On the cover: Adipose triglyceride lipase-mediated lipid catabolism is essential for bronchiolar regeneration
Kanti et al. report that adipose triglyceride lipase, the rate-limiting enzyme for intracellular lipolysis, is critical for club cell–driven regeneration of bronchiolar epithelia in mice. In the cover image, an electron micrograph of an Atgl-KO/cTg club cell shows substantial lipid accumulation within the intracellular lipid droplets.
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Research Articles
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Abstract
Uveal melanoma (UM) is a unique disease in that patients with primary UM are well stratified based on their risk of developing metastasis, yet there are limited effective treatments once metastases occur. There is an urgent need to better understand the distinct molecular pathogenesis of UM and the characteristics of patients at high risk for metastasis to identify neoantigenic targets that can be used in immunotherapy and to develop novel therapeutic strategies that may effectively target this lethal transition. An important and overlooked area of molecular pathogenesis and neoantigenic targets in UM comes from human endogenous retroviruses (HERVs). We investigated the HERV expression landscape in primary UM and found that tumors were stratified into 4 HERV-based subsets that provide clear delineation of risk outcome and support subtypes identified by other molecular indicators. Specific HERV loci are associated with the risk of uveal melanoma metastasis and may offer mechanistic insights into this process, including dysregulation of HERVs on chromosomes 3 and 8. A HERV signature composed of 17 loci was sufficient to classify tumors according to subtype with greater than 95% accuracy, including at least 1 intergenic HERV with coding potential (HERVE_Xp11.23) that could represent a potential HERV E target for immunotherapy.
Authors
Matthew L. Bendall, Jasmine H. Francis, Alexander N. Shoushtari, Douglas F. Nixon
×Abstract
Atrial natriuretic peptide (ANP), encoded by Nppa, is a vasodilatory hormone that promotes salt excretion. Genome-wide association studies identified Nppa as a causative factor of blood pressure development, and in humans, ANP levels were suggested as an indicator of salt sensitivity. This study aimed to provide insights into the effects of ANP on cardiorenal function in salt-sensitive hypertension. To address this question, hypertension was induced in SSNPPA–/– (KO of Nppa in the Dahl salt-sensitive [SS] rat background) or SSWT (WT Dahl SS) rats by a high-salt (HS) diet challenge (4% NaCl for 21 days). Chronic infusion of ANP in SSWT rats attenuated the increase in blood pressure and cardiorenal damage. Overall, the SSNPPA–/– strain demonstrated higher blood pressure and intensified cardiac fibrosis (with no changes in ejection fraction) compared with SSWT rats. Furthermore, SSNPPA–/– rats exhibited kidney hypertrophy and higher glomerular injury scores, reduced diuresis, and lower sodium and chloride excretion than SSWT when fed a HS diet. Additionally, the activity of epithelial Na+ channel (ENaC) was found to be increased in the collecting ducts of the SSNPPA–/– rats. Taken together, these data show promise for the therapeutic benefits of ANP and ANP-increasing drugs for treating salt-sensitive hypertension.
Authors
Daria V. Ilatovskaya, Vladislav Levchenko, Kristen Winsor, Gregory R. Blass, Denisha R. Spires, Elizaveta Sarsenova, Iuliia Polina, Adrian Zietara, Mark Paterson, Alison J. Kriegel, Alexander Staruschenko
×Abstract
The lung airways are constantly exposed to inhaled toxic substances, resulting in cellular damage that is repaired by local expansion of resident bronchiolar epithelial club cells. Disturbed bronchiolar epithelial damage repair lies at the core of many prevalent lung diseases, including chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, and lung cancer. However, it is still not known how bronchiolar club cell energy metabolism contributes to this process. Here, we show that adipose triglyceride lipase (ATGL), the rate-limiting enzyme for intracellular lipolysis, is critical for normal club cell function in mice. Deletion of the gene encoding ATGL, Pnpla2 (also known as Atgl), induced substantial triglyceride accumulation, decreased mitochondrial numbers, and decreased mitochondrial respiration in club cells. This defect manifested as bronchiolar epithelial thickening and increased airway resistance under baseline conditions. After naphthalene‑induced epithelial denudation, a regenerative defect was apparent. Mechanistically, dysfunctional PPARα lipid-signaling underlies this phenotype because (a) ATGL was needed for PPARα lipid-signaling in regenerating bronchioles and (b) administration of the specific PPARα agonist WY14643 restored normal bronchiolar club cell ultrastructure and regenerative potential. Our data emphasize the importance of the cellular energy metabolism for lung epithelial regeneration and highlight the significance of ATGL-mediated lipid catabolism for lung health.
Authors
Manu Manjunath Kanti, Isabelle Striessnig-Bina, Beatrix Irene Wieser, Silvia Schauer, Gerd Leitinger, Thomas O. Eichmann, Martina Schweiger, Margit Winkler, Elke Winter, Andrea Lana, Iris Kufferath, Leigh Matthew Marsh, Grazyna Kwapiszewska, Rudolf Zechner, Gerald Hoefler, Paul Willibald Vesely
×Abstract
Binding of the bromodomain and extraterminal domain proteins (BETs) to acetylated histone residues is critical for gene transcription. We sought to determine the antifibrotic efficacy and potential mechanisms of BET inhibition in systemic sclerosis (SSc). Blockade of BETs was done using a pan-BET inhibitor, JQ1; BRD2 inhibitor, BIC1; or BRD4 inhibitors AZD5153 or ARV825. BET inhibition, specifically BRD4 blockade, showed antifibrotic effects in an animal model of SSc and in patient-derived diffuse cutaneous SSc (dcSSc) fibroblasts. Transcriptome analysis of JQ1-treated dcSSc fibroblasts revealed differentially expressed genes related to extracellular matrix, cell cycle, and calcium (Ca2+) signaling. The antifibrotic effect of BRD4 inhibition was mediated at least in part by downregulation of Ca2+/calmodulin–dependent protein kinase II α and reduction of intracellular Ca2+ concentrations. On the basis of these results, we propose targeting Ca2+ pathways or BRD4 as potentially novel therapeutic approaches for progressive tissue fibrosis.
Authors
Sirapa Vichaikul, Mikel Gurrea-Rubio, M. Asif Amin, Phillip L. Campbell, Qi Wu, Megan N. Mattichak, William D. Brodie, Pamela J. Palisoc, Mustafa Ali, Sei Muraoka, Jeffrey H. Ruth, Ellen N. Model, Dallas M. Rohraff, Jonatan L. Hervoso, Yang Mao-Draayer, David A. Fox, Dinesh Khanna, Amr H. Sawalha, Pei-Suen Tsou
×Abstract
Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.
