Kaposi's sarcoma herpesvirus latency-associated nuclear antigen broadly regulates viral gene expression and is essential for lytic infection.

Kaposi's sarcoma herpesvirus (KSHV) is a leading cause of malignancy in AIDS and current therapies are limited. Like all herpesviruses, KSHV infection can be latent or lytic. KSHV latency-associated nuclear antigen (LANA) is essential for viral genome persistence during latent infection. LANA also maintains latency by antagonizing expression and function of the KSHV lytic switch protein, RTA. Here, we find LANA null KSHV is not capable of lytic replication, indicating a requirement for LANA. While LANA promoted both lytic and latent gene expression in cells partially permissive for lytic infection, it repressed expression in non-permissive cells. Importantly, forced RTA expression in non-permissive cells led to induction of lytic infection and LANA switched to promote, rather than repress, most lytic viral gene expression. When basal viral gene expression levels were high, LANA promoted expression, but repressed expression at low basal levels unless RTA expression was forcibly induced. LANA's effects were broad, but virus gene specific, extending to an engineered, recombinant viral GFP under control of host EF1α promoter, but not to host EF1α. Together, these results demonstrate that, in addition to its essential role in genome maintenance, LANA broadly regulates viral gene expression, and is required for high levels of lytic gene expression during lytic infection. Strategies that target LANA are expected to abolish KSHV infection.

Tox induces T cell IL-10 production in a BATF-dependent manner.

Tox is a member of the high mobility group (HMG)-Box transcription factors and plays important roles in thymic T cell development. Outside of the thymus, however, Tox is also highly expressed by CD8 and CD4 T cells in various states of activation and in settings of cancer and autoimmune disease. In CD4 T cells, Tox has been primarily studied in T follicular helper (TFH) cells where it, along with Tox2, promotes TFH differentiation by regulating key TFH-associated genes and suppressing CD4 cytotoxic T cell differentiation. However, the role of Tox in other T helper (Th) cell subtypes is less clear. Here, we show that Tox is expressed in several physiologically-activated Th subtypes and its ectopic expression enhances the in vitro differentiation of Th2 and T regulatory (Treg) cells. Tox overexpression in unpolarized Th cells also induced the expression of several genes involved in cell activation (Pdcd1), cellular trafficking (Ccl3, Ccl4, Xcl1) and suppressing inflammation (Il10) across multiple Th subtypes. We found that Tox binds the regulatory regions of these genes along with the transcription factors BATF, IRF4, and JunB and that Tox-induced expression of IL-10, but not PD-1, is BATF-dependent. Based on these data, we propose a model where Tox regulates Th cell chemotactic genes involved in facilitating dendritic cell-T cell interactions and aids in the resolution or prevention of inflammation through the production of IL-10.

RNA helicase DDX5 modulates sorafenib sensitivity in hepatocellular carcinoma via the Wnt/ß-catenin-ferroptosis axis.

Reduced expression of the RNA helicase DDX5 associated with increased hepatocellular carcinoma (HCC) tumor grade and poor patient survival following treatment with sorafenib. While immunotherapy is the first-line treatment for HCC, sorafenib and other multi-tyrosine kinase inhibitors (mTKIs) are widely used when immunotherapy is contra-indicated or fails. Herein, we elucidate the role of DDX5 in sensitizing HCC to sorafenib, offering new therapeutic strategies. Treatment of various human HCC cell lines with sorafenib/mTKIs downregulated DDX5 in vitro and in preclinical HCC models. Conversely, DDX5 overexpression reduced the viability of sorafenib-treated cells via ferroptosis, suggesting a role for DDX5 in sorafenib sensitivity. RNAseq of wild-type vs. DDX5-knockdown cells treated with or without sorafenib identified a set of common genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps with Wnt signaling genes, including Disheveled-1 (DVL1), an indispensable Wnt activator and prognostic indicator of poor survival for sorafenib-treated patients. DDX5-knockout (DDX5KO) HCC cells exhibited DVL1 induction, Wnt/ß-catenin pathway activation, and ferroptosis upon inhibition of canonical Wnt signaling. Consistently, xenograft HCC tumors exhibited reduced growth by inhibition of Wnt/ß-catenin signaling via induction of ferroptosis. Significantly, overexpression of DDX5 in HCC xenografts repressed DVL1 expression and increased ferroptosis, resulting in reduced tumor growth by sorafenib. We conclude that DDX5 downregulation by sorafenib mediates adaptive resistance by activating Wnt/ß-catenin signaling, leading to ferroptosis escape. Conversely, overexpression of DDX5 in vivo enhances the anti-tumor efficacy of sorafenib by suppressing Wnt/ß-catenin activation and induction of ferroptosis. Thus, DDX5 overexpression in combination with mTKIs is a promising therapeutic strategy for HCC.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

Importance of inner-sphere P-O-Fe bonds in natural and synthetic mineral-organic associations.

