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Abstract
Free light chains (FLCs) induce inflammatory pathways in proximal tubule cells (PTCs). The role of toll-like receptors (TLR) in these responses is unknown. Here we present findings on the role of TLRs in FLC-induced PTC injury. We exposed human kidney PTC cultures to κ and λ FLCs, and used cell supernatants and pellets for ELISA and gene expression studies. We also analyzed tissues from Stat1–/– and littermate control mice treated with daily intraperitoneal injections of a κ-FLC for 10 days. FLCs increased the expression of TLRs 2, 4, 6 via HMGB1, a damage-associated molecular pattern. Countering TLRs 2, 4, and 6 through GIT-27 or specific TLR-siRNAs reduced downstream cytokine responses. Blocking HMGB1 through siRNA or pharmacologic inhibition, or via STAT1 inhibition reduced FLC-induced TLRs 2, 4, and 6 expression. Blocking endocytosis of FLCs through silencing of megalin/cubilin, with bafilomycin-A1, or hypertonic sucrose attenuated FLC-induced cytokine responses in PTCs. Immunohistochemistry showed decreased TLR 4 and 6 expression in kidney sections from Stat1–/– mice compared to their littermate controls. PTCs exposed to FLCs released HMGB1, which induced TLRs 2, 4, 6 expression and downstream inflammation. Blocking FLCs’ endocytosis, Stat1 knock-down, HMGB1 inhibition, and TLR knock-down each rescued PTCs from FLC-induced injury.
Authors
Rohit Upadhyay, Wei-Zhong Ying, Zannatul Nasrin, Hana Safah, Edgar A. Jaimes, Wenguang Feng, Paul W. Sanders, Vecihi Batuman
Abstract
Wnt/β-catenin signaling is active in small subpopulations of Ewing sarcoma cells and these cells display a more metastatic phenotype, in part due to antagonism of EWS-FLI1-dependent transcriptional activity. Importantly, these β-catenin-activated Ewing cells also alter secretion of extracellular matrix (ECM) proteins. We thus hypothesized that, in addition to cell autonomous mechanisms, Wnt/β-catenin-active tumor cells might contribute to disease progression by altering the tumor microenvironment (TME). Analysis of transcriptomic data from primary patient biopsies and from β-catenin-active versus non-active tumor cells identified angiogenic switch genes as being highly and reproducibly upregulated in the context of β-catenin activation. In addition, in silico and in vitro analyses, along with chorioallantoic membrane assays, demonstrated that β-catenin-activated Ewing cells secrete factors that promote angiogenesis. In particular, activation of canonical Wnt signaling leads Ewing sarcoma cells to upregulate expression and secretion of pro-angiogenic ECM proteins, collectively termed the angiomatrix. Significantly, our data show that induction of the angiomatrix by Wnt-responsive tumor cells is indirect and is mediated by TGF-β. Mechanistically, Wnt/β-catenin signaling antagonizes EWS-FLI1-dependent repression of TGFBR2, thereby sensitizing tumor cells to TGF-β ligands. Together these findings suggest that Wnt/β-catenin active tumor cells can contribute to Ewing sarcoma progression by promoting angiogenesis in the local TME.
Authors
Allegra G. Hawkins, Elisabeth A. Pedersen, Sydney Treichel, Kelsey Temprine, Colin Sperring, Jay A. Read, Brian Magnuson, Rashmi Chugh, Elizabeth R. Lawlor
Abstract
Alcoholic liver disease is a spectrum of liver disorders with histopathological changes ranging from simple steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma. Recent data suggest that chronic-plus-binge ethanol intake induces steatohepatitis by promoting hepatocytes to release proinflammatory mitochondrial DNA (mtDNA)-enriched extracellular vesicles (EVs). The aim of this study was to investigate the role of the stress kinase apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinase (p38) in chronic-plus-binge ethanol-induced steatohepatitis and mtDNA-enriched EV release. Microarray analysis revealed the highest hepatic upregulation of metallothionein 1/2 (Mt1/2) which encode two most potent antioxidant proteins. Genetic deletion of the Mt1/2 gene aggravated ethanol-induced liver injury, as evidenced by elevation of serum ALT, neutrophil infiltration, oxidative stress and ASK1/p38 activation in the liver. Inhibition or genetic deletion of the Ask1 or p38 ameliorated ethanol-induced liver injury, inflammation, reactive oxygen species levels, and expression of phagocytic oxidase and ER stress markers in the liver. In addition, inhibition of ASK1 or p38 also attenuated ethanol-induced mtDNA-enriched EV secretion from hepatocytes. Taken together, these findings indicate that induction of hepatic mtDNA-enriched EVs by ethanol is dependent on ASK1 and p38, thereby promoting alcoholic steatohepatitis.
