Latest issue: October 15, 2020

In the issue

Abstract

BACKGROUND Patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) differ in the severity of disease. We hypothesized that characteristics of SARS-CoV-2–specific immunity correlate with disease severity.METHODS In this study, SARS-CoV-2–specific T cells and antibodies were characterized in uninfected controls and patients with different coronavirus disease 2019 (COVID-19) disease severity. SARS-CoV-2–specific T cells were flow cytometrically quantified after stimulation with SARS-CoV-2 peptide pools and analyzed for expression of cytokines (IFN-γ, IL-2, and TNF-α) and markers for activation, proliferation, and functional anergy. SARS-CoV-2–specific IgG and IgA antibodies were quantified using ELISA. Moreover, global characteristics of lymphocyte subpopulations were compared between patient groups and uninfected controls.RESULTS Despite severe lymphopenia affecting all major lymphocyte subpopulations, patients with severe disease mounted significantly higher levels of SARS-CoV-2–specific T cells as compared with convalescent individuals. SARS-CoV-2–specific CD4+ T cells dominated over CD8+ T cells and closely correlated with the number of plasmablasts and SARS-CoV-2–specific IgA and IgG levels. Unlike in convalescent patients, SARS-CoV-2–specific T cells in patients with severe disease showed marked alterations in phenotypical and functional properties, which also extended to CD4+ and CD8+ T cells in general.CONCLUSION Given the strong induction of specific immunity to control viral replication in patients with severe disease, the functionally altered characteristics may result from the need for contraction of specific and general immunity to counteract excessive immunopathology in the lung.FUNDING The study was supported by institutional funds to MS and in part by grants of Saarland University, the State of Saarland, and the Rolf M. Schwiete Stiftung.

Authors

David Schub, Verena Klemis, Sophie Schneitler, Janine Mihm, Philipp M. Lepper, Heinrike Wilkens, Robert Bals, Hermann Eichler, Barbara C. Gärtner, Sören L. Becker, Urban Sester, Martina Sester, Tina Schmidt

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Abstract

Impaired tolerance to innocuous particles during allergic asthma has been linked to increased plasticity of FoxP3+ regulatory T cells (Tregs) reprogramming into pathogenic effector cells, thus exacerbating airway disease. However, failure of tolerance mechanisms is driven by Th2 inflammatory signals. Therefore, the in vivo role of canonical IL-4 receptor α (IL-4Rα) signaling, an essential driver of Th2-type airway responses to allergens, on the regulatory function of FoxP3+ Tregs in allergic asthma was explored. Here, we used transgenic Foxp3cre IL-4Rα–/lox and littermate control mice to investigate the role of IL-4 and IL-13 signaling via Tregs in house dust mite–induced (HDM-induced) allergic airway disease. We sensitized mice intratracheally on day 0, challenged them on days 6–10, and analyzed airway hyperresponsiveness (AHR), airway inflammation, mucus production, and cellular profile on day 14. In the absence of IL-4Rα responsiveness on FoxP3+ Tregs, exacerbated AHR and airway inflammation were shown in HDM-sensitized mice. Interestingly, reduced induction of FoxP3+ Tregs accompanied increased IL-33 alarmin production and type 2 innate lymphoid cell activation in the lung, exacerbating airway hyperreactivity and lung eosinophilia. Taken together, our findings indicate that IL-4Rα–unresponsive FoxP3+ Tregs result in exaggerated innate Th2-type, IL-33–dependent airway inflammation and a break in tolerance during allergic asthma.

Authors

Jermaine Khumalo, Frank Kirstein, Sabelo Hadebe, Frank Brombacher

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Abstract

Focal adhesion kinase (FAK) is an important mediator of extracellular matrix–integrin mechano-signal transduction that regulates cell motility, survival, and proliferation. As such, FAK is being investigated as a potential therapeutic target for malignant and fibrotic diseases, and numerous clinical trials of FAK inhibitors are underway. The function of FAK in nonmalignant, nonmotile epithelial cells is not well understood. We previously showed that hepatocytes demonstrated activated FAK near stiff collagen tracts in fibrotic livers. In this study, we examined the role of liver epithelial FAK by inducing fibrotic liver disease in mice with liver epithelial FAK deficiency. We found that mice that lacked FAK in liver epithelial cells developed more severe liver injury and worse fibrosis as compared with controls. Increased fibrosis in liver epithelial FAK-deficient mice was linked to the activation of several profibrotic pathways, including the hedgehog/smoothened pathway. FAK-deficient hepatocytes produced increased Indian hedgehog in a manner dependent on matrix stiffness. Furthermore, expression of the hedgehog receptor, smoothened, was increased in macrophages and biliary cells of hepatocyte-specific FAK-deficient fibrotic livers. These results indicate that liver epithelial FAK has important regulatory roles in the response to liver injury and progression of fibrosis.

