| Primary biliary cirrhosis (PBC) is a chronic cholestatic autoimmune disease caused by immune response to intrahepatic small bile duct. PBC is characterized by loss of tolerance to mitochondrial autoantigens and destruction of small bile ducts. Terminal PBC patients have the development of liver fibrosis and cirrhosis, severely threatening life and health. In our previous work, we have developed IL-2Ra(-/-) mice as an animal model of human PBC. IL-2Ra(-/-) mice have appearance of antimitochondrial antibodies (AMAs) in serum, apparent portal inflammation and bile duct damage in histopathology, presence of CD4+and CD8+T cells infiltration in liver, and elevation of several inflammatory cytokines. These results show that IL-2Ra(-/-) mice develop spontaneous autoimmune cholangitis with several symptoms that resemble human PBC. These mice also spontaneously develop inflammatory bowel disease (IBD). So IL-2Ra(-/-) mice can also be used for studying the crosstalk between intestinal microenvironment (including intestinal microbe and intestinal inflammation) and liver inflammation. Based on previous studies, deletion of CD4in IL-2Ra(-/-) mice reduces colonic inflammation but not hepatic inflammation, whereas deletion of CD8attenuates bile duct damage and portal inflammation but not colonic inflammation. Enhanced Th1and Th17responses are likewise found in IL-2Rα(-/-) mice compared to wild type controls. Our earlier work demonstrated increased serum levels of IL-12p40in IL-2Ra(-/-) than in IL-2Ra(+/-)mice. IL-12p40is a subunit shared by IL-12and IL-23. IL-12and IL-23belong to IL-12family. There has been considerable interest in the IL-12family in a variety of autoimmune diseases, and in particular, in human PBC based on GWAS (genome wide association studies). But the specific role of IL-12p40in PBC still remains unclear.So we tried to knockout IL-12p40in IL-2Ra(-/-) mice to study the role of IL-12p40in PBC, and we found IL-12p40(-/-)IL-2Ra(-/-) mice as a better animal model of PBC compared with IL-2Ra(-/-) mice. Based on this model, we did intensive study for hematopoietic stem cells (HSCs), cytokines, and intestinal microbe.The results obtained in this study are as follows:1. Knockout of IL-12p40exacerbated autoimmune cholangitis and induced liver fibrosisSurprisngly, p40(-/-)IL-2Ra(-/-) mice manifested more severe portal inflammation and bile duct damage, but a significant reduction in colitis. p40(-/-)IL-2Ra(-/-) mice displayed high frequency of splenomegaly, and the number of spleen and liver cells is significant higher than IL-2Ra(-/-) mice, whereas the number of mesenteric lymph node (mLN) cells in p40(-/-)IL-2Ra(-/-) mice was lower than IL-2Ra(-/-) mice. Interestingly, in p40(-/-)IL-2Ra(-/-) mice, the number of spleen cells positively correlated with the number of liver cells, and in contrast these was no correlation between the numbers of of mLN cells and liver cells. Using several methods including fibrosis scoring, hyperproline content measurement, immunohistochemistry, real-time quantitative PCR, we demonstrated that distinct from IL-2Ra(-/-) mice, p40(-/-)IL-2Ra(-/-) mice spontaneously developed liver fibrosis, mimics symptoms of PBC patients in the late stage.We analyzed phenotype of p40(-/-)IL-2Rα(-/-) mice, and found the percent of T cells, CD4+T cells and CD8+T cells were increased. Major component of hepatic CD8+T cells in p40(-/-)IL-2Ra(-/-) mice were effector memory cells, and major component of hepatic CD8+T cells in IL-2Ra(-/-) mice were central memory cells. Major component of hepatic CD4+T and CD8+T cells in p40(-/-)IL-2Ra(-/-) mice were PD-1+cells, and major component of hepatic CD4+T and CD8+T cells in IL-2Ra(-/-) mice were PD-1-cells. Compared with IL-2Ra(-/-) mice, CD4+T cells in p40(-/-)IL-2Ra(-/-) mice had an enhanced ability to secrete IFN-γ and IL-10, but an imparied ability to secrect IL-17A. RT-PCR results also demonstrated that enhanced Th1and reduced Th17responses in the liver of p40(-/-)IL-2Ra(-/-) mice2. p40(-/-)IL-2Ra(-/-) mice displayed dysregulation of hematopoietic systemInterestingly, we found p40(-/-)IL-2Rα(-/-) mice had increased number and percentage of bone marrow Lineage-c-Kit+Sca-1+cells (LSK cells) compared with wild type mice. p40(-/-)IL-2Ra(-/-) mice also had extramedullary hematopoiesis because of the presence of LSK cells in spleen and liver. LSK cells were usually considered to contain hemopoietic stem cells (HSCs) and progenitor cells. The phenotype of bone marrow LSK cells in p40(-/-)IL-2Ra(-/-) mice was quite different from wild type mice, as they had larger size and granularity (high values of SSC and FSC), lower expression of CD86, and different expression patterns of CD150/CD48and CD34/CD135. The number and percent of erythrocytes, B cells, NK cells were reduced in the bone marrow of p40(-/-)IL-2Ra(-/-) mice, suggesting the hematopoiesis disorder of HSCs. Surprisingly, bone marrow LSK cells in p40(-/-)IL-2Ra(-/-) mice had high expression of MHCII molecules, indicating that these cells may act as antigen-presenting cells (APCs), but wild type mice did not have this characteristic.We found the percentage and number of bone marrow T cells, CD4+T cells, CD8+T cells, were significantly increased in p40(-/-)IL-2Ra(-/-) mice. We guessed bone marrow T cells played an important role in