Authors
Sydney Felker, Archana Shrestha, Jeff Bailey, Devin M Pillis, Dylan Siniard, Punam Malik
×Abstract
Greater than 25% of all men develop an inguinal hernia in their lifetime, and more than 20 million inguinal hernia repair surgeries are performed worldwide each year. The mechanisms causing abdominal muscle weakness, the formation of inguinal hernias, or their recurrence are largely unknown. We previously reported that excessively produced estrogen in the lower abdominal muscles (LAMs) triggers extensive LAM fibrosis, leading to hernia formation in a transgenic male mouse model expressing the human aromatase gene (Aromhum). To understand the cellular basis of estrogen-driven muscle fibrosis, we performed single-cell RNA sequencing on LAM tissue from Aromhum and wild-type littermates. We found a fibroblast-like cell group composed of 6 clusters, 2 of which were validated for their enrichment in Aromhum LAM tissue. One of the potentially novel hernia-associated fibroblast clusters in Aromhum was enriched for the estrogen receptor-α gene (Esr1hi). Esr1hi fibroblasts maximally expressed estrogen target genes and seemed to serve as the progenitors of another cluster expressing ECM-altering enzymes (Mmp3hi) and to upregulate expression of proinflammatory, profibrotic genes. The discovery of these 2 potentially novel and unique hernia-associated fibroblasts may lead to the development of novel treatments that can nonsurgically prevent or reverse inguinal hernias.
Authors
Tanvi Potluri, Matthew J. Taylor, Jonah J. Stulberg, Richard L. Lieber, Hong Zhao, Serdar E. Bulun
×Abstract
Arginine methylation mediated by protein arginine methyltransferases (PRMTs) has been shown to be an important posttranslational mechanism involved in various biological processes. Herein, we sought to investigate whether PRMT5, a major type II enzyme, is involved in pathological angiogenesis and, if so, to elucidate the molecular mechanism involved. Our results show that PRMT5 expression is significantly upregulated in ischemic tissues and hypoxic endothelial cells (ECs). Endothelial-specific Prmt5-KO mice were generated to define the role of PRMT5 in hindlimb ischemia–induced angiogenesis. We found that these mice exhibited impaired recovery of blood perfusion and motor function of the lower limbs, an impairment that was accompanied by decreased vascular density and increased necrosis as compared with their WT littermates. Furthermore, both pharmacological and genetic inhibition of PRMT5 significantly attenuated EC proliferation, migration, tube formation, and aortic ring sprouting. Mechanistically, we showed that inhibition of PRMT5 markedly attenuated hypoxia-induced factor 1-α (HIF-1α) protein stability and vascular endothelial growth factor–induced (VEGF-induced) signaling pathways in ECs. Our results provide compelling evidence demonstrating a crucial role of PRMT5 in hypoxia-induced angiogenesis and suggest that inhibition of PRMT5 may provide novel therapeutic strategies for the treatment of abnormal angiogenesis-related diseases, such as cancer and diabetic retinopathy.
Authors
Qing Ye, Jian Zhang, Chen Zhang, Bing Yi, Kyosuke Kazama, Wennan Liu, Xiaobo Sun, Yan Liu, Jianxin Sun
×Abstract
To elicit effective antitumor responses, CD8+ T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment (TME). Here, we evaluate the role of MNK activity in regulating CD8+ T cell infiltration and antitumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK activity are associated with decreased infiltration by CD8+ T cells. We then show that, while MNK inhibitors increase CD8+ T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of programmed cell death ligand 1 (PD-L1) but is caused by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8+ T cell exhaustion by an anti–PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a role of MNKs modulating a protumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance antitumor immune responses.
Authors
Thao N.D. Pham, Christina Spaulding, Mario A. Shields, Anastasia E. Metropulos, Dhavan N. Shah, Mahmoud G. Khalafalla, Daniel R. Principe, David J. Bentrem, Hidayatullah G. Munshi
×Abstract
Wound repair following acute injury requires a coordinated inflammatory response. Type I IFN signaling is important for regulating the inflammatory response after skin injury. IFN-κ, a type I IFN, has recently been found to drive skin inflammation in lupus and psoriasis; however, the role of IFN-κ in the context of normal or dysregulated wound healing is unclear. Here, we show that Ifnk expression is upregulated in keratinocytes early after injury and is essential for normal tissue repair. Under diabetic conditions, IFN-κ was decreased in wound keratinocytes, and early inflammation was impaired. Furthermore, we found that the histone methyltransferase mixed-lineage leukemia 1 (MLL1) is upregulated early following injury and regulates Ifnk expression in diabetic wound keratinocytes via an H3K4me3-mediated mechanism. Using a series of in vivo studies with a geneticall y engineered mouse model (Mll1fl/fl K14cre–) and human wound tissues from patients with T2D, we demonstrate that MLL1 controls wound keratinocyte–mediated Ifnk expression and that Mll1 expression is decreased in T2D keratinocytes. Importantly, we found the administration of IFN-κ early following injury improves diabetic tissue repair through increasing early inflammation, collagen deposition, and reepithelialization. These findings have significant implications for understanding the complex role type I IFNs play in keratinocytes in normal and diabetic wound healing. Additionally, they suggest that IFN may be a viable therapeutic target to improve diabetic wound repair.
Authors
Sonya J. Wolf, Christopher O. Audu, Amrita Joshi, Aaron denDekker, William J. Melvin, Frank M. Davis, Xianying Xing, Rachael Wasikowski, Lam C. Tsoi, Steven L. Kunkel, Johann E. Gudjonsson, Mary X. O’Riordan, J. Michelle Kahlenberg, Katherine A. Gallagher
×Abstract
The bromodomain and extraterminal (BET) family of chromatin reader proteins bind to acetylated histones and regulate gene expression. The development of BET inhibitors (BETi) has expanded our knowledge of BET protein function beyond transcriptional regulation and has ushered several prostate cancer (PCa) clinical trials. However, BETi as a single agent is not associated with antitumor activity in patients with castration-resistant prostate cancer (CRPC). We hypothesized novel combinatorial strategies are likely to enhance the efficacy of BETi. By using PCa patient-derived explants and xenograft models, we show that BETi treatment enhanced the efficacy of radiation therapy (RT) and overcame radioresistance. Mechanistically, BETi potentiated the activity of RT by blocking DNA repair. We also report a synergistic relationship between BETi and topoisomerase I (TOP1) inhibitors (TOP1i). We show that the BETi OTX015 synergized with the new class of synthetic noncamptothecin TOP1i, LMP400 (indotecan), to block tumor growth in aggressive CRPC xenograft models. Mechanistically, BETi potentiated the antitumor activity of TOP1i by disrupting replication fork stability. Longitudinal analysis of patient tumors indicated that TOP1 transcript abundance increased as patients progressed from hormone-sensitive prostate cancer to CRPC. TOP1 was highly expressed in metastatic CRPC, and its expression correlated with the expression of BET family genes. These studies open new avenues for the rational combinatorial treatment of aggressive PCa.