Authors

Yun Weng, Tyler J. Lieberthal, Vivian X. Zhou, Maya Lopez-Ichikawa, Manuel Armas-Phan, Tristan K. Bond, Miya C. Yoshida, Won-Tak Choi, Tammy T. Chang

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Abstract

In the aging population, lower urinary tract (LUT) dysfunction is common and often leads to storage and voiding difficulties classified into overlapping symptom syndromes. Despite prevalence and consequences of these syndromes, LUT disorders continue to be undertreated simply because there are few therapeutic options. LUT function and structure were assessed in aged (>25 months) male and female Fischer 344 rats randomized to oral treatment with a purine nucleoside phosphorylase (PNPase inhibitor) 8-aminoguanine (8-AG) or vehicle for 6 weeks. The bladders of aged rats exhibited multiple abnormalities: tactile insensitivity, vascular remodeling, reduced collagen-fiber tortuosity, increased bladder stiffness, abnormal smooth muscle morphology, swelling of mitochondria, and increases in urodamaging purine metabolites. Treatment of aged rats with 8-AG restored all evaluated histological, ultrastructural, and physiological abnormalities toward that of a younger state. 8-AG is an effective treatment that ameliorates key age-related structural and physiologic bladder abnormalities. Because PNPase inhibition blocks metabolism of inosine to hypoxanthine and guanosine to guanine, likely uroprotective effects of 8-AG are mediated by increased bladder levels of uroprotective inosine and guanosine and reductions in urodamaging hypoxanthine and xanthine. These findings demonstrate that 8-AG has translational potential for treating age-associated LUT dysfunctions and resultant syndromes in humans.

Authors

Lori A. Birder, Amanda Wolf-Johnston, Alan J. Wein, Fangzhou Cheng, Mara Grove-Sullivan, Anthony J. Kanai, Alan M. Watson, Donna Stoltz, Simon C. Watkins, Anne M. Robertson, Diane Newman, Roger R. Dmochowski, Edwin K. Jackson

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Abstract

The ability of HDL to inhibit inflammation in adipocytes and adipose tissue is reduced when HDL contains serum amyloid A (SAA) that is trapped by proteoglycans at the adipocyte surface. Because we recently found that the major extracellular matrix proteoglycan produced by hypertrophic adipocytes is versican, whereas activated adipose tissue macrophages produce mainly biglycan, we further investigated the role of proteoglycans in determining the antiinflammatory properties of HDL. The distributions of versican, biglycan, apolipoprotein A1 (the major apolipoprotein of HDL), and SAA were similar in adipose tissue from obese mice and obese human subjects. Colocalization of SAA-enriched HDL with versican and biglycan at the cell surface of adipocyte and peritoneal macrophages, respectively, was blocked by silencing these proteoglycans, which also restored the antiinflammatory property of SAA-enriched HDL despite the presence of SAA. Similar to adipocytes, normal HDL exerted its antiinflammatory function in macrophages by reducing lipid rafts, reactive oxygen species generation, and translocation of Toll-like receptor 4 and NADPH oxidase 2 into lipid rafts, effects that were not observed with SAA-enriched HDL. These findings imply that SAA present in HDL can be trapped by adipocyte-derived versican and macrophage-derived biglycan, thereby blunting HDL’s antiinflammatory properties.

Authors

Chang Yeop Han, Inkyung Kang, Mohamed Omer, Shari Wang, Tomasz Wietecha, Thomas N. Wight, Alan Chait

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Abstract

Hidradenitis suppurativa (HS) is a chronic skin disorder of unknown etiology that manifests as recurrent, painful lesions. Cutaneous dysbiosis and unresolved inflammation are hallmarks of active HS, but their origin and interplay remain unclear. Our metabolomic profiling of HS skin revealed an abnormal induction of the kynurenine pathway of tryptophan catabolism in dermal fibroblasts, correlating with the release of kynurenine pathway–inducing cytokines by inflammatory cell infiltrates. Notably, overactivation of the kynurenine pathway in lesional skin was associated with local and systemic depletion in tryptophan. Yet the skin microbiota normally degrades host tryptophan into indoles regulating tissue inflammation via engagement of the aryl hydrocarbon receptor (AHR). In HS skin lesions, we detected contextual defects in AHR activation coinciding with impaired production of bacteria-derived AHR agonists and decreased incidence of AHR ligand-producing bacteria in the resident flora. Dysregulation of tryptophan catabolism at the skin-microbiota interface thus provides a mechanism linking the immunological and microbiological features of HS lesions. In addition to revealing metabolic alterations in patients with HS, our study suggests that correcting AHR signaling would help restore immune homeostasis in HS skin.

