies or unaffected children, identifying C19MC miRNAs as the first circulating biomarkers of IH. Levels of circulating C19MC miRNAs correlated with IH tumor size and propranolol treatment response, and IH tissue from children treated with propranolol or from children with partially involuted tumors contained lower levels of C19MC miRNAs than untreated, proliferative tumors, implicating C19MC miRNAs as potential drivers of IH pathogenesis. Detection of C19MC miRNAs in the circulation of infants with IH may provide a specific and noninvasive means of IH diagnosis and identification of candidates for propranolol therapy as well as a means to monitor treatment response.
Graham M. Strub, Andrew L. Kirsh, Mark E. Whipple, Winston P. Kuo, Rachel B. Keller, Raj P. Kapur, Mark W. Majesky, Jonathan A. Perkins
Meningococcal factor H-binding protein (FHbp) is an antigen in 2 serogroup B meningococcal vaccines. FHbp specifically binds human and some nonhuman primate complement FH. To investigate the effect of binding of FH to FHbp on protective antibody responses, we immunized infant rhesus macaques with either a control recombinant FHbp antigen that bound macaque FH or a mutant antigen with 2 amino acid substitutions and >250-fold lower affinity for FH. The mutant antigen elicited 3-fold higher serum IgG anti-FHbp titers and up to 15-fold higher serum bactericidal titers than the control FHbp vaccine. When comparing sera with similar IgG anti-FHbp titers, the antibodies elicited by the mutant antigen gave greater deposition of complement component C4b on live meningococci (classical complement pathway) and inhibited binding of FH, while the anti-FHbp antibodies elicited by the control vaccine enhanced FH binding. Thus, the mutant FHbp vaccine elicited an anti-FHbp antibody repertoire directed at FHbp epitopes within the FH binding site, which resulted in greater protective activity than the antibodies elicited by the control vaccine, which targeted FHbp epitopes outside of the FH combining site. Binding of a host protein to a vaccine antigen impairs protective antibody responses, which can be overcome with low-binding mutant antigens.
Dan M. Granoff, Serena Giuntini, Flor A. Gowans, Eduardo Lujan, Kelsey Sharkey, Peter T. Beernink
Hypertension is nearly universal yet poorly controlled in the elderly despite proven benefits of intensive treatment. Mice lacking mineralocorticoid receptors in smooth muscle cells (SMC-MR-KO) are protected from rising blood pressure (BP) with aging, despite normal renal function. Vasoconstriction is attenuated in aged SMC-MR-KO mice, thus they were used to explore vascular mechanisms that may contribute to hypertension with aging. MicroRNA (miR) profiling identified miR-155 as the most down-regulated miR with vascular aging in MR-intact but not SMC-MR-KO mice. The aging-associated decrease in miR-155 in mesenteric resistance vessels was associated with increased mRNA abundance of MR and of predicted miR-155 targets Cav1.2 (L-type calcium channel (LTCC) subunit) and angiotensin type-1 receptor (AgtR1). SMC-MR-KO mice lacked these aging-associated vascular gene expression changes. In HEK293 cells, MR repressed miR-155 promoter activity. In cultured SMCs, miR-155 decreased Cav1.2 and AgtR1 mRNA. Compared to MR-intact littermates, aged SMC-MR-KO mice had decreased systolic BP, myogenic tone, SMC LTCC current, mesenteric vessel calcium influx, LTCC-induced vasoconstriction and angiotensin II-induced vasoconstriction and oxidative stress. Restoration of miR-155 specifically in SMCs of aged MR-intact mice decreased Cav1.2 and AgtR1 mRNA and attenuated LTCC-mediated and angiotensin II-induced vasoconstriction and oxidative stress. Finally, in a trial of MR blockade in elderly humans, changes in serum miR-155 predicted the BP treatment response. Thus, SMC-MR regulation of miR-155, Cav1.2 and AgtR1 impacts vasoconstriction with aging. This novel mechanism identifies potential new treatment strategies and biomarkers to improve and individualize antihypertensive therapy in the elderly.
Jennifer J. DuPont, Amy McCurley, Ana P. Davel, Joseph McCarthy, Shawn B. Bender, Kwangseok Hong, Yan Yang, Jeung-Ki Yoo, Mark Aronovitz, Wendy E. Baur, Demetra D. Christou, Michael A. Hill, Iris Z. Jaffe
Patrick H. Lizotte, Elena V. Ivanova, Mark M. Awad, Robert E. Jones, Lauren Keogh, Hongye Liu, Ruben Dries, Christina Almonte, Grit S. Herter-Sprie, Abigail Santos, Nora B. Feeney, Cloud P. Paweletz, Meghana M. Kulkarni, Adam J. Bass, Anil K. Rustgi, Guo-Cheng Yuan, Donald W. Kufe, Pasi A. Jänne, Peter S. Hammerman, Lynette M. Sholl, F. Stephen Hodi, William G. Richards, Raphael Bueno, Jessie M. English, Mark A. Bittinger, Kwok-Kin Wong
BM-derived DC (BMDC) are powerful antigen-presenting cells. When loaded with immune complexes (IC), consisting of tumor antigens bound to antitumor antibody, BMDC induce powerful antitumor immunity in mice. However, attempts to employ this strategy clinically with either tumor-associated DC (TADC) or monocyte-derived DC (MoDC) have been disappointing. To investigate the basis for this phenomenon, we compared the response of BMDC, TADC, and MoDC to tumor IgG-IC. Our findings revealed, in both mice and humans, that upon exposure to IgG-IC, BMDC internalized the IC, increased costimulatory molecule expression, and stimulated autologous T cells. In contrast, TADC and, surprisingly, MoDC remained inert upon contact with IC due to dysfunctional signaling following engagement of Fcγ receptors. Such dysfunction is associated with elevated levels of the Src homology region 2 domain–containing phosphatase-1 (SHP-1) and phosphatases regulating Akt activation. Indeed, concomitant inhibition of both SHP-1 and phosphatases that regulate Akt activation conferred upon TADC and MoDC the capacity to take up and process IC and induce antitumor immunity in vivo. This work identifies the molecular checkpoints that govern activation of MoDC and TADC and their capacity to elicit T cell immunity.
Yaron Carmi, Tyler R. Prestwood, Matthew H. Spitzer, Ian L. Linde, Jonathan Chabon, Nathan E. Reticker-Flynn, Nupur Bhattacharya, Hong Zhang, Xiangyue Zhang, Pamela A. Basto, Bryan M. Burt, Michael N. Alonso, Edgar G. Engleman
Secondary lymphedema is a common postcancer treatment complication, but the underlying pathological processes are poorly understood and no curative treatment exists. To investigate lymphedema pathomechanisms, a top-down approach was applied, using genomic data and validating the role of a single target. RNA sequencing of lymphedematous mouse skin indicated upregulation of many T cell–related networks, and indeed depletion of CD4+ cells attenuated lymphedema. The significant upregulation of Foxp3, a transcription factor specifically expressed by regulatory T cells (Tregs), along with other Treg-related genes, implied a potential role of Tregs in lymphedema. Indeed, increased infiltration of Tregs was identified in mouse lymphedematous skin and in human lymphedema specimens. To investigate the role of Tregs during disease progression, loss-of-function and gain-of-function studies were performed. Depletion of Tregs in transgenic mice with Tregs expressing the primate diphtheria toxin receptor and green fluorescent protein (
Epameinondas Gousopoulos, Steven T. Proulx, Samia B. Bachmann, Jeannette Scholl, Dimitris Dionyssiou, Efterpi Demiri, Cornelia Halin, Lothar C. Dieterich, Michael Detmar
Craig Balmforth, Job J.M.H. van Bragt, Titia Ruijs, James R. Cameron, Robert Kimmitt, Rebecca Moorhouse, Alicja Czopek, May Khei Hu, Peter J. Gallacher, James W. Dear, Shyamanga Borooah, Iain M. MacIntyre, Tom M.C. Pearson, Laura Willox, Dinesh Talwar, Muriel Tafflet, Christophe Roubeix, Florian Sennlaub, Siddharthan Chandran, Baljean Dhillon, David J. Webb, Neeraj Dhaun
CD4+ regulatory T cells (CD4Tregs) play a critical role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. IL-2 supports the proliferation and survival of CD4Tregs and previous studies have demonstrated that IL-2 induces selective expansion of CD4Tregs and improves clinical manifestations of chronic GVHD. However, mechanisms for selective activation of CD4Tregs and the effects of low-dose IL-2 on other immune cells are not well understood. Using mass cytometry, we demonstrate that low concentrations of IL-2 selectively induce STAT5 phosphorylation in Helios+ CD4Tregs and CD56brightCD16– NK cells in vitro. Preferential activation and expansion of Helios+ CD4Tregs and CD56brightCD16– NK cells was also demonstrated in patients with chronic GVHD receiving low-dose IL-2. With prolonged IL-2 treatment for 48 weeks, phenotypic changes were also observed in Helios– CD4Tregs. The effects of low-dose IL-2 therapy on conventional CD4+ T cells and CD8+ T cells were limited to increased expression of PD-1 on effector memory T cells. These studies reveal the selective effects of low-dose IL-2 therapy on Helios+ CD4Tregs and CD56bright NK cells that constitutively express high-affinity IL-2 receptors as well as the indirect effects of prolonged exposure to low concentrations of IL-2 in vivo.
Masahiro Hirakawa, Tiago R. Matos, Hongye Liu, John Koreth, Haesook T. Kim, Nicole E. Paul, Kazuyuki Murase, Jennifer Whangbo, Ana C. Alho, Sarah Nikiforow, Corey Cutler, Vincent T. Ho, Philippe Armand, Edwin P. Alyea, Joseph H. Antin, Bruce R. Blazar, Joao F. Lacerda, Robert J. Soiffer, Jerome Ritz
T cells that enter tumors are largely tolerized, but how that process is choreographed and how the ensuing “dysfunctional” tumor-infiltrating lymphocytes (TILs) are maintained are poorly understood and are difficult to assess in spontaneous disease. We exploited an autochthonous model of breast cancer for high-resolution imaging of the early and later stages of tumor residence to understand the relationships between cellular behaviors and cellular phenotypes. “Dysfunctional” differentiation began within the first days of tumor residence with an initial phase in which T cells arrest, largely on tumor-associated macrophages. Within 10 days, cellular motility increased and resembled a random walk, suggesting a relative absence of TCR signaling. We then studied the concurrent and apparently contradictory phenomenon that many of these cells express molecular markers of activation and were visualized undergoing active cell division. We found that whereas proliferation did not require ongoing TCR/ZAP70 signaling, instead this is driven in part by intratumoral IL-15 cytokine. Thus, TILs undergo sequential reprogramming by the tumor microenvironment and are actively retained, even while being antigen insensitive. We conclude that this program effectively fills the niche with ineffective yet cytokine-dependent TILs, and we propose that these might compete with new clones, when they arise.
Bijan Boldajipour, Amanda Nelson, Matthew F. Krummel
The challenge of translating findings from animal models to the clinic is well known. An example of this challenge is the striking effectiveness of neurokinin-1 receptor (NK-1R) antagonists in mouse models of inflammation coupled with their equally striking failure in clinical investigations in humans. Here, we provide an explanation for this dichotomy: Mas-related GPCRs (Mrgprs) mediate some aspects of inflammation that had been considered mediated by NK-1R. In support of this explanation, we show that conventional NK-1R antagonists have off-target activity on the mouse receptor MrgprB2 but not on the homologous human receptor MRGPRX2. An unrelated tripeptide NK-1R antagonist has dual activity on MRGPRX2. This tripeptide both suppresses itch in mice and inhibits degranulation from the LAD-2 human mast cell line elicited by basic secretagogue activation of MRGPRX2. Antagonists of Mrgprs may fill the void left by the failure of NK-1R antagonists.
Ehsan Azimi, Vemuri B. Reddy, Kai-Ting C. Shade, Robert M. Anthony, Sebastien Talbot, Paula Juliana Seadi Pereira, Ethan A. Lerner
Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645–1648) in the design of B-domain–deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector–based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.
Joshua I. Siner, Benjamin J. Samelson-Jones, Julie M. Crudele, Robert A. French, Benjamin J. Lee, Shanzhen Zhou, Elizabeth Merricks, Robin Raymer, Timothy C. Nichols, Rodney M. Camire, Valder R. Arruda
Psoriasis patients are at increased risk of heart attack and stroke and have elevated MRP8/14 levels that predict heart attack. The KC-Tie2 psoriasiform mouse model exhibits elevated MRP8/14 and is prothrombotic.
Yunmei Wang, Jackelyn B. Golden, Yi Fritz, Xiufen Zhang, Doina Diaconu, Maya I. Camhi, Huiyun Gao, Sean M. Dawes, Xianying Xing, Santhi K. Ganesh, Johann E. Gudjonsson, Daniel I. Simon, Thomas S. McCormick, Nicole L. Ward
The islet in type 2 diabetes (T2D) shares many features of the brain in protein misfolding diseases. There is a deficit of β cells with islet amyloid derived from islet amyloid polypeptide (IAPP), a protein coexpressed with insulin. Small intracellular membrane-permeant oligomers, the most toxic form of IAPP, are more frequent in β cells of patients with T2D and rodents expressing human IAPP. β Cells in T2D, and affected cells in neurodegenerative diseases, share a comparable pattern of molecular pathology, including endoplasmic reticulum stress, mitochondrial dysfunction, attenuation of autophagy, and calpain hyperactivation. While this adverse functional cascade in response to toxic oligomers is well described, the sequence of events and how best to intervene is unknown. We hypothesized that calpain hyperactivation is a proximal event and tested this in vivo by β cell–specific suppression of calpain hyperactivation with calpastatin overexpression in human IAPP transgenic mice. β Cell–specific calpastatin overexpression was remarkably protective against β cell dysfunction and loss and diabetes onset. The critical autophagy/lysosomal pathway for β cell viability was protected with calpain suppression, consistent with findings in models of neurodegenerative diseases. We conclude that suppression of calpain hyperactivation is a potentially beneficial disease-modifying strategy for protein misfolding diseases, including T2D.
Tatyana Gurlo, Safia Costes, Jonathan D. Hoang, Jacqueline F. Rivera, Alexandra E. Butler, Peter C. Butler
Marc A. Simon, Rebecca R. Vanderpool, Mehdi Nouraie, Timothy N. Bachman, Pamela M. White, Masataka Sugahara, John Gorcsan III, Ed L. Parsley, Mark T. Gladwin
Rebecca A. Sosa, Ali Zarrinpar, Maura Rossetti, Charles R. Lassman, Bita V. Naini, Nakul Datta, Ping Rao, Nicholas Harre, Ying Zheng, Roberto Spreafico, Alexander Hoffmann, Ronald W. Busuttil, David W. Gjertson, Yuan Zhai, Jerzy W. Kupiec-Weglinski, Elaine F. Reed
Muscle trauma is highly morbid due to intramuscular scarring, or fibrosis, and muscle atrophy. Studies have shown that bone morphogenetic proteins (BMPs) reduce muscle atrophy. However, increased BMP signaling at muscle injury sites causes heterotopic ossification, as seen in patients with fibrodysplasia ossificans progressiva (FOP), or patients with surgically placed BMP implants for bone healing. We use a genetic mouse model of hyperactive BMP signaling to show the development of intramuscular fibrosis surrounding areas of ectopic bone following muscle injury. Rapamycin, which we have previously shown to eliminate ectopic ossification in this model, also eliminates fibrosis without reducing osteogenic differentiation, suggesting clinical value for patients with FOP and with BMP implants. Finally, we use reporter mice to show that BMP signaling is positively associated with myofiber cross-sectional area. These findings underscore an approach in which 2 therapeutics (rapamycin and BMP ligand) can offset each other, leading to an improved outcome.
Shailesh Agarwal, David Cholok, Shawn Loder, John Li, Christopher Breuler, Michael T. Chung, Hsiao Hsin Sung, Kavitha Ranganathan, Joe Habbouche, James Drake, Joshua Peterson, Caitlin Priest, Shuli Li, Yuji Mishina, Benjamin Levi
Adipose tissue is a key endocrine organ that governs systemic homeostasis. PPARγ is a master regulator of adipose tissue signaling that plays an essential role in insulin sensitivity, making it an important therapeutic target. The selective PPARγ agonist rosiglitazone (RSG) has been used to treat diabetes. However, adverse cardiovascular effects have seriously hindered its clinical application. Experimental models have revealed that PPARγ activation increases cardiac hypertrophy. RSG stimulates cardiac hypertrophy and oxidative stress in cardiomyocyte-specific PPARγ knockout mice, implying that RSG might stimulate cardiac hypertrophy independently of cardiomyocyte PPARγ. However, candidate cell types responsible for RSG-induced cardiomyocyte hypertrophy remain unexplored. Utilizing cocultures of adipocytes and cardiomyocytes, we found that stimulation of PPARγ signaling in adipocytes increased miR-200a expression and secretion. Delivery of miR-200a in adipocyte-derived exosomes to cardiomyocytes resulted in decreased TSC1 and subsequent mTOR activation, leading to cardiomyocyte hypertrophy. Treatment with an antagomir to miR-200a blunted this hypertrophic response in cardiomyocytes. In vivo, specific ablation of PPARγ in adipocytes was sufficient to blunt hypertrophy induced by RSG treatment. By delineating mechanisms by which RSG elicits cardiac hypertrophy, we have identified pathways that mediate the crosstalk between adipocytes and cardiomyocytes to regulate cardiac remodeling.
Xi Fang, Matthew J. Stroud, Kunfu Ouyang, Li Fang, Jianlin Zhang, Nancy D. Dalton, Yusu Gu, Tongbin Wu, Kirk L. Peterson, Hsien-Da Huang, Ju Chen, Nanping Wang
The epigenome provides a substrate through which environmental exposures can exert their effects on gene expression and disease risk, but the relative importance of epigenetic variation on human disease onset and progression is poorly characterized. Asthma is a heterogeneous disease of the airways, for which both onset and clinical course result from interactions between host genotype and environmental exposures, yet little is known about the molecular mechanisms for these interactions. We assessed genome-wide DNA methylation using the Infinium Human Methylation 450K Bead Chip and characterized the transcriptome by RNA sequencing in primary airway epithelial cells from 74 asthmatic and 41 nonasthmatic adults. Asthma status was based on doctor’s diagnosis and current medication use. Genotyping was performed using various Illumina platforms. Our study revealed a regulatory locus on chromosome 17q12-21 associated with asthma risk and epigenetic signatures of specific asthma endotypes and molecular networks. Overall, these data support a central role for DNA methylation in lung cells, which promotes distinct molecular pathways of asthma pathogenesis and modulates the effects of genetic variation on disease risk and clinical heterogeneity.