Authors
Xiangyi Li, GuemHee Baek, Suzanne Carreira, Wei Yuan, Shihong Ma, Mia Hofstad, Sora Lee, Yunpeng Gao, Claudia Bertan, Maria de los Dolores Fenor de la Maza, Prasanna G. Alluri, Sandeep Burma, Benjamin P.C. Chen, Ganesh V. Raj, Johann de Bono, Yves Pommier, Ram S. Mani
×Abstract
Hematopoietic protein-1 (Hem-1) is a member of the actin-regulatory WASp family verprolin homolog (WAVE) complex. Loss-of-function variants in the NCKAP1L gene encoding Hem-1 were recently discovered to result in primary immunodeficiency disease (PID) in children, characterized by poor specific Ab responses, increased autoantibodies, and high mortality. However, the mechanisms of how Hem-1 deficiency results in PID are unclear. In this study, we utilized constitutive and B cell–specific Nckap1l-KO mice to dissect the importance of Hem-1 in B cell development and functions. B cell–specific disruption of Hem-1 resulted in reduced numbers of recirculating follicular (FO), marginal zone (MZ), and B1 B cells. B cell migration in response to CXCL12 and -13 were reduced. T-independent Ab responses were nearly abolished, resulting in failed protective immunity to Streptococcus pneumoniae challenge. In contrast, T-dependent IgM and IgG2c, memory B cell, and plasma cell responses were more robust relative to WT control mice. B cell–specific Hem-1–deficient mice had increased autoantibodies against multiple autoantigens, and this correlated with hyperresponsive BCR signaling and increased representation of CD11c+T-bet+ age-associated B cell (ABC cells) — alterations associated with autoimmune diseases. These results suggest that dysfunctional B cells may be part of a mechanism explaining why loss-of-function Hem-1 variants result in recurring infections and autoimmunity.
Authors
Alan Avalos, Jacob T. Tietsort, Nutthakarn Suwankitwat, Jonathan D. Woods, Shaun W. Jackson, Alexandra Christodoulou, Christopher Morrill, H. Denny Liggitt, Chengsong Zhu, Quan-Zhen Li, Kevin K. Bui, Heon Park, Brian M. Iritani
×Abstract
Severe COVID-19 disease is associated with dysregulation of the myeloid compartment during acute infection. Survivors frequently experience long-lasting sequelae, but little is known about the eventual persistence of this immune alteration. Herein, we evaluated TLR-induced cytokine responses in a cohort of mild to critical patients during acute or convalescent phases (n = 97). In the acute phase, we observed impaired cytokine production by monocytes in the patients with the most severe COVID-19. This capacity was globally restored in convalescent patients. However, we observed increased responsiveness to TLR1/2 ligation in patients who recovered from severe disease, indicating that these cells display distinct functional properties at the different stages of the disease. In patients with acute severe COVID-19, we identified a specific transcriptomic and epigenomic state in monocytes that can account for their functional refractoriness. The molecular profile of monocytes from recovering patients was distinct and characterized by increased chromatin accessibility at activating protein 1 (AP1) and MAF loci. These results demonstrate that severe COVID-19 infection has a profound impact on the differentiation status and function of circulating monocytes, during both the acute and the convalescent phases, in a completely distinct manner. This could have important implications for our understanding of short- and long-term COVID-19–related morbidity.
Authors
Elisa Brauns, Abdulkader Azouz, David Grimaldi, Hanxi Xiao, Séverine Thomas, Muriel Nguyen, Véronique Olislagers, Ines Vu Duc, Carmen Orte Cano, Véronique Del Marmol, Pieter Pannus, Frédérick Libert, Sven Saussez, Nicolas Dauby, Jishnu Das, Arnaud Marchant, Stanislas Goriely
×Abstract
Friedreich’s ataxia (FRDA) is an inherited disorder caused by reduced levels of frataxin (FXN), which is required for iron-sulfur cluster biogenesis. Neurological and cardiac comorbidities are prominent and have been a major focus of study. Skeletal muscle has received less attention despite indications that FXN loss affects it. Here, we show that lean mass is lower, whereas body mass index is unaltered, in separate cohorts of adults and children with FRDA. In adults, lower lean mass correlated with disease severity. To further investigate FXN loss in skeletal muscle, we used a transgenic mouse model of whole-body inducible and progressive FXN depletion. There was little impact of FXN loss when FXN was approximately 20% of control levels. When residual FXN was approximately 5% of control levels, muscle mass was lower along with absolute grip strength. When we examined mechanisms that can affect muscle mass, only global protein translation was lower, accompanied by integrated stress response (ISR) activation. Also in mice, aerobic exercise training, initiated prior to the muscle mass difference, improved running capacity, yet, muscle mass and the ISR remained as in untrained mice. Thus, FXN loss can lead to lower lean mass, with ISR activation, both of which are insensitive to exercise training.
Authors
César Vásquez-Trincado, Julia Dunn, Ji In Han, Briyanna Hymms, Jaclyn Tamaroff, Monika Patel, Sara Nguyen, Anna Dedio, Kristin Wade, Chinazo Enigwe, Zuzana Nichtova, David R. Lynch, Gyorgy Csordas, Shana E. McCormack, Erin L. Seifert
×Abstract
Patients with hereditary hemorrhagic telangiectasia (HHT) have arteriovenous malformations (AVMs) with genetic mutations involving the activin-A receptor like type 1 (ACVRL1 or ALK1) and endoglin (ENG). Recent studies have shown that Neuropilin-1 (NRP-1) inhibits ALK1. We investigated the expression of NRP-1 in livers of patients with HHT and found that there was a significant reduction in NRP-1 in perivascular smooth muscle cells (SMCs). We used Nrp1SM22KO mice (Nrp1 was ablated in SMCs) and found hemorrhage, increased immune cell infiltration with a decrease in SMCs, and pericyte lining in lungs and liver in adult mice. Histologic examination revealed lung arteriovenous fistulas (AVFs) with enlarged liver vessels. Evaluation of the retina vessels at P5 from Nrp1SM22KO mice demonstrated dilated capillaries with a reduction of pericytes. In inflow artery of surgical AVFs from the Nrp1SM22KO versus WT mice, there was a significant decrease in Tgfb1, Eng, and Alk1 expression and phosphorylated SMAD1/5/8 (pSMAD1/5/8), with an increase in apoptosis. TGF-β1–stimulated aortic SMCs from Nrp1SM22KO versus WT mice have decreased pSMAD1/5/8 and increased apoptosis. Coimmunoprecipitation experiments revealed that NRP-1 interacts with ALK1 and ENG in SMCs. In summary, NRP-1 deletion in SMCs leads to reduced ALK1, ENG, and pSMAD1/5/8 signaling and reduced cell death associated with AVM formation.
