We have previously reported that increased expression and activation of kidney cell complement components play an important role in the pathogenesis of renal scarring. Here, we used floxed green ...fluorescent protein (GFP)-C5a receptor 1 (C5aR1) knockin mice (GFP-
) and the model of folic acid (FA)-induced kidney injury to define the cell types and potential mechanisms by which increased C5aR1 activation leads to fibrosis. Using flow cytometry and confocal microscopy, we identified macrophages as the major interstitial cell type showing increased expression of C5aR1 in FA-treated mice.
.
Cre
mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control
mice. Examination of C5aR1-expressing macrophage transcriptomes by gene set enrichment analysis demonstrated that these cells were enriched in pathways corresponding to the complement cascade, collagen formation, and the NABA matrisome, strongly pointing to their critical roles in tissue repair/scarring. Since C5aR1 was also detected in a small population of platelet-derived growth factor receptor-β
GFP
cells, we developed
.
Cre
mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-β
pericytes isolated from FA-treated
.
Cre
mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-
mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis.
This study used novel green fluorescent protein C5a receptor 1 floxed mice and the model of folic acid-mediated kidney fibrosis to demonstrate the pathogenic role of increased expression of this complement receptor on macrophages.
Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the ...best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.
Systemic candidiasis is a common, high-mortality, nosocomial fungal infection. Unexpectedly, it has emerged as a complication of anti-complement C5-targeted monoclonal antibody treatment, indicating ...a critical niche for C5 in antifungal immunity. We identified transcription of complement system genes as the top biological pathway induced in candidemic patients and as predictive of candidemia. Mechanistically, C5a-C5aR1 promoted fungal clearance and host survival in a mouse model of systemic candidiasis by stimulating phagocyte effector function and ERK- and AKT-dependent survival in infected tissues. C5ar1 ablation rewired macrophage metabolism downstream of mTOR, promoting their apoptosis and enhancing mortality through kidney injury. Besides hepatocyte-derived C5, local C5 produced intrinsically by phagocytes provided a key substrate for antifungal protection. Lower serum C5a concentrations or a C5 polymorphism that decreases leukocyte C5 expression correlated independently with poor patient outcomes. Thus, local, phagocyte-derived C5 production licenses phagocyte antimicrobial function and confers innate protection during systemic fungal infection.
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•Complement component 5a drives phagocyte survival and effector functions for antifungal defense•Extrahepatic C5 production by phagocytes contributes to antifungal protection•Impaired complement activation correlates with poor outcomes in humans with candidemia•Transcriptional induction of a complement module is predictive of human candidemia
Beyond liver-derived complement component C5, locally produced C5 is a critical mediator of antifungal defense in infected tissues, and this finding explains the susceptibility of patients to systemic Candida infection after C5 inhibition therapy.
Abstract
Arginase 1 (ARG1), the enzyme catalyzing the conversion of arginine to ornithine and urea, is a hallmark of IL-10 producing immunosuppressive M2 macrophages, however ARG1 activity in T cells ...is disputed. Here we demonstrate that Arg1, but not Arg2, expression induction is a key feature of lung CD4 T cells during mouse in vivo influenza infection. Ablation of CD4 T cell-intrinsic Arg1 unexpectedly accelerated both the virus-specific Th1 effector response and its IL-10-associated contraction. Biologically, this led to efficient viral clearance, yet significantly reduced lung pathology. Surprisingly, loss of Arg1 in CD4 T cells did not result in disturbed intracellular ornithine or polyamine levels. Instead, by employing unbiased transcriptomic and metabolomic approaches, we found that Arg1 deficiency triggered altered glutamine metabolism, and rebalancing the glutamine flux normalized the Arg1 deficient T cell response. Further, the role of Arg1 in CD4 T cells was distinct from that of its’ isoenzyme Arg2, as ablation of CD4 T cell intrinsic Arg2 resulted in normal Th1 responses, and instead altered Th2 and Th17 responses. Finally, CD4 T cells from rare patients with a deficiency in ARG1, or from healthy donors with CRISPR-Cas9-mediated ARG1 deletion, recapitulated the mouse data, demonstrating that ARG1 also plays a CD4 T cell intrinsic role in human Th1 responses. Collectively, CD4 T cell-intrinsic ARG1, functions as an unexpected pace-keeper of human and mouse T helper 1 (Th1) responses with implications for Th1-associated tissue pathologies.
Supported by grants from NIH (5K22HL125593 to M.K.) the Intramural Research Program of the NIH (NIDDK ZIA/DK075149 to B.A. and NHLBI ZIA/HL006223 to C.K.)