Sorption of organic molecules on mineral surfaces can occur through several binding mechanisms of varying strength. Here, we investigated the importance of inner-sphere P-O-Fe bonds in synthetic and natural mineral-organic associations. Natural organic matter such as water extracted soil organic matter (WESOM) and extracellular polymeric substances (EPS) from liquid bacterial cultures were adsorbed to goethite and examined by FTIR spectroscopy and P K-edge NEXAFS spectroscopy. Natural particles from a Bg soil horizon (Gleysol) were subjected to X-ray fluorescence (XRF) mapping, NanoSIMS imaging, and NEXAFS spectro-microscopy at the P K-edge. Inner-sphere P-O-Fe bonds were identified for both, adsorbed EPS extracts and adsorbed WESOMs. Characteristic infrared peaks for P-O-Fe stretching vibrations are present but cannot unambiguously be interpreted due to possible interferences with mono- and polysaccharides. For the Bg horizon, P was only found on Fe oxides, covering the entire surface at different concentrations, but not on clay minerals. Linear combination fitting of NEXAFS spectra indicates that this adsorbed P is mainly a mixture of orthophosphate and organic P compounds. By combining atomic force microscopy (AFM) images with STXM-generated C and Fe distribution maps, we show that the Fe oxide surfaces were fully coated with organic matter. In contrast, clay minerals revealed a much lower C signal. The C NEXAFS spectra taken on the Fe oxides had a substantial contribution of carboxylic C, aliphatic C, and O-alkyl C, which is a composition clearly different from pure adsorbed EPS or aromatic-rich lignin-derived compounds. Our data show that inner-sphere P-O-Fe bonds are important for the association of Fe oxides with soil organic matter. In the Bg horizon, carboxyl groups and orthophosphate compete with the organic P compounds for adsorption sites.

Long-term organic carbon preservation enhanced by iron and manganese.

The balance between degradation and preservation of sedimentary organic carbon (OC) is important for global carbon and oxygen cycles1. The relative importance of different mechanisms and environmental conditions contributing to marine sedimentary OC preservation, however, remains unclear2-8. Simple organic molecules can be geopolymerized into recalcitrant forms by means of the Maillard reaction5, although reaction kinetics at marine sedimentary temperatures are thought to be slow9,10. More recent work in terrestrial systems suggests that the reaction can be catalysed by manganese minerals11-13, but the potential for the promotion of geopolymerized OC formation at marine sedimentary temperatures is uncertain. Here we present incubation experiments and find that iron and manganese ions and minerals abiotically catalyse the Maillard reaction by up to two orders of magnitude at temperatures relevant to continental margins where most preservation occurs4. Furthermore, the chemical signature of the reaction products closely resembles dissolved and total OC found in continental margin sediments globally. With the aid of a pore-water model14, we estimate that iron- and manganese-catalysed transformation of simple organic molecules into complex macromolecules might generate on the order of approximately 4.1 Tg C yr-1 for preservation in marine sediments. In the context of perhaps only about 63 Tg C yr-1 variation in sedimentary organic preservation over the past 300 million years6, we propose that variable iron and manganese inputs to the ocean could exert a substantial but hitherto unexplored impact on global OC preservation over geological time.

Combined Electrochemical, XPS, and STXM Study of Lithium Nitride as a Protective Coating for Lithium Metal and Lithium-Sulfur Batteries.

Li3N is an excellent protective coating material for lithium electrodes with very high lithium-ion conductivity and low electronic conductivity, but the formation of stable and homogeneous coatings is technically very difficult. Here, we show that protective Li3N coatings can be simply formed by the direct reaction of electrodeposited lithium electrodes with N2 gas, whereas using battery-grade lithium foil is problematic due to the presence of a native passivation layer that hampers that reaction. The protective Li3N coating is effective at preventing lithium dendrite formation, as found from unidirectional plating and plating-stripping measurements in Li-Li cells. The Li3N coating also efficiently suppresses the parasitic reactions of polysulfides and other electrolyte species with the lithium electrode, as demonstrated by scanning transmission X-ray microscopy, X-ray photoelectron spectroscopy, and optical microscopy. The protection of the lithium electrode against corrosion by polysulfides and other electrolyte species, as well as the promotion of smooth deposits without dendrites, makes the Li3N coating highly promising for applications in lithium metal batteries, such as lithium-sulfur batteries. The present findings show that the formation of Li3N can be achieved with lithium electrodes covered by a secondary electrolyte interface layer, which proves that the in situ formation of Li3N coatings inside the batteries is attainable.

Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection.

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.

The role of transcription factors in shaping regulatory T cell identity.

Forkhead box protein 3-expressing (FOXP3+) regulatory T cells (Treg cells) suppress conventional T cells and are essential for immunological tolerance. FOXP3, the master transcription factor of Treg cells, controls the expression of multiples genes to guide Treg cell differentiation and function. However, only a small fraction (<10%) of Treg cell-associated genes are directly bound by FOXP3, and FOXP3 alone is insufficient to fully specify the Treg cell programme, indicating a role for other accessory transcription factors operating upstream, downstream and/or concurrently with FOXP3 to direct Treg cell specification and specialized functions. Indeed, the heterogeneity of Treg cells can be at least partially attributed to differential expression of transcription factors that fine-tune their trafficking, survival and functional properties, some of which are niche-specific. In this Review, we discuss the emerging roles of accessory transcription factors in controlling Treg cell identity. We specifically focus on members of the basic helix-loop-helix family (AHR), basic leucine zipper family (BACH2, NFIL3 and BATF), CUT homeobox family (SATB1), zinc-finger domain family (BLIMP1, Ikaros and BCL-11B) and interferon regulatory factor family (IRF4), as well as lineage-defining transcription factors (T-bet, GATA3, RORγt and BCL-6). Understanding the imprinting of Treg cell identity and specialized function will be key to unravelling basic mechanisms of autoimmunity and identifying novel targets for drug development.

C5a-licensed phagocytes drive sterilizing immunity during systemic fungal infection.

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.

Non-coding RNAs in immunoregulation and autoimmunity: Technological advances and critical limitations.

Immune cell function is critically dependent on precise control over transcriptional output from the genome. In this respect, integration of environmental signals that regulate gene expression, specifically by transcription factors, enhancer DNA elements, genome topography and non-coding RNAs (ncRNAs), are key components. The first three have been extensively investigated. Even though non-coding RNAs represent the vast majority of cellular RNA species, this class of RNA remains historically understudied. This is partly because of a lag in technological and bioinformatic innovations specifically capable of identifying and accurately measuring their expression. Nevertheless, recent progress in this domain has enabled a profusion of publications identifying novel sub-types of ncRNAs and studies directly addressing the function of ncRNAs in human health and disease. Many ncRNAs, including circular and enhancer RNAs, have now been demonstrated to play key functions in the regulation of immune cells and to show associations with immune-mediated diseases. Some ncRNAs may function as biomarkers of disease, aiding in diagnostics and in estimating response to treatment, while others may play a direct role in the pathogenesis of disease. Importantly, some are relatively stable and are amenable to therapeutic targeting, for example through gene therapy. Here, we provide an overview of ncRNAs and review technological advances that enable their study and hold substantial promise for the future. We provide context-specific examples by examining the associations of ncRNAs with four prototypical human autoimmune diseases, specifically rheumatoid arthritis, psoriasis, inflammatory bowel disease and multiple sclerosis. We anticipate that the utility and mechanistic roles of these ncRNAs in autoimmunity will be further elucidated in the near future.

A comprehensive single cell data analysis of lymphoblastoid cells reveals the role of super-enhancers in maintaining EBV latency.

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.

EBV-associated diseases: Current therapeutics and emerging technologies.

EBV is a prevalent virus, infecting >90% of the world's population. This is an oncogenic virus that causes ~200,000 cancer-related deaths annually. It is, in addition, a significant contributor to the burden of autoimmune diseases. Thus, EBV represents a significant public health burden. Upon infection, EBV remains dormant in host cells for long periods of time. However, the presence or episodic reactivation of the virus increases the risk of transforming healthy cells to malignant cells that routinely escape host immune surveillance or of producing pathogenic autoantibodies. Cancers caused by EBV display distinct molecular behaviors compared to those of the same tissue type that are not caused by EBV, presenting opportunities for targeted treatments. Despite some encouraging results from exploration of vaccines, antiviral agents and immune- and cell-based treatments, the efficacy and safety of most therapeutics remain unclear. Here, we provide an up-to-date review focusing on underlying immune and environmental mechanisms, current therapeutics and vaccines, animal models and emerging technologies to study EBV-associated diseases that may help provide insights for the development of novel effective treatments.