Authors

Laure Guenin-Macé, Jean-David Morel, Jean-Marc Doisne, Angèle Schiavo, Lysiane Boulet, Véronique Mayau, Pedro Goncalves, Sabine Duchatelet, Alain Hovnanian, Vincent Bondet, Darragh Duffy, Marie-Noëlle Ungeheuer, Maïa Delage, Aude Nassif, James P. Di Santo, Caroline Demangel

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Abstract

Recently programmed death-ligand 1 (PD-L1) receptor PD-1 was found in dorsal root ganglion (DRG) neurons, and PD-L1 activates PD-1 to inhibit inflammatory and neuropathic pain by modulating neuronal excitability. However, the downstream signaling of PD-1 in sensory neurons remains unclear. Here, we show that PD-L1 activated Src homology 2 domain-containing tyrosine phosphatase-1 (SHP-1) to downregulate transient receptor potential vanilloid 1 (TRPV1) in DRG neurons and inhibit bone cancer pain in mice. Local injection of PD-L1 produced analgesia. PD-1 in DRG neurons colocalized with TRPV1 and SHP-1. PD-L1 induced the phosphorylation of SHP-1 in DRG TRPV1 neurons and inhibited TRPV1 currents. Loss of TRPV1 in mice abolished bone cancer–induced thermal hyperalgesia and PD-L1 analgesia. Conditioned deletion of SHP-1 in NaV1.8+ neurons aggravated bone cancer pain and diminished the inhibition of PD-L1 on TRPV1 currents and pain. Together, our findings suggest that PD-L1/PD-1 signaling suppresses bone cancer pain via inhibition of TRPV1 activity. Our results also suggest that SHP-1 in sensory neurons is an endogenous pain inhibitor and delays the development of bone cancer pain via suppressing TRPV1 function.

Authors

Ben-Long Liu, Qi-Lai Cao, Xin Zhao, Hui-Zhu Liu, Yu-Qiu Zhang

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Abstract

Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension–generating capacity. We demonstrated that cardiac twitch tension-time integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using multiscale computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L48Q can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L48Q cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.

Authors

Joseph D. Powers, Kristina B. Kooiker, Allison B. Mason, Abigail E. Teitgen, Galina V. Flint, Jil C. Tardiff, Steven D. Schwartz, Andrew D. McCulloch, Michael Regnier, Jennifer Davis, Farid Moussavi-Harami

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Abstract

Ischemia/reperfusion-induced edema (IRE), one of the most significant causes of mortality after lung transplantation, can be mimicked ex vivo in isolated perfused mouse lungs (IPL). Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel studied in endothelium; however, its role in the lung epithelium remains elusive. Here, we show enhanced IRE in TRPV4-deficient (TRPV4–/–) IPL compared with that of WT controls, indicating a protective role of TRPV4 in maintenance of the alveolar epithelial barrier. By immunohistochemistry, mRNA profiling, and electrophysiological characterization, we detected TRPV4 in bronchial epithelium, alveolar epithelial type I (ATI), and alveolar epithelial type II (ATII) cells. Genetic ablation of TRPV4 resulted in reduced expression of the water-conducting aquaporin-5 (AQP-5) channel in ATI cells. Migration of TRPV4–/– ATI cells was reduced, and cell barrier function was impaired. Analysis of isolated primary TRPV4–/– ATII cells revealed a reduced expression of surfactant protein C, and the TRPV4 activator GSK1016790A induced increases in current densities only in WT ATII cells. Moreover, TRPV4–/– lungs of adult mice developed significantly larger mean chord lengths and altered lung function compared with WT lungs. Therefore, our data illustrate essential functions of TRPV4 channels in alveolar epithelial cells and in protection from edema formation.