Abstract
Intracellularly generated and autocrine-functioning complement component C3 is a critical integral part of normal human CD4+ Th1 and cytotoxic CD8+ T cells responses. Increased or decreased ...intracellular C3 result in autoimmunity and infections, respectively. The mechanisms regulating intracellular C3 expression are, however, undefined. By comparing transcriptomes from blood and tissue, we identified the complement system, including C3, as one of the most significantly enriched biological pathways in tissue-occupying cells of human macrophages, CD4+ and CD8+ T cells. By leveraging a novel C3 reporter mouse, we confirmed that C3 gene transcription is a feature of immune cells in tissues, is induced during trans-endothelial diapedesis, and is dependent on the integrin intercellular adhesion molecule (ICAM)-1 liganding lymphocyte function-associated antigen (LFA)-1. Consequently, monocytes and T cells from patients with leukocyte adhesion deficiency (LAD)-1 had reduced C3 and diminished effector activities, which could be rescued proportionally by normalization of intracellular C3. In synovia of patients with rheumatoid arthritis (RA), C3 transcript expression by CD4+ T cells was linearly associated with disease severity and acted as a biomarker distinguishing inflamed versus uninflamed RA. Our study defines the integrin network as a novel and key controller of intracellular complement, demonstrates that perturbations in the LFA-1–C3 axis contribute to primary human immunodeficiency and identifies T cell C3 production as a biomarker of the severity of autoimmunity.
Abstract
Epstein-Barr Virus (EBV) is a complex oncovirus that infects around 90% of adults. Most primary infections remain asymptomatic but result in lifelong infection. EBV can drive lethal ...malignancies of both lymphoid (e.g. Burkitt lymphoma) and epithelial origins (e.g. Stomach Adenocarcinoma). Despite decades of research, the exact molecular mechanisms of EBV and host immune system interactions that lead to tumorigenesis remain elusive.
Through systematic analyses of high-throughput sequencing data from >1000 patients with different cancer types, we identified twelve EBV transcripts, including LMP1 and LMP2, which when overexpressed, significantly suppressed viral reactivation. These ‘virostatic genes’ had frequent missense and nonsense variations in cancer patients, disrupting their ability to restrict EBV reactivation. We also found that viral reactivation by classical chemical inducers up-regulates the expression of Programmed Death-Ligand 1 (PD-L1). Further analyses of the same RNA-seq dataset classified EBV+ tumors into two molecular subtypes based on expression pattern of upstream regulatory immune-related genes and Immune Checkpoint (IC) markers, including PD-L1. Overexpression of virostatic genes in lymphoblastic cells significantly repressed cellular PD-L1 transcription, leading us to hypothesize that a combination of IC and virostatic gene modulation may have synergistic benefits for immunotherapy of EBV-associated cancers.
Arginase 1 (Arg1), the enzyme catalyzing the conversion of arginine to ornithine, is a hallmark of IL-10-producing immunoregulatory M2 macrophages. However, its expression in T cells is disputed. ...Here, we demonstrate that induction of Arg1 expression is a key feature of lung CD4+ T cells during mouse in vivo influenza infection. Conditional ablation of Arg1 in CD4+ T cells accelerated both virus-specific T helper 1 (Th1) effector responses and its resolution, resulting in efficient viral clearance and reduced lung pathology. Using unbiased transcriptomics and metabolomics, we found that Arg1-deficiency was distinct from Arg2-deficiency and caused altered glutamine metabolism. Rebalancing this perturbed glutamine flux normalized the cellular Th1 response. CD4+ T cells from rare ARG1-deficient patients or CRISPR-Cas9-mediated ARG1-deletion in healthy donor cells phenocopied the murine cellular phenotype. Collectively, CD4+ T cell-intrinsic Arg1 functions as an unexpected rheostat regulating the kinetics of the mammalian Th1 lifecycle with implications for Th1-associated tissue pathologies.
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•CD4+ T cell-intrinsic arginase 1 controls Th1 induction and contraction kinetics•Loss of arginase 1 in CD4+ T cells results in reduced Th1-mediated tissue pathology•Th1-intrinsic arginase 1 ensures balanced glutamine vs. arginine metabolism•CD4+ T cell-intrinsic arginase 1 and 2 have distinct, non-overlapping functions
West et al. demonstrate that CD4+ T cell-intrinsic arginase 1 paces the transition of Th1 cells from their induction to their contraction program via balancing glutamine vs. arginine usage. They further show that Th1 cells lacking arginase 1 retain full pathogen clearance capacity but cause less Th1-associated tissue pathology.