LSK cell dysregulation. We examined the status of young mice, and found with3and4weeks old but not2weeks old, p40(-/-)IL-2Rα(-/-) mice displayed obvious splenomegaly, bone marrow T cell infiltration, and bone marrow LSK cell dysregulation. So we supposed that the changes of gut bacteria may influence bone marrow LSK cell dysregulation via T cells after weaning.To study whether the hematopoietic ability was changed in bone marrow from p40(-/-)IL-2Ra(-/-) mice, we did bone marrow chimera experiment. Bone marrow cells after treatment with red blood cell lysis buffer from Ly5.1p40(-/-)IL-2Ra(-/-) mice and Ly5.2p40(-/-)IL-2Ra(+/-) mice were mixed at the ratio1:1and transferred to lethal irradiated Ly5.1/5.2mice. After8weeks, we killed recipient mice and detected the Ly5.1or Ly5.2origins of cell subsets from different organs. We found the reconstitution ability of bone marrow cells from p40(-/-)IL-2Ra(-/-) mice was lower than p40(-/-)IL-2Ra(+/-) mice. We also did experiment of bone marrow LSK cells mixed at the ratio1:1, and demonstrated that the reconstitution ability of bone marrow LSK cells from p40(-/-)IL-2Ra(-/-) mice was much lower than p40(-/-)IL-2Ra(+/-) mice, indicating that much of bone marrow LSK cells in p40(-/-)IL-2Ra(-/-) mice were progenitor cells with weak reconstitution ability.3. IFN-y was the key cytokine in dysregulation of hematopoietic system in p40(-/-)IL-2Ra(-/-) miceAs p40(-/-)IL-2Ra(-/-) mice had enhanced T cell infiltration in bone marrow and T cells in p40(-/-)IL-2Ra(-/-) mice had very high expression of DFN-y, we supposed that T cell derived IFN-y may be the critical factor in LSK dysregulation. We generated p40(-/-)IFN-γ(-/-)IL-2Ra(-/-) mice by crossbacking p40(-/-)IL-2Ra(-/-) mice with IFN-y(-/-) mice, and compared to p40(-/-)IL-2Ra(-/-) mice. We found p40(-/-)IFN-γ(-/-)IL-2Ra(-/-) mice still contained liver inflammation, but did not have a huge spleen, In p40(-/-)IFN-y(-/-)IL-2Ra(-/-) mice, the percent of T cells in liver and spleen were not changed, and T cells still had a high activated phenotype. However, we found the percent and number of infiltrating T cells were decreased in the bone marrow of p40(-/-)IFN-γ(-/-)IL-2Ra(-/-) mice, and the percent and number of B cells and erythrocytes were increased. Importantly, the percent, number and phenotype of bone marrow LSK cells in p40(-/-)IFN-γ(-/-)IL-2Ra(-/-) mice were completely recovered and close to wild type mice, with almost completely disappearance of MHCII molecules. These results demonstrated that hematopoietic system hematopoietic system was totally dependent on IFN-y.4. Intestinal microorganisms participated in regulation of liver inflammation and hematopoietic system in p40(-/-)IL-2Ra(-/-) miceTo eliminate most of the symbiotic bacteria in gut, we fed p40(-/-)IL-2Ra(-/-) mice under SPF condition with water containing ampicillin, metronidazole, neomycin, vacomycin at4weeks old. After antibiotics feeding for8weeks, we sacrificed mice at12weeks old. We found clearance of intestinal microbe signifantly ameliorated splenomegaly and liver inflammation. The percent and number of T cells in p40(-/-)IL-2Ra(-/-) mice with clearance of intestinal bacteria were reduced, while the percent of effector memory cells in T cells was decreased, and the ability of IFN-y secretion in T cells was also decreased. Clearance of intestinal bacteria led to reduced percent and number of bone marrow LSK cells in p40(-/-)IL-2Ra(-/-) mice, characteristics of LSK cells dysregulation were also ameliorated. These data demonstrated that intestinal symbiotic bacteria in p40(-/-)IL-2Ra(-/-) mice could promote liver inflammation and influence bone marrow hematopoietic system.We extracted the genome DNA of faeces in colon from p40(-/-)IL-2Ra(-/-) mice and p40(-/-)IL-2Ra(+/-) mice, and measured the content of several kinds of bacteria by using qPCR to dectect16S RNA types. Interestingly, the content of Antinobacteria, Betaproteobacteria, Gammaproteobacteria and Lactobacillus group were increased in p40(-/-)IL-2Ra(-/-) mice without huge spleen compared with p40(-/-)IL-2Ra(-/-) mice with huge spleen and p40(-/-)IL-2Ra(+/-) mice, but we failed to observe the differences between p40(-/-)IL-2Ra(-/-) mice with huge spleen and p40(-/-)IL-2Rα(+/-) mice. Disruption of intestinal microbiota in p40(-/-)IL-2Ra(-/-) mice without huge spleen may contribute to inhibition of inflammation. But the definite role of which kinds of bacteria in p40(-/-)IL-2Ra(-/-) mice still remained unclear.In summary, this study discovered that p40(-/-)IL-2Ra(-/-) mice had an aggravated autoimmune cholangitis and an alleviated colitis, and spontaneously developed liver fibrosis. So compared with IL-2Ra(-/-) mice, p40(-/-)IL-2Ra(-/-) mice could better mimicing human PBC. This study also found p40(-/-)IL-2Ra(-/-) mice had dysregulation of hematopoietic system, and HSC-like cells with high expresssion of MHCII molecules. These phenomena were dependent on IFN-y and regulated by gut symbiotic bacteria. Using this model, we explored the relationship between liver inflammation, hemotopoietic system, cytokines and gut microenvironment in autoimmune liver disease, for studying the pathogenic mechnism of PBC and providing theory evidence to search effective therapic targets. |
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