Authors

Jonas Weber, Suhasini Rajan, Christian Schremmer, Yu-Kai Chao, Gabriela Krasteva-Christ, Martina Kannler, Ali Önder Yildirim, Monika Brosien, Johann Schredelseker, Norbert Weissmann, Christian Grimm, Thomas Gudermann, Alexander Dietrich

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Abstract

A critical response to lysosomal membrane permeabilization (LMP) is the clearance of damaged lysosomes through a selective form of macroautophagy known as lysophagy. Although regulators of this process are emerging, whether organ- and cell-specific components contribute to the control of lysophagy remains incompletely understood. Here, we examined LMP and lysophagy in Niemann-Pick type C (NPC) disease, an autosomal recessive disorder characterized by the accumulation of unesterified cholesterol within late endosomes and lysosomes, leading to neurodegeneration and early death. We demonstrated that NPC human fibroblasts show enhanced sensitivity to lysosomal damage as a consequence of lipid storage. Moreover, we described a role for the glycan-binding F-box protein 2 (Fbxo2) in CNS lysophagy. Fbxo2 functions as a component of the S phase kinase-associated protein 1–cullin 1–F-box protein (SKP1-CUL1-SCF) ubiquitin ligase complex. Loss of Fbxo2 in mouse primary cortical cultures delayed clearance of damaged lysosomes and decreased viability after lysosomal damage. Moreover, Fbxo2 deficiency in a mouse model of NPC exacerbated deficits in motor function, enhanced neurodegeneration, and reduced survival. Collectively, our data identified a role for Fbxo2 in CNS lysophagy and establish its functional importance in NPC.

Authors

Elaine A. Liu, Mark L. Schultz, Chisaki Mochida, Chan Chung, Henry L. Paulson, Andrew P. Lieberman

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Abstract

ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic SNPs identified in GWAS; A391T has associations with an increased risk of schizophrenia, obesity, Crohn’s disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knockin (KI) to generate a mouse model of ZIP8 A391T (Zip8 393T-KI mice). Recapitulating the SNP association with blood Mn, blood Mn was reduced in Zip8 393T-KI mice. There was restricted abnormal tissue Mn homeostasis, with decreases in liver and kidney Mn and a reciprocal increase in biliary Mn, providing in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemically induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of patients with Crohn’s disease. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in complex human disease.

Authors

Laxmi Sunuwar, Azra Frkatović, Sodbo Sharapov, Qinchuan Wang, Heather M. Neu, Xinqun Wu, Talin Haritunians, Fengyi Wan, Sarah Michel, Shaoguang Wu, Mark Donowitz, Dermot McGovern, Gordan Lauc, Cynthia Sears, Joanna Melia

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Abstract

The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue-resident, and/or follicular helper CD4+ T cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals. We observed minor differences in the frequency of nonintronic and nondistal intergenic sites, varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T cell subsets or tissue compartments was only observed in 8 unique sites of the 3540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule and indicate that additional studies are necessary to fully understand the heterogeneity of tissue-sequestered HIV reservoirs.

Authors

Vincent H. Wu, Christopher L. Nobles, Leticia Kuri-Cervantes, Kevin McCormick, John K. Everett, Son Nguyen, Perla M. del Rio Estrada, Mauricio González-Navarro, Santiago Ávila-Ríos, Gustavo Reyes-Terán, Frederic D. Bushman, Michael R. Betts

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Abstract

Giant cell arteritis (GCA) is a common form of primary systemic vasculitis in adults, with no reliable indicators of prognosis or treatment responses. We used single cell technologies to comprehensively map immune cell populations in the blood of patients with GCA and identified the CD66b+CD15+CD10lo/–CD64– band neutrophils and CD66bhiCD15+CD10lo/–CD64+/bright myelocytes/metamyelocytes to be unequivocally associated with both the clinical phenotype and response to treatment. Immature neutrophils were resistant to apoptosis, remained in the vasculature for a prolonged period of time, interacted with platelets, and extravasated into the tissue surrounding the temporal arteries of patients with GCA. We discovered that immature neutrophils generated high levels of extracellular reactive oxygen species, leading to enhanced protein oxidation and permeability of endothelial barrier in an in vitro coculture system. The same populations were also detected in other systemic vasculitides. These findings link functions of immature neutrophils to disease pathogenesis, establishing a clinical cellular signature of GCA and suggesting different therapeutic approaches in systemic vascular inflammation.

