Increased in vivo and in vitro TH17 differentiation in patients with Primary Sclerosing Cholangitis

Introduction: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease. Immune dysregulation is considered to be part of disease pathogenesis. Recently, we could show that in PSC patients, IL-17-producing T cells are located around bile ducts and the in vitro stimulation of peripheral blood mononuclear cells (PBMC) with pathogens resulted in increased Th17 responses. In this study, we further studied the role of Th17 cells in PSC disease pathogenesis and progression and investigated underlying mechanisms leading to increased Th17 responses.

Methods: 69 patients with PSC, 37 patients with primary biliary cholangitis (PBC) and 45 healthy controls were included in this study. Disease staging was defined by clinical and imaging signs of liver cirrhosis. IL-17 production of blood derived CD4+ T cells was analyzed by flow cytometry. IL-6 and IL-1β production from blood derived monocytes was analyzed by ELISA after in vitro stimulation with heat-inactivated C. albicans for 24 hours. For Th17 differentiation assays blood derived naïve CD4+ T cells were cultured for 12 days in the presence of IL-6, IL-1β, IL-23 and TGFβ. Th17 differentiation was analyzed by flow cytometry.

Results: Upon ex vivo stimulation of lymphocytes we observed significantly increased numbers of peripheral blood IL-17 producing CD4+ T cells in PSC patients compared to control groups (PSC: 2.52% vs. Healthy: 1.67% vs. PBC: 1.61%, p = 0.0007). The increased frequencies of Th17 cells in PSC patients correlated with advanced stage of disease (advanced PSC: 3.79% vs. PSC: 2.17% vs. Healthy: 1.70% vs. advanced PBC: 1.78% vs. PBC: 1.7%, p = 0.0006), indicating a potential role of IL-17 in disease progression. Monocytes from PSC patients produced significantly more IL-1β (PSC: 18901 pg/ml vs. Healthy: 14698, p = 0.046) and IL-6 (PSC: 46603 pg/ml vs. Healthy: 35903 pg/ml, p = 0.037) than healthy controls upon stimulation with C. albicans. Both cytokines are required for Th17 cell differentiation. Additionally, in in vitro conversion assays we found that naïve CD4+ T cells, isolated from PSC patients, showed an increased differentiation towards a

Th17 phenotype compared to control groups (PSC: 1.36% vs. Healthy: 0.41% vs. PBC:= 0.31%, p = 0.0002).

Conclusions: Patients with PSC showed higher frequencies of Th17 cells in peripheral blood and these frequencies were associated with disease stage. Monocytes from PSC patients produced more cytokines required for Th17 differentiation and the ability of naïve CD4+ T cells to convert into Th17 cells in vitro. Together these results should stimulate further research into the pathogenetic role of IL-17 in this enigmatic liver disease.