Increased Sensitivity of Treg Cells from Patients with PBC to Low Dose IL-12 Drives Their Differentiation into IFN-Gamma secreting Cells

IL-12 is a pro-inflammatory cytokine that induces the production of interferon-gamma (IFN gamma) and favours the differentiation of T helper 1 (Th1) cells. In the presence of IL-12 human Treg cells acquire a Th1-like phenotype with reduced suppressive activity in vitro. Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterised by high Th1 and Th17 infiltrating cells, reduced frequencies of Treg cells, and a genetic association with IL-12 signalling. Herein, we sought to evaluate the IL-12 signalling pathway in PBC pathology, by studying human samples from patients with PBC, alongside those with primary Sjogren's syndrome (pSS) (autoimmune disease with IL-12 signalling gene association), primary sclerosing cholangitis (PSC) (cholestatic liver disease without IL-12 gene association) and healthy individuals. Our data revealed that TLR stimulation of PBC (n = 17) and pSS monocytes (n = 6) resulted in significant induction of IL12A mRNA (p < 0.05, p < 0.01, respectively) compared to PSC monocytes (n = 13) and at similar levels to HC monocytes (n = 8). PSC monocytes expressed significantly less IL-12p70 (108 pg/ml, mean) and IL-23 (358 pg/ml) compared to HC (458 pg/ml and 951 pg/ml, respectively) (p < 0.01, p < 0.05). Treg cells from patients with PBC (n = 16) and pSS (n = 3) but not PSC (n = 10) and HC (n = 8) responded to low dose (10 ng/ml) IL-12 stimulation by significant upregulation of IFN gamma (mean 277 and 254 pg/ml, respectively) compared to PSC and HC Treg cells (mean 22 and 77 pg/ml, respectively)(p < 0.05). This effect was mediated by the rapid and strong phosphorylation of STAT4 on Treg cells from patients with PBC and pSS (p < 0.05) but not PSC and HC. In the liver of patients with PBC (n = 7) a significantly higher proportion of IL-12R beta 2(+)Tregs (16% on average) was detected (p < 0.05) compared to other liver disease controls (5%)(n = 18) which also showed ex vivo high IFNG and TBET expression. CONCLUSION: Our data show an increased sensitivity of PBC and pSS Treg cells to low dose IL-12 stimulation, providing ongoing support for the importance of the IL12-IL-12R beta 2-STAT4 pathway on Treg cells in disease pathogenesis and potentially treatment.