IL-1ß promotes IL-9-producing Th cell differentiation in IL-2-limiting conditions through the inhibition of BCL6.

IL-9-producing CD4+ T helper cells, termed Th9 cells, differentiate from naïve precursor cells in response to a combination of cytokine and cell surface receptor signals that are elevated in inflamed tissues. After differentiation, Th9 cells accumulate in these tissues where they exacerbate allergic and intestinal disease or enhance anti-parasite and anti-tumor immunity. Previous work indicates that the differentiation of Th9 cells requires the inflammatory cytokines IL-4 and TGF-ß and is also dependent of the T cell growth factor IL-2. While the roles of IL-4 and TGF-ß-mediated signaling are relatively well understood, how IL-2 signaling contributes to Th9 cell differentiation outside of directly inducing the Il9 locus remains less clear. We show here that murine Th9 cells that differentiate in IL-2-limiting conditions exhibit reduced IL-9 production, diminished NF-kB activation and a reduced NF-kB-associated transcriptional signature, suggesting that IL-2 signaling is required for optimal NF-kB activation in Th9 cells. Interestingly, both IL-9 production and the NF-kB transcriptional signature could be rescued by addition of the NF-kB-activating cytokine IL-1ß to IL-2-limiting cultures. IL-1ß was unique among NF-kB-activating factors in its ability to rescue Th9 differentiation as IL-2 deprived Th9 cells selectively induced IL-1R expression and IL-1ß/IL-1R1 signaling enhanced the sensitivity of Th9 cells to limiting amounts of IL-2 by suppressing expression of the Th9 inhibitory factor BCL6. These data shed new light on the intertwined nature of IL-2 and NF-kB signaling pathways in differentiating Th cells and elucidate the potential mechanisms that promote Th9 inflammatory function in IL-2-limiting conditions.

Investigation of the causal etiology in a patient with T-B+NK+ immunodeficiency.

Newborn screening for severe combined immunodeficiency (SCID) has not only accelerated diagnosis and improved treatment for affected infants, but also led to identification of novel genes required for human T cell development. A male proband had SCID newborn screening showing very low T cell receptor excision circles (TRECs), a biomarker for thymic output of nascent T cells. He had persistent profound T lymphopenia, but normal numbers of B and natural killer (NK) cells. Despite an allogeneic hematopoietic stem cell transplant from his brother, he failed to develop normal T cells. Targeted resequencing excluded known SCID genes; however, whole exome sequencing (WES) of the proband and parents revealed a maternally inherited X-linked missense mutation in MED14 (MED14V763A), a component of the mediator complex. Morpholino (MO)-mediated loss of MED14 function attenuated T cell development in zebrafish. Moreover, this arrest was rescued by ectopic expression of cDNA encoding the wild type human MED14 ortholog, but not by MED14V763A , suggesting that the variant impaired MED14 function. Modeling of the equivalent mutation in mouse (Med14V769A) did not disrupt T cell development at baseline. However, repopulation of peripheral T cells upon competitive bone marrow transplantation was compromised, consistent with the incomplete T cell reconstitution experienced by the proband upon transplantation with bone marrow from his healthy male sibling, who was found to have the same MED14V763A variant. Suspecting that the variable phenotypic expression between the siblings was influenced by further mutation(s), we sought to identify genetic variants present only in the affected proband. Indeed, WES revealed a mutation in the L1 cell adhesion molecule (L1CAMQ498H); however, introducing that mutation in vivo in mice did not disrupt T cell development. Consequently, immunodeficiency in the proband may depend upon additional, unidentified gene variants.

The cytoplasmic LSm1-7 and nuclear LSm2-8 complexes exert opposite effects on Hepatitis B virus biosynthesis and interferon responses.