Authors
Sreenivasulu Kilari, Ying Wang, Avishek Singh, Rondell P. Graham, Vivek Iyer, Scott M. Thompson, Michael S. Torbenson, Debabrata Mukhopadhyay, Sanjay Misra
×Abstract
It is currently thought that UVB radiation drives photoaging of the skin primarily by generating ROS. In this model, ROS purportedly activates activator protein-1 to upregulate MMPs 1, 3, and 9, which then degrade collagen and other extracellular matrix components to produce wrinkles. However, these MMPs are expressed at relatively low levels and correlate poorly with wrinkles, suggesting that another mechanism distinct from ROS and MMP1/3/9 may be more directly associated with photoaging. Here we show that MMP2, which degrades type IV collagen, is abundantly expressed in human skin, increases with age in sun-exposed skin, and correlates robustly with aryl hydrocarbon receptor (AhR), a transcription factor directly activated by UV-generated photometabolites. Through mechanistic studies with HaCaT human immortalized keratinocytes, we found that AhR, specificity protein 1 (SP1), and other pathways associated with DNA damage are required for the induction of both MMP2 and MMP11 (another MMP implicated in photoaging), but not MMP1/3. Last, we found that topical treatment with AhR antagonists vitamin B12 and folic acid ameliorated UVB-induced wrinkle formation in mice while dampening MMP2 expression in the skin. These results directly implicate DNA damage in photoaging and reveal AhR as a potential target for preventing wrinkles.
Authors
Daniel J. Kim, Akiko Iwasaki, Anna L. Chien, Sewon Kang
×Abstract
BACKGROUND Potent synthetic opioids, such as fentanyl, are increasingly abused, resulting in unprecedented numbers of fatalities from respiratory depression. Treatment with the high-affinity mu-opioid receptor partial agonist buprenorphine may prevent fatalities by reducing binding of potent opioids to the opioid receptor, limiting respiratory depression.METHODS To characterize buprenorphine-fentanyl interaction at the level of the mu-opioid receptor in 2 populations (opioid-naive individuals and individuals who chronically use high-dose opioids), the effects of escalating i.v. fentanyl doses with range 0.075–0.35 mg/70 kg (opioid naive) and 0.25–0.70 mg/70 kg (chronic opioid use) on iso-hypercapnic ventilation at 2–3 background doses of buprenorphine (target plasma concentrations range: 0.2–5 ng/mL) were quantified using receptor association/dissociation models combined with biophase distribution models.RESULTS Buprenorphine produced mild respiratory depression, while high doses of fentanyl caused pronounced respiratory depression and apnea in both populations. When combined with fentanyl, buprenorphine produced a receptor binding–dependent reduction of fentanyl-induced respiratory depression in both populations. In individuals with chronic opioid use, at buprenorphine plasma concentrations of 2 ng/mL or higher, a protective effect against high-dose fentanyl was observed.CONCLUSION Overall, the results indicate that when buprenorphine mu-opioid receptor occupancy is sufficiently high, fentanyl is unable to activate the mu-opioid receptor and consequently will not cause further respiratory depression in addition to the mild respiratory effects of buprenorphine.TRIAL REGISTRATION Trialregister.nl, no. NL7028 (https://www.trialregister.nl/trial/7028)FUNDING Indivior Inc., North Chesterfield, Virginia, USA.
Authors
Erik Olofsen, Marijke Hyke Algera, Laurence Moss, Robert L. Dobbins, Geert J. Groeneveld, Monique van Velzen, Marieke Niesters, Albert Dahan, Celine M. Laffont
×Abstract
Long-term impairment in T cell–mediated adaptive immunity is a major clinical obstacle following treatment of blood disorders with hematopoietic stem cell transplantation. Although T cell development in the thymus has been extensively characterized, there are significant gaps in our understanding of prethymic processes that influence early T cell potential. We have uncovered a Notch/IL-21 signaling axis in bone marrow common lymphoid progenitor (CLP) cells. IL-21 receptor expression was driven by Notch activation in CLPs, and in vivo treatment with IL-21 induced Notch-dependent CLP proliferation. Taking advantage of this potentially novel signaling axis, we generated T cell progenitors ex vivo, which improved repopulation of the thymus and peripheral lymphoid organs of mice in an allogeneic transplant model. Importantly, Notch and IL-21 activation were equally effective in the priming and expansion of human cord blood cells toward the T cell fate, confirming the translational potential of the combined treatment.
Authors
Kilian Sottoriva, Na Yoon Paik, Zachary White, Thilinie Bandara, Lijian Shao, Teruyuki Sano, Kostandin V. Pajcini
×Abstract
Background The value of the soluble receptor for advanced glycation end-products (sRAGE) as a biomarker in COVID-19 is not well understood. We tested the association between plasma sRAGE and illness severity, viral burden, and clinical outcomes in hospitalized patients with COVID-19 who were not mechanically ventilated.Methods Baseline sRAGE was measured among participants enrolled in the ACTIV-3/TICO trial of bamlanivimab for hospitalized patients with COVID-19. Spearman’s rank correlation was used to assess the relationship between sRAGE and other plasma biomarkers, including viral nucleocapsid antigen. Fine-Gray models adjusted for baseline supplemental oxygen requirement, antigen level, positive endogenous anti-nucleocapsid antibody response, sex, age, BMI, diabetes mellitus, renal impairment, corticosteroid treatment, and log2-transformed IL-6 level were used to assess the association between baseline sRAGE and time to sustained recovery. Cox regression adjusted for the same factors was used to assess the association between sRAGE and mortality.Results Among 277 participants, baseline sRAGE was strongly correlated with viral plasma antigen concentration (ρ = 0.57). There was a weaker correlation between sRAGE and biomarkers of systemic inflammation, such as IL-6 (ρ = 0.36) and CRP (ρ = 0.20). Participants with plasma sRAGE in the highest quartile had a significantly lower rate of sustained recovery (adjusted recovery rate ratio, 0.64 [95% CI, 0.43–0.90]) and a higher unadjusted risk of death (HR, 4.70 [95% CI, 2.01–10.99]) compared with participants in the lower quartiles.Conclusion Elevated plasma sRAGE in hospitalized, nonventilated patients with COVID-19 was an indicator of both clinical illness severity and plasma viral load. Plasma sRAGE in the highest quartile was associated with a lower likelihood of sustained recovery and higher unadjusted risk of death. These findings, which we believe to be novel, indicate that plasma sRAGE may be a promising biomarker for COVID-19 prognostication and clinical trial enrichment.Trial Registration ClinicalTrials.gov NCT04501978.Funding NIH (5T32GM008440-24, 18X107CF6, HHSN261201500003I, R35HL140026, and OT2HL156812).