Authors
Jing Ma, Haixia Cao, Robim M. Rodrigues, Mingjiang Xu, Tianyi Ren, Yong He, Seonghwan Hwang, Dechun Feng, Ruixue Ren, Peixin Yang, Suthat Liangpunsakul, Jian Sun, Bin Gao
Abstract
During the growth of lymphatic vessels (lymphangiogenesis), lymphatic endothelial cells (LECs) at the growing front sprout by forming filopodia. Those tip cells are not exposed to circulating lymph, as they are not lumenized. In contrast, LECs that trail the growing front are exposed to shear stress, become quiescent and remodel into stable vessels. The mechanisms that coordinate the opposed activities of lymphatic sprouting and maturation remain poorly understood. Here we show that the canonical tip cell marker Delta-Like 4 (DLL4) promotes sprouting lymphangiogenesis by enhancing Vascular Endothelial Growth Factor C (VEGF-C) /VEGF Receptor 3 (VEGFR3) signaling. However, in lumenized lymphatic vessels laminar shear stress (LSS) inhibits the expression of DLL4, as well as additional tip cell markers. Paradoxically, LSS also upregulates VEGF-C/VEGFR3 signaling in LECs, but sphingosine 1-phosphate (S1P) receptor 1 (S1PR1) activity antagonizes LSS-mediated VEGF-C signaling to promote lymphatic vascular quiescence. Correspondingly, S1pr1 loss in LECs induced lymphatic vascular hypersprouting and hyperbranching, which could be rescued by reducing Vegfr3 gene dosage in vivo. In addition, S1PR1 regulates lymphatic vessel maturation by inhibiting RhoA activity to promote membrane localization of the tight junction molecule Claudin-5. Our findings suggest a new paradigm in which LSS induces quiescence and promotes the survival of LECs by downregulating DLL4 and enhancing VEGF-C signaling, respectively. S1PR1 dampens LSS/VEGF-C signaling, thereby preventing sprouting from quiescent lymphatic vessels. These results also highlight the distinct roles that S1PR1 and DLL4 play in LECs when compared to their known roles in the blood vasculature.
Authors
Xin Geng, Keisuke Yanagida, Racheal G. Akwii, Dongwon Choi, Lijuan Chen, YenChun Ho, Boksik Cha, Md. Riaj Mahamud, Karen Berman de Ruiz, Hirotake Ichise, Hong Chen, Joshua Wythe, Constantinos M. Mikelis, Timothy Hla, R. Sathish Srinivasan
Abstract
BACKGROUND. The numbers of fatal cases of Coronavirus Disease 2019 (COVID-19) continue to increase rapidly around the world. We aim to retrospectively investigate potential roles of factors, mainly immunologic parameters, in early predicting outcomes of patients with COVID-19. METHODS. A total of 1,018 patients confirmed COVID-19 were enrolled in our retrospective study from two centers. The data of clinical features, laboratory tests, immunological tests, radiological findings, and outcomes were collected. Univariate and multivariable logistic regression analysis were performed to evaluate factors associated with in-hospital mortality. Receiver operator characteristic (ROC) curves and survival curves were plotted to evaluate the clinical usefulness. RESULTS. Compared to the survival patients, the counts of all T lymphocytes subsets were markedly lower in non-survivors(P < 0.001), especially in CD8+ T cells (96.89 vs 203.98 cells/μl, P < 0.001) . Among all tested cytokines, IL-6 elevated most significantly with an upward trend of more than ten times (56.16 vs 5.36 pg/mL, P < 0.001). By a multivariable logistic regression analysis, two immunological indicators were found to be associated with in-hospital mortality, including IL-6 > 20 pg/mL (OR = 9.781; 95%CI, 6.304–15.174; P < 0.001) and CD8+ T cell count < 165 cells/μl (OR = 5.930; 95%CI, 3.677–9.562; P < 0.001), after adjusting confounding factors (age, gender, and underlying diseases). All the patients were divided into four groups according to levels of IL-6 and CD8+ T cells. The group with IL-6 > 20 pg/mL and CD8+ T cell count < 165 cells/μl had more old and male patients, as well as more proportion of patients with comorbidities, ventilation, ICU admission, shock, and death than those of any other group (P < 0.001). Furthermore, the ROC curve of the model combining IL-6 (>20 pg/mL) and CD8+ T cell count(<165 cells/μl) displayed more favorable discrimination than that of CURB-65 score (area under curve (AUC) = 0.907 vs 0.843, P < 0.001). Hosmer-Lemeshow test showed a good fitting of the model with no statistical significance (P = 0.581). CONCLUSIONS. We firstly identify two reliable prognostic indicators, IL-6 (>20 pg/mL) and CD8+ T cell count (<165 cells/μl), which can accurately stratify patients into risk categories and predict mortality of patients with COVID-19. Those two indicators combined may guide clinicians to evaluate patient prognosis and make appropriate decisions.