Authors

Lihui Wang, Zhichao Ai, Tariq Khoyratty, Kristina Zec, Hayley L. Eames, Erinke van Grinsven, Alison Hudak, Susan Morris, David Ahern, Claudia Monaco, Evgeniy B. Eruslanov, Raashid Luqmani, Irina A. Udalova

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Abstract

Schwannomas are tumors of the Schwann cells that cause chronic pain, numbness, and potentially life-threatening impairment of vital organs. Despite the identification of causative genes, including NF2 (Merlin), INI1/SMARCB1, and LZTR1, the exact molecular mechanism of schwannoma development is still poorly understood. Several studies have identified Merlin as a key regulator of the Hippo, MAPK, and PI3K signaling pathways; however, definitive evidence demonstrating the importance of these pathways in schwannoma pathogenesis is absent. Here, we provide direct genetic evidence that dysregulation of the Hippo pathway in the Schwann cell lineage causes development of multiple schwannomas in mice. We found that canonical Hippo signaling through the effectors YAP/TAZ is required for schwannomagenesis and that MAPK signaling modifies schwannoma formation. Furthermore, cotargeting YAP/TAZ transcriptional activity and MAPK signaling demonstrated a synergistic therapeutic effect on schwannomas. Our new model provides a tractable platform to dissect the molecular mechanisms underpinning schwannoma formation and the role of combinatorial targeted therapy in schwannoma treatment.

Authors

Zhiguo Chen, Stephen Li, Juan Mo, Eric Hawley, Yong Wang, Yongzheng He, Jean-Philippe Brosseau, Tracey Shipman, D. Wade Clapp, Thomas J. Carroll, Lu Q. Le

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Abstract

Myeloid cells are increasingly recognized as major players in transplant rejection. Here, we used a murine kidney transplantation model and single cell transcriptomics to dissect the contribution of myeloid cell subsets and their potential signaling pathways to kidney transplant rejection. Using a variety of bioinformatic techniques, including machine learning, we demonstrate that kidney allograft–infiltrating myeloid cells followed a trajectory of differentiation from monocytes to proinflammatory macrophages, and they exhibited distinct interactions with kidney allograft parenchymal cells. While this process correlated with a unique pattern of myeloid cell transcripts, a top gene identified was Axl, a member of the receptor tyrosine kinase family Tyro3/Axl/Mertk (TAM). Using kidney transplant recipients with Axl gene deficiency, we further demonstrate that Axl augmented intragraft differentiation of proinflammatory macrophages, likely via its effect on the transcription factor Cebpb. This, in turn, promoted intragraft recruitment, differentiation, and proliferation of donor-specific T cells, and it enhanced early allograft inflammation evidenced by histology. We conclude that myeloid cell Axl expression identified by single cell transcriptomics of kidney allografts in our study plays a major role in promoting intragraft myeloid cell and T cell differentiation, and it presents a potentially novel therapeutic target for controlling kidney allograft rejection and improving kidney allograft survival.

Authors

Anil Dangi, Naveen R. Natesh, Irma Husain, Zhicheng Ji, Laura Barisoni, Jean Kwun, Xiling Shen, Edward B. Thorp, Xunrong Luo

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Abstract

Mottled skin pigmentation and solar lentigines from chronic photodamage with aging involve complex interactions between keratinocytes and melanocytes. However, the precise signaling mechanisms that could serve as therapeutic targets are unclear. Herein, we report that expression of nuclear factor erythroid 2–related factor 2 (NRF2), which regulates reduction-oxidation reactions, is altered in solar lentigines and photodamaged skin. Moreover, mottled skin pigmentation in humans could be treated with topical application of the NRF2 inducer sulforaphane (SF). Similarly, UV light–induced pigmentation of WT mouse ear skin could be treated or prevented with SF treatment. Conversely, SF treatment was unable to reduce UV-induced ear skin pigmentation in mice deficient in NRF2 or in mice with keratinocyte-specific conditional deletion of IL-6Rα. Taken together, NRF2 and IL-6Rα signaling are involved in the pathogenesis of UV-induced skin pigmentation, and specific enhancement of NRF2 signaling could represent a potential therapeutic target.

Authors

Michelle L. Kerns, Robert J. Miller, Momina Mazhar, Angel S. Byrd, Nathan K. Archer, Bret L. Pinkser, Lance Lew, Carly A. Dillen, Ruizhi Wang, Lloyd S. Miller, Anna L. Chien, Sewon Kang