Jessie Nicodemus-Johnson, Rachel A. Myers, Noburu J. Sakabe, Debora R. Sobreira, Douglas K. Hogarth, Edward T. Naureckas, Anne I. Sperling, Julian Solway, Steven R. White, Marcelo A. Nobrega, Dan L. Nicolae, Yoav Gilad, Carole Ober
Noonan syndrome (NS) is a common autosomal dominant disorder that presents with short stature, craniofacial dysmorphism, and cardiac abnormalities. Activating mutations in the
Jae-Sung Yi, Yan Huang, Andrea T. Kwaczala, Ivana Y. Kuo, Barbara E. Ehrlich, Stuart G. Campbell, Frank J. Giordano, Anton M. Bennett
Maladaptive epithelial repair from chronic injury is a common feature in fibrotic diseases, which in turn activates a pathogenic fibroblast response that produces excessive matrix deposition. Dysregulated microRNAs (miRs) can regulate expression of multiple genes and fundamentally alter cellular phenotypes during fibrosis. Although several miRs have been shown to be associated with lung fibrosis, the mechanisms by which miRs modulate epithelial behavior in lung fibrosis are lacking. Here, we identified miR-323a-3p to be downregulated in the epithelium of lungs with bronchiolitis obliterans syndrome (BOS) after lung transplantation, idiopathic pulmonary fibrosis (IPF), and murine bleomycin-induced fibrosis. Antagomirs for miR-323a-3p augment, and mimics suppress, murine lung fibrosis after bleomycin injury, indicating that this miR may govern profibrotic signals. We demonstrate that miR-323a-3p attenuates TGF-α and TGF-β signaling by directly targeting key adaptors in these important fibrogenic pathways. Moreover, miR-323a-3p lowers caspase-3 expression, thereby limiting programmed cell death from inducers of apoptosis and ER stress. Finally, we find that epithelial expression of miR-323a-3p modulates inhibitory crosstalk with fibroblasts. These studies demonstrate that miR-323a-3p has a central role in lung fibrosis that spans across murine and human disease, and downregulated expression by the lung epithelium releases inhibition of various profibrotic pathways to promote fibroproliferation.
Lingyin Ge, David M. Habiel, Phil M. Hansbro, Richard Y. Kim, Sina A. Gharib, Jeffery D. Edelman, Melanie Königshoff, Tanyalak Parimon, Rena Brauer, Ying Huang, Jenieke Allen, Dianhua Jiang, Adrianne A. Kurkciyan, Takako Mizuno, Barry R. Stripp, Paul W. Noble, Cory M. Hogaboam, Peter Chen
Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease.
Yan Xu, Takako Mizuno, Anusha Sridharan, Yina Du, Minzhe Guo, Jie Tang, Kathryn A. Wikenheiser-Brokamp, Anne-Karina T. Perl, Vincent A. Funari, Jason J. Gokey, Barry R. Stripp, Jeffrey A. Whitsett
A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase,
Ryan E. Feaver, Banumathi K. Cole, Mark J. Lawson, Stephen A. Hoang, Svetlana Marukian, Brett R. Blackman, Robert A. Figler, Arun J. Sanyal, Brian R. Wamhoff, Ajit Dash
Preeclampsia (PE) is a disorder of pregnancy that manifests as late gestational maternal hypertension and proteinuria and can be life-threatening to both the mother and baby. It is believed that abnormal placentation is responsible for the cascade of events leading to the maternal syndrome. Embryo implantation is critical to establishing a healthy pregnancy. Defective implantation can cause adverse “ripple effects,” leading to abnormal decidualization and placentation, retarded fetal development, and poor pregnancy outcomes, such as PE and fetal growth restriction. The precise mechanism(s) of implantation defects that lead to PE remain elusive. BPH/5 mice, which spontaneously develop the cardinal features of PE, show peri-implantation defects including upregulation of Cox2 and IL-15 at the maternal-fetal interface. This was associated with decreased decidual natural killer (dNK) cells, which have important roles in establishing placental perfusion. Interestingly, a single administration of a Cox2 inhibitor (celecoxib) during decidualization restrained Cox2 and IL-15 expression, restored dNK cell numbers, improved fetal growth, and attenuated late gestational hypertension in BPH/5 female mice. This study provides evidence that decidual overexpression of Cox2 and IL-15 may trigger the adverse pregnancy outcomes reflected in the preeclamptic syndrome, underscoring the idea that Cox2 inhibitor treatment is an effective strategy for the prevention of PE-associated fetal and maternal morbidity and mortality.
Jenny L. Sones, Jeeyeon Cha, Ashley K. Woods, Amanda Bartos, Christa Y. Heyward, Heinrich E. Lob, Catherine E. Isroff, Scott D. Butler, Stephanie E. Shapiro, Sudhansu K. Dey, Robin L. Davisson
The capacity of pancreatic β cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote β cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, β cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and β cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes.
Alexandra Kuznetsova, Yue Yu, Jennifer Hollister-Lock, Lynn Opare-Addo, Aldo Rozzo, Marianna Sadagurski, Lisa Norquay, Jessica E. Reed, Ilham El Khattabi, Susan Bonner-Weir, Gordon C. Weir, Arun Sharma, Morris F. White
Obesity is an increasing health problem worldwide, and nonsurgical strategies to treat obesity have remained rather inefficient. We here show that acute loss of TGF-β–activated kinase 1 (TAK1) in adipocytes results in an increased rate of apoptotic adipocyte death and increased numbers of M2 macrophages in white adipose tissue. Mice with adipocyte-specific TAK1 deficiency have reduced adipocyte numbers and are resistant to obesity induced by a high-fat diet or leptin deficiency. In addition, adipocyte-specific TAK1-deficient mice under a high-fat diet showed increased energy expenditure, which was accompanied by enhanced expression of the uncoupling protein UCP1. Interestingly, acute induction of adipocyte-specific TAK1 deficiency in mice already under a high-fat diet was able to stop further weight gain and improved glucose tolerance. Thus, loss of TAK1 in adipocytes reduces the total number of adipocytes, increases browning of white adipose tissue, and may be an attractive strategy to treat obesity, obesity-dependent diabetes, and other associated complications.
Antonia Sassmann-Schweda, Pratibha Singh, Cong Tang, Astrid Wietelmann, Nina Wettschureck, Stefan Offermanns
Development of novel treatments for lymphedema has been limited by the fact that the pathophysiology of this disease is poorly understood. It remains unknown, for example, why limb swelling resulting from surgical injury resolves initially, but recurs in some cases months or years later. Finding answers for these basic questions has been hampered by the lack of adequate animal models. In the current study, we used
Jason C. Gardenier, Geoffrey E. Hespe, Raghu P. Kataru, Ira L. Savetsky, Jeremy S. Torrisi, Gabriela D. García Nores, Joseph J. Dayan, David Chang, Jamie Zampell, Inés Martínez-Corral, Sagrario Ortega, Babak J. Mehrara
Despite the rare appearance of potent HIV-neutralizing mAbs in infected individuals requiring prolonged affinity maturation, little is known regarding this process in the majority of viremic individuals. HIV-infected individuals with chronic HIV viremia have elevated numbers of nonconventional tissue-like memory (TLM) B cells that predominate in blood over conventional resting memory (RM) B cells. Accordingly, we investigated affinity maturation in these 2 memory B cell populations. Analysis of IgG-expressing TLM B cells revealed a higher number of cell divisions compared with RM B cells; however, TLM B cells paradoxically displayed significantly lower frequencies of somatic hypermutation (SHM). To assess Ab reactivity in TLM and RM B cells, single-cell cloning was performed on HIV envelope CD4–binding site–sorted (CD4bs-sorted) B cells from 3 individuals with chronic HIV viremia. Several clonal families were present among the 127 cloned recombinant mAbs, with evidence of crosstalk between TLM and RM B cell populations that was largely restricted to non-VH4 families. Despite evidence of common origins, SHM frequencies were significantly decreased in TLM-derived mAbs compared with SHM frequencies in RM-derived mAbs. However, both cell populations had lower frequencies of SHMs than did broadly neutralizing CD4bs–specific mAbs. There was a significant correlation between SHM frequencies and the HIV-neutralizing capacities of the mAbs. Furthermore, HIV neutralization was significantly higher in the RM-derived mAbs compared with that seen in the TLM-derived mAbs, and both SHM frequencies and neutralizing capacity were lowest in TLM-derived mAbs with high polyreactivity. Thus, deficiencies in memory B cells that arise during chronic HIV viremia provide insight into the inadequacy of the Ab response in viremic individuals.
Eric Meffre, Aaron Louie, Jason Bannock, Leo J.Y. Kim, Jason Ho, Cody C. Frear, Lela Kardava, Wei Wang, Clarisa M. Buckner, Yimeng Wang, Olivia R. Fankuchen, Kathleen R. Gittens, Tae-Wook Chun, Yuxing Li, Anthony S. Fauci, Susan Moir
The prevalence of fatty liver reaches alarming proportions. Fatty liver increases the risk for insulin resistance, cardiovascular disease, and nonalcoholic steatohepatitis (NASH). Although extensively studied in a preclinical setting, the lack of noninvasive methodologies hampers our understanding of which pathways promote hepatic fat accumulation in humans. Dietary fat retention is one of the pathways that may lead to fatty liver. The low (1.1%) natural abundance (NA) of carbon-13 (13C) allows use of 13C-enriched lipids for in vivo MR studies. Successful implementation of such methodology, however, is challenging due to low sensitivity of 13C-magnetic resonance spectroscopy (13C-MRS). Here, we investigated the use of 1-dimensional gradient enhanced heteronuclear single quantum coherence (ge-HSQC) spectroscopy for the in vivo detection of hepatic 1H-[13C]-lipid signals after a single high-fat meal with 13C-labeled fatty acids in 5 lean and 6 obese subjects. Postprandial retention of orally administered 13C-labeled fatty acids was significant (
Lucas Lindeboom, Robin A. de Graaf, Christine I. Nabuurs, Petronella A. van Ewijk, Matthijs K.C. Hesselink, Joachim E. Wildberger, Patrick Schrauwen, Vera B. Schrauwen-Hinderling
Ab-producing plasma cells (PCs) serve as key participants in countering pathogenic challenges as well as being contributors to autoimmune and malignant disorders. Thus far, only a limited number of PC–specific markers have been identified. The characterization of the unique variable lymphocyte receptor (VLR) Abs that are made by evolutionarily distant jawless vertebrates prompted us to investigate whether VLR Abs could detect novel PC antigens that have not been recognized by conventional Abs. Here, we describe a monoclonal lamprey Ab, VLRB MM3, that was raised against primary multiple myeloma cells. VLRB MM3 recognizes a unique epitope of the CD38 ectoenzyme that is present on plasmablasts and PCs from healthy individuals and on most, but not all, multiple myelomas. Binding by the VLRB MM3 Ab coincides with CD38 dimerization and NAD glycohydrolase activity. Our data demonstrate that the lamprey VLRB MM3 Ab is a unique reagent for the identification of plasmablasts and PCs, with potential applications in the diagnosis and therapeutic intervention of PC or autoimmune disorders.
Cuiling Yu, Yanling Liu, Justin Tze Ho Chan, Jiefei Tong, Zhihua Li, Mengyao Shi, Dariush Davani, Marion Parsons, Srijit Khan, Wei Zhan, Shuya Kyu, Eyal Grunebaum, Paolo Campisi, Evan J. Propst, David L. Jaye, Suzanne Trudel, Michael F. Moran, Mario Ostrowski, Brantley R. Herrin, F. Eun-Hyung Lee, Ignacio Sanz, Max D. Cooper, Götz R.A. Ehrhardt
Myocardial fuel and energy metabolic derangements contribute to the pathogenesis of heart failure. Recent evidence implicates posttranslational mechanisms in the energy metabolic disturbances that contribute to the pathogenesis of heart failure. We hypothesized that accumulation of metabolite intermediates of fuel oxidation pathways drives posttranslational modifications of mitochondrial proteins during the development of heart failure. Myocardial acetylproteomics demonstrated extensive mitochondrial protein lysine hyperacetylation in the early stages of heart failure in well-defined mouse models and the in end-stage failing human heart. To determine the functional impact of increased mitochondrial protein acetylation, we focused on succinate dehydrogenase A (SDHA), a critical component of both the tricarboxylic acid (TCA) cycle and respiratory complex II. An acetyl-mimetic mutation targeting an SDHA lysine residue shown to be hyperacetylated in the failing human heart reduced catalytic function and reduced complex II–driven respiration. These results identify alterations in mitochondrial acetyl-CoA homeostasis as a potential driver of the development of energy metabolic derangements that contribute to heart failure.
Julie L. Horton, Ola J. Martin, Ling Lai, Nicholas M. Riley, Alicia L. Richards, Rick B. Vega, Teresa C. Leone, David J. Pagliarini, Deborah M. Muoio, Kenneth C. Bedi Jr., Kenneth B. Margulies, Joshua J. Coon, Daniel P. Kelly
Multiple myeloma (MM) is a relapsed and refractory disease, one that highlights the need for developing new molecular therapies for overcoming of drug resistance. Addition of panobinostat, a histone deacetylase (HDAC) inhibitor, to bortezomib and dexamethasone improved progression-free survival (PFS) in relapsed and refractory MM patients. Here, we demonstrate how calcineurin, when inhibited by immunosuppressive drugs like FK506, is involved in myeloma cell growth and targeted by panobinostat. mRNA expression of
Yoichi Imai, Eri Ohta, Shu Takeda, Satoko Sunamura, Mariko Ishibashi, Hideto Tamura, Yan-hua Wang, Atsuko Deguchi, Junji Tanaka, Yoshiro Maru, Toshiko Motoji
Noelia Escobedo, Steven T. Proulx, Sinem Karaman, Miriam E. Dillard, Nicole Johnson, Michael Detmar, Guillermo Oliver
The intrathecal (IT) dosing route offers a seemingly obvious solution for delivering drugs directly to the central nervous system. However, gaps in understanding drug molecule behavior within the anatomically and kinetically unique environment of the mammalian IT space have impeded the establishment of pharmacokinetic principles for optimizing regional drug exposure along the neuraxis. Here, we have utilized high-resolution single-photon emission tomography with X-ray computed tomography to study the behavior of multiple molecular imaging tracers following an IT bolus injection, with supporting histology, autoradiography, block-face tomography, and MRI. Using simultaneous dual-isotope imaging, we demonstrate that the regional CNS tissue exposure of molecules with varying chemical properties is affected by IT space anatomy, cerebrospinal fluid (CSF) dynamics, CSF clearance routes, and the location and volume of the injected bolus. These imaging approaches can be used across species to optimize the safety and efficacy of IT drug therapy for neurological disorders.
Daniel A. Wolf, Jacob Y. Hesterman, Jenna M. Sullivan, Kelly D. Orcutt, Matthew D. Silva, Merryl Lobo, Tyler Wellman, Jack Hoppin, Ajay Verma
BACKGROUND. Kidney transplant biopsies offer an opportunity to understand the pathogenesis of organ fibrosis. We studied the relationships between the time of biopsy after transplant (TxBx), histologic fibrosis, diseases, and transcript expression.
METHODS. Expression microarrays from 681 kidney transplant indication biopsies taken either early (
RESULTS. Fibrosis was absent at transplantation but was present in some early biopsies by 4 months after transplant, apparently as a self-limited response to donation implantation injury not associated with progression to failure. The molecular phenotype of early biopsies represented the time sequence of the response to wounding: immediate expression of acute kidney injury transcripts, followed by fibrillar collagen transcripts after several weeks, then by the appearance of immunoglobulin and mast cell transcripts after several months as fibrosis appeared. Fibrosis in late biopsies correlated with injury, fibrillar collagen, immunoglobulin, and mast cell transcripts, but these were independent of time. Pathway analysis revealed epithelial response-to-wounding pathways such as Wnt/β-catenin.
CONCLUSION. Fibrosis in late biopsies had different associations because many kidneys had potentially progressive diseases and subsequently failed. Molecular correlations with fibrosis in late biopsies were independent of time, probably because ongoing injury obscured the response-to-wounding time sequence. The results indicate that fibrosis in kidney transplants is driven by nephron injury and that progression to failure reflects continuing injury, not autonomous fibrogenesis.
TRIAL REGISTRATION. INTERCOM study (www.clinicalTrials.gov; NCT01299168).
FUNDING. Canada Foundation for Innovation and Genome Canada.
Jeffery M. Venner, Konrad S. Famulski, Jeff Reeve, Jessica Chang, Philip F. Halloran
Secreted by activated cells or passively released by damaged cells, extracellular HMGB1 is a prototypical damage-associated molecular pattern (DAMP) inflammatory mediator. During the course of developing extracorporeal approaches to treating injury and infection, we inadvertently discovered that haptoglobin, the acute phase protein that binds extracellular hemoglobin and targets cellular uptake through CD163, also binds HMGB1. Haptoglobin-HMGB1 complexes elicit the production of antiinflammatory enzymes (heme oxygenase-1) and cytokines (e.g., IL-10) in WT but not in CD163-deficient macrophages. Genetic disruption of haptoglobin or CD163 expression significantly enhances mortality rates in standardized models of intra-abdominal sepsis in mice. Administration of haptoglobin to WT and to haptoglobin gene-deficient animals confers significant protection. These findings reveal a mechanism for haptoglobin modulation of the inflammatory action of HMGB1, with significant implications for developing experimental strategies targeting HMGB1-dependent inflammatory diseases.
Huan Yang, Haichao Wang, Yaakov A. Levine, Manoj K. Gunasekaran, Yongjun Wang, Meghan Addorisio, Shu Zhu, Wei Li, Jianhua Li, Dominique P.V. de Kleijn, Peder S. Olofsson, H. Shaw Warren, Mingzhu He, Yousef Al-Abed, Jesse Roth, Daniel J. Antoine, Sangeeta S. Chavan, Ulf Andersson, Kevin J. Tracey
Progressive HIV-1 infection leads to both profound immune suppression and pathologic inflammation in the majority of infected individuals. While adaptive immune dysfunction, as evidenced by CD4+ T cell depletion and exhaustion, has been extensively studied, less is known about the functional capacity of innate immune cell populations in the context of HIV-1 infection. Given the broad susceptibility to opportunistic infections and the dysregulated inflammation observed in progressive disease, we hypothesized that there would be significant changes in the innate cellular responses. Using a cohort of patients with multiple samplings before and after antiretroviral therapy (ART) initiation, we demonstrated increased responses to innate immune stimuli following viral suppression, as measured by the production of inflammatory cytokines. Plasma viral load itself had the strongest association with this change in innate functional capacity. We further identified epigenetic modifications in the
Eileen P. Scully, Ainsley Lockhart, Wilfredo Garcia-Beltran, Christine D. Palmer, Chelsey Musante, Eric Rosenberg, Todd M. Allen, J. Judy Chang, Ronald J. Bosch, Marcus Altfeld
The 2p15p16.1 microdeletion syndrome has a core phenotype consisting of intellectual disability, microcephaly, hypotonia, delayed growth, common craniofacial features, and digital anomalies. So far, more than 20 cases of 2p15p16.1 microdeletion syndrome have been reported in the literature; however, the size of the deletions and their breakpoints vary, making it difficult to identify the candidate genes. Recent reports pointed to 4 genes (
Hani Bagheri, Chansonette Badduke, Ying Qiao, Rita Colnaghi, Iga Abramowicz, Diana Alcantara, Christopher Dunham, Jiadi Wen, Robert S. Wildin, Malgorzata J.M. Nowaczyk, Jennifer Eichmeyer, Anna Lehman, Bruno Maranda, Sally Martell, Xianghong Shan, Suzanne M.E. Lewis, Mark O’Driscoll, Cheryl Y. Gregory-Evans, Evica Rajcan-Separovic
The transforming growth factor-β (TGF-β) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-β network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature. Quantitative proteomic and transcriptomic analyses of young-adult muscles identified a transcription/translation signature elicited by follistatin exposure, which included repression of ankyrin repeat and SOCS box protein 2 (
Jonathan R. Davey, Kevin I. Watt, Benjamin L. Parker, Rima Chaudhuri, James G. Ryall, Louise Cunningham, Hongwei Qian, Vittorio Sartorelli, Marco Sandri, Jeffrey Chamberlain, David E. James, Paul Gregorevic
We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α–activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B–deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition.