Despite many studies on host or viral gene expression, how the cellular proteome responds to internal or external cues during the infection process remains unclear. In this study, we used a Hepatitis B Virus (HBV) replication model and performed proteomic analyses to understand how HBV evades innate immunity as a function of cell cycle progression. Specifically, we performed proteomic analyses of HBV-replicating cells in G1/S and G2/M phases, as a function of IFN-α treatment. We identified that the conserved LSm (Like-Sm1-8) proteins were differentially regulated in HBV replicating cells treated with IFN-α. Specifically, in G2/M phase, IFN-α increased protein level of LSm1, the unique subunit of cytoplasmic LSm1-7 complex involved in mRNA decay. By contrast, IFN-α decreased LSm8, the unique subunit of nuclear LSm2-8 complex, a chaperone of U6 spliceosomal RNA, suggesting the cytoplasmic LSm1-7 complex is antiviral, whereas the nuclear LSm2-8 complex is pro-viral. In HBV replication and infection models, siRNA-mediated knockdown of LSm1 increased all viral RNAs. Conversely, LSm8 knockdown reduced viral RNA levels, dependent on N6-adenosine methylation (m6A) of the epsilon stem-loop at the 5' end of pre-Core/pregenomic (preC/pg) RNA. Methylated RNA immunoprecipitation (MeRIP) assays demonstrated reduced viral RNA methylation by LSm8 knockdown, dependent on the 5' m6A modification, suggesting the LSm2-8 complex has a role in mediating this modification. Interestingly, splicing inhibitor Cp028 acting upstream of the LSm2-8 complex suppressed viral RNA levels without reducing the 5' m6A modification. This observation suggests Cp028 has novel antiviral effects, likely potentiating IFN-α-mediated suppression of HBV biosynthesis.

GSTM2 is a key molecular determinant of resistance to SG-ARIs.

Prostate cancer (PCa) continues to threaten men's health, and treatment targeting the androgen receptor (AR) pathway is the major therapy for PCa patients. Several second-generation androgen receptor inhibitors (SG-ARIs), including enzalutamide (ENZ), apalutamide (APA) and darolutamide (DARO), have been developed to better block the activity of AR. Unavoidably, emergence of resistance to these novel drugs still persists. Herein, we identified glutathione S-transferase Mu 2 (GSTM2) as an important determinant in the acquisition of resistance to SG-ARIs. Elevated GSTM2 was detected in enzalutamide-resistant (ENZ-R) PCa, and overexpression of GSTM2 in naïve enzalutamide-sensitive (ENZ-S) cells effectively transformed them to ENZ-R PCa. Aryl hydrocarbon receptor (AhR), the upstream transcription factor, was implicated in the overexpression of GSTM2 in ENZ-R cells. Mechanistically, GSTM2 antagonized the effect of ENZ by rescuing cells from oxidative stress-associated damage and activation of p38 MAPK pathway. Surprisingly, high GSTM2 levels also associated with cross-resistance to APA and DARO. Taking together, these results provide new insight to ameliorate resistance to SG-ARIs and improve treatment outcome.

Folic acid-mediated fibrosis is driven by C5a receptor 1-mediated activation of kidney myeloid cells.

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-C5ar1fl/fl) 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. C5ar1fl/fl.Lyz2Cre+/- mice, in which C5aR1 has been specifically deleted in lysozyme M-expressing myeloid cells, experienced reduced fibrosis compared with control C5ar1fl/fl 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-ß+ GFP+ cells, we developed C5ar1fl/fl.Foxd1Cre+/- mice, in which C5aR1 is deleted specifically in pericytes, and found reduced FA-induced fibrosis. Primary cell cultures of platelet-derived growth factor receptor-ß+ pericytes isolated from FA-treated C5ar1fl/fl.Foxd1Cre+/- mice showed reduced secretion of several cytokines, including IL-6 and macrophage inflammatory protein-2, compared with pericytes isolated from FA-treated control GFP-C5ar1fl/fl mice. Collectively, these data imply that C5a/C5aR1 axis activation primarily in interstitial cells contributes to the development of renal fibrosis.NEW & NOTEWORTHY 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.

Autocrine vitamin D signaling switches off pro-inflammatory programs of TH1 cells.

The molecular mechanisms governing orderly shutdown and retraction of CD4+ type 1 helper T (TH1) cell responses remain poorly understood. Here we show that complement triggers contraction of TH1 responses by inducing intrinsic expression of the vitamin D (VitD) receptor and the VitD-activating enzyme CYP27B1, permitting T cells to both activate and respond to VitD. VitD then initiated the transition from pro-inflammatory interferon-γ+ TH1 cells to suppressive interleukin-10+ cells. This process was primed by dynamic changes in the epigenetic landscape of CD4+ T cells, generating super-enhancers and recruiting several transcription factors, notably c-JUN, STAT3 and BACH2, which together with VitD receptor shaped the transcriptional response to VitD. Accordingly, VitD did not induce interleukin-10 expression in cells with dysfunctional BACH2 or STAT3. Bronchoalveolar lavage fluid CD4+ T cells of patients with COVID-19 were TH1-skewed and showed de-repression of genes downregulated by VitD, from either lack of substrate (VitD deficiency) and/or abnormal regulation of this system.