Authors
Katherine D. Wick, Lianne Siegel, James D. Neaton, Cathryn Oldmixon, Jens Lundgren, Robin L. Dewar, H. Clifford Lane, B. Taylor Thompson, Michael A. Matthay, on behalf of the ACTIV-3/TICO study group
×Abstract
BACKGROUND Immune cell profiling of primary and metastatic CNS tumors has been focused on the tumor, not the tumor microenvironment (TME), or has been analyzed via biopsies.METHODS En bloc resections of gliomas (n = 10) and lung metastases (n = 10) were analyzed via tissue segmentation and high-dimension Opal 7-color multiplex imaging. Single-cell RNA analyses were used to infer immune cell functionality.RESULTS Within gliomas, T cells were localized in the infiltrating edge and perivascular space of tumors, while residing mostly in the stroma of metastatic tumors. CD163+ macrophages were evident throughout the TME of metastatic tumors, whereas in gliomas, CD68+, CD11c+CD68+, and CD11c+CD68+CD163+ cell subtypes were commonly observed. In lung metastases, T cells interacted with CD163+ macrophages as dyads and clusters at the brain-tumor interface and within the tumor itself and as clusters within the necrotic core. In contrast, gliomas typically lacked dyad and cluster interactions, except for T cell CD68+ cell dyads within the tumor. Analysis of transcriptomic data in glioblastomas revealed that innate immune cells expressed both proinflammatory and immunosuppressive gene signatures.CONCLUSION Our results show that immunosuppressive macrophages are abundant within the TME and that the immune cell interactome between cancer lineages is distinct. Further, these data provide information for evaluating the role of different immune cell populations in brain tumor growth and therapeutic responses.FUNDING This study was supported by the NIH (NS120547), a Developmental research project award (P50CA221747), ReMission Alliance, institutional funding from Northwestern University and the Lurie Comprehensive Cancer Center, and gifts from the Mosky family and Perry McKay. Performed in the Flow Cytometry & Cellular Imaging Core Facility at MD Anderson Cancer Center, this study received support in part from the NIH (CA016672) and the National Cancer Institute (NCI) Research Specialist award 1 (R50 CA243707). Additional support was provided by CCSG Bioinformatics Shared Resource 5 (P30 CA046592), a gift from Agilent Technologies, a Research Scholar Grant from the American Cancer Society (RSG-16-005-01), a Precision Health Investigator Award from University of Michigan (U-M) Precision Health, the NCI (R37-CA214955), startup institutional research funds from U-M, and a Biomedical Informatics & Data Science Training Grant (T32GM141746).
Authors
Hinda Najem, Martina Ott, Cynthia Kassab, Arvind Rao, Ganesh Rao, Anantha Marisetty, Adam M. Sonabend, Craig Horbinski, Roel Verhaak, Anand Shankar, Santhoshi N. Krishnan, Frederick S. Varn, Víctor A. Arrieta, Pravesh Gupta, Sherise D. Ferguson, Jason T. Huse, Gregory N. Fuller, James P. Long, Daniel E. Winkowski, Ben A. Freiberg, Charles David James, Leonidas C. Platanias, Maciej S. Lesniak, Jared K. Burks, Amy B. Heimberger
×Abstract
Post-exertional malaise (PEM) is a hallmark symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). We monitored the evolution of 1157 plasma metabolites in 60 ME/CFS (45 female, 15 male) and 45 matched healthy control participants (30 female, 15 male) before and after 2 maximal cardiopulmonary exercise test (CPET) challenges separated by 24 hours, with the intent of provoking PEM in patients. Four time points allowed exploration of the metabolic response to maximal energy-producing capacity and the recovery pattern of participants with ME/CFS compared with the healthy control group. Baseline comparison identified several significantly different metabolites, along with an enriched percentage of yet-to-be identified compounds. Additionally, temporal measures demonstrated an increased metabolic disparity between cohorts, including unknown metabolites. The effects of exertion in the ME/CFS cohort predominantly highlighted lipid-related as well as energy-related pathways and chemical structure clusters, which were disparately affected by the first and second exercise sessions. The 24-hour recovery period was distinct in the ME/CFS cohort, with over a quarter of the identified pathways statistically different from the controls. The pathways that are uniquely different 24 hours after an exercise challenge provide clues to metabolic disruptions that lead to PEM. Numerous altered pathways were observed to depend on glutamate metabolism, a crucial component of the homeostasis of many organs in the body, including the brain.
Authors
Arnaud Germain, Ludovic Giloteaux, Geoffrey E. Moore, Susan M. Levine, John K. Chia, Betsy A. Keller, Jared Stevens, Carl J. Franconi, Xiangling Mao, Dikoma C. Shungu, Andrew Grimson, Maureen R. Hanson
×Immunogenic epitope panel for accurate detection of non-cross-reactive T cell response to SARS-CoV-2
Abstract
The ongoing COVID-19 pandemic calls for more effective diagnostic tools. T cell response assessment serves as an independent indicator of prior COVID-19 exposure while also contributing to a more comprehensive characterization of SARS-CoV-2 immunity. In this study, we systematically assessed the immunogenicity of 118 epitopes with immune cells collected from multiple cohorts of vaccinated, convalescent, healthy unexposed, and SARS-CoV-2–exposed donors. We identified 75 immunogenic epitopes, 24 of which were immunodominant. We further confirmed HLA restriction for 49 epitopes and described association with more than 1 HLA allele for 14 of these. Exclusion of 2 cross-reactive epitopes that generated a response in prepandemic samples left us with a 73-epitope set that offered excellent diagnostic specificity without losing sensitivity compared with full-length antigens, and this evoked a robust cross-reactive response. We subsequently incorporated this set of epitopes into an in vitro diagnostic Corona-T-test, which achieved a diagnostic accuracy of 95% in a clinical trial. In a cohort of asymptomatic seronegative individuals with a history of prolonged SARS-CoV-2 exposure, we observed a complete absence of T cell response to our epitope panel. In combination with strong reactivity to full-length antigens, this suggests that a cross-reactive response might protect these individuals.