Yi Zhang, Yan Huang, Anna Cantalupo, Paula S. Azevedo, Mauro Siragusa, Jacek Bielawski, Frank J. Giordano, Annarita Di Lorenzo
Over one-fifth of North American women of childbearing age are obese, putting these women at risk for a variety of detrimental chronic diseases. In addition, obesity increases the risk for developing major complications during pregnancy. The mechanisms by which obesity contributes to pregnancy complications and loss remain unknown. Increasing evidence indicates that obesity results in major changes to adipose tissue immune cell composition and function; whether or not obesity also affects immune function in the uterus has not been explored. Here we investigated the effect of obesity on uterine natural killer (uNK) cells, which are essential for uterine artery remodeling and placental development. Using a cohort of obese or lean women, we found that obesity led to a significant reduction in uNK cell numbers accompanied with impaired uterine artery remodeling. uNK cells isolated from obese women had altered expression of genes and pathways associated with extracellular matrix remodeling and growth factor signaling. Specifically, uNK cells were hyper-responsive to PDGF, resulting in overexpression of decorin. Functionally, decorin strongly inhibited placental development by limiting trophoblast survival. Together, these findings establish a potentially new link between obesity and poor pregnancy outcomes, and indicate that obesity-driven changes to uterine-resident immune cells critically impair placental development.
Sofie Perdu, Barbara Castellana, Yoona Kim, Kathy Chan, Lauren DeLuca, Alexander G. Beristain
Over the past 8 years, the discovery of 11 new human polyomaviruses (HPyVs) has revived interest in this DNA tumor virus family. Although HPyV infection is widespread and largely asymptomatic, one of these HPyVs, Merkel cell polyomavirus (MCV), is a bona fide human tumor virus. JC virus (JCV), BK virus, HPyV7, and trichodysplasia-spinulosa virus (TSV) can cause nonneoplastic diseases in the setting of immunosuppression. Few specific reagents are available to study the biology of the newly discovered HPyVs. We developed a pan-HPyV-screening method using a cocktail of 3 antibodies that, when combined, recognize T antigen proteins of all HPyVs. We validated detection characteristics of the antibody cocktail by immunoblotting and immunohistochemistry and screened 1,184 cases, including well-defined diseases and tumor tissue microarrays. This assay robustly detected MCV, TSV, JCV, and HPyV7 in etiologically related diseases. We further identified WU polyomavirus in a case of chronic lymphocytic lymphoma-associated bronchitis. Except for scattered, incidentally infected cells in 5% of lung squamous cell carcinomas and colon adenocarcinomas, a broad panel of tumor tissues was largely negative for infection by any HPyV. This method eliminates known HPyVs as suspected causes of cancers investigated in this study. Pan-HPyV survey can be applied to identify diseases associated with recently discovered polyomaviruses.
Tuna Toptan, Samuel A. Yousem, Jonhan Ho, Yuki Matsushima, Laura P. Stabile, Maria-Teresa Fernández-Figueras, Rohit Bhargava, Akihide Ryo, Patrick S. Moore, Yuan Chang
FMS-like tyrosine kinase 3–targeted (FLT3-targeted) therapies have shown initial promise for the treatment of acute myeloid leukemia (AML) expressing FLT3-activating mutations; however, resistance emerges rapidly. Furthermore, limited options exist for the treatment of FLT3-independent AML, demonstrating the need for novel therapies that reduce toxicity and improve survival. MERTK receptor tyrosine kinase is overexpressed in 80% to 90% of AMLs and contributes to leukemogenesis. Here, we describe MRX-2843, a type 1 small-molecule tyrosine kinase inhibitor that abrogates activation of both MERTK and FLT3 and their downstream effectors. MRX-2843 treatment induces apoptosis and inhibits colony formation in AML cell lines and primary patient samples expressing MERTK and/or FLT3-ITD, with a wide therapeutic window compared with that of normal human cord blood cells. In murine orthotopic xenograft models, once-daily oral therapy prolonged survival 2- to 3-fold over that of vehicle-treated controls. Additionally, MRX-2843 retained activity against quizartinib-resistant FLT3-ITD–mutant proteins with clinically relevant alterations at the D835 or F691 loci and prolonged survival in xenograft models of quizartinib-resistant AML. Together, these observations validate MRX-2843 as a translational agent and support its clinical development for the treatment of AML.
Katherine A. Minson, Catherine C. Smith, Deborah DeRyckere, Clara Libbrecht, Alisa B. Lee-Sherick, Madeline G. Huey, Elisabeth A. Lasater, Gregory D. Kirkpatrick, Michael A. Stashko, Weihe Zhang, Craig T. Jordan, Dmitri Kireev, Xiaodong Wang, Stephen V. Frye, H. Shelton Earp, Neil P. Shah, Douglas K. Graham
Juvenile idiopathic arthritis (JIA) is the most common pediatric rheumatological condition. Although it has been proposed that JIA has an autoimmune component, the autoantigens are still unknown. Using biochemical and proteomic approaches, we identified the molecular chaperone transthyretin (TTR) as an antigenic target for B and T cell immune responses. TTR was eluted from IgG complexes and affinity purified from 3 JIA patients, and a statistically significant increase in TTR autoantibodies was observed in a group of 43 JIA patients. Three cryptic, HLA-DR1–restricted TTR peptides, which induced CD4+ T cell expansion and IFN-γ and TNF-α production in 3 out of 17 analyzed patients, were also identified. Misfolding, aggregation and oxidation of TTR, as observed in the synovial fluid of all JIA patients, enhanced its immunogenicity in HLA-DR1 transgenic mice. Our data point to TTR as an autoantigen potentially involved in the pathogenesis of JIA and to oxidation and aggregation as a mechanism facilitating TTR autoimmunity.
Cristina C. Clement, Halima Moncrieffe, Aditi Lele, Ginger Janow, Aniuska Becerra, Francesco Bauli, Fawzy A. Saad, Giorgio Perino, Cristina Montagna, Neil Cobelli, John Hardin, Lawrence J. Stern, Norman Ilowite, Steven A. Porcelli, Laura Santambrogio
The cardioprotective inducible enzyme heme oxygenase-1 (HO-1) degrades prooxidant heme into equimolar quantities of carbon monoxide, biliverdin, and iron. We hypothesized that HO-1 mediates cardiac protection, at least in part, by regulating mitochondrial quality control. We treated WT and HO-1 transgenic mice with the known mitochondrial toxin, doxorubicin (DOX). Relative to WT mice, mice globally overexpressing human HO-1 were protected from DOX-induced dilated cardiomyopathy, cardiac cytoarchitectural derangement, and infiltration of CD11b+ mononuclear phagocytes. Cardiac-specific overexpression of HO-1 ameliorated DOX-mediated dilation of the sarcoplasmic reticulum as well as mitochondrial disorganization in the form of mitochondrial fragmentation and increased numbers of damaged mitochondria in autophagic vacuoles. HO-1 overexpression promotes mitochondrial biogenesis by upregulating protein expression of NRF1, PGC1α, and TFAM, which was inhibited in WT animals treated with DOX. Concomitantly, HO-1 overexpression inhibited the upregulation of the mitochondrial fission mediator Fis1 and resulted in increased expression of the fusion mediators, Mfn1 and Mfn2. It also prevented dynamic changes in the levels of key mediators of the mitophagy pathway, PINK1 and parkin. Therefore, these findings suggest that HO-1 has a novel role in protecting the heart from oxidative injury by regulating mitochondrial quality control.
Travis D. Hull, Ravindra Boddu, Lingling Guo, Cornelia C. Tisher, Amie M. Traylor, Bindiya Patel, Reny Joseph, Sumanth D. Prabhu, Hagir B. Suliman, Claude A. Piantadosi, Anupam Agarwal, James F. George
Glioblastomas are highly infiltrated by diverse immune cells, including microglia, macrophages, and myeloid-derived suppressor cells (MDSCs). Understanding the mechanisms by which glioblastoma-associated myeloid cells (GAMs) undergo metamorphosis into tumor-supportive cells, characterizing the heterogeneity of immune cell phenotypes within glioblastoma subtypes, and discovering new targets can help the design of new efficient immunotherapies. In this study, we performed a comprehensive battery of immune phenotyping, whole-genome microarray analysis, and microRNA expression profiling of GAMs with matched blood monocytes, healthy donor monocytes, normal brain microglia, nonpolarized M0 macrophages, and polarized M1, M2a, M2c macrophages. Glioblastoma patients had an elevated number of monocytes relative to healthy donors. Among CD11b+ cells, microglia and MDSCs constituted a higher percentage of GAMs than did macrophages. GAM profiling using flow cytometry studies revealed a continuum between the M1- and M2-like phenotype. Contrary to current dogma, GAMs exhibited distinct immunological functions, with the former aligned close to nonpolarized M0 macrophages.
Konrad Gabrusiewicz, Benjamin Rodriguez, Jun Wei, Yuuri Hashimoto, Luke M. Healy, Sourindra N. Maiti, Ginu Thomas, Shouhao Zhou, Qianghu Wang, Ahmed Elakkad, Brandon D. Liebelt, Nasser K. Yaghi, Ravesanker Ezhilarasan, Neal Huang, Jeffrey S. Weinberg, Sujit S. Prabhu, Ganesh Rao, Raymond Sawaya, Lauren A. Langford, Janet M. Bruner, Gregory N. Fuller, Amit Bar-Or, Wei Li, Rivka R. Colen, Michael A. Curran, Krishna P. Bhat, Jack P. Antel, Laurence J. Cooper, Erik P. Sulman, Amy B. Heimberger
In human myocarditis and its sequela dilated cardiomyopathy (DCM), the mechanisms and immune phenotype governing disease and subsequent heart failure are not known. Here, we identified a Th17 cell immunophenotype of human myocarditis/DCM with elevated CD4+IL17+ T cells and Th17-promoting cytokines IL-6, TGF-β, and IL-23 as well as GM-CSF–secreting CD4+ T cells. The Th17 phenotype was linked with the effects of cardiac myosin on CD14+ monocytes, TLR2, and heart failure. Persistent heart failure was associated with high percentages of IL-17–producing T cells and IL-17–promoting cytokines, and the myocarditis/DCM phenotype included significantly low percentages of FOXP3+ Tregs, which may contribute to disease severity. We demonstrate a potentially novel mechanism in human myocarditis/DCM in which TLR2 peptide ligands from human cardiac myosin stimulated exaggerated Th17-related cytokines including TGF-β, IL-6, and IL-23 from myocarditic CD14+ monocytes in vitro, and an anti-TLR2 antibody abrogated the cytokine response. Our translational study explains how an immune phenotype may be initiated by cardiac myosin TLR ligand stimulation of monocytes to generate Th17-promoting cytokines and development of pathogenic Th17 cells in human myocarditis and heart failure, and provides a rationale for targeting IL-17A as a therapeutic option.
Jennifer M. Myers, Leslie T. Cooper, David C. Kem, Stavros Stavrakis, Stanley D. Kosanke, Ethan M. Shevach, DeLisa Fairweather, Julie A. Stoner, Carol J. Cox, Madeleine W. Cunningham
We report the discovery of a claudin-low molecular subtype of high-grade bladder cancer that shares characteristics with the homonymous subtype of breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features including increased rates of
Jordan Kardos, Shengjie Chai, Lisle E. Mose, Sara R. Selitsky, Bhavani Krishnan, Ryoichi Saito, Michael D. Iglesia, Matthew I. Milowsky, Joel S. Parker, William Y. Kim, Benjamin G. Vincent
The mechanisms by which exercise mediates its multiple cardiac benefits are only partly understood. Prior comprehensive analyses of the cardiac transcriptional components and microRNAs dynamically regulated by exercise suggest that the CBP/p300-interacting protein CITED4 is a downstream effector in both networks. While CITED4 has documented functional consequences in neonatal cardiomyocytes in vitro, nothing is known about its effects in the adult heart. To investigate the impact of cardiac CITED4 expression in adult animals, we generated transgenic mice with regulated, cardiomyocyte-specific CITED4 expression. Cardiac CITED4 expression in adult mice was sufficient to induce an increase in heart weight and cardiomyocyte size with normal systolic function, similar to the effects of endurance exercise training. After ischemia-reperfusion, CITED4 expression did not change initial infarct size but mediated substantial functional recovery while reducing ventricular dilation and fibrosis. Forced cardiac expression of CITED4 also induced robust activation of the mTORC1 pathway after ischemic injury. Moreover, pharmacological inhibition of mTORC1 abrogated CITED4’s effects in vitro and in vivo. Together, these data establish CITED4 as a regulator of mTOR signaling that is sufficient to induce physiologic hypertrophy at baseline and mitigate adverse ventricular remodeling after ischemic injury.
Vassilios J. Bezzerides, Colin Platt, Carolin Lerchenmüller, Kaavya Paruchuri, Nul Loren Oh, Chunyang Xiao, Yunshan Cao, Nina Mann, Bruce M. Spiegelman, Anthony Rosenzweig
Central clonal deletion has been considered the critical factor responsible for the robust state of tolerance achieved by chimerism-based experimental protocols, but split-tolerance models and the clinical experience are calling this assumption into question. Although clone-size reduction through deletion has been shown to be universally required for achieving allotolerance, it remains undetermined whether it is sufficient by itself. Therapeutic Treg treatment induces chimerism and tolerance in a stringent murine BM transplantation model devoid of myelosuppressive recipient treatment. In contrast to irradiation chimeras, chronic rejection (CR) of skin and heart allografts in Treg chimeras was permanently prevented, even in the absence of complete clonal deletion of donor MHC-reactive T cells. We show that minor histocompatibility antigen mismatches account for CR in irradiation chimeras without global T cell depletion. Furthermore, we show that Treg therapy–induced tolerance prevents CR in a linked suppression–like fashion, which is maintained by active regulatory mechanisms involving recruitment of thymus-derived Tregs to the graft. These data suggest that highly efficient intrathymic and peripheral deletion of donor-reactive T cells for specificities expressed on hematopoietic cells preclude the expansion of donor-specific Tregs and, hence, do not allow for spreading of tolerance to minor specificities that are not expressed by donor BM.
Nina Pilat, Benedikt Mahr, Lukas Unger, Karin Hock, Christoph Schwarz, Andreas M. Farkas, Ulrike Baranyi, Fritz Wrba, Thomas Wekerle
Rheumatoid arthritis (RA) is a debilitating disease characterized by persistent accumulation of leukocytes within the articular cavity and synovial tissue. Metabololipidomic profiling of arthritic joints from omega-3 supplemented mice identified elevated levels of specialized proresolving lipid mediators (SPM) including resolvin D1 (RvD1). Profiling of human RA synovial fluid revealed physiological levels of RvD1, which — once applied to human neutrophils — attenuated chemotaxis. These results prompted analyses of the antiarthritic properties of RvD1 in a model of murine inflammatory arthritis. The stable epimer 17
Lucy V. Norling, Sarah E. Headland, Jesmond Dalli, Hildur H. Arnardottir, Oliver Haworth, Hefin R. Jones, Daniel Irimia, Charles N. Serhan, Mauro Perretti
Arrhythmogenic cardiomyopathy (ACM) is characterized by redistribution of junctional proteins, arrhythmias, and progressive myocardial injury. We previously reported that SB216763 (SB2), annotated as a GSK3β inhibitor, reverses disease phenotypes in a zebrafish model of ACM. Here, we show that SB2 prevents myocyte injury and cardiac dysfunction in vivo in two murine models of ACM at baseline and in response to exercise. SB2-treated mice with desmosome mutations showed improvements in ventricular ectopy and myocardial fibrosis/inflammation as compared with vehicle-treated (Veh-treated) mice. GSK3β inhibition improved left ventricle function and survival in sedentary and exercised
Stephen P. Chelko, Angeliki Asimaki, Peter Andersen, Djahida Bedja, Nuria Amat-Alarcon, Deeptankar DeMazumder, Ravirasmi Jasti, Calum A. MacRae, Remo Leber, Andre G. Kleber, Jeffrey E. Saffitz, Daniel P. Judge
Immunotherapies targeting the immune checkpoint receptor programmed cell death protein 1 (PD-1) have shown remarkable efficacy in treating cancer. CD4+CD25hiFoxP3+ Tregs are critical regulators of immune responses in autoimmunity and malignancies, but the functional status of human Tregs expressing PD-1 remains unclear. We examined functional and molecular features of PD-1hi Tregs in healthy subjects and patients with glioblastoma multiforme (GBM), combining functional assays, RNA sequencing, and cytometry by time of flight (CyTOF). In both patients with GBM and healthy subjects, circulating PD-1hi Tregs displayed reduced suppression of CD4+ effector T cells, production of IFN-γ, and molecular signatures of exhaustion. Transcriptional profiling of tumor-resident Tregs revealed that several genes coexpressed with PD-1 and associated with IFN-γ production and exhaustion as well as enrichment in exhaustion signatures compared with circulating PD-1hi Tregs. CyTOF analysis of circulating and tumor-infiltrating Tregs from patients with GBM treated with PD-1-blocking antibodies revealed that treatment shifts the profile of circulating Tregs toward a more exhausted phenotype reminiscent of that of tumor-infiltrating Tregs, further increasing IFN-γ production. Thus, high PD-1 expression on human Tregs identifies dysfunctional, exhausted Tregs secreting IFN-γ that exist in healthy individuals and are enriched in tumor infiltrates, possibly losing function as they attempt to modulate the antitumoral immune responses.
