Monocytes as a potential trigger of Th17-differentiation in primary sclerosing cholangitis (PSC)

Background:

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease and immune dysregulation is considered to be part of disease pathogenesis. We could recently show that in patients with PSC, IL-17 producing T cells were located around the bile ducts. In vitro stimulation of peripheral blood mononuclear cells (PBMC) with pathogens, that were previously identified to be of clinical relevance for PSC, resulted in increased Th17-responses compared to controls and healthy individuals. In this study, we analyzed whether Th17-differentiation in PSC already occurs in vivo and aimed to identify potentialmechanisms involved in the differentiation to Th17 cells.

Methods:

Blood samples of patients with PSC (n = 37), PBC (n = 29) and healthy controls (n = 33) 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. Blood derived monocytes were stimulated in vitro with heat-inactivated Candida albicans for 24 hours. Trans-well experiments were performed with the human biliary epithelial cell line H69, or isolated PSC-derived cholangiocytes, together with monocytes and C. albicans for 24 hours. Cytokine production was analyzed by ELISA and gene expression was determined in cholangiocytes using TaqMan analysis.

Results:

Upon ex vivo stimulation with PMA/Ionomycin, patients with PSC showed significantly increased numbers of IL-17 producing CD4+ T cells in peripheral blood compared to patients with primary biliary cholangitis (PBC) and healthy controls (PSC: 2.25% vs. PBC: 1.56% vs. Healthy: 1.55%, p = 0.0007). Upon stimulation with C.albicans, monocytes from PSC patients produced significantly more cytokines required for Th17-differentiation, such as IL-1β and IL-6, compared to healthy controls (IL-1β= PSC: 18901 pg/ml vs. Healthy: 14698 pg/ml, p = 0.046 and IL-6 = PSC: 46603 pg/ml vs. Healthy: 35903 pg/ml, p = 0.037). Moreover, IL-1β, which was secreted by pathogen-activated monocytes, stimulated the secretion of IL-17 and the monocyte-recruiting chemokines CCL-20 and CCL-2 as well as IL-6 by cholangiocytes. Of note, CD14 positive monocytes were found to be located close to bile ducts within inflamed portal tracts of PSC patients and immunohistochemical RNAscope® staining of liver tissue from PSC patients revealed mRNA expression of IL-6 and IL-1β in cholangiocytes within inflamed portal tracts.

Conclusion:

PSC patients show increased Th17-differentiation already in vivo. Monocytes may contribute to that finding, since they produced increased amounts of cytokines required for Th17-differentiation, locate around bile ducts and were able to stimulate the release of Th17 recruiting chemokines by cholangiocytes. Taken together, these results should stimulate further research into the functional role of monocyte/Th-17 interaction in the pathogenesis of PSC.