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Abstract

BACKGROUND Physical frailty in older individuals is characterized by subjective symptoms of fatigue and exercise intolerance (EI). Objective abnormalities in skeletal muscle (SM) mitochondrial high-energy phosphate (HEP) metabolism contribute to EI in inherited myopathies; however, their presence or link to EI in the frail older adult is unknown.METHODS Here, we studied 3 groups of ambulatory, community-dwelling adults with no history of significant coronary disease: frail older (FO) individuals (81 ± 2.7 years, mean ± SEM), nonfrail older (NFO) individuals (79 ± 2.0 years), and healthy middle-aged individuals, who served as controls (CONT, 51 ± 2.1 years). Lower extremity SM HEP levels and mitochondrial function were measured with 31P magnetic resonance (MR) techniques during graded multistage plantar flexion exercise (PFE). EI was quantified by a 6-minute walk (6MW) and peak oxygen consumption during cardiopulmonary testing (peak VO2).RESULTS During graded exercise, FO, NFO, and CONT individuals all fatigued at similar SM HEP levels, as measured by 31P-MR. However, FO individuals fatigued fastest, with several-fold higher rates of PFE-induced HEP decline that correlated closely with shorter exercise duration in the MR scanner and with 6MW distance and lower peak oxygen consumption on cardiopulmonary testing (P < 0.001 for all). SM mitochondrial oxidative capacity was lower in older individuals and correlated with rapid HEP decline but less closely with EI.CONCLUSION Several-fold faster SM energetic decline during exercise occurs in FO individuals and correlates closely with multiple measures of EI. Rapid energetic decline represents an objective, functional measure of SM metabolic changes and a potential new target for mitigating frailty-associated physical limitations.FUNDING This work was supported by NIH R21 AG045634, R01 AG063661, R01 HL61912, the Johns Hopkins University Claude D. Pepper Older Americans Independence Center P30AG021334, and the Clarence Doodeman Endowment in Cardiology at Johns Hopkins.

Authors

Sabra C. Lewsey, Kilian Weiss, Michael Schär, Yi Zhang, Paul A. Bottomley, T. Jake Samuel, Qian-Li Xue, Angela Steinberg, Jeremy D. Walston, Gary Gerstenblith, Robert G. Weiss

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Abstract

Despite the efficacy of antiretroviral therapy (ART), HIV persists in a latent form and remains a hurdle to eradication. CD4+ T lymphocytes harbor the majority of the HIV reservoir, but the role of individual subsets remains unclear. CD4+ T cells were sorted into central, transitional, effector memory, and naive T cells. We measured HIV DNA and performed proviral sequencing of more than 1900 proviruses in 2 subjects at 2 and 9 years after ART initiation to estimate the contribution of each subset to the reservoir. Although our study was limited to 2 subjects, we obtained comparable findings with publicly available sequences. While the HIV integration levels were lower in naive compared with memory T cells, naive cells were a major contributor to the intact proviral reservoir. Notably, proviral sequences isolated from naive cells appeared to be unique, while those retrieved from effector memory cells were mainly clonal. The number of clones increased as cells differentiated from a naive to an effector memory phenotype, suggesting naive cells repopulate the effector memory reservoir as previously shown for central memory cells. Naive T cells contribute substantially to the intact HIV reservoir and represent a significant hurdle for HIV eradication.

Authors

Emmanuele Venanzi Rullo, Marilia Rita Pinzone, LaMont Cannon, Sam Weissman, Manuela Ceccarelli, Ryan Zurakowski, Giuseppe Nunnari, Una O’Doherty

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Abstract

Although congenital heart defects (CHDs) represent the most common birth defect, a comprehensive understanding of disease etiology remains unknown. This is further complicated since CHDs can occur in isolation or as a feature of another disorder. Analyzing disorders with associated CHDs provides a powerful platform to identify primary pathogenic mechanisms driving disease. Aberrant localization and expression of cathepsin proteases can perpetuate later-stage heart diseases, but their contribution toward CHDs is unclear. To investigate the contribution of cathepsins during cardiovascular development and congenital disease, we analyzed the pathogenesis of cardiac defects in zebrafish models of the lysosomal storage disorder mucolipidosis II (MLII). MLII is caused by mutations in the GlcNAc-1-phosphotransferase enzyme (Gnptab) that disrupt carbohydrate-dependent sorting of lysosomal enzymes. Without Gnptab, lysosomal hydrolases, including cathepsin proteases, are inappropriately secreted. Analyses of heart development in gnptab-deficient zebrafish show cathepsin K secretion increases its activity, disrupts TGF-β–related signaling, and alters myocardial and valvular formation. Importantly, cathepsin K inhibition restored normal heart and valve development in MLII embryos. Collectively, these data identify mislocalized cathepsin K as an initiator of cardiac disease in this lysosomal disorder and establish cathepsin inhibition as a viable therapeutic strategy.

