IFNγ and CTLA-4 Drive Hepatic CD4 T-Cell Tolerance and Protection From Autoimmunity in Mice.

BACKGROUND & AIMS: The liver has a distinct capacity to induce immune tolerance to hepatic antigens. Although liver tolerance can be advantageous for preventing autoimmune and inflammatory diseases, it also can be detrimental by preventing immune surveillance of infected or malignant cells. Here, we investigated the immune mechanisms that establish hepatic tolerance. METHODS: Tolerance was investigated in C-reactive protein (CRP)-myelin basic protein (MBP) mice expressing the neuroantigen MBP in hepatocytes, providing profound resistance to MBP-induced neuroinflammation. Tolerance induction was studied after transfer of MBP-specific CD4 T cells into CRP-MBP mice, and tolerance mechanisms were tested using depleting or blocking antibodies. RESULTS: Although tolerant CRP-MBP mice display increased numbers of forkhead box P3+ regulatory T cells, we here found them not essential for the maintenance of hepatic tolerance. Instead, upon MBP recognition in the liver, MBP-specific T cells became activated to produce interferon (IFN)γ, which, in turn, induced local up-regulation of recruitment molecules, including Chemokine (C-X-C motif) ligand9 and its receptor C-X-C motif chemokine receptor3, facilitating endothelial translocation and redirection of MBP-specific T cells into the hepatic parenchyma. There, the translocated MBP-specific CD4 T cells partly converted into interleukin 10-producing type 1 regulatory T cells, and significantly up-regulated the expression of immune checkpoint molecules, notably cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Intriguingly, although liver tolerance was not affected by impairment of interleukin 10 signaling, concomitant blockade of IFNγ and CTLA-4 abrogated hepatic tolerance induction to MBP, resulting in neuroinflammatory autoimmune disease in these mice. CONCLUSIONS: IFNγ-mediated redirection of autoreactive CD4 T cells into the liver and up-regulation of checkpoint molecules, including CTLA-4, were essential for tolerance induction in the liver, hence representing a potential treatment target for boosting or preventing liver tolerance.

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.

Harnessing the liver to induce antigen-specific immune tolerance.

Autoimmune diseases develop when the adaptive immune system attacks the body's own antigens leading to tissue damage. At least 80 different conditions are believed to have an autoimmune aetiology, including rheumatoid arthritis, type I diabetes, multiple sclerosis or systemic lupus erythematosus. Collectively, autoimmune diseases are a leading cause of severe health impairment along with substantial socioeconomic costs. Current treatments are mostly symptomatic and non-specific, and it is typically not possible to cure these diseases. Thus, the development of more causative treatments that suppress only the pathogenic immune responses, but spare general immunity is of great biomedical interest. The liver offers considerable potential for development of such antigen-specific immunotherapies, as it has a distinct physiological capacity to induce immune tolerance. Indeed, the liver has been shown to specifically suppress autoimmune responses to organ allografts co-transplanted with the liver or to autoantigens that were transferred to the liver. Liver tolerance is established by a unique microenvironment that facilitates interactions between liver-resident antigen-presenting cells and lymphocytes passing by in the low blood flow within the hepatic sinusoids. Here, we summarise current concepts and mechanisms of liver immune tolerance, and review present approaches to harness liver tolerance for antigen-specific immunotherapy.

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.

Cell-autonomous hepatocyte-specific GP130 signaling is sufficient to trigger a robust innate immune response in mice.

BACKGROUND & AIMS: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling. METHODS: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model. RESULTS: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling. CONCLUSIONS: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity. LAY SUMMARY: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.

Aryl Hydrocarbon Receptor Activity in Hepatocytes Sensitizes to Hyperacute Acetaminophen-Induced Hepatotoxicity in Mice.

BACKGROUND & AIMS: Acetaminophen (APAP)-induced liver injury is one of the most common causes of acute liver failure, however, a clear definition of sensitizing risk factors is lacking. Here, we investigated the role of the ligand-activated transcription factor aryl hydrocarbon receptor (Ahr) in APAP-induced liver injury. We hypothesized that Ahr, which integrates environmental, dietary, microbial and metabolic signals into complex cellular transcriptional programs, might act as a rheostat for APAP-toxicity. METHODS: Wildtype or conditional Ahr knockout mice lacking Ahr in hepatocytes (AlbΔ/ΔAhr) or myeloid cells (LysMΔ/ΔAhr) were treated with the specific Ahr ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) together with APAP. RESULTS: Ahr activation by ITE, which by itself was non-toxic, exacerbated APAP-induced hepatotoxicity compared to vehicle-treated controls, causing 80% vs. 0% mortality after administration of a normally sublethal APAP overdose. Of note, Ahr activation induced hepatocyte death even at APAP doses within the therapeutic range. Aggravated liver injury was associated with significant neutrophil infiltration; however, lack of Ahr in myeloid cells did not protect LysMΔ/ΔAhr mice from exacerbated APAP hepatotoxicity. In contrast, AlbΔ/ΔAhr mice were largely protected from ITE-induced aggravated liver damage, indicating that Ahr activation in hepatocytes, but not in myeloid cells, was instrumental for disease exacerbation. Mechanistically, Ahr activation fueled hepatic accumulation of toxic APAP metabolites by up-regulating expression of the APAP-metabolizing enzyme Cyp1a2, a direct Ahr downstream target. CONCLUSIONS: Ahr activation in hepatocytes potentiates APAP-induced hepatotoxicity. Thus, individual exposition to environmental Ahr ligands might explain individual sensitivity to hyperacute liver failure.