Daniel E. Lowther, Brittany A. Goods, Liliana E. Lucca, Benjamin A. Lerner, Khadir Raddassi, David van Dijk, Amanda L. Hernandez, Xiangguo Duan, Murat Gunel, Vlad Coric, Smita Krishnaswamy, J. Christopher Love, David A. Hafler
We created and tested multi-epitope DNA or protein vaccines with TLR4 ligand emulsion adjuvant (gluco glucopyranosyl lipid adjuvant in a stable emulsion [GLA-SE]) for their ability to protect against Toxoplasma gondii in HLA transgenic mice. Our constructs each included 5 of our best down-selected CD8+ T cell–eliciting epitopes, a universal CD4+ helper T lymphocyte epitope (PADRE), and a secretory signal, all arranged for optimal MHC-I presentation. Their capacity to elicit immune and protective responses was studied using immunization of HLA-A*11:01 transgenic mice. These multi-epitope vaccines increased memory CD8+ T cells that produced IFN-γ and protected mice against parasite burden when challenged with T. gondii. Endocytosis of emulsion-trapped protein and cross presentation of the antigens must account for the immunogenicity of our adjuvanted protein. Thus, our work creates an adjuvanted platform assembly of peptides resulting in cross presentation of CD8+ T cell–eliciting epitopes in a vaccine that prevents toxoplasmosis.
Kamal El Bissati, Aziz A. Chentoufi, Paulette A. Krishack, Ying Zhou, Stuart Woods, Jitender P. Dubey, Lo Vang, Joseph Lykins, Kate E. Broderick, Ernest Mui, Yasuhiro Suzuki, Qila Sa, Stephanie Bi, Nestor Cardona, Shiv K. Verma, Laura Fraczek, Catherine A. Reardon, John Sidney, Jeff Alexander, Alessandro Sette, Tom Vedvick, Chris Fox, Jeffrey A. Guderian, Steven Reed, Craig W. Roberts, Rima McLeod
Michael P. Whyte, Katherine L. Madson, Dawn Phillips, Amy L. Reeves, William H. McAlister, Amy Yakimoski, Karen E. Mack, Kim Hamilton, Kori Kagan, Kenji P. Fujita, David D. Thompson, Scott Moseley, Tatjana Odrljin, Cheryl Rockman-Greenberg
Tregs imprint an early immunotolerant tumor environment that prevents effective antitumor immune responses. Using transcriptomics of tumor tissues, we identified early upregulation of VEGF and TGF-β pathways compatible with tolerance imprinting. Silencing of VEGF or TGF-β in tumor cells induced early and pleiotropic modulation of immune-related transcriptome signatures in tumor tissues. These were surprisingly similar for both silenced tumors and related to common downstream effects on Tregs. Silencing of VEGF or TGF-β resulted in dramatically delayed tumor growth, associated with decreased Tregs and myeloid-derived suppressor cells and increased effector T cell activation in tumor infiltrates. Strikingly, co-silencing of TGF-β and VEGF led to a substantial spontaneous tumor eradication rate and the combination of their respective inhibitory drugs was synergistic. VEGF and/or TGF-β silencing also restored tumor sensitivity to tumor-specific cell therapies and markedly improved the efficacy of anti–PD-1/anti–CTLA-4 treatment. Thus, TGF-β and VEGF cooperatively control the tolerant environment of tumors and are targets for improved cancer immunotherapies.
Tristan Courau, Djamel Nehar-Belaid, Laura Florez, Béatrice Levacher, Thomas Vazquez, Faustine Brimaud, Bertrand Bellier, David Klatzmann
Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.
Florian Grahammer, Christoph Wigge, Christoph Schell, Oliver Kretz, Jaakko Patrakka, Simon Schneider, Martin Klose, Julia Kind, Sebastian J. Arnold, Anja Habermann, Ricarda Bräuniger, Markus M. Rinschen, Linus Völker, Andreas Bregenzer, Dennis Rubbenstroth, Melanie Boerries, Dontscho Kerjaschki, Jeffrey H. Miner, Gerd Walz, Thomas Benzing, Alessia Fornoni, Achilleas S. Frangakis, Tobias B. Huber
Suresh Gopi Kalathil, Amit Anand Lugade, Austin Miller, Renuka Iyer, Yasmin Thanavala
Mst1 is a central Ser-Thr kinase in the Hippo pathway, which promotes apoptosis and inhibits cell proliferation. We have shown previously that, in cardiomyocytes, oxidative stress activates Mst1 at mitochondria, where Mst1 phosphorylates Bcl-xL at Ser14, inducing dissociation of Bcl-xL from Bax and thereby promoting apoptosis. However, the functional significance of Ser14 phosphorylation of endogenous Bcl-xL in vivo remains elusive. We generated knockin (KI) mice in which Ser14 of Bcl-xL is replaced with Ala. KI mice were born at the expected Mendelian ratio, and adult KI mice exhibited normal cardiac morphology and function at baseline. However, KI mice were protected from myocardial ischemia/reperfusion (I/R) injury and exhibited reduced cardiomyocyte apoptosis. Although suppression of endogenous Mst1 also reduced I/R injury, there was no additive protective effect when Mst1 was inhibited in KI mice. The development of dilated cardiomyopathy induced by cardiac-specific overexpression of Mst1 was also ameliorated in KI mice. Lats2 and YAP, two other key components of the Hippo pathway, were not affected in KI mice. These results suggest that Ser14 phosphorylation of Bcl-xL plays an essential role in mediating both cardiomyocyte apoptosis and myocardial injury by acting as a key downstream mediator of Mst1 independently of the canonical Hippo pathway.
Michinari Nakamura, Peiyong Zhai, Dominic P. Del Re, Yasuhiro Maejima, Junichi Sadoshima
Posttransplantation cyclophosphamide (PTCy) effectively prevents graft-versus-host disease (GVHD), but its immunologic impact is poorly understood. We assessed lymphocyte reconstitution via flow cytometry (
Christopher G. Kanakry, David G. Coffey, Andrea M.H. Towlerton, Ante Vulic, Barry E. Storer, Jeffrey Chou, Cecilia C.S. Yeung, Christopher D. Gocke, Harlan S. Robins, Paul V. O’Donnell, Leo Luznik, Edus H. Warren
Mutations of the
Toshifumi Fujiwara, Shiqiao Ye, Thiago Castro-Gomes, Caylin G. Winchell, Norma W. Andrews, Daniel E. Voth, Kottayil I. Varughese, Samuel G. Mackintosh, Yunfeng Feng, Nathan Pavlos, Takashi Nakamura, Stavros C. Manolagas, Haibo Zhao
In idiopathic pulmonary fibrosis (IPF), the fibroblast focus is a key histological feature representing active fibroproliferation. On standard 2D pathologic examination, fibroblast foci are considered small, distinct lesions, although they have been proposed to form a highly interconnected reticulum as the leading edge of a “wave” of fibrosis. Here, we characterized fibroblast focus morphology and interrelationships in 3D using an integrated micro-CT and histological methodology. In 3D, fibroblast foci were morphologically complex structures, with large variations in shape and volume (range, 1.3 × 104 to 9.9 × 107 μm3). Within each tissue sample numerous multiform foci were present, ranging from a minimum of 0.9 per mm3 of lung tissue to a maximum of 11.1 per mm3 of lung tissue. Each focus was an independent structure, and no interconnections were observed. Together, our data indicate that in 3D fibroblast foci form a constellation of heterogeneous structures with large variations in shape and volume, suggesting previously unrecognized plasticity. No evidence of interconnectivity was identified, consistent with the concept that foci represent discrete sites of lung injury and repair.
Mark G. Jones, Aurélie Fabre, Philipp Schneider, Francesco Cinetto, Giacomo Sgalla, Mark Mavrogordato, Sanjay Jogai, Aiman Alzetani, Ben G. Marshall, Katherine M.A. O’Reilly, Jane A. Warner, Peter M. Lackie, Donna E. Davies, David M. Hansell, Andrew G. Nicholson, Ian Sinclair, Kevin K. Brown, Luca Richeldi
Airway and/or lung remodeling, involving exaggerated extracellular matrix (ECM) protein deposition, is a critical feature common to pulmonary diseases including chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). Fibulin-1 (Fbln1), an important ECM protein involved in matrix organization, may be involved in the pathogenesis of these diseases. We found that Fbln1 was increased in COPD patients and in cigarette smoke–induced (CS-induced) experimental COPD in mice. Genetic or therapeutic inhibition of
Gang Liu, Marion A. Cooley, Andrew G. Jarnicki, Alan C-Y. Hsu, Prema M. Nair, Tatt Jhong Haw, Michael Fricker, Shaan L. Gellatly, Richard Y. Kim, Mark D. Inman, Gavin Tjin, Peter A.B. Wark, Marjorie M. Walker, Jay C. Horvat, Brian G. Oliver, W. Scott Argraves, Darryl A. Knight, Janette K. Burgess, Philip M. Hansbro
Reperfusion injury can exacerbate tissue damage in ischemic stroke, but little is known about the mechanisms linking ROS to stroke severity. Here, we tested the hypothesis that protein methionine oxidation potentiates NF-κB activation and contributes to cerebral ischemia/reperfusion injury. We found that overexpression of methionine sulfoxide reductase A (MsrA), an antioxidant enzyme that reverses protein methionine oxidation, attenuated ROS-augmented NF-κB activation in endothelial cells, in part, by protecting against the oxidation of methionine residues in the regulatory domain of calcium/calmodulin-dependent protein kinase II (CaMKII). In a murine model, MsrA deficiency resulted in increased NF-κB activation and neutrophil infiltration, larger infarct volumes, and more severe neurological impairment after transient cerebral ischemia/reperfusion injury. This phenotype was prevented by inhibition of NF-κB or CaMKII. MsrA-deficient mice also exhibited enhanced leukocyte rolling and upregulation of E-selectin, an endothelial NF-κB–dependent adhesion molecule known to contribute to neurovascular inflammation in ischemic stroke. Finally, bone marrow transplantation experiments demonstrated that the neuroprotective effect was mediated by MsrA expressed in nonhematopoietic cells. These findings suggest that protein methionine oxidation in nonmyeloid cells is a key mechanism of postischemic oxidative injury mediated by NF-κB activation, leading to neutrophil recruitment and neurovascular inflammation in acute ischemic stroke.
Sean X. Gu, Ilya O. Blokhin, Katina M. Wilson, Nirav Dhanesha, Prakash Doddapattar, Isabella M. Grumbach, Anil K. Chauhan, Steven R. Lentz
Fingolimod (FTY720, Gilenya), a sphingosine-1-phosphate receptor (S1PR) modulator, is one of the first-line immunomodulatory therapies for treatment of relapsing-remitting multiple sclerosis (MS). Human
Hsing-Chuan Tsai, Yingxiang Huang, Christopher S. Garris, Monica A. Moreno, Christina W. Griffin, May H. Han
The deubiquitinase-encoding gene
Yingai Jane Jin, Sally Wang, Joshua Cho, M. Angelica Selim, Tim Wright, George Mosialos, Jennifer Y. Zhang
The
Keisuke Maeshima, Stephanie M. Stanford, Deepa Hammaker, Cristiano Sacchetti, Li-fan Zeng, Rizi Ai, Vida Zhang, David L. Boyle, German R. Aleman Muench, Gen-Sheng Feng, John W. Whitaker, Zhong-Yin Zhang, Wei Wang, Nunzio Bottini, Gary S. Firestein
Levodopa-induced dyskinesia (LID) is the most common, disruptive complication of Parkinson’s disease (PD) pharmacotherapy, yet despite decades of research, the changes in regional brain function underlying LID remain largely unknown. We previously found that the cerebral vasomotor and metabolic responses to levodopa are dissociated in PD subjects. Nonetheless, it is unclear whether levodopa-mediated dissociation is exaggerated in LID or distinguishes LID from non-LID subjects. To explore this possibility, we used dual-tracer positron emission tomography to quantify regional cerebral blood flow and metabolic activity in 28 PD subjects (14 LID, 14 non-LID), scanned before and during intravenous levodopa infusion. Levodopa-mediated dissociation was most prominent in the posterior putamen (
Vincent A. Jourdain, Chris C. Tang, Florian Holtbernd, Christian Dresel, Yoon Young Choi, Yilong Ma, Vijay Dhawan, David Eidelberg
Lymphangioleiomyomatosis (LAM) is a progressive lung disease that primarily affects young women. Genetic evidence suggests that LAM cells bearing
Chenggang Li, Na Li, Xiaolei Liu, Erik Y. Zhang, Yang Sun, Kouhei Masuda, Jing Li, Julia Sun, Tasha Morrison, Xiangke Li, Yuanguang Chen, Jiang Wang, Nagla A. Karim, Yi Zhang, John Blenis, Mauricio J. Reginato, Elizabeth P. Henske, Jane J. Yu
Within the CNS, a dysregulated hemostatic response contributes to both hemorrhagic and ischemic strokes. Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, plays an essential role in hemostasis and also contributes to thrombosis. Using both genetic and pharmacologic approaches, we characterized the contribution of neuroectodermal (NE) cell TF to the pathophysiology of stroke. We used mice with various levels of TF expression and found that astrocyte TF activity reduced to ~5% of WT levels was still sufficient to maintain hemostasis after hemorrhagic stroke but was also low enough to attenuate inflammation, reduce damage to the blood-brain barrier, and improve outcomes following ischemic stroke. Pharmacologic inhibition of TF during the reperfusion phase of ischemic stroke attenuated neuronal damage, improved behavioral deficit, and prevented mortality of mice. Our data demonstrate that NE cell TF limits bleeding complications associated with the transition from ischemic to hemorrhagic stroke and also contributes to the reperfusion injury after ischemic stroke. The high level of TF expression in the CNS is likely the result of selective pressure to limit intracerebral hemorrhage (ICH) after traumatic brain injury but, in the modern era, poses the additional risk of increased ischemia-reperfusion injury after ischemic stroke.
Shaobin Wang, Brandi Reeves, Erica M. Sparkenbaugh, Janice Russell, Zbigniew Soltys, Hua Zhang, James E. Faber, Nigel S. Key, Daniel Kirchhofer, D. Neil Granger, Nigel Mackman, Rafal Pawlinski
Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding–mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.
Arjun Saha, Susan Buntz, Paula Scotland, Li Xu, Pamela Noeldner, Sachit Patel, Amy Wollish, Aruni Gunaratne, Tracy Gentry, Jesse Troy, Glenn K. Matsushima, Joanne Kurtzberg, Andrew E. Balber
Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and
Tao Huang, Meredith Hazen, Yonglei Shang, Meijuan Zhou, Xiumin Wu, Donghong Yan, Zhonghua Lin, Margaret Solon, Elizabeth Luis, Hai Ngu, Yongchang Shi, Arna Katewa, David F. Choy, Nandhini Ramamoorthi, Erick R. Castellanos, Mercedesz Balazs, Min Xu, Wyne P. Lee, Marissa L. Matsumoto, Jian Payandeh, Joseph R. Arron, Jo-Anne Hongo, Jianyong Wang, Isidro Hötzel, Cary D. Austin, Karin Reif
Mucopolysaccharidosis type II (MPSII) is an X-linked lysosomal storage disease characterized by severe neurologic and somatic disease caused by deficiency of iduronate-2-sulfatase (IDS), an enzyme that catabolizes the glycosaminoglycans heparan and dermatan sulphate. Intravenous enzyme replacement therapy (ERT) currently constitutes the only approved therapeutic option for MPSII. However, the inability of recombinant IDS to efficiently cross the blood-brain barrier (BBB) limits ERT efficacy in treating neurological symptoms. Here, we report a gene therapy approach for MPSII through direct delivery of vectors to the CNS. Through a minimally invasive procedure, we administered adeno-associated virus vectors encoding IDS (AAV9-
Sandra Motas, Virginia Haurigot, Miguel Garcia, Sara Marcó, Albert Ribera, Carles Roca, Xavier Sánchez, Víctor Sánchez, Maria Molas, Joan Bertolin, Luca Maggioni, Xavier León, Jesús Ruberte, Fatima Bosch
Zoheb B. Kazi, Sean N. Prater, Joyce A. Kobori, David Viskochil, Carrie Bailey, Renuka Gera, David W. Stockton, Paul McIntosh, Amy S. Rosenberg, Priya S. Kishnani
Regulatory T cells (Tregs) play a crucial role in the maintenance of peripheral tolerance. Quantitative and/or qualitative defects in Tregs result in diseases such as autoimmunity, allergy, malignancy, and graft-versus-host disease (GVHD), a serious complication of allogeneic stem cell transplantation (SCT). We recently reported increased expression of autophagy-related genes (
Laëtitia Le Texier, Katie E. Lineburg, Benjamin Cao, Cameron McDonald-Hyman, Lucie Leveque-El Mouttie, Jemma Nicholls, Michelle Melino, Blessy C. Nalkurthi, Kylie A. Alexander, Bianca Teal, Stephen J. Blake, Fernando Souza-Fonseca-Guimaraes, Christian R. Engwerda, Rachel D. Kuns, Steven W. Lane, Michele Teng, Charis Teh, Daniel Gray, Andrew D. Clouston, Susan K. Nilsson, Bruce R. Blazar, Geoffrey R. Hill, Kelli P.A. MacDonald
Despite identification of causal genes for various lipodystrophy syndromes, the molecular basis of some peculiar lipodystrophies remains obscure. In an African-American pedigree with a novel autosomal dominant, atypical familial partial lipodystrophy (FPLD), we performed linkage analysis for candidate regions and whole-exome sequencing to identify the disease-causing mutation. Affected adults reported marked loss of fat from the extremities, with excess fat in the face and neck at age 13–15 years, and developed metabolic complications later. A heterozygous g.112837956C>T mutation on chromosome 10 (c.202C>T, p.Leu68Phe) affecting a highly conserved residue in adrenoceptor α 2A (
Abhimanyu Garg, Shireesha Sankella, Chao Xing, Anil K. Agarwal
Despite major advances in early detection and prognosis, chemotherapy resistance is a major hurdle in the battle against breast cancer. Identifying predictive markers and understanding the mechanisms are key steps to overcoming chemoresistance. Methylation-controlled J protein (MCJ, also known as DNAJC15) is a negative regulator of mitochondrial respiration and has been associated with chemotherapeutic drug sensitivity in cancer cell lines. Here we show, in a retrospective study of a large cohort of breast cancer patients, that low MCJ expression in breast tumors predicts high risk of relapse in patients treated with chemotherapy; however, MCJ expression does not correlate with response to endocrine therapy. In a prospective study in breast cancer patients undergoing neoadjuvant therapy, low MCJ expression also correlates with poor clinical response to chemotherapy and decreased disease-free survival. Using MCJ-deficient mice, we demonstrate that lack of MCJ is sufficient to induce mammary tumor chemoresistance in vivo. Thus, loss of expression of this endogenous mitochondrial modulator in breast cancer promotes the development of chemoresistance.
Maria J. Fernández-Cabezudo, Issam Faour, Kenneth Jones, Devin P. Champagne, Mohammed A. Jaloudi, Yassir A. Mohamed, Ghada Bashir, Saeeda Almarzooqi, Alia Albawardi, M. Jawad Hashim, Thomas S. Roberts, Haytham El-Salhat, Hakam El-Taji, Adnan Kassis, Dylan E. O’Sullivan, Brock C. Christensen, James DeGregori, Basel K. al-Ramadi, Mercedes Rincon
BACKGROUND. Kidney function decreases with age. A potential mechanistic explanation for kidney and allograft half-life has evolved through the realization that linear reduction in glomerular podocyte density could drive progressive glomerulosclerosis to impact both native kidney and allograft half-lives.
