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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
Abstract
Genetic or acquired defects of the lymphatic vasculature often result in disfiguring, disabling and, occasionally, life-threatening clinical consequences. Advanced forms of lymphedema are readily diagnosed clinically, but more subtle presentations often require invasive imaging or other technologies for a conclusive diagnosis. On the other hand, lipedema, a chronic lymphatic microvascular disease with pathological accumulation of subcutaneous adipose tissue is often misdiagnosed as obesity or lymphedema; currently there are no biomarkers or imaging criteria available for a conclusive diagnosis. Recent evidence suggests that otherwise asymptomatic defective lymphatic vasculature likely contributes to an array of other pathologies, including obesity, inflammatory bowel disease and neurological disorders, among others. Accordingly, identification of biomarkers of lymphatic malfunction will provide a valuable resource for the diagnosis and clinical discrimination of lymphedema, lipedema, obesity and other potential lymphatic-related pathologies. In this paper we profiled and compared blood plasma exosomes isolated from mouse models and from human subjects with and without symptomatic lymphatic pathologies. We identified platelet factor 4 (PF4/CXCL4) as a biomarker that could be used to diagnose lymphatic vasculature dysfunction. Furthermore, we determined that PF4 levels in circulating blood plasma exosomes were also elevated in lipedema patients, supporting current claims arguing that at least some of the underlying attributes of this disease are also the consequence of lymphatic defects.
Authors
Wanshu Ma, Hyea Jin Gil, Noelia Escobedo, Alberto Benito-Martín, Pilar Ximénez-Embún, Javier Muñoz, Héctor Peinado, Stanley G. Rockson, Guillermo Oliver
Abstract
Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder caused by reduced expression of the survival motor neuron (SMN) protein. SMN has key functions in multiple RNA pathways, including the biogenesis of small nuclear ribonucleoproteins (snRNPs) that are essential components of both major (U2-dependent) and minor (U12-dependent) spliceosomes. Here we investigated the specific contribution of U12 splicing dysfunction to SMA pathology through selective restoration of this RNA pathway in mouse models of varying phenotypic severity. We show that viral-mediated delivery of minor snRNA genes specifically improves select U12 splicing defects induced by SMN deficiency in cultured mammalian cells as well as in the spinal cord and dorsal root ganglia of SMA mice without increasing SMN expression. This approach resulted in a moderate amelioration of several parameters of the disease phenotype in SMA mice including survival, weight gain and motor function. Importantly, minor snRNA gene delivery improved aberrant splicing of the U12 intron-containing gene Stasimon and rescued the severe loss of proprioceptive sensory synapses on SMA motor neurons, which are early signatures of motor circuit dysfunction in mouse models. Taken together, these findings establish the direct contribution of U12 splicing dysfunction to synaptic deafferentation and motor circuit pathology in SMA.
Authors
Erkan Y. Osman, Meaghan Van Alstyne, Pei-Fen Yen, Francesco Lotti, Zhihua Feng, Karen K.Y. Ling, Chien-Ping Ko, Livio Pellizzoni, Christian L. Lorson
Abstract
Increased microvascular leakage is a cardinal feature of many critical diseases. Regular exercise is associated with improved endothelial function and reduced risk of cardiovascular disease. Irisin, secreted during exercise, contributes to many health benefits of exercise. However, the effects of irisin on endothelial function and microvascular leakage remain unknown. In this study, we found that irisin remarkably strengthened endothelial junctions and barrier function via binding to integrin αVβ5 receptor in LPS-treated endothelial cells. The beneficial effect of irisin was associated with suppression of the Src-MLCK-β-catenin pathway, activation of the AMPK-Cdc42/Rac1 pathway and improvement of mitochondrial function. In preclinical models of microvascular leakage, exogenous irisin improved pulmonary function, decreased lung edema and injury, suppressed inflammation, and increased survival. In ARDS patients, serum irisin levels were decreased and inversely correlated with disease severity and mortality. In conclusion, irisin enhances endothelial barrier function and mitigates microvascular leakage related diseases.