Nanoparticle-mediated targeting of autoantigen peptide to cross-presenting liver sinusoidal endothelial cells protects from CD8 T-cell-driven autoimmune cholangitis.

Autoimmune diseases are caused by adaptive immune responses to self-antigens. The development of antigen-specific therapies that suppress disease-related, but not unrelated immune responses in general, is an important goal of biomedical research. We have previously shown that delivery of myelin peptides to liver sinusoidal endothelial cells (LSECs) using LSEC-targeting nanoparticles provides effective protection from CD4 T-cell-driven autoimmune encephalomyelitis. Here, we investigated whether this methodology might also serve antigen-specific treatment of a CD8 T-cell-driven autoimmune disease. As a model for CD8 T-cell-mediated autoimmunity, we used OT-1 T-cell-driven cholangitis in K14-OVAp mice expressing the cognate MHC I-restricted SIINFEKL peptide in cholangiocytes. To study whether peptide delivery to LSECs could modulate cholangitis, SIINFEKL peptide-conjugated nanoparticles were administered intravenously one day before transfer of OT-1 T cells; five days after cell transfer, liver pathology and hepatic infiltrates were analysed. SIINFEKL peptide-conjugated nanoparticles were rapidly taken up by LSECs in vivo, which effectively cross-presented the delivered peptide on MHC I molecules. Intriguingly, K14-OVAp mice receiving SIINFEKL-loaded nanoparticles manifested significantly reduced liver damage compared with vehicle-treated K14-OVAp mice. Mechanistically, treatment with LSEC-targeting SIINFEKL-loaded nanoparticles significantly reduced the number of liver-infiltrating OT-1 T cells, which up-regulated expression of the co-inhibitory receptor PD-1 and down-regulated cytotoxic effector function and inflammatory cytokine production. These findings show that tolerogenic LSECs can effectively internalize circulating nanoparticles and cross-present nanoparticle-bound peptides on MHC I molecules. Therefore, nanoparticle-mediated autoantigen peptide delivery to LSECs might serve the antigen-specific treatment of CD8 T-cell-driven autoimmune disease.

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.

Inflammatory Phenotype of Intrahepatic Sulfatide-Reactive Type II NKT Cells in Humans With Autoimmune Hepatitis.

Background: Natural Killer T (NKT) cells are CD1d-restricted innate-like T cells that can rapidly release stored cytokines upon recognition of lipid antigens. In mice, type I NKT cells seem to promote liver inflammation, whereas type II NKT cells seem to restrict hepatitis. Here, we aimed at characterizing the role of human type I and type II NKT in patients with autoimmune hepatitis (AIH). Methods: NKT cells were analyzed by flow cytometry in peripheral blood and liver of AIH patients and control groups. α-galactosylceramide-loaded or sulfatide-loaded tetramers were used to detect type I or II NKT cells, respectively. Hepatic CD1d was stained by in situ-hybridization of liver biopsies. Results and Conclusions: Type II NKT cells were more prevalent in human peripheral blood and liver than type I NKT cells. In AIH patients, the frequency of sulfatide-reactive type II NKT cells was significantly increased in peripheral blood (0.11% of peripheral blood leukocytes) and liver (3.78% of intrahepatic leukocytes) compared to healthy individuals (0.05% and 1.82%) and patients with drug-induced liver injury (0.06% and 2.03%; p < 0.05). Intrahepatic type II NKT cells of AIH patients had a different cytokine profile than healthy subjects with an increased frequency of TNFα (77.8% vs. 59.1%, p < 0.05), decreased IFNγ (32.7% vs. 63.0%, p < 0.05) and a complete lack of IL-4 expressing cells (0% vs. 2.1%, p < 0.05). T cells in portal tracts expressed significantly more CD1d-RNA in AIH livers compared to controls. This study supports that in contrast to their assumed protective role in mice, human intrahepatic, sulfatide-reactive type II NKT cells displayed a proinflammatory cytokine profile in patients with AIH. Infiltrating T cells in portal areas of AIH patients overexpressed CD1d and could thereby activate type II NKT cells.

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.

T-cell repertoire profiling by next-generation sequencing reveals tissue migration dynamics of TRBV13-family clonotypes in a common experimental autoimmune encephalomyelitis mouse model.

The experimental autoimmune encephalomyelitis (EAE) model is indispensable for autoimmunity research, but model-specific T cell dynamics are sparsely studied. We used next-generation immunosequencing across lymphoid organs, blood and spinal cord in response to immunization with myelin basic protein (MBP) to study T cell repertoires and migration patterns. Surprisingly, most spinal cord T cells were unique to the individual animal despite the existence of shared MBP-specific clones, suggesting a previously underestimated T cell diversity. Almost complete emigration of pathogenic clones from blood to spinal cord indicates that blood is not a suitable compartment to study EAE-mediating T cells.