Authors

Po-Nien Lu, Trevor Moreland, Courtney J. Christian, Troy C. Lund, Richard A. Steet, Heather Flanagan-Steet

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Abstract

Peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes that are involved in a variety of human disorders, including cancer and autoimmune diseases. Although targeting PAD4 has shown no benefit in sepsis, the role of PAD2 remains unknown. Here, we report that PAD2 is engaged in sepsis and sepsis-induced acute lung injury in both human patients and mice. Pad2–/– or selective inhibition of PAD2 by a small molecule inhibitor increased survival and improved overall outcomes in mouse models of sepsis. Pad2 deficiency decreased neutrophil extracellular trap (NET) formation. Importantly, Pad2 deficiency inhibited Caspase-11–dependent pyroptosis in vivo and in vitro. Suppression of PAD2 expression reduced inflammation and increased macrophage bactericidal activity. In contrast to Pad2–/–, Pad4 deficiency enhanced activation of Caspase-11–dependent pyroptosis in BM-derived macrophages and displayed no survival improvement in a mouse sepsis model. Collectively, our findings highlight the potential of PAD2 as an indicative marker and therapeutic target for sepsis.

Authors

Yuzi Tian, Shibin Qu, Hasan B. Alam, Aaron M. Williams, Zhenyu Wu, Qiufang Deng, Baihong Pan, Jing Zhou, Baoling Liu, Xiuzhen Duan, Jianjie Ma, Santanu Mondal, Paul R. Thompson, Kathleen A. Stringer, Theodore J. Standiford, Yongqing Li

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Abstract

In search of new prognostic markers, many mutation analyses of the HBV genome were performed. However, the Kozak sequence preceding precore was covered only infrequently in these analyses. In this study, HBV core promoter/precore region was sequenced in serum samples of European inactive HBV carriers (n=560). Quadruple mutation GCAC1809-1812TTCT was found with a high prevalence of 42% in the Kozak sequence preceding precore among all HBV genotypes. GCAC1809-1812TTCT was strongly associated with coexistence of basal core promoter (BCP) double mutation A1762T/G1764A and lower HBV DNA levels (p<0.0001). In vitro GCAC1809-1812TTCT leads to drastically diminished synthesis of pregenomic(pg)RNA, precore mRNA, core, HBsAg and HBeAg. Calculation of the pgRNA secondary structure suggests a destabilization of the pgRNA structure by A1762T/G1764A that is compensated by GCAC1809-1812TTCT. In 125 patients with HBV-related cirrhosis, GCAC1809-1812TTCT was not detected. While a strong association of GCAC1809-1812TTCT with inactive carrier status (p<0.0001) was observed, BCP double mutation was strongly correlated with cirrhosis (p<0.0001), but this was only observed in absence of GCAC1809-1812TTCT. In conclusion, our data reveal that GCAC1809-1812TTCT is highly prevalent in inactive carriers, and acts as a compensatory mutation for BCP double mutation. GCAC1809-1812TTCT seems to be a biomarker of good prognosis in HBV infection.

Authors

Kai-Henrik Peiffer, Catrina Spengler, Michael Basic, Bingfu Jiang, Lisa Kuhnhenn, Wiebke Obermann, Tobias Zahn, Mirco Glitscher, Alessandro Loglio, Floriana Facchetti, Gert Carra, Alica Kubesch, Johannes Vermehren, Viola Knop, Christiana Graf, Julia Dietz, Fabian Finkelmeier, Eva Herrmann, Jonel Trebicka, Arnold Grünweller, Stefan Zeuzem, Christoph Sarrazin, Pietro Lampertico, Eberhard Hildt

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Abstract

Heart failure is often accompanied by titin-dependent myocardial stiffness. Phosphorylation of titin by cGMP-dependent protein kinase (PKG)I increases cardiomyocyte distensibility. The upstream pathways stimulating PKGI-mediated titin phosphorylation are unclear. We studied whether C-type natriuretic peptide (CNP), via its guanylyl cyclase-B (GC-B) receptor and cGMP/PKGI signalling, modulates titin-based ventricular compliance. To dissect GC-B-mediated effects of endogenous CNP in cardiomyocytes, we generated mice with cardiomyocyte (CM)-restricted GC-B deletion. The impact on heart morphology and function, myocyte passive tension and titin isoform expression and phosphorylation was studied at baseline and after increased afterload induced by transverse aortic constriction (TAC).Pressure-overload increased left ventricular, especially endothelial CNP expression, with an early peak after 3 days. Concomitantly, titin phosphorylation at Ser4080, the site phosphorylated by PKGI, was augmented. Notably, in CM GC-B KO mice this titin response was abolished. TAC-induced hypertrophy and fibrosis were not different between genotypes. However, the KO mice presented mild systolic and diastolic dysfunction together with myocyte stiffness, which were not observed in control littermates. In vitro, recombinant PKGI rescued reduced Titin-Ser4080 phosphorylation and reverted passive stiffness of GC-B-deficient cardiomyocytes. CNP-induced activation of GC-B/cGMP/PKGI signalling in cardiomyocytes provides a protecting regulatory circuit preventing titin-based myocyte stiffening during early phases of pressure-overload.

