Role of cell autophagy in the generation of IgM and hepatic fibrosis in primary biliary cholangitis

Objective Autophagy in the immune and autoimmune diseases has been concerned more and more. We investigated the potential role of cell autophagy of B cells generating IgM in primary biliary cholangitis (PBC), and explored the relationship between the cell autophagy of hepatic stellate cells (HSCs) and hepatic fibrosis in PBC. Methods We examined the aggregation degree of microtubule-associated protein light chain 3II (LC3II) in B cells of PBC (n = 21), health control (HC, n = 15) and disease control (DC, n = 8, active hepatitis B). The expression level of p62/SQSTM1 (p62) in B cells was detected by flow cytometry. ELISA was adopted to detect the level of IgM in the B cell culture supernatant under the basal condition, activated condition(stimulated by pokeweed mitogen, PWM) and inhibited condition(inhibited by autophagy inhibitor, 3-methyladenine, 3-MA) respectively. We detected the expression of alpha-smooth muscle actin (alpha-SMA), the infiltration level of LC3II, transforming growth factor-beta (TGF-beta), platelet-derived growth factor-bb (PDGF-bb), and collagen I in the hepatic tissues of five PBC patients and two HC individuals. Results When B cells were stimulated and activated by PWM, the expression of p62 increased, and the mean fluorescence intensity (MFI, 2404.8 +/- 689.0) of p62 in PBC was lower than that in HC (2966.8 +/- 488.9,P = 0.0227). Under 3-MA treatment, the MFI expressed of p62 in B cells weakened. The reduction difference in PBC (466.4 +/- 214.9) was smaller than that in HC (1166.6 +/- 231.2, P = 0.0000) and DC (1184.8 +/- 197.7, P = 0.0001). The level of generating IgM in the PBC group was obviously higher than that in the HC group (65.7 +/- 15.4 vs. 49.5 +/- 20.4 ng/ml, P = 0.0357). Before and after B cells were treated with 3-MA, the peak level of IgM did not significantly diminish in PBC and DC (65.7 +/- 15.4 vs. 59.6 +/- 18.7 ng/ml, P = 0.2965; 50.1 +/- 17.4 vs. 48.4 +/- 12.3 ng/ml, P = 0.8336). The immunohistochemical result revealed that the expression level of collagen I, alpha-SMA, TGF-beta and PDGF-bb in PBC was higher than that in HC, but the expression of LC3II was lower. Similarly, immunofluorescence assay revealed that the fluorescence intensity of collagen I was higher but LC3II was lower in PBC than that in HC. Conclusion The high level autophagy of B cells from PBC patients is one important factor to synthesize and secrete IgM. Hepatic fibrosis of PBC is probably associated with the weakened autophagy of HSCs.