Autoantigen-selected B cells are bystanders in spontaneous T cell-driven experimental autoimmune hepatitis.

Autoreactive B cells are considered pathogenic drivers in many autoimmune diseases; however, it is not clear whether autoimmune B cells are invariably pathogenic or whether they can also arise as bystanders of T cell-driven autoimmune pathology. Here, we studied the B cell response in an autoantigen- and CD4+ T cell-driven model of autoimmune hepatitis (AIH), the Alb-iGP_Smarta mouse in which expression of a viral model antigen (GP) in hepatocytes and its recognition by GP-specific CD4+ T cells causes spontaneous AIH-like disease. T cell-driven AIH in Alb-iGP_Smarta mice was marked by autoantibodies and hepatic infiltration of plasma cells and B cells, particularly of isotype-switched memory B cells, indicating antigen-driven selection and activation. Immunosequencing of B cell receptor repertoires confirmed B cell expansion selectively in the liver, which was most likely driven by the hepatic GP model antigen, as indicated by branched networks of connected sequences and elevated levels of IgG antibodies to GP. However, intrahepatic B cells did not produce increased levels of cytokines and their depletion with anti-CD20 antibody did not alter the CD4+ T cell response in Alb-iGP_Smarta mice. Moreover, B cell depletion did not prevent spontaneous liver inflammation and AIH-like disease in Alb-iGP_Smarta mice. In conclusion, selection and isotype-switch of liver-infiltrating B cells was dependent on the presence of CD4+ T cells recognizing liver antigen. However, recognition of hepatic antigen by CD4+ T cells and CD4+ T cell-mediated hepatitis was not dependent on B cells. Thus, autoreactive B cells can be bystanders and need not be drivers of liver inflammation in AIH.

Inflammatory type 2 conventional dendritic cells contribute to murine and human cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a progressive cholangiopathy characterised by fibrotic stricturing and inflammation of bile ducts, which seems to be driven by a maladaptive immune response to bile duct injury. The histological finding of dendritic cell expansion in portal fields of patients with PSC prompted us to investigate the role of dendritic cells in orchestrating the immune response to bile duct injury. METHODS: Dendritic cell numbers and subtypes were determined in different mouse models of cholangitis by flow cytometry based on lineage-imprinted markers. Findings were confirmed by immunofluorescence microscopy of murine livers, and liver samples from patients with PSC were compared to control samples from bariatric surgery patients. Using genetic tools, selected dendritic cell subsets were depleted in murine cholangitis. The dendritic cell response to bile duct injury was determined by single-cell transcriptomics. RESULTS: Cholangitis mouse models were characterised by selective intrahepatic expansion of type 2 conventional dendritic cells, whereas plasmacytoid and type 1 conventional dendritic cells were not expanded. Expansion of type 2 conventional dendritic cells in human PSC lesions was confirmed by histology. Depletion studies revealed a proinflammatory role of type 2 conventional dendritic cells. Single-cell transcriptomics confirmed inflammatory maturation of the intrahepatic type 2 conventional dendritic cells and identified dendritic cell-derived inflammatory mediators. CONCLUSIONS: Cholangitis is characterised by intrahepatic expansion and inflammatory maturation of type 2 conventional dendritic cells in response to biliary injury. Therefore, type 2 conventional dendritic cells and their inflammatory mediators might be potential therapeutic targets for the treatment of PSC. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease of the bile ducts for which there is no effective treatment. Herein, we show that the inflammatory immune response to bile duct injury is organised by a specific subtype of immune cell called conventional type 2 dendritic cells. Our findings suggest that this cell subtype and the inflammatory molecules it produces are potential therapeutic targets for PSC.

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.

Liver infiltrating T cells regulate bile acid metabolism in experimental cholangitis.

BACKGROUND & AIMS: T cells are central mediators of liver inflammation and represent potential treatment targets in cholestatic liver disease. Whereas emerging evidence shows that bile acids (BAs) affect T cell function, the role of T cells for the regulation of BA metabolism is unknown. In order to understand this interplay, we investigated the influence of T cells on BA metabolism in a novel mouse model of cholangitis. METHODS: Mdr2-/- mice were crossed with transgenic K14-OVAp mice, which express an MHC class I restricted ovalbumin peptide on biliary epithelial cells (Mdr2-/-xK14-OVAp). T cell-mediated cholangitis was induced by the adoptive transfer of antigen-specific CD8+ T cells. BA levels were quantified using a targeted liquid chromatography-mass spectrometry-based approach. RESULTS: T cell-induced cholangitis resulted in reduced levels of unconjugated BAs in the liver and significantly increased serum and hepatic levels of conjugated BAs. Genes responsible for BA synthesis and uptake were downregulated and expression of the bile salt export pump was increased. The transferred antigen-specific CD8+ T cells alone were able to induce these changes, as demonstrated using Mdr2-/-xK14-OVAp recipient mice on the Rag1-/- background. Mechanistically, we showed by depletion experiments that alterations in BA metabolism were partly mediated by the proinflammatory cytokines TNF and IFN-γ in an FXR-dependent manner, a process that in vitro required cell contact between T cells and hepatocytes. CONCLUSION: Whereas it is known that BA metabolism is dysregulated in sepsis and related conditions, we have shown that T cells are able to control the synthesis and metabolism of BAs, a process which depends on TNF and IFN-γ. Understanding the effect of lymphocytes on BA metabolism will help in the design of combined treatment strategies for cholestatic liver diseases. LAY SUMMARY: Dysregulation of bile acid metabolism and T cells can contribute to the development of cholangiopathies. Before targeting T cells for the treatment of cholangiopathies, it should be determined whether they exert protective effects on bile acid metabolism. Herein, we demonstrate that T cell-induced cholangitis resulted in decreased levels of harmful unconjugated bile acids. T cells were able to directly control synthesis and metabolism of bile acids, a process which was dependent on the proinflammatory cytokines TNF and IFN-γ. Understanding the effect of lymphocytes on bile acid metabolism will help in the design of combined treatment strategies for cholestatic liver diseases.