METHODS. Predictions from podometrics (quantitation of podocyte parameters) were tested using independent pathologic, functional, and outcome data for native kidneys and allografts derived from published reports and large registries.
RESULTS. With age, native kidneys exponentially develop glomerulosclerosis, reduced renal function, and end-stage kidney disease, projecting a finite average kidney life span. The slope of allograft failure rate versus age parallels that of reduction in podocyte density versus age. Quantitative modeling projects allograft half-life at any donor age, and rate of podocyte detachment parallels the observed allograft loss rate.
CONCLUSION. Native kidneys are designed to have a limited average life span of about 100–140 years. Allografts undergo an accelerated aging-like process that accounts for their unexpectedly short half-life (about 15 years), the observation that older donor age is associated with shorter allograft half-life, and the fact that long-term allograft survival has not substantially improved. Podometrics provides potential readouts for these processes, thereby offering new approaches for monitoring and intervention.
FUNDING: National Institutes of Health.
Abhijit S. Naik, Farsad Afshinnia, Diane Cibrik, Jeffrey B. Hodgin, Fan Wu, Min Zhang, Masao Kikuchi, Larysa Wickman, Milagros Samaniego, Markus Bitzer, Jocelyn E. Wiggins, Akinlolu Ojo, Yi Li, Roger C. Wiggins
Diabetes is associated with altered cellular metabolism, but how altered metabolism contributes to the development of diabetic complications is unknown. We used the BKS
Kelli M. Sas, Pradeep Kayampilly, Jaeman Byun, Viji Nair, Lucy M. Hinder, Junguk Hur, Hongyu Zhang, Chengmao Lin, Nathan R. Qi, George Michailidis, Per-Henrik Groop, Robert G. Nelson, Manjula Darshi, Kumar Sharma, Jeffrey R. Schelling, John R. Sedor, Rodica Pop-Busui, Joel M. Weinberg, Scott A. Soleimanpour, Steven F. Abcouwer, Thomas W. Gardner, Charles F. Burant, Eva L. Feldman, Matthias Kretzler, Frank C. Brosius III, Subramaniam Pennathur
Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates’ receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor–treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis.
Eirini Kefaloyianni, Muthu Lakshmi Muthu, Jakob Kaeppler, Xiaoming Sun, Venkata Sabbisetti, Athena Chalaris, Stefan Rose-John, Eitan Wong, Irit Sagi, Sushrut S. Waikar, Helmut Rennke, Benjamin D. Humphreys, Joseph V. Bonventre, Andreas Herrlich
Lynette M. Sholl, Khanh Do, Priyanka Shivdasani, Ethan Cerami, Adrian M. Dubuc, Frank C. Kuo, Elizabeth P. Garcia, Yonghui Jia, Phani Davineni, Ryan P. Abo, Trevor J. Pugh, Paul van Hummelen, Aaron R. Thorner, Matthew Ducar, Alice H. Berger, Mizuki Nishino, Katherine A. Janeway, Alanna Church, Marian Harris, Lauren L. Ritterhouse, Joshua D. Campbell, Vanesa Rojas-Rudilla, Azra H. Ligon, Shakti Ramkissoon, James M. Cleary, Ursula Matulonis, Geoffrey R. Oxnard, Richard Chao, Vanessa Tassell, James Christensen, William C. Hahn, Philip W. Kantoff, David J. Kwiatkowski, Bruce E. Johnson, Matthew Meyerson, Levi A. Garraway, Geoffrey I. Shapiro, Barrett J. Rollins, Neal I. Lindeman, Laura E. MacConaill
DC-based vaccines that initiate T cell responses are well tolerated and have demonstrated efficacy for tumor immunotherapy, with the potential to be combined with other therapies. Targeting vaccine antigens (Ag) directly to the DCs in vivo is more effective than cell-based therapies in mouse models and is therefore a promising strategy to translate to humans. The human CD141+ DCs are considered the most clinically relevant for initiating CD8+ T cell responses critical for killing tumors or infected cells, and they specifically express the C-type lectin-like receptor CLEC9A that facilitates presentation of Ag by these DCs. We have therefore developed a human chimeric Ab that specifically targets CLEC9A on CD141+ DCs in vitro and in vivo. These human chimeric Abs are highly effective at delivering Ag to DCs for recognition by both CD4+ and CD8+ T cells. Given the importance of these cellular responses for antitumor or antiviral immunity, and the superior specificity of anti-CLEC9A Abs for this DC subset, this approach warrants further development for vaccines.
Kirsteen M. Tullett, Ingrid M. Leal Rojas, Yoshihito Minoda, Peck S. Tan, Jian-Guo Zhang, Corey Smith, Rajiv Khanna, Ken Shortman, Irina Caminschi, Mireille H. Lahoud, Kristen J. Radford
Dermal white adipose tissue (dWAT) was recently recognized for its potential to modify whole body metabolism. Here, we show that dWAT can be quantified using a high-resolution, fat-specific magnetic resonance imaging (MRI) technique. Noninvasive MRI has been used to describe adipocyte depots for many years; the MRI technique we describe uses an advanced fat-specific method to measure the thickness of dWAT, together with the total volume of WAT and the relative activation/fat depletion of brown adipose tissues (BAT). Since skin-embedded adipocytes may provide natural insulation, they provide an important counterpoint to the activation of thermogenic brown and beige adipose tissues, whereby these distinct depots are functionally interrelated and require simultaneous assay. This method was validated using characterized mouse cohorts of a lipodystrophic, dWAT-deficient strain (syndecan-1 KO) and 2 obese models (diet-induced obese mice and genetically obese animals,
Ildiko Kasza, Diego Hernando, Alejandro Roldán-Alzate, Caroline M. Alexander, Scott B. Reeder
Significant morbidity in cystic fibrosis (CF) results from chronic lung inflammation, most commonly due to
Kong Chen, Brian T. Campfield, Sally E. Wenzel, Jeremy P. McAleer, James L. Kreindler, Geoffrey Kurland, Radha Gopal, Ting Wang, Wei Chen, Taylor Eddens, Kathleen M. Quinn, Mike M. Myerburg, William T. Horne, Jose M. Lora, Brian K. Albrecht, Joseph M. Pilewski, Jay K. Kolls
Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease driven by both innate and adaptive immune cells. African Americans tend to present with more severe disease at an earlier age compared with patients of European ancestry. In order to better understand the immunological differences between African American and European American patients, we analyzed the frequencies of B cell subsets and the expression of B cell activation markers from a total of 68 SLE patients and 69 normal healthy volunteers. We found that B cells expressing the activation markers CD86, CD80, PD1, and CD40L, as well as CD19+CD27–IgD– double-negative B cells, were enriched in African American patients vs. patients of European ancestry. In addition to increased expression of CD40L, surface levels of CD40 on B cells were lower, suggesting the engagement of the CD40 pathway. In vitro experiments confirmed that CD40L expressed by B cells could lead to CD40 activation and internalization on adjacent B cells. To conclude, these results indicate that, compared with European American patients, African American SLE patients present with a particularly active B cell component, possibly via the activation of the CD40/CD40L pathway. These data may help guide the development of novel therapies.
Laurence C. Menard, Sium Habte, Waldemar Gonsiorek, Deborah Lee, Dana Banas, Deborah A. Holloway, Nataly Manjarrez-Orduno, Mark Cunningham, Dawn Stetsko, Francesca Casano, Selena Kansal, Patricia M. Davis, Julie Carman, Clarence K. Zhang, Ferva Abidi, Richard Furie, Steven G. Nadler, Suzanne J. Suchard
Marlise R. Luskin, Phyllis A. Gimotty, Catherine Smith, Alison W. Loren, Maria E. Figueroa, Jenna Harrison, Zhuoxin Sun, Martin S. Tallman, Elisabeth M. Paietta, Mark R. Litzow, Ari M. Melnick, Ross L. Levine, Hugo F. Fernandez, Selina M. Luger, Martin Carroll, Stephen R. Master, Gerald B.W. Wertheim
Liver fibrosis, a consequence of chronic liver injury and a way station to cirrhosis and hepatocellular carcinoma, lacks effective treatment. Endocannabinoids acting via cannabinoid-1 receptors (CB1R) induce profibrotic gene expression and promote pathologies that predispose to liver fibrosis. CB1R antagonists produce opposite effects, but their therapeutic development was halted due to neuropsychiatric side effects. Inducible nitric oxide synthase (iNOS) also promotes liver fibrosis and its underlying pathologies, but iNOS inhibitors tested to date showed limited therapeutic efficacy in inflammatory diseases. Here, we introduce a peripherally restricted, orally bioavailable CB1R antagonist, which accumulates in liver to release an iNOS inhibitory leaving group. In mouse models of fibrosis induced by CCl4 or bile duct ligation, the hybrid CB1R/iNOS antagonist surpassed the antifibrotic efficacy of the CB1R antagonist rimonabant or the iNOS inhibitor 1400W, without inducing anxiety-like behaviors or CB1R occupancy in the CNS. The hybrid inhibitor also targeted CB1R-independent, iNOS-mediated profibrotic pathways, including increased PDGF, Nlrp3/Asc3, and integrin αvβ6 signaling, as judged by its ability to inhibit these pathways in cnr1–/– but not in nos2–/– mice. Additionally, it was able to slow fibrosis progression and to attenuate established fibrosis. Thus, dual-target peripheral CB1R/iNOS antagonists have therapeutic potential in liver fibrosis.
Resat Cinar, Malliga R. Iyer, Ziyi Liu, Zongxian Cao, Tony Jourdan, Katalin Erdelyi, Grzegorz Godlewski, Gergő Szanda, Jie Liu, Joshua K. Park, Bani Mukhopadhyay, Avi Z. Rosenberg, Jeih-San Liow, Robin G. Lorenz, Pal Pacher, Robert B. Innis, George Kunos
Radiation therapy (RT), a critical modality in the treatment of lung cancer, induces direct tumor cell death and augments tumor-specific immunity. However, despite initial tumor control, most patients suffer from locoregional relapse and/or metastatic disease following RT. The use of immunotherapy in non–small-cell lung cancer (NSCLC) could potentially change this outcome by enhancing the effects of RT. Here, we report significant (up to 70% volume reduction of the target lesion) and durable (up to 12 weeks) tumor regressions in conditional
Grit S. Herter-Sprie, Shohei Koyama, Houari Korideck, Josephine Hai, Jiehui Deng, Yvonne Y. Li, Kevin A. Buczkowski, Aaron K. Grant, Soumya Ullas, Kevin Rhee, Jillian D. Cavanaugh, Neermala Poudel Neupane, Camilla L. Christensen, Jan M. Herter, G. Mike Makrigiorgos, F. Stephen Hodi, Gordon J. Freeman, Glenn Dranoff, Peter S. Hammerman, Alec C. Kimmelman, Kwok-Kin Wong
Studies in rodents and newborn humans demonstrate the influence of brown adipose tissue (BAT) in temperature control and energy balance and a critical role in the regulation of body weight. Here, we obtained samples of epicardial adipose tissue (EAT) from neonates, infants, and children in order to evaluate changes in their transcriptional landscape by applying a systems biology approach. Surprisingly, these analyses revealed that the transition to infancy is a critical stage for changes in the morphology of EAT and is reflected in unique gene expression patterns of a substantial proportion of thermogenic gene transcripts (~10%). Our results also indicated that the pattern of gene expression represents a distinct developmental stage, even after the rebound in abundance of thermogenic genes in later childhood. Using weighted gene coexpression network analyses, we found precise anthropometric-specific correlations with changes in gene expression and the decline of thermogenic capacity within EAT. In addition, these results indicate a sequential order of transcriptional events affecting cellular pathways, which could potentially explain the variation in the amount, or activity, of BAT in adulthood. Together, these results provide a resource to elucidate gene regulatory mechanisms underlying the progressive development of BAT during early life.
Shalini Ojha, Hernan P. Fainberg, Victoria Wilson, Giuseppe Pelella, Marcos Castellanos, Sean T. May, Attilio A. Lotto, Harold Sacks, Michael E. Symonds, Helen Budge
Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US. The majority of COPD patients have symptoms of chronic bronchitis, which lacks specific therapies. A major impediment to therapeutic development has been the absence of animal models that recapitulate key clinical and pathologic features of human disease. Ferrets are well suited for the investigation of the significance of respiratory diseases, given prior data indicating similarities to human airway physiology and submucosal gland distribution. Here, we exposed ferrets to chronic cigarette smoke and found them to approximate complex clinical features of human COPD. Unlike mice, which develop solely emphysema, smoke-exposed ferrets exhibited markedly higher numbers of early-morning spontaneous coughs and sporadic infectious exacerbations as well as a higher level of airway obstruction accompanied by goblet cell metaplasia/hyperplasia and increased mucus expression in small airways, indicative of chronic bronchitis and bronchiolitis. Overall, we demonstrate the first COPD animal model exhibiting clinical and pathologic features of chronic bronchitis to our knowledge, providing a key advance that will greatly facilitate the preclinical development of novel treatments for this disease.
S. Vamsee Raju, Hyunki Kim, Stephen A. Byzek, Li Ping Tang, John E. Trombley, Patricia Jackson, Lawrence Rasmussen, J. Michael Wells, Emily Falk Libby, Erik Dohm, Lindy Winter, Sharon L. Samuel, Kurt R. Zinn, J. Edwin Blalock, Trenton R. Schoeb, Mark T. Dransfield, Steven M. Rowe
Novel, tumor-specific drugs are urgently needed for a breakthrough in cancer therapy. Herein, we generated a first-in-class humanized antibody (PRL3-zumab) against PRL-3, an intracellular tumor-associated phosphatase upregulated in multiple human cancers, for unconventional cancer immunotherapies. We focused on gastric cancer (GC), wherein elevated
Min Thura, Abdul Qader Omer Al-Aidaroos, Wei Peng Yong, Koji Kono, Abhishek Gupta, You Bin Lin, Kousaku Mimura, Jean Paul Thiery, Boon Cher Goh, Patrick Tan, Ross Soo, Cheng William Hong, Lingzhi Wang, Suling Joyce Lin, Elya Chen, Sun Young Rha, Hyun Cheol Chung, Jie Li, Sayantani Nandi, Hiu Fung Yuen, Shu-Dong Zhang, Yeoh Khay Guan, Jimmy So, Qi Zeng
Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of
Ghayda Mirzaa, Andrew E. Timms, Valerio Conti, Evan August Boyle, Katta M. Girisha, Beth Martin, Martin Kircher, Carissa Olds, Jane Juusola, Sarah Collins, Kaylee Park, Melissa Carter, Ian Glass, Inge Krägeloh-Mann, David Chitayat, Aditi Shah Parikh, Rachael Bradshaw, Erin Torti, Stephen Braddock, Leah Burke, Sondhya Ghedia, Mark Stephan, Fiona Stewart, Chitra Prasad, Melanie Napier, Sulagna Saitta, Rachel Straussberg, Michael Gabbett, Bridget C. O’Connor, Catherine E. Keegan, Lim Jiin Yin, Angeline Hwei Meeng Lai, Nicole Martin, Margaret McKinnon, Marie-Claude Addor, Luigi Boccuto, Charles E. Schwartz, Agustina Lanoel, Robert L. Conway, Koenraad Devriendt, Katrina Tatton-Brown, Mary Ella Pierpont, Michael Painter, Lisa Worgan, James Reggin, Raoul Hennekam, Karen Tsuchiya, Colin C. Pritchard, Mariana Aracena, Karen W. Gripp, Maria Cordisco, Hilde Van Esch, Livia Garavelli, Cynthia Curry, Anne Goriely, Hulya Kayserilli, Jay Shendure, John Graham Jr., Renzo Guerrini, William B. Dobyns
Asthma is a chronic inflammatory airways disease that usually begins in early life and involves gene-environment interactions. Although most asthma exhibits allergic inflammation, many allergic individuals do not have asthma. Here, we report how the asthma gene a disintegrin and metalloprotease 33 (
Elizabeth R. Davies, Joanne F.C. Kelly, Peter H. Howarth, David I. Wilson, Stephen T. Holgate, Donna E. Davies, Jeffrey A. Whitsett, Hans Michael Haitchi
There continues to be a need for immunotherapies to treat type 1 diabetes in the clinic. We previously reported that nondepleting anti-CD4 and -CD8 Ab treatment effectively reverses diabetes in new-onset NOD mice. A key feature of the induction of remission is the egress of the majority of islet-resident T cells. How this occurs is undefined. Herein, the effects of coreceptor therapy on islet T cell retention were investigated. Bivalent Ab binding to CD4 and CD8 blocked TCR signaling and T cell cytokine production, while indirectly downregulating islet chemokine expression. These processes were required for T cell retention, as ectopic IFN-γ or CXCL10 inhibited Ab-mediated T cell purging. Importantly, treatment of humanized mice with nondepleting anti–human CD4 and CD8 Ab similarly reduced tissue-infiltrating human CD4+ and CD8+ T cells. These findings demonstrate that Ab binding of CD4 and CD8 interrupts a feed-forward circuit by suppressing T cell–produced cytokines needed for expression of chemotactic cues, leading to rapid T cell egress from the islets. Coreceptor therapy therefore offers a robust approach to suppress T cell–mediated pathology by purging T cells in an inflammation-dependent manner.
Aaron J. Martin, Matthew Clark, Gregory Gojanovich, Fatima Manzoor, Keith Miller, Douglas E. Kline, Y. Maurice Morillon, Bo Wang, Roland Tisch
Acute kidney injury (AKI) is a common clinical condition defined as a rapid decline in kidney function. AKI is a global health burden, estimated to cause 2 million deaths annually worldwide. Unlike AKI in the young, which is reversible, AKI in the elderly often leads to end-stage renal disease, and the mechanism that prevents kidney repair in the elderly is unclear. Here we demonstrate that aged but not young mice developed multiple tertiary lymphoid tissues (TLTs) in the kidney after AKI. TLT size was associated with impaired renal function and increased expression of proinflammatory cytokines and homeostatic chemokines, indicating a possible contribution of TLTs to sustained inflammation after injury. Notably, resident fibroblasts from a single lineage diversified into p75 neurotrophin receptor+ (p75NTR+) fibroblasts and homeostatic chemokine–producing fibroblasts inside TLTs, and retinoic acid–producing fibroblasts around TLTs. Deletion of CD4+ cells as well as late administration of dexamethasone abolished TLTs and improved renal outcomes. Importantly, aged but not young human kidneys also formed TLTs that had cellular and molecular components similar to those of mouse TLTs. Therefore, the inhibition of TLT formation may offer a novel therapeutic strategy for AKI in the elderly.
