Tanja Schoknecht

UNLABELLED: T helper (Th)17 cells are important for host defense against bacteria and fungi, but are also involved in the pathogenesis of autoimmune diseases. In primary sclerosing cholangitis (PSC), bile fluid is frequently colonized with pathogens and its strong association with inflammatory bowel disease suggests the contribution of pathogen responses to disease pathogenesis. Interleukin (IL)-17A, the signature cytokine of Th17 cells, was recently described to promote inflammation and fibrosis within the liver. Therefore, we investigated Th17 immune response to pathogens in patients with PSC. Bile fluid was obtained by endoscopic retrograde cholangiography, and bacterial and fungal species grew in the majority of samples. In addition, bacterial RNA was stained in liver sections using 16sRNA fluorescence in situ hybridization and was detected in the portal tracts in 12 of 13 tested PSC patients. Bacteria grown from patients' bile fluid were then used to stimulate peripheral blood mononuclear cells (PBMCs) and to assess their Th17 response. Compared to healthy controls or primary biliary cirrhosis patients, PBMCs from PSC patients manifested significantly higher frequencies of Th17 and Th1/Th17 cells after pathogen stimulation. The highest frequencies of Th17 cells were detected after stimulation with Candida albicans, a pathogen that has been linked to disease progression. Immunohistochemically, IL-17A-expressing lymphocytes were detected within the periductal areas of PSC patients. Th17 induction was also noted after stimulation of Toll-like receptor 5 or 7, but not of other pattern recognition receptors tested, pointing to signaling pathways potentially involved in Th17 induction in PSC. CONCLUSION: We demonstrate an increased Th17 response to microbial stimulation in patients with PSC. These data should prompt further studies investigating the link between pathogen responses, inflammation, and fibrosis in patients with 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 e x 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‐1β 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, CD14 hi CD16 int and CD14 lo CD16 hi 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.

Abstract The pathogenesis of the progressive liver disease, primary sclerosing cholangitis (PSC), remains largely elusive. The strong genetic association with HLA loci suggests that T cell–dependent, adaptive immune reactions could contribute to disease pathogenesis. Recent studies have indicated that PSC is also associated with polymorphisms in the locus encoding for proapoptotic Bim (BCL2L11). Bim is crucial for the maintenance of immunologic tolerance through induction of apoptosis in activated T cells. Of interest with regard to PSC is the finding that BCL2L11-deficient mice develop periductular infiltrates. We, therefore, investigated, whether defective apoptosis of T cells might contribute to the phenotype of PSC. Thus, we induced apoptosis of T cells from patients with PSC and controls by repeated T cell receptor (TCR) stimulation or cytokine withdrawal. We found that CD4+ T cells, but not CD8+ T cells, from patients with PSC exhibited significantly reduced apoptosis in response to both, TCR restimulation or cytokine withdrawal. This increased apoptosis resistance was associated with significantly reduced up-regulation of proapoptotic Bim in T cells from patients with PSC. However, T cell apoptosis did not seem to be influenced by the previously described BCL2L11 polymorphisms. Reduced CD4+ T cell apoptosis in patients with PSC was not due to reduced cell activation, as indicated by a similar surface expression of the activation markers CD69, CD25, and CD28 in T cells from patients and controls. Thus, decreased apoptosis of activated CD4+ T cells may be part of the immune dysregulation observed in patients with PSC.