Human liver-resident CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways.

RATIONALE: The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. OBJECTIVES: We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. FINDINGS: Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. CONCLUSION: Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will require modification of hepatic CCR5 and CXCR6.

Failure of thymic deletion and instability of autoreactive Tregs drive autoimmunity in immune-privileged liver.

The liver is an immune-privileged organ that can deactivate autoreactive T cells. Yet in autoimmune hepatitis (AIH), autoreactive T cells can defy hepatic control and attack the liver. To elucidate how tolerance to self-antigens is lost during AIH pathogenesis, we generated a spontaneous mouse model of AIH, based on recognition of an MHC class II-restricted model peptide in hepatocytes by autoreactive CD4+ T cells. We found that the hepatic peptide was not expressed in the thymus, leading to deficient thymic deletion and resulting in peripheral abundance of autoreactive CD4+ T cells. In the liver, autoreactive CD4+ effector T cells accumulated within portal ectopic lymphoid structures and maturated toward pathogenic IFN-γ and TNF coproducing cells. Expansion and pathogenic maturation of autoreactive effector T cells was enabled by a selective increase of plasticity and instability of autoantigen-specific Tregs but not of nonspecific Tregs. Indeed, antigen-specific Tregs were reduced in frequency and manifested increased IL-17 production, reduced epigenetic demethylation, and reduced expression of Foxp3. As a consequence, autoantigen-specific Tregs had a reduced suppressive capacity, as compared with that of nonspecific Tregs. In conclusion, loss of tolerance and the pathogenesis of AIH were enabled by combined failure of thymic deletion and peripheral regulation.

Increased Infiltration of Natural Killer and T Cells in Colorectal Liver Metastases Improves Patient Overall Survival.

INTRODUCTION: Cancer heterogeneity and degree of intra-tumoral immune cells represent variables affecting overall survival (OS). The present study investigated the impact of natural killer (NK) and T cells infiltrating colorectal liver metastases (CLM) in patients undergoing hepatectomy after neoadjuvant chemotherapy. METHODS: The frequencies of intra-tumoral, marginal, and peritumoral CD3+ T and NKp46+ NK cells were determined for 121 patients. OS was assessed in relation to prognostic factors. RESULTS: At univariate analysis, several variables, including T and N of the primary tumor, metachronous CLM, radiological response, and higher density of intra-tumoral CD3+ T cell (>1%/mm2) and of NKp46+ NK cells (>1 cell/mm2), were associated with OS. Only increased frequencies of intra-tumoral CD3+ T cells (p = 0.005) and NKp46+ NK cells (p = 0.004) correlated with OS at multivariate analysis. The logistic regression revealed that metachronous CLM (OR = 2.781; p = 0.002), the use of an epidermal growth factor receptor inhibitor (OR = 3.891; p = 0.001), and radiological response (OR = 3.219; p = 0.001) were associated with higher infiltration of these cells. CONCLUSIONS: High frequencies of NK and T cells in response to chemotherapy predict OS in CLM patients. These findings provide important insights that can help physicians to choose the best treatment option and adopt more predictive follow-up strategies for patients with CLM.

Attenuated viral hepatitis in Trem1-/- mice is associated with reduced inflammatory activity of neutrophils.

TREM1 (Triggering Receptor Expressed on Myeloid Cells 1) is a pro-inflammatory receptor expressed by phagocytes, which can also be released as a soluble molecule (sTREM1). The roles of TREM1 and sTREM1 in liver infection and inflammation are not clear. Here we show that patients with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection manifest elevated serum levels of sTREM1. In mice, experimental viral hepatitis induced by infection with Lymphocytic Choriomeningitis Virus (LCMV)-WE was likewise associated with increased sTREM1 in serum and urine, and with increased TREM1 and its associated adapter molecule DAP12 in the liver. Trem1-/- mice showed accelerated clearance of LCMV-WE and manifested attenuated liver inflammation and injury. TREM1 expression in the liver of wild-type mice was mostly confined to infiltrating neutrophils, which responded to LCMV by secretion of CCL2 and TNF-α, and release of sTREM1. Accordingly, the production of CCL2 and TNF-α was decreased in the livers of LCMV-infected Trem1-/- mice, as compared to LCMV-infected wildtype mice. These findings indicate that TREM1 plays a role in viral hepatitis, in which it seems to aggravate the immunopathology associated with viral clearance, mainly by increasing the inflammatory activity of neutrophils.