Authors
Aleksei Titov, Regina Shaykhutdinova, Olga V. Shcherbakova, Yana V. Serdyuk, Savely A. Sheetikov, Ksenia V. Zornikova, Alexandra V. Maleeva, Alexandra Khmelevskaya, Dmitry V. Dianov, Naina T. Shakirova, Dmitry B. Malko, Maxim Shkurnikov, Stepan Nersisyan, Alexander Tonevitsky, Ekaterina Khamaganova, Anton V. Ershov, Elena Y. Osipova, Ruslan V. Nikolaev, Dmitry E. Pershin, Viktoria A. Vedmedskia, Michael Maschan, Victoria R. Ginanova, Grigory A. Efimov
×Abstract
Transplant recipients exhibit an impaired protective immunity after SARS-CoV-2 vaccination, potentially caused by mycophenolate (MPA) immunosuppression. Recent data from patients with autoimmune disorders suggest that temporary MPA hold might greatly improve booster vaccination outcomes. We applied a fourth dose of SARS-CoV-2 vaccine to 29 kidney transplant recipients during a temporary (5 weeks) MPA/azathioprine hold, who had not mounted a humoral immune response to previous vaccinations. Seroconversion until day 32 after vaccination was observed in 76% of patients, associated with acquisition of virus-neutralizing capacity. Interestingly, 21/25 (84%) calcineurin inhibitor–treated patients responded, but only 1/4 belatacept-treated patients responded. In line with humoral responses, counts and relative frequencies of spike receptor binding domain–specific (RBD-specific) B cells were markedly increased on day 7 after vaccination, with an increase in RBD-specific CD27++CD38+ plasmablasts. Whereas overall proportions of spike-reactive CD4+ T cells remained unaltered after the fourth dose, frequencies were positively correlated with specific IgG levels. Importantly, antigen-specific proliferating Ki67+ and in vivo–activated programmed cell death 1–positive T cells significantly increased after revaccination during MPA hold, whereas cytokine production and memory differentiation remained unaffected. In summary, antimetabolite hold augmented all arms of immunity during booster vaccination. These data suggest further studies of antimetabolite hold in kidney transplant recipients.
Authors
Eva Schrezenmeier, Hector Rincon-Arevalo, Annika Jens, Ana-Luisa Stefanski, Charlotte Hammett, Bilgin Osmanodja, Nadine Koch, Bianca Zukunft, Julia Beck, Michael Oellerich, Vanessa Proß, Carolin Stahl, Mira Choi, Friederike Bachmann, Lutz Liefeldt, Petra Glander, Ekkehard Schütz, Kirsten Bornemann-Kolatzki, Covadonga López del Moral, Hubert Schrezenmeier, Carolin Ludwig, Bernd Jahrsdörfer, Kai-Uwe Eckardt, Nils Lachmann, Katja Kotsch, Thomas Dörner, Fabian Halleck, Arne Sattler, Klemens Budde
×Abstract
Bronchoalveolar lavage is commonly performed to assess inflammation and identify responsible pathogens in lung diseases. Findings from bronchoalveolar lavage might be used to evaluate the immune profile of the lung tumor microenvironment (TME). To investigate whether bronchoalveolar lavage fluid (BALF) analysis can help identify patients with non–small cell lung cancer (NSCLC) who respond to immune checkpoint inhibitors (ICIs), BALF and blood were prospectively collected before initiating nivolumab. The secreted molecules, microbiome, and cellular profiles based on BALF and blood analysis of 12 patients were compared with regard to therapeutic effect. Compared with ICI nonresponders, responders showed significantly higher CXCL9 levels and a greater diversity of the lung microbiome profile in BALF, along with a greater frequency of the CD56+ subset in blood T cells, whereas no significant difference in PD-L1 expression was found in tumor cells. Antibiotic treatment in a preclinical lung cancer model significantly decreased CXCL9 in the lung TME, resulting in reduced sensitivity to anti–PD-1 antibody, which was reversed by CXCL9 induction in tumor cells. Thus, CXCL9 might be associated with the lung TME microbiome, and the balance of CXCL9 and lung TME microbiome could contribute to nivolumab sensitivity in patients with NSCLC. BALF analysis can help predict the efficacy of ICIs when performed along with currently approved examinations.
Authors
Kentaro Masuhiro, Motohiro Tamiya, Kosuke Fujimoto, Shohei Koyama, Yujiro Naito, Akio Osa, Takashi Hirai, Hidekazu Suzuki, Norio Okamoto, Takayuki Shiroyama, Kazumi Nishino, Yuichi Adachi, Takuro Nii, Yumi Kinugasa-Katayama, Akiko Kajihara, Takayoshi Morita, Seiya Imoto, Satoshi Uematsu, Takuma Irie, Daisuke Okuzaki, Taiki Aoshi, Yoshito Takeda, Toru Kumagai, Tomonori Hirashima, Atsushi Kumanogoh
×Abstract
Arterial stiffness predicts cardiovascular disease and all-cause mortality, but its treatment remains challenging. Mice treated with angiotensin II (Ang II) develop hypertension, arterial stiffness, vascular dysfunction, and a downregulation of Rho-related BTB domain–containing protein 1 (RhoBTB1) in the vasculature. RhoBTB1 is associated with blood pressure regulation, but its function is poorly understood. We tested the hypothesis that restoring RhoBTB1 can attenuate arterial stiffness, hypertension, and vascular dysfunction in Ang II–treated mice. Genetic complementation of RhoBTB1 in the vasculature was achieved using mice expressing a tamoxifen-inducible, smooth muscle–specific RhoBTB1 transgene. RhoBTB1 restoration efficiently and rapidly alleviated arterial stiffness but not hypertension or vascular dysfunction. Mechanistic studies revealed that RhoBTB1 had no substantial effect on several classical arterial stiffness contributors, such as collagen deposition, elastin content, and vascular smooth muscle remodeling. Instead, Ang II increased actin polymerization in the aorta, which was reversed by RhoBTB1. Changes in the levels of 2 regulators of actin polymerization, cofilin and vasodilator-stimulated phosphoprotein, in response to RhoBTB1 were consistent with an actin depolymerization mechanism. Our study reveals an important function of RhoBTB1, demonstrates its vital role in antagonizing established arterial stiffness, and further supports a functional and mechanistic separation among hypertension, vascular dysfunction, and arterial stiffness.