Authors

Konstanze Michel, Melissa Herwig, Franziska Werner, Katarina Špiranec Spes, Marco Abeßer, Kai Schuh, Swati Dabral, Andreas Mügge, Hideo A. Baba, Boris V. Skryabin, Nazha Hamdani, Michaela Kuhn

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Abstract

The challenge of discovering a completely new human tumor virus of unknown phylogeny or sequence depends on detecting viral molecules and differentiating them from host molecules in the virus-associated neoplasm. We developed differential peptide subtraction (DPS) using differential mass-spectrometry (dMS) followed by targeted analysis to facilitate this discovery. We validated this approach by analyzing Merkel cell carcinoma (MCC), an aggressive human neoplasm, in which ~80% of cases are caused by the human Merkel cell polyomavirus (MCV). Approximately 20% of MCC have a high mutational burden and are negative for MCV, but are microscopically indistinguishable from virus positive cases. Using 23 (12 MCV positive, 11 MCV negative) formalin-fixed MCC, DPS identified both viral and human biomarkers (MCV Large T antigen, CDKN2AIP, SERPINB5 and TRIM29) that discriminates MCV positive and negative MCC. Statistical analysis of 498,131 dMS features not matching the human proteome by DPS revealed 562 (0.11%) to be up-regulated in virus-infected samples. Remarkably, four (20%) of the top 20 candidate MS spectra originated from MCV T oncoprotein peptides and confirmed by reverse translation degenerate oligonucleotide sequencing. DPS is a robust proteomic approach to identify novel viral sequences in infectious tumors when nucleic acid-based methods are not feasible.

Authors

Tuna Toptan, Pamela S. Cantrell, Xuemei Zeng, Yang Liu, Mai Sun, Nathan A. Yates, Yuan Chang, Patrick S. Moore

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Abstract

Infections caused by multi-drug resistant Staphylococcus aureus, especially MRSA, are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals, but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared to today’s antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of reactive oxygen species by an appropriate photosensitizer. Here we conjugated the near-infrared photosensitizer IRDye700DX to a fully human monoclonal antibody, specific for the invariantly expressed staphylococcal antigen IsaA. The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human post-mortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the non-toxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.

Authors

Mafalda Bispo, Andrea Anaya-Sanchez, Sabrina Suhani, Elisa J.M. Raineri, Marina López-Álvarez, Marjolein Heuker, Wiktor Szymański, Francisco Romero Pastrana, Girbe Buist, Alexander R. Horswill, Kevin P. Francis, Gooitzen M. van Dam, Marleen van Oosten, Jan Maarten van Dijl

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Abstract

Intratumoral immune infiltrate was recently reported in Gastrointestinal Stromal Tumors (GIST). However, what tumor-intrinsic factors dictate GIST immunogenicity is still largely undefined. To shed light on this issue a large cohort (82 samples) of primary untreated GIST, representative of major clinicopathological variables, was investigated by an integrated immunohistochemical, transcriptomic and computational approach. Our results indicate that tumor genotype, location and malignant potential concur to shape the immunogenicity of primary naïve GIST. Immune infiltration was greater in overt GIST than in lesions with limited malignant potential (miniGIST), in KIT/PDGFRA mutated than in KIT/PDGFRA wild-type tumors and in PDGFRA versus KIT mutated GIST. Within the KIT mutated subset, a higher degree of immune colonization was detected in the intestine. Immune hot tumors showed expression patterns compatible with a potentially proficient but curbed antigen-specific immunity, hinting at sensitivity to immunomodulatory treatments. Poorly infiltrated GIST, primarily KIT/PDGFRA wild-type intestinal tumors, showed activation of Hedgehog and WNT/β-catenin immune excluding pathways. This finding discloses a potential therapeutic vulnerability, as the targeting of these pathways might prove effective by both inhibiting pro-oncogenic signals and fostering anti-tumor immune responses. Finally, an intriguing anticorrelation between immune infiltration and ANO1/DOG1 expression was observed, suggesting an immunomodulatory activity for anoctamin-1.

Authors

Daniela Gasparotto, Marta Sbaraglia, Sabrina Rossi, Davide Baldazzi, Monica Brenca, Alessia Mondello, Federica Nardi, Dominga Racanelli, Matilde Cacciatore, Angelo Paolo Dei Tos, Roberta Maestro

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