Yuki Sato, Akiko Mii, Yoko Hamazaki, Harumi Fujita, Hirosuke Nakata, Kyoko Masuda, Shingo Nishiyama, Shinsuke Shibuya, Hironori Haga, Osamu Ogawa, Akira Shimizu, Shuh Narumiya, Tsuneyasu Kaisho, Makoto Arita, Masashi Yanagisawa, Masayuki Miyasaka, Kumar Sharma, Nagahiro Minato, Hiroshi Kawamoto, Motoko Yanagita
Obesity is associated with increased classically activated M1 adipose tissue macrophages (ATMs) and decreased alternatively activated M2 ATMs, both of which contribute to obesity-induced inflammation and insulin resistance. However, the underlying mechanism remains unclear. We find that inhibiting DNA methylation pharmacologically using 5-aza-2′-deoxycytidine or genetically by DNA methyltransferase 1 (DNMT1) deletion promotes alternative activation and suppresses inflammation in macrophages. Consistently, mice with myeloid DNMT1 deficiency exhibit enhanced macrophage alternative activation, suppressed macrophage inflammation, and are protected from obesity-induced inflammation and insulin resistance. The promoter and 5′-untranslated region of peroxisome proliferator-activated receptor γ1 (PPARγ1) are enriched with CpGs and are epigenetically regulated. The saturated fatty acids stearate and palmitate and the inflammatory cytokine TNF-α significantly increase, whereas the TH2 cytokine IL-4 significantly decreases PPARγ1 promoter DNA methylation. Accordingly, inhibiting PPARγ1 promoter DNA methylation pharmacologically using 5-aza-2′-deoxycytidine or genetically by DNMT1 deletion promotes macrophage alternative activation. Our data therefore establish DNA hypermethylation at the PPARγ1 promoter induced by obesity-related factors as a critical determinant of ATM proinflammatory activation and inflammation, which contributes to insulin resistance in obesity.
Xianfeng Wang, Qiang Cao, Liqing Yu, Huidong Shi, Bingzhong Xue, Hang Shi
Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2–targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.
Lingegowda S. Mangala, Hongyu Wang, Dahai Jiang, Sherry Y. Wu, Anoma Somasunderam, David E. Volk, Ganesh L. R. Lokesh, Xin Li, Sunila Pradeep, Xianbin Yang, Monika Haemmerle, Cristian Rodriguez-Aguayo, Archana S Nagaraja, Rajesha Rupaimoole, Emine Bayraktar, Recep Bayraktar, Li Li, Takemi Tanaka, Wei Hu, Cristina Ivan, Kshipra M Gharpure, Michael H. McGuire, Varatharasa Thiviyanathan, Xinna Zhang, Sourindra N. Maiti, Nataliya Bulayeva, Hyun-Jin Choi, Piotr L. Dorniak, Laurence J.N. Cooper, Kevin P. Rosenblatt, Gabriel Lopez-Berestein, David G. Gorenstein, Anil K. Sood
Oxidative stress is implicated in various neurodegenerative disorders, including retinitis pigmentosa (RP), an inherited disease that causes blindness. The biological and cellular mechanisms by which oxidative stress mediates neuronal cell death are largely unknown. In a mouse model of RP (rd10 mice), we show that oxidative DNA damage activates microglia through MutY homolog–mediated (MUYTH-mediated) base excision repair (BER), thereby exacerbating retinal inflammation and degeneration. In the early stage of retinal degeneration, oxidative DNA damage accumulated in the microglia and caused single-strand breaks (SSBs) and poly(ADP-ribose) polymerase activation. In contrast,
Shunji Nakatake, Yusuke Murakami, Yasuhiro Ikeda, Noriko Morioka, Takashi Tachibana, Kohta Fujiwara, Noriko Yoshida, Shoji Notomi, Toshio Hisatomi, Shigeo Yoshida, Tatsuro Ishibashi, Yusaku Nakabeppu, Koh-Hei Sonoda
Constitutively active splice variants of androgen receptor (AR-Vs) lacking ligand-binding domain (LBD) are a mechanism of resistance to androgen receptor LBD–targeted (AR LBD–targeted) therapies for metastatic castration-resistant prostate cancer (CRPC). There is a strong unmet clinical need to identify prostate cancer patients with AR-V–positive lesions to determine whether they will benefit from further AR LBD–targeting therapies or should receive taxanes or investigational drugs like EPI-506 or galeterone. Both EPI-506 (NCT02606123) and galeterone (NCT02438007) are in clinical trials and are proposed to have efficacy against lesions that are positive for AR-Vs. AR activation function-1 (AF-1) is common to the N-terminal domains of full-length AR and AR-Vs. Here, we provide proof of concept for developing imaging compounds that directly bind AR AF-1 to detect both AR-Vs and full-length AR. 123I-EPI-002 had specific binding to AR AF-1, which enabled direct visualization of CRPC xenografts that express full-length AR and AR-Vs. Our findings highlight the potential of 123I-EPI-002 as an imaging agent for the detection of full-length AR and AR-Vs in CRPC.
Yusuke Imamura, Amy H. Tien, Jinhe Pan, Jacky K. Leung, Carmen A. Banuelos, Kunzhong Jian, Jun Wang, Nasrin R. Mawji, Javier Garcia Fernandez, Kuo-Shyan Lin, Raymond J. Andersen, Marianne D. Sadar
To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites.
Rintaro Saito, Anaïs Rocanin-Arjo, Young-Hyun You, Manjula Darshi, Benjamin Van Espen, Satoshi Miyamoto, Jessica Pham, Minya Pu, Simone Romoli, Loki Natarajan, Wenjun Ju, Matthias Kretzler, Robert Nelson, Keiichiro Ono, Dana Thomasova, Shrikant R. Mulay, Trey Ideker, Vivette D’Agati, Ergin Beyret, Juan Carlos Izpisua Belmonte, Hans Joachim Anders, Kumar Sharma
GWAS have linked SNPs to risk of inflammatory bowel disease (IBD), but a systematic characterization of disease-associated genes has been lacking. Prior studies utilized microarrays that did not capture many genes encoded within risk loci or defined expression quantitative trait loci (eQTLs) using peripheral blood, which is not the target tissue in IBD. To address these gaps, we sought to characterize the expression of IBD-associated risk genes in disease-relevant tissues and in the setting of active IBD. Terminal ileal (TI) and colonic mucosal tissues were obtained from patients with Crohn’s disease or ulcerative colitis and from healthy controls. We developed a NanoString code set to profile 678 genes within IBD risk loci. A subset of patients and controls were genotyped for IBD-associated risk SNPs. Analyses included differential expression and variance analysis, weighted gene coexpression network analysis, and eQTL analysis. We identified 116 genes that discriminate between healthy TI and colon samples and uncovered patterns in variance of gene expression that highlight heterogeneity of disease. We identified 107 coexpressed gene pairs for which transcriptional regulation is either conserved or reversed in an inflammation-independent or -dependent manner. We demonstrate that on average approximately 60% of disease-associated genes are differentially expressed in inflamed tissue. Last, we identified eQTLs with either genotype-only effects on expression or an interaction effect between genotype and inflammation. Our data reinforce tissue specificity of expression in disease-associated candidate genes, highlight genes and gene pairs that are regulated in disease-relevant tissue and inflammation, and provide a foundation to advance the understanding of IBD pathogenesis.
Joanna M. Peloquin, Gautam Goel, Lingjia Kong, Hailiang Huang, Talin Haritunians, R. Balfour Sartor, Mark J. Daly, Rodney D. Newberry, Dermot P. McGovern, Vijay Yajnik, Sergio A. Lira, Ramnik J. Xavier
The autosomal recessive neuromuscular disease spinal muscular atrophy (SMA) is caused by loss of survival motor neuron (SMN) protein. Molecular pathways that are disrupted downstream of SMN therefore represent potentially attractive therapeutic targets for SMA. Here, we demonstrate that therapeutic targeting of ubiquitin pathways disrupted as a consequence of SMN depletion, by increasing levels of one key ubiquitination enzyme (ubiquitin-like modifier activating enzyme 1 [UBA1]), represents a viable approach for treating SMA. Loss of UBA1 was a conserved response across mouse and zebrafish models of SMA as well as in patient induced pluripotent stem cell–derive motor neurons. Restoration of UBA1 was sufficient to rescue motor axon pathology and restore motor performance in SMA zebrafish. Adeno-associated virus serotype 9–UBA1 (AAV9-UBA1) gene therapy delivered systemic increases in UBA1 protein levels that were well tolerated over a prolonged period in healthy control mice. Systemic restoration of UBA1 in SMA mice ameliorated weight loss, increased survival and motor performance, and improved neuromuscular and organ pathology. AAV9-UBA1 therapy was also sufficient to reverse the widespread molecular perturbations in ubiquitin homeostasis that occur during SMA. We conclude that UBA1 represents a safe and effective therapeutic target for the treatment of both neuromuscular and systemic aspects of SMA.
Rachael A. Powis, Evangelia Karyka, Penelope Boyd, Julien Côme, Ross A. Jones, Yinan Zheng, Eva Szunyogova, Ewout J.N. Groen, Gillian Hunter, Derek Thomson, Thomas M. Wishart, Catherina G. Becker, Simon H. Parson, Cécile Martinat, Mimoun Azzouz, Thomas H. Gillingwater
Primary pigmented nodular adrenocortical disease (PPNAD) is a rare cause of ACTH-independent hypercortisolism. The disease is primarily caused by germline mutations of the protein kinase A (PKA) regulatory subunit 1A (
Zakariae Bram, Estelle Louiset, Bruno Ragazzon, Sylvie Renouf, Julien Wils, Céline Duparc, Isabelle Boutelet, Marthe Rizk-Rabin, Rossella Libé, Jacques Young, Dennis Carson, Marie-Christine Vantyghem, Eva Szarek, Antoine Martinez, Constantine A. Stratakis, Jérôme Bertherat, Hervé Lefebvre
Regulation of lineage-restricted transcription factors has been shown to influence malignant transformation in several types of cancer. Whether similar mechanisms are involved in ovarian cancer pathogenesis is unknown. PAX8 is a nuclear transcription factor that controls the embryologic development of the Müllerian system, including the fallopian tubes. Recent studies have shown that fallopian tube secretory epithelial cells (FTSECs) give rise to the most common form of ovarian cancer, high-grade serous ovarian carcinomas (HGSOCs). We designed the present study in order to understand whether changes in gene expression between FTSECs and HGSOCs relate to alterations in PAX8 binding to chromatin. Using whole transcriptome shotgun sequencing (RNA-Seq) after PAX8 knockdown and ChIP-Seq, we show that FTSECs and HGSOCs are distinguished by marked reprogramming of the PAX8 cistrome. Genes that are significantly altered between FTSECs and HGSOCs are enriched near PAX8 binding sites. These sites are also near TEAD binding sites, and these transcriptional changes may be related to PAX8 interactions with the TEAD/YAP1 signaling pathway. These data suggest that transcriptional changes after transformation in ovarian cancer are closely related to epigenetic remodeling in lineage-specific transcription factors.
Kevin M. Elias, Megan M. Emori, Thomas Westerling, Henry Long, Anna Budina-Kolomets, Fugen Li, Emily MacDuffie, Michelle R. Davis, Alexander Holman, Brian Lawney, Matthew L. Freedman, John Quackenbush, Myles Brown, Ronny Drapkin
Motile airway cilia that propel contaminants out of the lung are oriented in a common direction by planar cell polarity (PCP) signaling, which localizes PCP protein complexes to opposite cell sides throughout the epithelium to orient cytoskeletal remodeling. In airway epithelia, PCP is determined in a 2-phase process. First, cell-cell communication via PCP complexes polarizes all cells with respect to the proximal-distal tissue axis. Second, during ciliogenesis, multiciliated cells (MCCs) undergo cytoskeletal remodeling to orient their cilia in the proximal direction. The second phase not only directs cilium polarization, but also consolidates polarization across the epithelium. Here, we demonstrate that in airway epithelia, PCP depends on MCC differentiation. PCP mutant epithelia have misaligned cilia, and also display defective barrier function and regeneration, indicating that PCP regulates multiple aspects of airway epithelial homeostasis. In humans, MCCs are often sparse in chronic inflammatory diseases, and these airways exhibit PCP dysfunction. The presence of insufficient MCCs impairs mucociliary clearance in part by disrupting PCP-driven polarization of the epithelium. Consistent with defective PCP, barrier function and regeneration are also disrupted. Pharmacological stimulation of MCC differentiation restores PCP and reverses these defects, suggesting its potential for broad therapeutic benefit in chronic inflammatory disease.
Eszter K. Vladar, Jayakar V. Nayak, Carlos E. Milla, Jeffrey D. Axelrod
Catheter-associated urinary tract infections (CAUTI) are the most common hospital-associated infections. Here, we report that bladder catheterization initiated a persistent sterile inflammatory reaction within minutes of catheter implantation. Catheterization resulted in increased expression of genes associated with defense responses and cellular migration, with ensuing rapid and sustained innate immune cell infiltration into the bladder. Catheterization also resulted in hypersensitivity to
Matthieu Rousseau, H.M. Sharon Goh, Sarah Holec, Matthew L. Albert, Rohan B.H. Williams, Molly A. Ingersoll, Kimberly A. Kline
Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross-species activities of stem cell factor (SCF), IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple-transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to nontransgenic controls. Shortly after engraftment with UCB, these mice develop a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis. This manifestation of MAS does not respond to lymphocyte-suppressive therapies such as steroids, i.v. immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte-independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease. Additionally, the IL-6R antibody tocilizumab delayed progression and prolonged lifespan. This new model of MAS provides an opportunity for investigation of the mechanisms driving this disease and for the testing of directed therapies in a humanized mouse.
Mark Wunderlich, Courtney Stockman, Mahima Devarajan, Navin Ravishankar, Christina Sexton, Ashish R. Kumar, Benjamin Mizukawa, James C. Mulloy
Using mice rendered insulin resistant with high fat diets (HFD), we examined blood glucose levels and insulin resistance after i.v. delivery of an adeno-associated virus type 8 encoding murine urocortin 2 (AAV8.UCn2). A single i.v. injection of AAV8.UCn2-normalized blood glucose and glucose disposal within weeks, an effect that lasted for months. Hyperinsulinemic-euglycemic clamps showed reduced plasma insulin, increased glucose disposal rates, and increased insulin sensitivity following UCn2 gene transfer. Mice with corticotropin-releasing hormone type 2-receptor deletion that were rendered insulin resistant by HFD showed no improvement in glucose disposal after UCn2 gene transfer, indicating that the effect requires UCn2’s cognate receptor. We also demonstrated increased glucose disposal after UCn2 gene transfer in db/db mice, a second model of insulin resistance. UCn2 gene transfer reduced fatty infiltration of the liver in both models of insulin resistance. UCn2 increases Glut4 translocation to the plasma membrane in skeletal myotubes in a manner quantitatively similar to insulin, indicating a mechanism through which UCn2 operates to increase insulin sensitivity. UCn2 gene transfer, in a dose-dependent manner, is insulin sensitizing and effective for months after a single injection. These findings suggest a potential long-term therapy for clinical type-2 diabetes.
Mei Hua Gao, Dimosthenis Giamouridis, N. Chin Lai, Evelyn Walenta, Vivian Almeida Paschoal, Young Chul Kim, Atsushi Miyanohara, Tracy Guo, Min Liao, Li Liu, Zhen Tan, Theodore P. Ciaraldi, Simon Schenk, Aditi Bhargava, Da Young Oh, H. Kirk Hammond
Urine concentration is regulated by vasopressin. Congenital nephrogenic diabetes insipidus (NDI) is caused by vasopressin type 2 receptor (V2R) mutations. We studied whether metformin could improve urine concentration in rodent models of congenital NDI by stimulating AMPK. To block the V2R in rats, tolvaptan (10 mg/kg/d) was given by oral gavage with or without metformin (800 mg/kg/d). Control rats received vehicle with or without metformin. Tamoxifen-induced V2R KO mice were given metformin (600 mg/kg) or vehicle twice daily. Urine osmolality in tolvaptan-treated rats (1,303 ± 126 mOsM) was restored to control levels by metformin (2,335 ± 273 mOsM) within 3 days and was sustained for up to 10 days. Metformin increased protein abundance of inner medullary urea transporter UT-A1 by 61% and aquaporin 2 (AQP2) by 44% in tolvaptan-treated rats, and immunohistochemistry showed increased membrane accumulation of AQP2 with acute and chronic AMPK stimulation. Outer medullary Na+-K+-2Cl– cotransporter 2 (NKCC2) abundance increased (117%) with AMPK stimulation in control rats but not in V2R-blocked rats. Metformin increased V2R KO mouse urine osmolality within 3 hours, and the increase persisted for up to 12 hours. Metformin increased AQP2 in the V2R KO mice similar to the tolvaptan-treated rats. These results indicate that AMPK activators, such as metformin, might provide a promising treatment for congenital NDI.
Orhan Efe, Janet D. Klein, Lauren M. LaRocque, Huiwen Ren, Jeff M. Sands
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient–derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.
Mahlet B. Abera, Jingbo Xiao, Jonathan Nofziger, Steve Titus, Noel Southall, Wei Zheng, Kasey E. Moritz, Marc Ferrer, Jonathan J. Cherry, Elliot J. Androphy, Amy Wang, Xin Xu, Christopher Austin, Kenneth H. Fischbeck, Juan J. Marugan, Barrington G. Burnett
The strong association of Zika virus infection with congenital defects has led to questions of how a flavivirus is capable of crossing the placental barrier to reach the fetal brain. Here, we demonstrate permissive Zika virus infection of primary human placental macrophages, commonly referred to as Hofbauer cells, and placental villous fibroblasts. We also demonstrate Zika virus infection of Hofbauer cells within the context of the tissue ex vivo using term placental villous explants. In addition to amplifying infectious virus within a usually inaccessible area, the putative migratory activities of Hofbauer cells may aid in dissemination of Zika virus to the fetal brain. Understanding the susceptibility of placenta-specific cell types will aid future work around and understanding of Zika virus–associated pregnancy complications.
Kellie Ann Jurado, Michael K. Simoni, Zhonghua Tang, Ryuta Uraki, Jesse Hwang, Sarah Householder, Mingjie Wu, Brett D. Lindenbach, Vikki M. Abrahams, Seth Guller, Erol Fikrig
Low-grade chronic inflammation is a key etiological phenomenon responsible for the initiation and perpetuation of obesity and diabetes. Novel therapeutic approaches that can specifically target inflammatory pathways are needed to avert this looming epidemic of metabolic disorders. Genetic and chemical inhibition of SH2-containing inositol 5′ phosphatase 1 (SHIP1) has been associated with systemic expansion of immunoregulatory cells that promote a lean-body state; however, SHIP1 function in immunometabolism has never been assessed. This led us to investigate the role of SHIP1 in metabolic disorders during excess caloric intake in mice. Using a small-molecule inhibitor of SHIP1 (SHIPi), here we show that SHIPi treatment in mice significantly reduces body weight and fat content, improves control of blood glucose and insulin sensitivity, and increases energy expenditure, despite continued consumption of a high-fat diet. Additionally, SHIPi reduces age-associated fat in mice. We found that SHIPi treatment reverses diet-associated obesity by attenuating inflammation in the visceral adipose tissue (VAT). SHIPi treatment increases IL-4–producing eosinophils in VAT and consequently increases both alternatively activated macrophages and myeloid-derived suppressor cells. In addition, SHIPi decreases the number of IFN-γ–producing T cells and NK cells in VAT. Thus, SHIPi represents an approach that permits control of obesity and diet-induced metabolic syndrome without apparent toxicity.
