IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis by upregulating PD-L1 expression.

BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.

Monocytes as Potential Mediators of Pathogen-Induced T-Helper 17 Differentiation in Patients With Primary Sclerosing Cholangitis (PSC).

BACKGROUND AND AIMS: T cells from patients with primary sclerosing cholangitis (PSC) show a prominent interleukin (IL)-17 response upon stimulation with bacteria or fungi, yet the reasons for this dominant T-helper 17 (Th17) response in PSC are not clear. Here, we analyzed the potential role of monocytes in microbial recognition and in skewing the T-cell response toward Th17. APPROACH AND RESULTS: Monocytes and T cells from blood and livers of PSC patients and controls were analyzed ex vivo and in vitro using transwell experiments with cholangiocytes. Cytokine production was measured using flow cytometry, enzyme-linked immunosorbent assay, RNA in situ hybridization, and quantitative real-time PCR. Genetic polymorphisms were obtained from ImmunoChip analysis. Following ex vivo stimulation with phorbol myristate acetate/ionomycin, PSC patients showed significantly increased numbers of IL-17A-producing peripheral blood CD4+ T cells compared to PBC patients and healthy controls, indicating increased Th17 differentiation in vivo. Upon stimulation with microbes, monocytes from PSC patients produced significantly more IL-1ß and IL-6, cytokines known to drive Th17 cell differentiation. Moreover, microbe-activated monocytes induced the secretion of Th17 and monocyte-recruiting chemokines chemokine (C-C motif) ligand (CCL)-20 and CCL-2 in human primary cholangiocytes. In livers of patients with PSC cirrhosis, CD14hiCD16int and CD14loCD16hi monocytes/macrophages were increased compared to alcoholic cirrhosis, and monocytes were found to be located around bile ducts. CONCLUSIONS: PSC patients show increased Th17 differentiation already in vivo. Microbe-stimulated monocytes drive Th17 differentiation in vitro and induce cholangiocytes to produce chemokines mediating recruitment of Th17 cells and more monocytes into portal tracts. Taken together, these results point to a pathogenic role of monocytes in patients with PSC.

TNF-Producing Th1 Cells Are Selectively Expanded in Liver Infiltrates of Patients with Autoimmune Hepatitis.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that is believed to be driven by a CD4+ T cell response to liver Ags. However, the pathogenic function of CD4+ effector T cells in AIH is not fully understood. To characterize liver-infiltrating lymphocytes in AIH, we determined the cytokine production of infiltrating cells obtained from biopsy material by quantitative RT-PCR and flow cytometry. A cytokine quantitiative RT-PCR array of AIH specimens revealed that TNF was the most strongly upregulated cytokine, as compared with control livers. To confirm this finding, we determined the frequencies of TNF-producing CD4+ T cells in peripheral blood and in liver biopsy specimens in comparison with those of CD4+ T cells producing IFN-γ or IL-17. In AIH, TNF-producing CD4+ T cells were significantly expanded, both in blood and liver, whereas IL-17-producing CD4+ T cells were not. However, the majority of the TNF-producing CD4+ T cells in AIH also produced IFN-γ, suggesting that TNF producers might represent a pathogenic activation state of Th1 cells. Ag-specific stimulation of PBMC from AIH patients with the AIH-associated autoantigen SEPSECS resulted in significant TNF production only in patients manifesting SLA/LP autoantibodies targeting SEPSEC but not in healthy individuals who do not manifest this reactivity. Taken together, our findings indicated that TNF-producing CD4+ T cells are expanded in AIH, both in blood and in liver. TNF-producing CD4+ T cells in AIH seem to be aberrantly activated Th1 cells. Our findings provide a rationale for therapeutic efforts using TNF blockade in AIH.

Human γδ T cell identification from single-cell RNA sequencing datasets by modular TCR expression.

Accurately identifying γδ T cells in large single-cell RNA sequencing (scRNA-seq) datasets without additional single-cell γδ T cell receptor sequencing (sc-γδTCR-seq) or CITE-seq (cellular indexing of transcriptomes and epitopes sequencing) data remains challenging. In this study, we developed a TCR module scoring strategy for human γδ T cell identification (i.e. based on modular gene expression of constant and variable TRA/TRB and TRD genes). We evaluated our method using 5' scRNA-seq datasets comprising both sc-αßTCR-seq and sc-γδTCR-seq as references and demonstrated that it can identify γδ T cells in scRNA-seq datasets with high sensitivity and accuracy. We observed a stable performance of this strategy across datasets from different tissues and different subtypes of γδ T cells. Thus, we propose this analysis method, based on TCR gene module scores, as a standardized tool for identifying and reanalyzing γδ T cells from 5'-end scRNA-seq datasets.

A prospective pilot study of a gluten-free diet for primary sclerosing cholangitis and associated colitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a progressive bile duct disease associated with inflammatory bowel disease (PSC-IBD). AIM: To investigate whether patients with PSC-IBD benefit from a gluten-free and amylase trypsin inhibitor (ATI)-free diet (GFD). METHODS: We performed a prospective clinical pilot study administering an eight-week GFD. The primary outcomes were colonic inflammation assessed by proctosigmoidoscopy, and liver stiffness (surrogate for fibrosis, inflammation and cholestasis) measured by transient elastography before and after GFD. Amongst the secondary (exploratory) outcomes were colonic mucosal and serum cytokine/chemokine changes, the intestinal microbiome and transcriptome dynamics, and shifts in serum markers of hepatic fibrogenesis. RESULTS: Fifteen patients with PSC-IBD completed the study. The study did not meet its primary outcome: the endoscopic score and liver stiffness remained unchanged. However, the expression of pro-inflammatory mucosal cytokines and chemokines such as IL6, IL8, CCL2, and TNFα was significantly down-regulated. Two critical markers of liver fibrosis and matrix remodelling, thrombospondin-2 and -4, decreased significantly. The microbiota composition changed slightly, including a decrease in the pathogen Romboutsia ilealis. The intestinal transcriptome indicated a gut barrier improvement. Pruritus, fatigue, overall well-being, faecal calprotectin levels, and serum alkaline phosphatase did not change significantly. CONCLUSIONS: This study did not demonstrate a clinical improvement with short-term GFD in patients with PSC-IBD. However, a gluten/ATI-free diet may improve biomarkers of intestinal inflammation and barrier function in these patients with associated changes in the enteric microbiota. Further investigation of the therapeutic potential of the GFD in PSC-IBD is warranted.

The Effects of Androgens on T Cells: Clues to Female Predominance in Autoimmune Liver Diseases?

The immune system responds differently in women and in men. Generally speaking, adult females show stronger innate and adaptive immune responses than males. This results in lower risk of developing most of the infectious diseases and a better ability to clear viral infection in women (1-5). On the other hand, women are at increased risk of developing autoimmune diseases (AID) such as rheumatoid arthritis, multiple sclerosis (MS), systemic lupus erythematosus (SLE), Sjögren's syndrome, and the autoimmune liver diseases autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) (6). Factors contributing to the female sex bias in autoimmune diseases include environmental exposure, e.g., microbiome, behavior, and genetics including X chromosomal inactivation of genes. Several lines of evidence and clinical observations clearly indicate that sex hormones contribute significantly to disease pathogenesis, and the role of estrogen in autoimmune diseases has been extensively studied. In many of these diseases, including the autoimmune liver diseases, T cells are thought to play an important pathogenetic role. We will use this mini-review to focus on the effects of androgens on T cells and how the two major androgens, testosterone and dihydrotestosterone, potentially contribute to the pathogenesis of autoimmune liver diseases (AILD).