CCL21-expression and accumulation of CCR7+ NK cells in livers of patients with primary sclerosing cholangitis.

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease, remains unknown. The aim of this study was to characterize peripheral blood and intrahepatic NK cells from patients with PSC. Peripheral blood samples from patients with PSC, other autoimmune liver diseases, and from healthy control individuals were used, as well as liver tissues from PSC patients undergoing liver transplantation. Multiparameter flow cytometry showed that peripheral blood NK cells from PSC patients were significantly enriched for CCR7+ and CXCR3+ cells, and CCR7+ but not CXCR3+ cells were also significantly increased within intrahepatic NK cells. PSC patients undergoing liver transplantation furthermore had significantly higher plasma levels of the CCR7-ligand CCL21, and the CXCR3-ligands CXCL10 and CXCL11, and significantly higher levels of CCL21, but not CXCL10, were detected in liver tissues. CCR7+ and CXCR3+ NK cells from PSC patients exhibited significantly higher functional capacity in peripheral blood, but not liver tissues, consistent with chronic activation of these NK cells in the inflamed liver. These data show that PSC is characterized by intrahepatic CCL21 expression and accumulation of CCR7+ NK cells in the inflamed liver tissue.

Review article: unanswered clinical and research questions in autoimmune hepatitis-conclusions of the International Autoimmune Hepatitis Group Research Workshop.

BACKGROUND: Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease that results in substantial morbidity and mortality with many unanswered clinical and research questions. Improved understanding of disease pathogenesis, including the extra-hepatic manifestations of AIH, may allow targeted treatments with greater efficacy and fewer associated adverse events. AIM: To identify the spectrum of unanswered clinical and research questions facing care providers in the management of patients with autoimmune hepatitis (AIH). METHODS: The International Autoimmune Hepatitis Group initiated a series of research workshops to start to address these questions. Key issues were discussed in small group sessions with collation of all discussions to be summarised in this manuscript. RESULTS: Key issues were identified as: the need for better understanding of disease pathogenesis, standardisation of the methods and assays used to evaluate autoantibodies in AIH, refinement of the histopathological criteria for "typical" or "compatible" AIH, focus on the interaction with non-alcohol related fatty liver disease, how to treat acute severe AIH, better assessment of quality of life in adults and paediatrics, standardising use of standard, third-line and experimental therapies in AIH and search for biomarkers early in the disease course that predict outcome. CONCLUSION: This workshop has outlined the key unanswered clinical and research questions to help to define the research agenda in AIH.

Colitis Promotes a Pathological Condition of the Liver in the Absence of Foxp3+ Regulatory T Cells.

Inflammatory bowel disease is associated with extraintestinal diseases such as primary sclerosing cholangitis in the liver. Interestingly, it is known that an imbalance between Foxp3+ regulatory T cells (Treg) and Th17 cells is involved in inflammatory bowel disease and also in primary sclerosing cholangitis. To explain these associations, one hypothesis is that intestinal inflammation and barrier defects promote liver disease because of the influx of bacteria and inflammatory cells to the liver. However, whether and how this is linked to the Treg and Th17 cell imbalance is unclear. To address this, we used dextran sodium sulfate (DSS) and T cell transfer colitis mouse models. We analyzed the pathological conditions of the intestine and liver on histological, cellular, and molecular levels. We observed bacterial translocation and an influx of inflammatory cells, in particular Th17 cells, to the liver during colitis. In the DSS colitis model, in which Treg were concomitantly increased in the liver, we did not observe an overt pathological condition of the liver. In contrast, the T cell-mediated colitis model, in which Treg are not abundant, was associated with marked liver inflammation and a pathological condition. Of note, upon depletion of Treg in DEREG mice, DSS colitis promotes accumulation of Th17 cells and a pathological condition of the liver. Finally, we studied immune cell migration using KAEDE mice and found that some of these cells had migrated directly from the inflamed intestine into the liver. Overall, these data indicate that colitis can promote a pathological condition of the liver and highlight an important role of Treg in controlling colitis-associated liver inflammation.

Dietary and metabolic modulators of hepatic immunity.

The liver is the central metabolic organ of the organism and is thus constantly exposed to gut-derived dietary and microbial antigens. The liver maintains homoeostatic tolerance to these mostly harmless antigens. However, the liver also functions as a barrier organ to harmful pathogens and is thus permissive to liver inflammation. The regulation of the delicate balance between liver tolerance and liver inflammation is of vital importance for the organism. In recent years, a general role for dietary components and metabolites as immune mediators has been emerging. However, although the liver is exposed to a great deal of metabolic mediators, surprisingly, little is known about their actual role in the regulation of hepatic immune responses. Here, we will explore the possible impacts of metabolic mediators for homoeostatic and pathological immunity in the liver, by highlighting selected examples of metabolic immune regulation in the liver.