We probed the lifecycle of Epstein‐Barr virus (EBV) on a cell‐by‐cell basis using single cell RNA sequencing (scRNA‐seq) data from nine publicly available lymphoblastoid cell lines (LCLs). While the ...majority of LCLs comprised cells containing EBV in the latent phase, two other clusters of cells were clearly evident and were distinguished by distinct expression of host and viral genes. Notably, both were high expressors of EBV LMP1/BNLF2 and BZLF1 compared to another cluster that expressed neither gene. The two novel clusters differed from each other in their expression of EBV lytic genes, including glycoprotein gene GP350. The first cluster, comprising GP350–LMP1hi cells, expressed high levels of HIF1A and was transcriptionally regulated by HIF1‐α. Treatment of LCLs with Pevonedistat, a drug that enhances HIF1‐α signaling, markedly induced this cluster. The second cluster, containing GP350+LMP1hi cells, expressed EBV lytic genes. Host genes that are controlled by super‐enhancers (SEs), such as transcription factors MYC and IRF4, had the lowest expression in this cluster. Functionally, the expression of genes regulated by MYC and IRF4 in GP350+LMP1hi cells were lower compared to other cells. Indeed, induction of EBV lytic reactivation in EBV+ AKATA reduced the expression of these SE‐regulated genes. Furthermore, CRISPR‐mediated perturbation of the MYC or IRF4 SEs in LCLs induced the lytic EBV gene expression, suggesting that host SEs and/or SE target genes are required for maintenance of EBV latency. Collectively, our study revealed EBV‐associated heterogeneity among LCLs that may have functional consequence on host and viral biology.
Abstract
Background
Pro-inflammatory CD4+ T helper (Th)1 cells clear pathogens effectively but cause excessive tissue injury if not shut down appropriately. The complement (C’) system both induces ...Th1 differentiation and their shutdown, but the mechanisms regulating orderly shutdown remain unknown.
Hypothesis
C’ receptor engagement recruits transcriptional regulators essential to Th1 shutdown.
Methods
Multi-modal profiling of activated, or patient-derived Th cells, psoriatic skin, and SARS-CoV2-infected tissues was carried out by epigenome profiling, RNAseq, network modeling, phospho-arrays, confocal, and regulator knockdown.
Results
C’ receptor signaling induced the vitamin D (VitD) receptor (VDR) and CYP27B1, the enzyme that activates VitD, allowing T cells to both fully activate and respond to VitD. Active VitD shut down IFN-γ production by Th1 cells and induced IL-10. This was mediated by activation of IL-6 production by T cells and signaling through STAT3. Mechanistically, VitD reprogrammed the Th1 transcriptomes by forming super-enhancers and recruiting a transcription factor (TF) network consisting of VDR, c-JUN, STAT3, and BACH2. We mapped genome-wide targets of these TFs by CUT&RUN/Tag. As proof of principal, psoriatic skin treated with VitD induced BACH2 in Th cells, and genetic deficiency of either BACH2 or STAT3 inhibited IL-10 produced in response to VitD. Bronchoalveolar lavage fluid of COVID-19 patients, a C’-rich environment, showed excessive Th1 skewing and perturbation of the VitD-regulated program of genes.
Conclusion
We identified a C’-recruited autocrine VitD system as key to Th1 shutdown and indicate the potential for adjunct therapy with VitD in hyper-inflammatory syndromes, e.g. COVID-19.
This work was supported by the Wellcome Trust (grant 097261/Z/11/Z to B.A.), the Crohn’s and Colitis Foundation of America (grant CCFA no. 3765 — CCFA genetics initiative to A.L.), British Heart Foundation (grant RG/13/12/30395 to G.L.), the National Institute of General Medical Sciences (R35GM138283 to M.K.), the Showalter Trust (research award to M.K.), German Research Foundation (DFG scholarship to T.F.; FR 3851/2-1), the NIDDK (DK12262401A1 to D.P.) and the National Agency of Research and Development of Chile (grant PAI79170073 to E.N.L.). Research was also supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London and/or the NIHR Clinical Research Facility. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. This research was supported (in part) by the Intramural Research Programs of the NIDDK (project no. ZIA/DK075149 to B.A), the National Heart, Lung and Blood Institute (project nos. ZIA/Hl006223 to C.K. and ZIA/HL006193 to N.M.), the NIAID (project no. ZIA/AI001175 to M.S.L.) of the NIH. D.C. is supported by an NIH Office of Dietary Supplements research scholar award.
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