Authors
Shi Fang, Jing Wu, John J. Reho, Ko-Ting Lu, Daniel T. Brozoski, Gaurav Kumar, Alec M. Werthman, Sebastiao Donato Silva Jr., Patricia C. Muskus Veitia, Kelsey K. Wackman, Angela J. Mathison, Bi Qing Teng, Chien-Wei Lin, Frederick W. Quelle, Curt D. Sigmund
×Abstract
Background Some clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.Methods In an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.Results Anti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38–42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6–21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.Conclusion We identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDING Bill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891)
Authors
Livia Casciola-Rosen, David R. Thiemann, Felipe Andrade, Maria I. Trejo-Zambrano, Elissa K. Leonard, Jamie B. Spangler, Nicole E. Skinner, Justin Bailey, Srinivasan Yegnasubramanian, Rulin Wang, Ajay M. Vaghasia, Anuj Gupta, Andrea L. Cox, Stuart C. Ray, Raleigh M. Linville, Zhaobin Guo, Peter C. Searson, Carolyn E. Machamer, Stephen Desiderio, Lauren M. Sauer, Oliver Laeyendecker, Brian T. Garibaldi, Li Gao, Mahendra Damarla, Paul M. Hassoun, Jody E. Hooper, Christopher A. Mecoli, Lisa Christopher-Stine, Laura Gutierrez-Alamillo, Qingyuan Yang, David Hines, William A. Clarke, Richard E. Rothman, Andrew Pekosz, Katherine Z.J. Fenstermacher, Zitong Wang, Scott L. Zeger, Antony Rosen
×Abstract
Identifying predictive biomarkers at early stages of inflammatory arthritis is crucial for starting appropriate therapies to avoid poor outcomes. Monocytes (MOs) and macrophages, largely associated with arthritis, are contributors and sensors of inflammation through epigenetic modifications. In this study, we investigated associations between clinical features and DNA methylation in blood and synovial fluid (SF) MOs in a prospective cohort of patients with early inflammatory arthritis. DNA methylation profiles of undifferentiated arthritis (UA) blood MOs exhibited marked alterations in comparison with those from healthy donors. We identified additional differences both in blood and SF MOs after comparing patients with UA grouped by their future outcomes, i.e., good versus poor. Patient profiles in subsequent visits revealed a reversion toward a healthy level in both groups, those requiring disease-modifying antirheumatic drugs and those who remitted spontaneously. Changes in disease activity between visits also affected DNA methylation, which was partially concomitant in the SF of UA and in blood MOs of patients with rheumatoid arthritis. Epigenetic similarities between arthritis types allow a common prediction of disease activity. Our results constitute a resource of DNA methylation–based biomarkers of poor prognosis, disease activity, and treatment efficacy for the personalized clinical management of early inflammatory arthritis.
Authors
Carlos de la Calle-Fabregat, Javier Rodríguez-Ubreva, Laura Ciudad, Julio Ramírez, Raquel Celis, Ana Belén Azuaga, Andrea Cuervo, Eduard Graell, Carolina Pérez-García, César Díaz-Torné, Georgina Salvador, José A. Gómez-Puerta, Isabel Haro, Raimon Sanmartí, Juan D. Cañete, Esteban Ballestar
×Abstract
HIV-1 vaccine efforts are primarily directed toward eliciting neutralizing antibodies (nAbs). However, vaccine trials and mother-to-child natural history cohort investigations indicate that antibody-dependent cellular cytotoxicity (ADCC), not nAbs, correlate with prevention. The ADCC characteristics associated with lack of HIV-1 acquisition remain unclear. Here, we examine ADCC and nAb properties in pretransmission plasma from HIV-1–exposed infants and from the corresponding transmitting and nontransmitting mothers’ breast milk and plasma. Breadth and potency (BP) were assessed against a panel of heterologous, nonmaternal variants. ADCC and neutralization sensitivity were estimated for the strains in the infected mothers. Infants who eventually acquired HIV-1 and those who remained uninfected had similar pretransmission ADCCBP. Viruses circulating in the transmitting and nontransmitting mothers had similar ADCC susceptibility. Infants with higher pretransmission ADCCBP and exposure to more ADCC-susceptible strains were less likely to acquire HIV-1. In contrast, higher preexisting infant neutralization BP and greater maternal virus neutralization sensitivity did not associate with transmission. Infants had higher ADCCBP closer to birth and in the presence of high plasma IgG relative to IgA levels. Mothers with potent humoral responses against their autologous viruses harbored more ADCC-sensitive strains. ADCC sensitivity of the exposure variants and preexisting ADCCBP influenced mother-to-child HIV-1 transmission during breastfeeding. Vaccination strategies that enhance ADCC are likely insufficient to prevent HIV-1 transmission because some strains may have low ADCC susceptibility.
Authors
Allison S. Thomas, Carolyn Coote, Yvetane Moreau, John E. Isaac, Alexander C. Ewing, Athena P. Kourtis, Manish Sagar
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Review
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Abstract
MicroRNAs (miRNAs) belong to a class of endogenous small noncoding RNAs that regulate gene expression at the posttranscriptional level, through both translational repression and mRNA destabilization. They are key regulators of kidney morphogenesis, modulating diverse biological processes in different renal cell lineages. Dysregulation of miRNA expression disrupts early kidney development and has been implicated in the pathogenesis of developmental kidney diseases. In this Review, we summarize current knowledge of miRNA biogenesis and function and discuss in detail the role of miRNAs in kidney morphogenesis and developmental kidney diseases, including congenital anomalies of the kidney and urinary tract and Wilms tumor. We conclude by discussing the utility of miRNAs as potentially novel biomarkers and therapeutic agents.
