Abstract

Background: Autoimmune hepatitis is a serious chronic liver disease with immune disorders, histological lesions and liver dysfunction, with a gradually increasing prevalence. Yet the cellular and molecular mechanisms of immune dysregulation in AIH are poorly understood.

Methods: Using proteomic analysis, we comprehensively profiled the differentially expressed proteins and signaling pathways of liver during human AIH. Then, monocytes and macrophage from blood and livers of AIH patients and controls were analyzed. The recruitment and polarization of monocyte-derived macrophages in AIH and the mechanism of CCL2/CCR2 axis activation were investigated by Concanavalin induced experimental AIH (EAH). The CCL2/CCR2 axis was blocked by CCL2 neutralizing antibody and CCR2 antagonist to determine its effect on AIH mice. Finally, M2 macrophage-derived extracellular vesicles (M2-EVs) were isolated and extracted as drug delivery tool of CCR2 antagonist, and its therapeutic effect on AIH mice was determined.

Results: Proteomic analysis took expression ratio (FC) > 1.5 times and P<0.05 as screening criteria, and a total of 1028 proteins were identified as increased or decreased. KEEG analysis suggested that differential expressed proteins were mainly associated with metabolic processes. The expression of mononuclear macrophage system marker proteins were increased in the liver tissue of AIH patients as revealed by proteomic analysis and immunohistochemistry. The proportion of classical monocytes in peripheral blood of AIH was increased, which was positively correlated with the levels of ALT and AST of AIH patients. The co-localization analysis of liver tissue suggested that CCL2 originated from Kupffer cells (KC), and the expression of CCR2 increased after circulating monocytes infiltrated liver. The expression of M1 marker in AIH liver tissue increased. AIH mouse models suggest mobilization of inflammatory monocytes on the bone marrow-liver axis and spleen-liver axis. Blocking-up of CCL2/CCR2 axis with CCL2 neutralizing antibody or CCR2 antagonist, respectively, alleviated liver injury in AIH mice, while recombinant CCL2 injection increased recruitment of inflammatory monocytes with bone marrow-liver axis and spleen-liver axis to liver, aggravating liver injury. CCR2 antagonist-M2-EV can target circulating mononuclear cells and activated mononuclear macrophages in the liver, respectively, to reduce liver injury in AIH mice.

Conclusion: AIH mediates the recruitment of inflammatory monocytes from bone marrow and spleen to liver through the CCL2/CCR2 axis, which can be inhibited by different methods to reduce liver inflammatory injury in AIH mice. M2- EVs delivers CCR2 antagonists targeting activated pro-inflammatory monocytes and hepato-splenic mononuclear macrophage system in the circulating pool, indicating that CCR2 antagonists-EVs could be a potential agent for liver and monocyte targeted therapy for AIH.