Authors
Miao Luo, Jing Liu, Weiling Jiang, Shuang Yue, Huiguo Liu, Shuang Wei
Abstract
Patients with hereditary or acquired hemolytic anemias have a high risk of developing in-situ thrombosis of the pulmonary vasculature. While pulmonary thrombosis is a major morbidity associated with hemolytic disorders, the etiological mechanism underlying hemolysis-induced pulmonary thrombosis remains largely unknown. Here, we use intravital lung microscopy in mice for the first time to assess the pathogenesis of pulmonary thrombosis following deionized-water induced acute intravascular hemolysis. Acute hemolysis triggered the development of αIIbβ3-dependent platelet-rich thrombi in precapillary pulmonary arterioles, which led to the transient impairment of pulmonary blood flow. The hemolysis-induced pulmonary thrombosis was phenocopied with intravenous ADP- but not thrombin-triggered pulmonary thrombosis. Consistent with a mechanism involving ADP release from hemolyzing erythrocytes, the inhibition of platelet-P2Y12 purinergic-receptor signaling attenuated pulmonary thrombosis and rescued blood flow in the pulmonary arterioles of mice following intravascular hemolysis. These findings are the first in vivo studies to suggest that acute intravascular hemolysis promotes ADP-dependent platelet activation leading to thrombosis in the pre-capillary pulmonary arterioles and that thrombin generation most likely does not play a significant role in the pathogenesis of acute hemolysis-triggered pulmonary thrombosis.
Authors
Tomasz Brzoska, Ravi Vats, Margaret F. Bennewitz, Egemen Tutuncuoglu, Simon C. Watkins, Margaret V. Ragni, Matthew D. Neal, Mark T. Gladwin, Prithu Sundd
Abstract
Musculoskeletal disorders represent the 3rd greatest burden on health in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million in the UK and US respectively. Osteoarthritis commonly occurs after joint injury, particularly affecting younger patients. Painful joints are often treated with injections of steroid or hyaluronic acid (HA), but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in ligament and meniscus after knee injury and synovial fluid glutamate concentrations ranged from 19–129 µM. Intra-articular injection of NBQX (GluR antagonist), administered at the time of injury, substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect and depo-medrone reduced swelling for 1 day, but increased degeneration by 50%. Intra-articular administration of NBQX was both symptom and disease modifying to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders, with proven safety in man, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis.
Authors
Cleo S. Bonnet, Sophie J. Gilbert, Emma J. Blain, Anwen S. Williams, Deborah J. Mason
Abstract
Tissue regeneration capacity declines with aging in association with heightened oxidative stress. Expression of the oxidant-generating enzyme, NADPH oxidase 4 (Nox4) is elevated in aged mice with diminished capacity for fibrosis resolution. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal (BET) family of proteins that function as epigenetic “readers” of acetylated lysine groups on histones. In this study, we explored the role of Brd4 and its interaction with the p300 acetyltransferase in the regulation of Nox4, and the in-vivo efficacy of a BET inhibitor to reverse established age-associated lung fibrosis. BET inhibition interferes with the association of Brd4, p300, and acetylated histone H4K16 with the Nox4 promoter in lung fibroblasts stimulated with the pro-fibrotic cytokine, transforming growth factor-β1 (TGF-β1). This Brd4-Nox4 epigenetic axis is constitutively upregulated in fibroblasts from human subjects with idiopathic pulmonary fibrosis. A number of BET inhibitors, including I-BET-762, JQ1, and OTX015, downregulate Nox4 gene expression and activity. Aged mice with established and persistent lung fibrosis recovered capacity for fibrosis resolution with OTX015 treatment. This study implicates epigenetic regulation of Nox4 by Brd4 and p300, and supports BET/Brd4 inhibition as an effective strategy for the treatment of age-related fibrotic lung disease.
Authors
Yan Y. Sanders, Xing Lyu, Q. Jennifer Zhou, Zheyi Xiang, Denise Stanford, Sandeep Bodduluri, Steven M. Rowe, Victor J. Thannickal
Abstract
Purinergic modulators, such as dipyridamole, target multiple pathways that have been implicated in COVID-19 pathogenesis, and thus the therapeutic benefit of these should be explored.
Authors
Yogendra Kanthi, Jason S. Knight, Yu Zuo, David J. Pinsky
Abstract
Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human peripheral blood mononuclear cells (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127lowCD45RA+CD45RO–. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including a high expression of the latent form of TGF-β. CD19-CAR Tregs suppressed IgG antibody production from primary B cell differentiation in vitro via a TGF-β-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs with reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.
Authors
Yuki Imura, Makoto Ando, Taisuke Kondo, Minako Ito, Akihiko Yoshimura
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