Authors
Jianbin Bi, Jia Zhang, Yifan Ren, Zhaoqing Du, Yuanyuan Zhang, Chang Liu, Yawen Wang, Lin Zhang, Zhihong Shi, Zheng Wu, Yi Lv, Rongqian Wu
Abstract
Apelin is a well-established mediator of survival and mitogenic signalling through apelin receptor (Aplnr) and have been implicated in various cancers, however little is known regarding Elabela (ELA/APELA) signalling, also mediated by Aplnr, and its role and the role of the conversion of its precursor proELA into mature ELA in cancer are unknown. Here we identify a function of mTORC1 signalling as an essential mediator of ELA that represses kidney tumour cells growth, migration and survival. Moreover, sunitinib and ELA show synergistic effect in repressing tumour growth and angiogenesis in mice. The use of site directed mutagenesis and pharmacologic experiments provide evidence that the alteration of the cleavage site of proELA by Furin induced improved ELA anti-tumorigenic activity. Finally, cohort of tumours and public data sets revealed that ELA is only repressed in the main human kidney cancer subtypes namely clear cell, papillary, and chromophobe renal cell carcinoma. While Aplnr is expressed by various kidney cells, ELA is generally expressed by epithelial cells. Collectively, these results show the tumour-suppressive role of mTORC1 signalling mediated by ELA and establish the potential use of ELA or derivatives in kidney cancers treatment.
Authors
Fabienne Soulet, Clement Bodineau, Katarzyna B. Hooks, Jean Descarpentrie, Isabel D. Alves, Marielle Dubreuil, Amandine Mouchard, Malaurie Eugenie, Jean-Luc Hoepffner, José Javier Lopez, Juan A. Rosado, Isabelle Soubeyran, Mercedes Tomé, Raúl V. Durán, Macha Nikolski, Bruno O. Villoutreix, Serge Evrard, Geraldine Siegfried, Abdel-Majid Khatib
Abstract
Background Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation and drug development. Here we present a temporal analysis of key immune mediators, cytokine and chemokines in blood of hospitalised COVID-19 patients from serial sampling and follow up over four weeks. Methods A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You’an hospital in China with either mild (53 patients) or severe disease (18 patients) were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines and chemokines in peripheral blood every 4-7 days over one month per patient using a bio-plex multiplex immunoassay. Results We found that the chemokine RANTES(CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1Ra and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-gamma were only significantly elevated in the late stage of severe COVID-19 illness. TNF- alpha and GM-CSF showed no significant differences between severe and mild cases. Conclusion Together our data suggest early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1Ra, IL-10 in blood individually and in combination might be useful prognostic bio-markers to guide treatment strategies.
Authors
Yan Zhao, Ling Qin, Ping Zhang, Kang Li, Lianchun Liang, Jianping Sun, Bin Xu, Yanchao Dai, Xuemei Li, Chi Zhang, Yanchun Peng, Yingmei Feng, Ang Li, Zhongjie Hu, Haiping Xiang, Graham Ogg, Ling-Pei Ho, Andrew J. McMichael, Ronghua Jin, Julian C. Knight, Tao Dong, Yonghong Zhang
Abstract
BACKGROUND. Dysregulation of L-arginine metabolism has been proposed to occur in severe asthma patients. The effects of L-arginine supplementation on L-arginine metabolite profiles in these patients is unknown. We hypothesized that severe asthmatics with low fractional exhaled nitric oxide (FeNO) would have fewer asthma exacerbations with the addition of L-arginine to their standard asthma medications compared to placebo and would demonstrate the greatest changes in metabolite profiles. METHODS. Participants were enrolled in a single-center, cross-over, double-blinded, L-arginine intervention trial at the University of California-Davis (NCT01841281). Subjects received placebo or L-arginine, dosed orally at 0.05mg/kg (ideal body weight) twice daily. The primary endpoint was moderate asthma exacerbations. Longitudinal plasma metabolite levels were measured using mass spectrometry. A linear mixed-effect model with subject-specific intercepts was used for testing treatment effects. RESULTS. A cohort of 50 subjects was included in the final analysis. L-arginine did not significantly decrease asthma exacerbations in the overall cohort. Higher citrulline levels and a lower arginine availability index (AAI) were associated with higher FeNO (P-value = 0.005 and 2.51 x 10–9 respectively). Higher AAI was associated with lower exacerbation events. The eicosanoid prostaglandin H2 (PGH2) and Nα-Acetyl-L-arginine were found to be good predictors for differentiating clinical responders and non-responders. CONCLUSIONS. There was no statistically significant decrease in asthma exacerbations in the overall cohort with L-arginine intervention. PGH2, Nα-Acetyl-L-arginine and the AAI could serve as predictive biomarkers in future clinical trials that intervene in the arginine metabolome.
