The Regulatory Function Of Peritoneal Cavity B Cells In Murine Primary Biliary Cholangitis

Primary biliary cholangitis (PBC) is a liver autoimmune disease, characterized by lymphocytic infiltrations in portal tracts and anti-mitochondrial antibodies (AMAs) secreted by autoreactive B cells. Multiple factors are involved in the onset of PBC, including immunological dysfunction, genetic predisposition and environmental factors. Previous work had reported that the role of B cells in the pathogenesis of PBC was complicated and contradictory, including pathogenic effect and regulatory function. However, further investigation and exploration are still needed as the detailed mechanisms remain unknown.B cells are major immune cell subsets, which contain two distinct B cell lineages, B1 (including B1a and B1b) and B2 cells. They arise from distinct progenitors, and differ in development, tissue distribution, phenotype and function. During autoimmune disease, B cells are generally considered to play a pathogenic role through antibody production. But recent studies demonstrated that B cells contained functionally distinct subsets which performed pathogenic or regulatory functions respectively. The mechanisms of regulatory B cells include IL-10 and TGF-0 production, secondary Ag presentation, interaction with pathogenic T cells and induction of regulatory T (Treg) cell generation. B1 cells are unique B cell populations that are enriched in the peritoneal and pleural cavities and contribute to immune protection through spontaneous production of natural antibodies and take part in immune regulation by acting as antigen-presenting cells and secreting cytokines. B1 cells derived natural antibodies are essential for protective immunity in bacterial and viral infections. B1a cells are major parts of B1 cells in peritoneal cavity. Classic B1a cell functions include production of serum IgM, secretion of intestinal IgA, and immuno-regulation. B1a cells are also major sources of anti-inflammatory cytokine IL-10 and have been shown to play regulatory roles in contact hypersensitivity model and pathological conditions.To better define the functions of B cell subsets in the disease progress of PBC, we took advantage of our recently-generated p40-/-IL-2Rα-/- mice and well-studied dnTGF-βRⅡmice as PBC models. We mainly investigated the deregulation of peritoneal cavity (PC) B1a cells and murine primary biliary cholangitis in p40-/-IL-2Rα-/- mice, meanwhile co-transfered the peritoneal cavity B cells from dnTGF-βRⅡ mice to prove the regulatory function of peritoneal cavity B cells in PBC models. The results were as follows:1. Altered PC environment affected PC B cell number and was correlated with the severity of liver inflammation in p40-/-IL-2Rα-/- miceTo investigate the change of PC environment under liver injury, we used FACS to detect the change of cell subsets in PC, and found the total number of PC cells was increased in p40-/-IL-2Rα-/- mice. Majority of them were T cells, especially CD8+T cells, while B cell number dramatically decreased. In PC CD4+ and CD8+ T cells, the Th1 cell associated cytokine IFN-y producing ability was higher in p40-/-IL-2Rα-/-mice compared with p40-/-IL-2Rα-/- mice. The concentrations of TNF and MCP-1 were significantly increased in PC lavage fluid of p40-/-IL-2Rα-/- mice compared with p40-/-IL-2Rα-/- mice. Thence, we speculated that the change of PC cell subsets in p40-/-IL-2Rα-/- mice might resulted from the inflammatory environment of PC. Using correlation analysis, we found PC cell number was positively correlated with the number of liver mononuclear cells (MNCs) in p40-/-IL-2Rα-/- mice, and the frequency of B1a in B cells was negatively correlated with PC and liver MNC numbers. In addition, there was a negative correlation between the frequency of B1a in B cells and the frequencies of pathogenic CD8+ T cells in PC and liver.2. Bla cell number decreased and functional molecules changed in p40-/-IL-2Rα-/-miceBy detecting the change of B cell subsets during the disease progress in detail, we found the frequencies and cell numbers of B1a and B1b cells were much lower in p40-/-IL-2Rα-/- mice compared with p40-/-IL-2Rα-/- mice. And the frequency of B1a cells decreased gradually with age in p40-/-IL-2Rα-/- mice, which was due to the decreased proliferation profile and increased apoptosis level of B1a cells from p40-/-IL-2Rα-/- mice. As the Bla cell number in PC significantly decreased in p40-/-IL-2Rα-/- mice, the level of regulatory markers, like IL-10, CTLA-4 and GITR, also decreased in B1a cells of p40-/-IL-2Rα-/- mice. The expression of CD44 and CD80, which correlated with B cell activation and co-stimulation, were downregulated significantly in B1a cells of p40-/-IL-2Rα-/- mice compared with p40-/-IL-2Rα-/- mice. Similar results were also detected in the PC B1a cells of dnTGF-PRII mice compared with their littermates, including the decrease of PC B1a cell frequency and the lower expression of IL-10, CTLA-4, CD44 and CD80.3. Transcription profile analysis of Bla cells in PC of p40-/-IL-2Rα-/- miceWe further comprehensively studied the transcription profile differed from B1a cells in the PC of p40-/-IL-2Rα-/- mice and p40-/-IL-2Rα+/- mice and revealed the inner mechanism. We used microarrays and verification by RT-PCR, found that there was a differential transcription of 381 genes which changed greater than two fold in B1a cells of p40-/-IL-2Rα-/- mice compared with p40-/-IL-2Rα-/- mice. The differential changed genes including the cytokine-cytokine receptor interaction, transcription factor activity and cell cycle realted genes. Amongst B cell function related genes, the expression of ITIM domain containing protein, such as CD72 (Cd72) and CD22 (Cd22) was up-regulated. The expression of Cd44 and Ctla4 was down-regulated and it was consistent with FACS results. In addition, the expression of Ahr, which could inhibit IL-10 secretion, was up-regulated. These findings exhibited the difference between B1a cells from p40-/-IL-2Rα-/- mice and p40-/-IL-2Rα-/- mice. These results revealed the importance of B1a cell disorder during the pathogenesis of PBC, and suggested some genes/pathways that was worth considering in the study of human PBC.4. The assessment of therapeutic potential of PC B cells to cholangitis in PBC mice modelTo investigate the therapeutic potential of the PC B cells to cholangitis in PBC mice model, using adoptive transfer experiments, we found that adoptive transfered CD8+T cells in spleen of dnTGF-βRⅡ mice induced severe autoimmune cholangitis in RAG1-/- recipient mice. But the liver lymphocyte infiltration was lower in some co-transfer CD8+T cells and PC B cells recipient mice. The B cells were either from dnTGF-βRⅡ mice or WT mice. These experiments proved that the PC B cells in PBC mice model exhibited powerful regulatory function under inflammatory scenario.In summary, we found Bla cells dramatically decreased in PC of p40-/-IL-2Rα-/-mice under inflammatory scenario. The regulatory function of B1a cells was disrupted and the expression of immune regulatory molecule, including IL-10 and CTLA-4 were downregulated in B1a cells from p40-/-IL-2Rα-/- mice. These led to the downregulation of suppression on pathogenetic T cells by PC B1a cells. Similar phenotypes were also detected in the PC B1a cells of dnTGF-PRⅡ mice compared with p40-/-IL-2Rα-/- mice. We suggest that the dysfunction of B1a cells in the PC of this murine model of autoimmune cholangitis results in aggravated cholangitis. This highlights a new potential therapeutic target in PBC.