Neetu Srivastava, Sonia Iyer, Raki Sudan, Christie Youngs, Robert W. Engelman, Kyle T. Howard, Christopher M. Russo, John D. Chisholm, William G. Kerr
Diana Golden, Antonina Kolmakova, Sunitha Sura, Anthony T. Vella, Ani Manichaikul, Xin-Qun Wang, Suzette J. Bielinski, Kent D. Taylor, Yii-Der Ida Chen, Stephen S. Rich, Annabelle Rodriguez
The creation of a humanized chimeric mouse nervous system permits the study of human neural development and disease pathogenesis using human cells in vivo. Humanized glial chimeric mice with the brain and spinal cord being colonized by human glial cells have been successfully generated. However, generation of humanized chimeric mouse brains repopulated by human neurons to possess a high degree of chimerism have not been well studied. Here we created humanized neuronal chimeric mouse brains by neonatally engrafting the distinct and highly neurogenic human induced pluripotent stem cell (hiPSC)–derived rosette-type primitive neural progenitors. These neural progenitors predominantly differentiate to neurons, which disperse widely throughout the mouse brain with infiltration of the cerebral cortex and hippocampus at 6 and 13 months after transplantation. Building upon the hiPSC technology, we propose that this potentially unique humanized neuronal chimeric mouse model will provide profound opportunities to define the structure, function, and plasticity of neural networks containing human neurons derived from a broad variety of neurological disorders.
Chen Chen, Woo-Yang Kim, Peng Jiang
Obesity-related insulin resistance is associated with fatty liver, dyslipidemia, and low plasma adiponectin. Insulin resistance due to insulin receptor (INSR) dysfunction is associated with none of these, but when due to dysfunction of the downstream kinase AKT2 phenocopies obesity-related insulin resistance. We report 5 patients with SHORT syndrome and C-terminal mutations in
Isabel Huang-Doran, Patsy Tomlinson, Felicity Payne, Alexandra Gast, Alison Sleigh, William Bottomley, Julie Harris, Allan Daly, Nuno Rocha, Simon Rudge, Jonathan Clark, Albert Kwok, Stefano Romeo, Emma McCann, Barbara Müksch, Mehul Dattani, Stefano Zucchini, Michael Wakelam, Lazaros C. Foukas, David B. Savage, Rinki Murphy, Stephen O’Rahilly, Inês Barroso, Robert K. Semple
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare primary immunodeficiency disorder typically caused by homozygous
Elise M.N. Ferre, Stacey R. Rose, Sergio D. Rosenzweig, Peter D. Burbelo, Kimberly R. Romito, Julie E. Niemela, Lindsey B. Rosen, Timothy J. Break, Wenjuan Gu, Sally Hunsberger, Sarah K. Browne, Amy P. Hsu, Shakuntala Rampertaap, Muthulekha Swamydas, Amanda L. Collar, Heidi H. Kong, Chyi-Chia Richard Lee, David Chascsa, Thomas Simcox, Angela Pham, Anamaria Bondici, Mukil Natarajan, Joseph Monsale, David E. Kleiner, Martha Quezado, Ilias Alevizos, Niki M. Moutsopoulos, Lynne Yockey, Cathleen Frein, Ariane Soldatos, Katherine R. Calvo, Jennifer Adjemian, Morgan N. Similuk, David M. Lang, Kelly D. Stone, Gulbu Uzel, Jeffrey B. Kopp, Rachel J. Bishop, Steven M. Holland, Kenneth N. Olivier, Thomas A. Fleisher, Theo Heller, Karen K. Winer, Michail S. Lionakis
The molecular determinants of lung cancer risk remain largely unknown. Airway epithelial cells are prone to assault by risk factors and are considered to be the primary cell type involved in the field of cancerization. To investigate risk-associated changes in the bronchial epithelium proteome that may offer new insights into the molecular pathogenesis of lung cancer, proteins were identified in the airway epithelial cells of bronchial brushing specimens from risk-stratified individuals by shotgun proteomics. Differential expression of selected proteins was validated by parallel reaction monitoring mass spectrometry in an independent set of individual bronchial brushings. We identified 2,869 proteins, of which 312 proteins demonstrated a trend in expression. Pathway analysis revealed enrichment of carbohydrate metabolic enzymes in high-risk individuals. Glucose consumption and lactate production were increased in human bronchial epithelial BEAS2B cells treated with cigarette smoke condensate for 7 months. Increased lipid biosynthetic capacity and net reductive carboxylation were revealed by metabolic flux analyses of [U-13C5] glutamine in this in vitro model, suggesting profound metabolic reprogramming in the airway epithelium of high-risk individuals. These results provide a rationale for the development of potentially new chemopreventive strategies and selection of patients for surveillance programs.
S.M. Jamshedur Rahman, Xiangming Ji, Lisa J. Zimmerman, Ming Li, Bradford K. Harris, Megan D. Hoeksema, Irina A. Trenary, Yong Zou, Jun Qian, Robbert J.C. Slebos, Jennifer Beane, Avrum Spira, Yu Shyr, Rosana Eisenberg, Daniel C. Liebler, Jamey D. Young, Pierre P. Massion
A number of studies in model animal systems and in the clinic have established that RANKL promotes bone resorption. Paradoxically, we found that pulsing ovariectomized mice with low-dose RANKL suppressed bone resorption, decreased the levels of proinflammatory effector T cells and led to increased bone mass. This effect of RANKL is mediated through the induction of FoxP3+CD25+ regulatory CD8+ T cells (TcREG) by osteoclasts. Here, we show that pulses of low-dose RANKL are needed to induce TcREG, as continuous infusion of identical doses of RANKL by pump did not induce TcREG. We also show that low-dose RANKL can induce TcREG at 2, 3, 6, and 10 weeks after ovariectomy. Our results show that low-dose RANKL treatment in ovariectomized mice is optimal at once-per-month doses to maintain the bone mass. Finally, we found that treatment of ovariectomized mice with the Cathepsin K inhibitor odanacatib also blocked TcREG induction by low-dose RANKL. We interpret this result to indicate that antigens presented to CD8+ T cells by osteoclasts are derived from the bone protein matrix because Cathepsin K degrades collagen in the bone. Taken together, our studies provide a basis for using low-dose RANKL as a potential therapeutic for postmenopausal osteoporosis.
Anna Cline-Smith, Jesse Gibbs, Elena Shashkova, Zachary S. Buchwald, Rajeev Aurora
Transcriptome profiles derived from the site of human disease have led to the identification of genes that contribute to pathogenesis, yet the complex mixture of cell types in these lesions has been an obstacle for defining specific mechanisms. Leprosy provides an outstanding model to study host defense and pathogenesis in a human infectious disease, given its clinical spectrum, which interrelates with the host immunologic and pathologic responses. Here, we investigated gene expression profiles derived from skin lesions for each clinical subtype of leprosy, analyzing gene coexpression modules by cell-type deconvolution. In lesions from tuberculoid leprosy patients, those with the self-limited form of the disease, dendritic cells were linked with
Megan S. Inkeles, Rosane M.B. Teles, Delila Pouldar, Priscila R. Andrade, Cressida A. Madigan, David Lopez, Mike Ambrose, Mahdad Noursadeghi, Euzenir N. Sarno, Thomas H. Rea, Maria T. Ochoa, M. Luisa Iruela-Arispe, William R. Swindell, Tom H.M. Ottenhoff, Annemieke Geluk, Barry R. Bloom, Matteo Pellegrini, Robert L. Modlin
While respiratory failure in cystic fibrosis (CF) frequently associates with chronic infection by
Sladjana Skopelja, B. JoNell Hamilton, Jonathan D. Jones, Mei-Ling Yang, Mark Mamula, Alix Ashare, Alex H. Gifford, William F.C. Rigby
Alveolar epithelial cell (AEC) dysfunction underlies the pathogenesis of pulmonary fibrosis in Hermansky-Pudlak syndrome (HPS) and other genetic syndromes associated with interstitial lung disease; however, mechanisms linking AEC dysfunction and fibrotic remodeling are incompletely understood. Since increased macrophage recruitment precedes pulmonary fibrosis in HPS, we investigated whether crosstalk between AECs and macrophages determines fibrotic susceptibility. We found that AECs from HPS mice produce excessive MCP-1, which was associated with increased macrophages in the lungs of unchallenged HPS mice. Blocking MCP-1/CCR2 signaling in HPS mice with genetic deficiency of CCR2 or targeted deletion of MCP-1 in AECs normalized macrophage recruitment, decreased AEC apoptosis, and reduced lung fibrosis in these mice following treatment with low-dose bleomycin. We observed increased TGF-β production by HPS macrophages, which was eliminated by CCR2 deletion. Selective deletion of TGF-β in myeloid cells or of TGF-β signaling in AECs through deletion of TGFBR2 protected HPS mice from AEC apoptosis and bleomycin-induced fibrosis. Together, these data reveal a feedback loop in which increased MCP-1 production by dysfunctional AECs results in recruitment and activation of lung macrophages that produce TGF-β, thus amplifying the fibrotic cascade through AEC apoptosis and stimulation of fibrotic remodeling.
Lisa R. Young, Peter M. Gulleman, Chelsi W. Short, Harikrishna Tanjore, Taylor Sherrill, Aidong Qi, Andrew P. McBride, Rinat Zaynagetdinov, John T. Benjamin, William E. Lawson, Sergey V. Novitskiy, Timothy S. Blackwell
Inositol 1, 4, 5-trisphosphate receptor–mediated (IP3R-mediated) calcium (Ca2+) release has been proposed to play an important role in regulating vascular smooth muscle cell (VSMC) contraction for decades. However, whether and how IP3R regulates blood pressure in vivo remains unclear. To address these questions, we have generated a smooth muscle–specific IP3R triple-knockout (smTKO) mouse model using a tamoxifen-inducible system. In this study, the role of IP3R-mediated Ca2+ release in adult VSMCs on aortic vascular contractility and blood pressure was assessed following tamoxifen induction. We demonstrated that deletion of IP3Rs significantly reduced aortic contractile responses to vasoconstrictors, including phenylephrine, U46619, serotonin, and endothelin 1. Deletion of IP3Rs also dramatically reduced the phosphorylation of MLC20 and MYPT1 induced by U46619. Furthermore, although the basal blood pressure of smTKO mice remained similar to that of wild-type controls, the increase in systolic blood pressure upon chronic infusion of angiotensin II was significantly attenuated in smTKO mice. Taken together, our results demonstrate an important role for IP3R-mediated Ca2+ release in VSMCs in regulating vascular contractility and hypertension.
Qingsong Lin, Guiling Zhao, Xi Fang, Xiaohong Peng, Huayuan Tang, Hong Wang, Ran Jing, Jie Liu, W. Jonathan Lederer, Ju Chen, Kunfu Ouyang
Adaptive changes in the genome of a locally predominant clinical isolate of the multidrug-resistant
Danielle Ahn, Hernán Peñaloza, Zheng Wang, Matthew Wickersham, Dane Parker, Purvi Patel, Antonius Koller, Emily I. Chen, Susan M. Bueno, Anne-Catrin Uhlemann, Alice Prince
BACKGROUND. Alopecia areata (AA) is an autoimmune disease characterized by hair loss mediated by CD8+ T cells. There are no reliably effective therapies for AA. Based on recent developments in the understanding of the pathomechanism of AA, JAK inhibitors appear to be a therapeutic option; however, their efficacy for the treatment of AA has not been systematically examined.
METHODS. This was a 2-center, open-label, single-arm trial using the pan-JAK inhibitor, tofacitinib citrate, for AA with >50% scalp hair loss, alopecia totalis (AT), and alopecia universalis (AU). Tofacitinib (5 mg) was given twice daily for 3 months. Endpoints included regrowth of scalp hair, as assessed by the severity of alopecia tool (SALT), duration of hair growth after completion of therapy, and disease transcriptome.
RESULTS. Of 66 subjects treated, 32% experienced 50% or greater improvement in SALT score. AA and ophiasis subtypes were more responsive than AT and AU subtypes. Shorter duration of disease and histological peribulbar inflammation on pretreatment scalp biopsies were associated with improvement in SALT score. Drug cessation resulted in disease relapse in 8.5 weeks. Adverse events were limited to grade I and II infections. An AA responsiveness to JAK/STAT inhibitors score was developed to segregate responders and nonresponders, and the previously developed AA disease activity index score tracked response to treatment.
CONCLUSIONS. At the dose and duration studied, tofacitinib is a safe and effective treatment for severe AA, though it does not result in a durable response. Transcriptome changes reveal unexpected molecular complexity within the disease.
TRIAL REGISTRATION. ClinicalTrials.gov NCT02197455 and NCT02312882.
FUNDING. This work was supported by the US Department of Veterans Affairs Office of Research and Development, National Institute of Arthritis and Musculoskeletal and Skin Diseases National Institutes of Health grant R01 AR47223 and U01 AR67173, the National Psoriasis Foundation, the Swedish Society of Medicine, the Fernström Foundation, the Locks of Love Foundation, the National Alopecia Areata Foundation, and the Ranjini and Ajay Poddar Resource Fund for Dermatologic Diseases Research.
Milène Kennedy Crispin, Justin M. Ko, Brittany G. Craiglow, Shufeng Li, Gautam Shankar, Jennifer R. Urban, James C. Chen, Jane E. Cerise, Ali Jabbari, Mårten C.G. Winge, M. Peter Marinkovich, Angela M. Christiano, Anthony E. Oro, Brett A. King
Julian Mackay-Wiggan, Ali Jabbari, Nhan Nguyen, Jane E. Cerise, Charlotte Clark, Grace Ulerio, Megan Furniss, Roger Vaughan, Angela M. Christiano, Raphael Clynes
Counteracting the progressive neurological disability caused by neuronal and axonal loss is the major unmet clinical need in multiple sclerosis therapy. However, the mechanisms underlying irreversible neuroaxonal degeneration in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE) are not well understood. A long-standing hypothesis holds that the distribution of voltage-gated sodium channels along demyelinated axons contributes to neurodegeneration by increasing neuroaxonal sodium influx and energy demand during CNS inflammation. Here, we tested this hypothesis in vivo by inserting a human gain-of-function mutation in the mouse NaV1.2-encoding gene
Benjamin Schattling, Walid Fazeli, Birgit Engeland, Yuanyuan Liu, Holger Lerche, Dirk Isbrandt, Manuel A. Friese
Recent clinical trials have demonstrated a clear survival advantage in advanced head and neck squamous cell carcinoma (HNSCC) patients treated with immune checkpoint blockade. These emerging results reveal that HNSCC is one of the most promising frontiers for immunotherapy research. However, further progress in head and neck immuno-oncology will require a detailed understanding of the immune infiltrative landscape found in these tumors. We leveraged transcriptome data from 280 tumors profiled by The Cancer Genome Atlas (TCGA) to comprehensively characterize the immune landscape of HNSCC in order to develop a rationale for immunotherapeutic strategies in HNSCC and guide clinical investigation. We find that both HPV+ and HPV– HNSCC tumors are among the most highly immune-infiltrated cancer types. Strikingly, HNSCC had the highest median Treg/CD8+ T cell ratio and the highest levels of CD56dim NK cell infiltration, in our pan-cancer analysis of the most immune-infiltrated tumors. CD8+ T cell infiltration and CD56dim NK cell infiltration each correlated with superior survival in HNSCC. Tumors harboring genetic smoking signatures had lower immune infiltration and were associated with poorer survival, suggesting these patients may benefit from immune agonist therapy. These findings illuminate the immune landscape of HPV+ and HPV– HNSCC. Additionally, this landscape provides a potentially novel rationale for investigation of agents targeting modulators of Tregs (e.g., CTLA-4, GITR, ICOS, IDO, and VEGFA) and NK cells (e.g., KIR, TIGIT, and 4-1BB) as adjuncts to anti–PD-1 in the treatment of advanced HNSCC.
Rajarsi Mandal, Yasin Şenbabaoğlu, Alexis Desrichard, Jonathan J. Havel, Martin G. Dalin, Nadeem Riaz, Ken-Wing Lee, Ian Ganly, A. Ari Hakimi, Timothy A. Chan, Luc G.T. Morris
Viral hepatitis remains a global health challenge despite recent progress in the development of more effective therapies. Although virus-specific CD8+ and CD4+ T cell responses are essential for viral clearance, it remains largely unknown what regulates T cell–mediated viral clearance. Thus, a better understanding of the regulation of anti-viral T cell immunity would be critical for the design of more effective therapies for viral hepatitis. Using a model of adenovirus-induced hepatitis, here we showed that adenoviral infection induced recruitment of Ly6Chi monocytes to the liver in a CCR2-dependent manner. These recruited Ly6Chi monocytes suppressed CD8+ and CD4+ T cell responses to adenoviral infection, leading to a delay in viral clearance. In vivo depletion of Ly6Chi monocytes markedly enhanced anti-viral T cell responses and promoted viral clearance. Mechanistically, we showed that induction of iNOS and the production of NO by Ly6Chi monocytes are critical for the suppression of T cell responses. In addition, a contact-dependent mechanism mediated by PD-1 and PD-L1 interaction is also required for T cell suppression by Ly6Chi monocytes. These findings suggest a critical role for Ly6Chi monocytes in the regulation of T cell immunity in viral hepatitis and may provide new insights into development of more effective therapies for treating viral hepatitis based on targeting the immunosuppressing monocytes.
Jiangao Zhu, Huiyao Chen, Xiaopei Huang, Songfu Jiang, Yiping Yang
Invasive pulmonary aspergillosis is a life-threatening mycosis that only affects patients with immunosuppression, chemotherapy-induced neutropenia, transplantation, or congenital immunodeficiency. We studied the clinical, genetic, histological, and immunological features of 2 unrelated patients without known immunodeficiency who developed extrapulmonary invasive aspergillosis at the ages of 8 and 18. One patient died at age 12 with progressive intra-abdominal aspergillosis. The other patient had presented with intra-abdominal candidiasis at age 9, and developed central nervous system aspergillosis at age 18 and intra-abdominal aspergillosis at age 25. Neither patient developed
Nikolaus Rieber, Roel P. Gazendam, Alexandra F. Freeman, Amy P. Hsu, Amanda L. Collar, Janyce A. Sugui, Rebecca A. Drummond, Chokechai Rongkavilit, Kevin Hoffman, Carolyn Henderson, Lily Clark, Markus Mezger, Muthulekha Swamydas, Maik Engeholm, Rebecca Schüle, Bettina Neumayer, Frank Ebel, Constantinos M. Mikelis, Stefania Pittaluga, Vinod K. Prasad, Anurag Singh, Joshua D. Milner, Kelli W. Williams, Jean K. Lim, Kyung J. Kwon-Chung, Steven M. Holland, Dominik Hartl, Taco W. Kuijpers, Michail S. Lionakis
A unique feature of rheumatoid arthritis (RA) is the presence of anti-citrullinated protein antibodies (ACPA). Several risk factors for RA are known to increase the expression or activity of peptidyl arginine deiminases (PADs), which catalyze citrullination and, when dysregulated, can result in hypercitrullination. However, the consequence of hypercitrullination is unknown and the function of each PAD has yet to be defined. Th cells of RA patients are hypoglycolytic and hyperproliferative due to impaired expression of PFKFB3 and ATM, respectively. Here, we report that these features are also observed in peripheral blood mononuclear cells (PBMCs) from healthy at-risk individuals (ARIs). PBMCs of ARIs are also hypercitrullinated and produce more IL-2 and Th17 cytokines but fewer Th2 cytokines. These abnormal features are due to impaired induction of PTPN22, a phosphatase that also suppresses citrullination independently of its phosphatase activity. Attenuated phosphatase activity of PTPN22 results in aberrant expression of IL-2, ATM, and PFKFB3, whereas diminished nonphosphatase activity of PTPN22 leads to hypercitrullination mediated by PADs. PAD2- or PAD4-mediated hypercitrullination reduces the expression of Th2 cytokines. By contrast, only PAD2-mediated hypercitrullination can increase the expression of Th17 cytokines. Taken together, our data depict a molecular signature of preclinical RA that is triggered by impaired induction of PTPN22.