Authors
Débora Malta Cerqueira, Maliha Tayeb, Jacqueline Ho
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Abstract
Macrophages play a crucial role in the inflammatory response to the human stomach pathogen Helicobacter pylori, which infects half of the world’s population and causes gastric cancer. Recent studies have highlighted the importance of macrophage immunometabolism in their activation state and function. We have demonstrated that the cysteine-producing enzyme, cystathionine g-lyase (CTH), is upregulated in humans and mice with H. pylori infection. Here we show that induction of CTH in macrophages by H. pylori promotes persistent inflammation. Cth–/– mice have reduced macrophage and T-cell activation in H. pylori-infected tissues, an altered metabolome, and decreased enrichment of immune-associated gene networks, culminating in decreased H. pylori-induced-gastritis. CTH is downstream of the proposed anti-inflammatory molecule, S-adenosylmethionine (SAM). While Cth–/– mice exhibit gastric SAM accumulation, WT mice treated with SAM did not display protection against H. pylori-induced inflammation. Instead, we demonstrate that Cth-deficient macrophages exhibit alterations in the proteome, decreased NF-kB activation, diminished expression of macrophage activation markers, and impaired oxidative phosphorylation and glycolysis. Thus, through altering cellular respiration, CTH is a key enhancer of macrophage activation contributing to a pathogenic inflammatory response that is the universal precursor for the development of H. pylori-induced gastric disease.
Authors
Yvonne L. Latour, Johanna C. Sierra, Jordan L. Finley, Mohammad Asim, Daniel P. Barry, Margaret M. Allaman, Thaddeus M. Smith, Kara M. McNamara, Paula B. Luis, Claus Schneider, Justin Jacobse, Jeremy A. Goettel, M. Wade Calcutt, Kristie L. Rose, Kevin L Schey, Ginger L. Milne, Alberto G. Delgado, M. Blanca Piazuelo, Bindu D. Paul, Solomon Snyder, Alain P. Gobert, Keith T. Wilson
Abstract
Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC) although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side-effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular co-culture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Further, CN06 preserves the viability and function of human iPSC-derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy:toxicity ratio of CPA/DOX-containing chemotherapy.
Authors
Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang
Abstract
BACKGROUND. Sudden cardiac death (SCD) remains a worldwide public health problem in need of better noninvasive predictive tools. Current guidelines for primary preventive SCD therapies such as implantable cardioverter defibrillators (ICDs) are based on left ventricular ejection fraction (LVEF), but these are imprecise with fewer than 5% of ICDs delivering life-saving therapy per year. Impaired cardiac metabolism and ATP depletion cause arrhythmias in experimental models, but a link between arrhythmias and cardiac energetic abnormalities in people has not been explored, nor the potential for metabolically predicting clinical SCD risk. METHODS. We prospectively measured myocardial energy metabolism noninvasively with phosphorus magnetic resonance spectroscopy in patients with no history of significant arrhythmias prior to scheduled ICD implantation for primary prevention in the setting of reduced LVEF (≤35%). RESULTS. By two different analyses, low myocardial ATP significantly predicted the composite of subsequent appropriate ICD firings for life-threatening arrhythmias and cardiac death over ~10 years. Life-threatening arrhythmia risk was ~3-fold higher in low ATP patients and independent of established risk factors including LVEF. In patients with normal ATP, rates of appropriate ICD firings were several-fold lower than reported rates of ICD complications and inappropriate firings. CONCLUSION. These first data linking in vivo myocardial ATP depletion and subsequent significant arrhythmic events in people suggest an energetic component to clinical life-threatening ventricular arrhythmogenesis. The findings support investigation of metabolic strategies that limit ATP loss to treat or prevent life-threatening cardiac arrhythmias and herald non-invasive metabolic imaging as a complementary SCD risk stratification tool. TRIAL REGISTRATION. NCT00181233. FUNDING. This work was supported by DW Reynolds Foundation, the National Institutes of Health (grants HL61912, HL056882, HL103812, HL132181, HL140034), and the Russell H. Morgan (P.A.B.) and Clarence Doodeman (R.G.W.) Endowments at Johns Hopkins.
Authors
T. Jake Samuel, Shenghan Lai, Michael Schär, Katherine C. Wu, Angela M. Steinberg, An-Chi Wei, Mark Anderson, Gordon F. Tomaselli, Gary Gerstenblith, Paul A. Bottomley, Robert G. Weiss
Abstract
Nemaline Myopathy (NM) is the most common congenital myopathy, characterized by extreme weakness of the respiratory, limb, and facial muscles. Pathogenic variants in Tropomyosin 2 (TPM2), which encodes a skeletal muscle specific actin binding protein essential for sarcomere function, cause a spectrum of musculoskeletal disorders that include NM as well as Cap Myopathy, congenital fiber type disproportion, and distal arthrogryposis (DA). The in vivo pathomechanisms underlying TPM2-related disorders are unknown, so we expressed a series of dominant, pathogenic TPM2 variants in Drosophila embryos and found four variants significantly affected muscle development and muscle function. Transient overexpression of the four variants also disrupted the morphogenesis of mouse myotubes in vitro, and negatively affected zebrafish muscle development in vivo. We used transient overexpression assays in zebrafish to characterize two novel TPM2 variants and one recurring variant that we identified in DA patients (V129A, E139K, A155T), and found these variants caused musculoskeletal defects similar to those of known pathogenic variants. The consistency of musculoskeletal phenotypes in our assays correlated with the severity of clinical phenotypes observed in our DA patients, suggesting disrupted myogenesis is a novel pathomechanism of TPM2 disorders, and that our myogenic assays can predict the clinical severity of TPM2 variants.
Authors
Jennifer McAdow, Shuo Yang, Tiffany Ou, Gary Huang, Matthew B. Dobbs, Christina A. Gurnett, Michael J. Greenberg, Aaron N. Johnson
Abstract
In situ vaccination has demonstrated the feasibility of priming local immunity for systemic antitumor responses. Although direct intratumoral delivery of adjuvant is the mainstay, tumor-draining lymph nodes (TDLNs) also play essential roles in antitumor immunity. We report that directing an adjuvant to both tumors and TDLNs during in situ vaccination can induce robust antitumor responses. Conventional intratumoral dosing leads to tumor-limited delivery of agents; however, delivery to both tumors and TDLNs can be ensured through a micellar formation. The peritumoral delivery of micellar MEDI9197 (mcMEDI), a toll-like receptor 7/8 agonist, induced significantly stronger innate and adaptive immune responses than those on conventional dosing. Optimal dosing was crucial because excessive or insufficient accumulation of the adjuvant in the TDLNs compromised therapeutic efficacy. The combination of local mcMEDI therapy significantly improved the efficacy of systemic anti-programmed death receptor-1 therapy. These data suggest that rerouting adjuvants to tumors and TDLNs can augment the therapeutic efficacy of in situ vaccination.
Authors
Moonkyoung Jeong, Heegon Kim, Junyong Yoon, Dong-Hyun Kim, Ji-Ho Park