Authors
Shu-Yi Liao, Megan R. Showalter, Angela L. Linderholm, Lisa M. Franzi, Celeste Kivler, Yao Li, Michael R. Sa, Zachary A. Kons, Oliver Fiehn, Lihong Qi, Amir A. Zeki, Nicholas J. Kenyon
Activation of CAR and non-CAR T cells within the tumor microenvironment following CAR T-cell therapy
Abstract
The mechanisms of CAR-T cell mediated anti-tumor immunity and toxicity remain poorly characterized due to few studies examining the intact tumor microenvironment (TME) following CAR T-cell infusion. Axicabtagene ciloleucel is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for patients with large B-cell lymphoma. We devised multiplex immunostaining and in-situ hybridization assays to interrogate CAR T cells and other immune cell infiltrates in biopsies of diffuse large B-cell lymphoma following axicabtagene ciloleucel infusion. We found a majority of intratumoral CAR T cells expressed markers of T-cell activation but, unexpectedly, comprised ≤ 5% of all T cells within the TME five days or more after therapy. T cells lacking CAR (non-CAR T cells) were also activated within the TME after axicabtagene ciloleucel infusion, being positive for Ki-67, interferon-γ, granzyme B and/or PD-1, and highest in biopsies with CAR T cells. Additionally, non-CAR immune cells were the exclusive source of IL-6, a cytokine associated with cytokine release syndrome, and highest in biopsies with CAR T cells. These data indicate that intratumoral CAR T cells are associated with generalized immune cell activation within the TME with both beneficial and pathological effects.
Authors
Pei-Hsuan Chen, Mikel Lipschitz, Jason L. Weirather, Caron Jacobson, Philippe Armand, Kyle Wright, F. Stephen Hodi, Zachary J. Roberts, Stuart A. Sievers, John Rossi, Adrian Bot, William Y. Go, Scott J. Rodig
Abstract
Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin resulting in pathologic cartilage and bone matrix deposition. CCN family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1(also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1/CCN4 expression across traumatic and genetic HO mouse models, as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1 null tenocytes revealed enrichment in signaling pathways such as STAT3 and PCP signaling that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.
Authors
Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase A. Pagani, Charles Hwang, David M. Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James
Abstract
Rheumatoid arthritis (RA) is characterized by synovial joint inflammation, cartilage damage and dysregulation of the adaptive immune system. While neutrophil extracellular traps (NETs) have been proposed to play a role in the generation of modified autoantigens and in the activation of synovial fibroblasts, it remains unknown whether NETs are directly involved in cartilage damage. Here, we report a new mechanism by which NET-derived elastase disrupts cartilage matrix and induces release membrane-bound peptidylarginine deiminase-2 (PAD2) by fibroblast-like synoviocytes (FLS). Cartilage fragments are subsequently citrullinated, internalized by FLS, and then presented to antigen-specific CD4+ T cells. Furthermore, immune-complexes containing citrullinated cartilage components can activate macrophages to release pro-inflammatory cytokines. HLA-DRB1*04:01 transgenic mice immunized with NETs develop autoantibodies to citrullinated cartilage proteins and display enhanced cartilage damage. Inhibition of NET-elastase rescues NET-mediated cartilage damage. These results show that NETs and neutrophil elastase externalized in these structures play fundamental pathogenic roles in promoting cartilage damage and synovial inflammation. Strategies targeting neutrophil elastase and NETs could have a therapeutic role in RA and in other inflammatory diseases associated with inflammatory joint damage.
Authors
Carmelo Carmona-Rivera, Philip M. Carlucci, Rishi R. Goel, Eddie A. James, Stephen R. Brooks, Cliff R. Rims, Victoria Hoffmann, David A. Fox, Jane H. Buckner, Mariana J. Kaplan
Abstract
Whole sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage-arresting replication-competent (LARC) GAPs confer superior protection when compared to early liver stage-arresting replication-deficient (EARD) GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (Pf) has been challenging. Here we report the generation and characterization of an unprecedented Pf LARC GAP generated by targeted gene deletion of the Mei2 gene; Pf mei2–. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for Pf mei2- liver stages in human liver-chimeric mice. However, Pf mei2– liver stages failed to complete development and did not form infectious exo-erythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, Pf mei2– is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against Pf malaria infection.
Authors
Debashree Goswami, William Betz, Navin K. Locham, Chaitra Parthiban, Carolyn Brager, Carola Schäfer, Nelly Camargo, Thao Nguyen, Spencer Y. Kennedy, Sean C. Murphy, Ashley M. Vaughan, Stefan H.I. Kappe