Hui-Hsin Chang, Guang-Yaw Liu, Nishant Dwivedi, Bo Sun, Yuko Okamoto, Jennifer D. Kinslow, Kevin D. Deane, M. Kristen Demoruelle, Jill M. Norris, Paul R. Thompson, Jeffrey A. Sparks, Deepak A. Rao, Elizabeth W. Karlson, Hui-Chih Hung, V. Michael Holers, I-Cheng Ho
Cullin-3 (
Larry N. Agbor, Stella-Rita C. Ibeawuchi, Chunyan Hu, Jing Wu, Deborah R. Davis, Henry L. Keen, Frederick W. Quelle, Curt D. Sigmund
The placenta is a barrier against maternal-fetal transmission of pathogens. Placental infections can cause several adverse pregnancy outcomes, including preterm birth (PTB). Yet, we have limited knowledge regarding the mechanisms the placenta uses to control infections. Here, we show that autophagy, a cellular recycling pathway important for host defense against pathogens, and the autophagy gene Atg16L1 play a key role in placental defense and are negatively associated with PTB in pregnant women. First, we demonstrate that placentas from women who delivered preterm exhibit reduced autophagy activity and are associated with higher infection indicators. Second, we identify the cellular location of the autophagy activity as being in syncytial trophoblasts. Third, we demonstrate that higher levels of autophagy and ATG16L1 in human trophoblasts were associated with increased resistance to infection. Accordingly, loss of autophagy or ATG16L1 impaired trophoblast antibacterial defenses. Fourth, we show that
Bin Cao, Colin Macones, Indira U. Mysorekar
The repulsive guidance cue SLIT2 and its receptor ROBO2 are required for kidney development and podocyte foot process structure, but the SLIT2/ROBO2 signaling mechanism regulating podocyte function is not known. Here we report that a potentially novel signaling pathway consisting of SLIT/ROBO Rho GTPase activating protein 1 (SRGAP1) and nonmuscle myosin IIA (NMIIA) regulates podocyte adhesion downstream of ROBO2. We found that the myosin II regulatory light chain (MRLC), a subunit of NMIIA, interacts directly with SRGAP1 and forms a complex with ROBO2/SRGAP1/NMIIA in the presence of SLIT2. Immunostaining demonstrated that SRGAP1 is a podocyte protein and is colocalized with ROBO2 on the basal surface of podocytes. In addition, SLIT2 stimulation inhibits NMIIA activity, decreases focal adhesion formation, and reduces podocyte attachment to collagen. In vivo studies further showed that podocyte-specific knockout of
Xueping Fan, Hongying Yang, Sudhir Kumar, Kathleen E. Tumelty, Anna Pisarek-Horowitz, Hila Milo Rasouly, Richa Sharma, Stefanie Chan, Edyta Tyminski, Michael Shamashkin, Mostafa Belghasem, Joel M. Henderson, Anthony J. Coyle, David J. Salant, Stephen P. Berasi, Weining Lu
Regeneration of hepatic sinusoidal vasculature is essential for non-fibrotic liver regrowth and restoration of its metabolic capacity. However, little is known about how this specialized vascular niche is regenerated. Here we show that activation of endothelial sphingosine-1-phosphate receptor-1 (S1P1) by its natural ligand bound to HDL (HDL-S1P) induces liver regeneration and curtails fibrosis. In mice lacking HDL-S1P, liver regeneration after partial hepatectomy was impeded and associated with aberrant vascular remodeling, thrombosis and peri-sinusoidal fibrosis. Notably, this “maladaptive repair” phenotype was recapitulated in mice that lack S1P1 in the endothelium. Reciprocally, enhanced plasma levels of HDL-S1P or administration of SEW2871, a pharmacological agonist specific for S1P1 enhanced regeneration of metabolically functional vasculature and alleviated fibrosis in mouse chronic injury and cholestasis models. This study shows that natural and pharmacological ligands modulate endothelial S1P1 to stimulate liver regeneration and inhibit fibrosis, suggesting that activation of this pathway may be a novel therapeutic strategy for liver fibrosis.
Bi-Sen Ding, Catherine H. Liu, Yue Sun, Yutian Chen, Steven L. Swendeman, Bongnam Jung, Deebly Chavez, Zhongwei Cao, Christina Christoffersen, Lars Bo Nielsen, Susan R. Schwab, Shahin Rafii, Timothy Hla
Heterogeneity of tumor cells and their microenvironment can affect outcome in cancer. Blockade of immune checkpoints (ICPs) expressed only on a subset of immune cells leads to durable responses in advanced melanoma. Tissue-resident memory T (TRM) cells have recently emerged as a distinct subset of memory T cells in nonlymphoid tissues. Here, we show that functional properties and expression of ICPs within tumor-infiltrating lymphocytes (TILs) differ from those of blood T cells. TILs secrete less IL-2, IFN-γ, and TNF-α compared with circulating counterparts, and expression of VEGF correlated with reduced TIL infiltration. Within tumors, ICPs are particularly enriched within T cells with phenotype and genomic features of TRM cells and the CD16+ subset of myeloid cells. Concurrent T cell receptor (TCR) and tumor exome sequencing of individual metastases in the same patient revealed that interlesional diversity of TCRs exceeded differences in mutation/neoantigen load in tumor cells. These findings suggest that the TRM subset of TILs may be the major target of ICP blockade and illustrate interlesional diversity of tissue-resident TCRs within individual metastases, which did not equilibrate between metastases and may differentially affect the outcome of immune therapy at each site.
Chandra Sekhar Boddupalli, Noffar Bar, Krishna Kadaveru, Michael Krauthammer, Natopol Pornputtapong, Zifeng Mai, Stephan Ariyan, Deepak Narayan, Harriet Kluger, Yanhong Deng, Rakesh Verma, Rituparna Das, Antonella Bacchiocchi, Ruth Halaban, Mario Sznol, Madhav V. Dhodapkar, Kavita M. Dhodapkar
Systemic sclerosis (SSc) is a rare autoimmune disease with the highest case-fatality rate of all connective tissue diseases. Current efforts to determine patient response to a given treatment using the modified Rodnan skin score (mRSS) are complicated by interclinician variability, confounding, and the time required between sequential mRSS measurements to observe meaningful change. There is an unmet critical need for an objective metric of SSc disease severity. Here, we performed an integrated, multicohort analysis of SSc transcriptome data across 7 datasets from 6 centers composed of 515 samples. Using 158 skin samples from SSc patients and healthy controls recruited at 2 centers as a discovery cohort, we identified a 415-gene expression signature specific for SSc, and validated its ability to distinguish SSc patients from healthy controls in an additional 357 skin samples from 5 independent cohorts. Next, we defined the SSc skin severity score (4S). In every SSc cohort of skin biopsy samples analyzed in our study, 4S correlated significantly with mRSS, allowing objective quantification of SSc disease severity. Using transcriptome data from the largest longitudinal trial of SSc patients to date, we showed that 4S allowed us to objectively monitor individual SSc patients over time, as (a) the change in 4S of a patient is significantly correlated with change in the mRSS, and (b) the change in 4S at 12 months of treatment could predict the change in mRSS at 24 months. Our results suggest that 4S could be used to distinguish treatment responders from nonresponders prior to mRSS change. Our results demonstrate the potential clinical utility of a novel robust molecular signature and a computational approach to SSc disease severity quantification.
Shane Lofgren, Monique Hinchcliff, Mary Carns, Tammara Wood, Kathleen Aren, Esperanza Arroyo, Peggie Cheung, Alex Kuo, Antonia Valenzuela, Anna Haemel, Paul J. Wolters, Jessica Gordon, Robert Spiera, Shervin Assassi, Francesco Boin, Lorinda Chung, David Fiorentino, Paul J. Utz, Michael L. Whitfield, Purvesh Khatri
Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion.
Øystein Fluge, Olav Mella, Ove Bruland, Kristin Risa, Sissel E. Dyrstad, Kine Alme, Ingrid G. Rekeland, Dipak Sapkota, Gro V. Røsland, Alexander Fosså, Irini Ktoridou-Valen, Sigrid Lunde, Kari Sørland, Katarina Lien, Ingrid Herder, Hanne Thürmer, Merete E. Gotaas, Katarzyna A. Baranowska, Louis M.L.J. Bohnen, Christoph Schäfer, Adrian McCann, Kristian Sommerfelt, Lars Helgeland, Per M. Ueland, Olav Dahl, Karl J. Tronstad
Monique A.J. van Eijndhoven, Josée M. Zijlstra, Nils J. Groenewegen, Esther E.E. Drees, Stuart van Niele, S. Rubina Baglio, Danijela Koppers-Lalic, Hans van der Voorn, Sten F.W.M. Libregts, Marca H.M. Wauben, Renee X. de Menezes, Jan R.T. van Weering, Rienk Nieuwland, Lydia Visser, Anke van den Berg, Daphne de Jong, D. Michiel Pegtel
Current therapies to treat non–small cell lung carcinoma (NSCLC) have proven ineffective owing to transient, variable, and incomplete responses. Here we show that ABL kinases, ABL1 and ABL2, promote metastasis of lung cancer cells harboring EGFR or KRAS mutations. Inactivation of ABL kinases suppresses NSCLC metastasis to brain and bone, and other organs. ABL kinases are required for expression of prometastasis genes. Notably, ABL1 and ABL2 depletion impairs extravasation of lung adenocarcinoma cells into the lung parenchyma. We found that ABL-mediated activation of the TAZ and β-catenin transcriptional coactivators is required for NSCLC metastasis. ABL kinases activate TAZ and β-catenin by decreasing their interaction with the β-TrCP ubiquitin ligase, leading to increased protein stability. High-level expression of
Jing Jin Gu, Clay Rouse, Xia Xu, Jun Wang, Mark W. Onaitis, Ann Marie Pendergast
The second-generation HIV-1 integrase strand transfer inhibitor (InSTI) dolutegravir (DTG) has had a major impact on the treatment of HIV-1 infection. Here we describe important but previously undetermined pharmacodynamic parameters for DTG. We show that the dose-response curve slope, which indicates cooperativity and is a major determinant of antiviral activity, is higher for DTG than for first-generation InSTIs. This steepness does not reflect inhibition of multiple steps in the HIV-1 life cycle, as is the case for allosteric integrase inhibitors and HIV-1 protease inhibitors. We also show that degree of independence, a metric of interaction favorability between antiretroviral drugs, is high for DTG and nucleoside reverse transcriptase inhibitors. Finally, we demonstrate poor selective advantage for HIV-1 bearing InSTI resistance mutations. Selective advantage, which incorporates both the magnitude of resistance conferred by a mutation and its fitness cost, explains the high genetic barrier to DTG resistance. Together, these parameters provide an explanation for the remarkable clinical success of DTG.
Sarah B. Laskey, Robert F. Siliciano
Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.
Yogindra Vedvyas, Enda Shevlin, Marjan Zaman, Irene M. Min, Alejandro Amor-Coarasa, Spencer Park, Susan Park, Keon-Woo Kwon, Turner Smith, Yonghua Luo, Dohyun Kim, Young Kim, Benedict Law, Richard Ting, John Babich, Moonsoo M. Jin
Life-sustaining responses to low oxygen, or hypoxia, depend on signal transduction by HIFs, but the underlying mechanisms by which cells sense hypoxia are not completely understood. Based on prior studies suggesting a link between the β-adrenergic receptor (β-AR) and hypoxia responses, we hypothesized that the β-AR mediates hypoxia sensing and is necessary for HIF-1α accumulation. Beta blocker treatment of mice suppressed hypoxia induction of renal HIF-1α accumulation, erythropoietin production, and erythropoiesis in vivo. Likewise, beta blocker treatment of primary human endothelial cells in vitro decreased hypoxia-mediated HIF-1α accumulation and binding to target genes and the downstream hypoxia-inducible gene expression. In mechanistic studies, cAMP-activated PKA and/or GPCR kinases (GRK), which both participate in β-AR signal transduction, were investigated. Direct activation of cAMP/PKA pathways did not induce HIF-1α accumulation, and inhibition of PKA did not blunt HIF-1α induction by hypoxia. In contrast, pharmacological inhibition of GRK, or expression of a GRK phosphorylation–deficient β-AR mutant in cells, blocked hypoxia-mediated HIF-1α accumulation. Mass spectrometry–based quantitative analyses revealed a hypoxia-mediated β-AR phosphorylation barcode that was different from the classical agonist phosphorylation barcode. These findings indicate that the β-AR is fundamental to the molecular and physiological responses to hypoxia.
Hoi I. Cheong, Kewal Asosingh, Olivia R. Stephens, Kimberly A. Queisser, Weiling Xu, Belinda Willard, Bo Hu, Josephine Kam Tai Dermawan, George R. Stark, Sathyamangla V. Naga Prasad, Serpil C. Erzurum
Duchenne muscular dystrophy (DMD) is a devastating muscle disease characterized by progressive muscle deterioration and replacement with an aberrant fatty, fibrous matrix. Chronic upregulation of nuclear factor κB (NF-κB) is implicated as a driver of the dystrophic pathogenesis. Herein, 2 members of a novel class of NF-κB inhibitors, edasalonexent (formerly CAT-1004) and CAT-1041, were evaluated in both
David W. Hammers, Margaret M. Sleeper, Sean C. Forbes, Cora C. Coker, Michael R. Jirousek, Michael Zimmer, Glenn A. Walter, H. Lee Sweeney
Three-dimensional cardiac mapping is important for optimal visualization of the heart during cardiac ablation for the treatment of certain arrhythmias. However, many hospitals and clinics worldwide cannot afford the high cost of the current mapping systems. We set out to determine if, using predefined algorithms, comparable 3D cardiac maps could be created by a new device that relies on data generated from single-plane fluoroscopy and patient recording and monitoring systems, without the need for costly equipment, infrastructure changes, or specialized catheters. The study included phantom and animal experiments to compare the prototype test device, Navik 3D, with the existing CARTO 3 System. The primary endpoint directly compared: (a) the 3D distance between the Navik 3D–simulated ablation location and the back-projected ground truth location of the pacing and mapping catheter electrode, and (b) the same distance for CARTO. The study’s primary objective was considered met if the 95% confidence lower limit was greater than 0.75% for the Navik 3D–CARTO difference between the 2 distances, or less than or equal to 2 mm. Study results showed that the Navik 3D performance was equivalent to the CARTO system, and that accurate 3D cardiac maps can be created using data from equipment that already exists in all electrophysiology labs.
Jasbir Sra, David Krum, Indrajit Choudhuri, Barry Belanger, Mark Palma, Donald Brodnick, Daniel B. Rowe
Angiogenesis and co-optive vascular remodeling are prerequisites of solid tumor growth. Vascular heterogeneity, notably perivascular composition, may play a critical role in determining the rate of cancer progression. The contribution of vascular pericyte heterogeneity to cancer progression and therapy response is unknown. Here, we show that angiopoietin-2 (Ang2) orchestrates pericyte heterogeneity in breast cancer with an effect on metastatic disease and response to chemotherapy. Using multispectral imaging of human breast tumor specimens, we report that perivascular composition, as defined by the ratio of PDGFRβ– and desmin+ pericytes, provides information about the response to epirubicin but not paclitaxel. Using 17 distinct patient-derived breast cancer xenografts, we demonstrate a cancer cell–derived influence on stromal Ang2 production and a cancer cell–defined control over tumor vasculature and perivascular heterogeneity. The aggressive features of tumors and their distinct response to therapies may thus emerge by the cancer cell–defined engagement of distinct and heterogeneous angiogenic programs.
Jiha Kim, Pedro Correa de Sampaio, Donna Marie Lundy, Qian Peng, Kurt W. Evans, Hikaru Sugimoto, Mihai Gagea, Yvonne Kienast, Nayra Soares do Amaral, Rafael Malagoli Rocha, Hans Petter Eikesdal, Per Eystein Lønning, Funda Meric-Bernstam, Valerie S. LeBleu
Bcl-2–associated athanogene 3 (BAG3) is an evolutionarily conserved protein expressed at high levels in the heart and the vasculature and in many cancers. While altered BAG3 expression has been associated with cardiac dysfunction, its role in ischemia/reperfusion (I/R) is unknown. To test the hypothesis that BAG3 protects the heart from reperfusion injury, in vivo cardiac function was measured in hearts infected with either recombinant adeno-associated virus serotype 9–expressing (rAAV9-expressing) BAG3 or GFP and subjected to I/R. To elucidate molecular mechanisms by which BAG3 protects against I/R injury, neonatal mouse ventricular cardiomyocytes (NMVCs) in which BAG3 levels were modified by adenovirus expressing (Ad-expressing) BAG3 or siBAG3 were exposed to hypoxia/reoxygenation (H/R). H/R significantly reduced NMVC BAG3 levels, which were associated with enhanced expression of apoptosis markers, decreased expression of autophagy markers, and reduced autophagy flux. The deleterious effects of H/R on apoptosis and autophagy were recapitulated by knockdown of BAG3 with Ad-siBAG3 and were rescued by Ad-BAG3. In vivo, treatment of mice with rAAV9-BAG3 prior to I/R significantly decreased infarct size and improved left ventricular function when compared with mice receiving rAAV9-GFP and improved markers of autophagy and apoptosis. These findings suggest that BAG3 may provide a therapeutic target in patients undergoing reperfusion after myocardial infarction.
Feifei Su, Valerie D. Myers, Tijana Knezevic, JuFang Wang, Erhe Gao, Muniswamy Madesh, Farzaneh G. Tahrir, Manish K. Gupta, Jennifer Gordon, Joseph Rabinowitz, Frederick V. Ramsey, Douglas G. Tilley, Kamel Khalili, Joseph Y. Cheung, Arthur M. Feldman