Effect And Mechanism Of Toll-like Receptors In The Pathogenesis Of Murine Autoimmune Liver Injury

Toll-like receptors (TLRs), one of the pattern recognition receptors (PRRs), recognize a diverse range of pathogen associated molecular patterns. TLRs not only play a critical role in antimicrobial host defense, but also participate in the initiation and progression of autoimmune diseases. The liver is the largest organ for toxic elimination and is continuously exposed to gut-derived pathogen products. Increasing evidences suggest that TLRs and pathogen products are involved in the pathogenesis of liver diseases; however, the underlying mechanisms remain obscure. Our present study aims to investigate the modulating mechanisms of TLRs in the pathogenesis of liver injury via animal model of liver injury.We examined the liver injury by checking serum transaminase level, cytokine release and the liver pathologic changes in this study. In experiments of cell transfer, cell depletion, cell purification, and AST cytotoxicity assay, we investigated the effects of the specific lymphocyte population in the process of liver injury. The FACS analysis provided us with the precise information about specific molecular expression, cell phenotype, activation and cytokine secretion. The phophorlation of intracellular signaling molecular was detected by Western blotting. Moreover, we also applied specific blocking with antibodies to explore the potential communication of TLRs inside cell. In the following, we summarize the major results of our experiments:TLR3 ligand attenuates LPS-induced liver injury via down-regulation of TLR4 expression on MacrophagesThe low dose of TLR3 ligand, poly I C, pretreatment prevented LPS/D-GalN-induced fulminant liver injury. The protective effect of poly I:C was also confirmed by the results of the decreased mortality and alleviated hepatic necrosis. Poly I:C pretreatment inhibited elevation of serum ALT and AST levels and inflammatory cytokines in LPS/D-GalN-treated mice. Furthermore, pathology analysis showed that poly I:C pretreatment completely inhibited hepatocytes destruction induced by co-injection of LPS and D-GalN. The evidences from specific cell depletion and cell adoptive transfer experiments showed that the inhibitory effect of poly I:C on LPS/D-GalN-induced hepatitis was due to directly targeting macrophages. Depletion of NK cells, NKT cells,αβT cells, andγδT cells did not abolish the protective effects of poly I:C on LPS/D-GalN-induced liver injury. Adoptive transfer of PBS-treated macrophages restored LPS/D-GalN-induced liver injury in macrophage-depleted mice, while transfer of macrophages treated with poly I.C in vitro failed to restore such injury.This study also showed that poly I:C treatment might down-regulate TLR4 (the ligand of LPS) expression on macrophage, and diminished the ability of macrophages to respond to LPS-induced signaling activation, which was possibly the primary mechanism for poly I:C to exert the inhibitory effect on liver injury. Treatment with poly I:C down-regulated the expression of TLR4 on Kupffer cells in mice, and similar results were found in peritoneal macrophages from C57BL/6 mice, or RAW264.7 cells in vitro. And poly I:C pretreatment impaired the signaling of MAPKs and NF-κB induced by LPS in RAW264.7 cells. Blockade of TLR3 with a TLR3 antibody abolished poly I.C down-regulation of TLR4 expression on RAW264.7 cells. Taken together, our findings suggested that activation of TLR3 by its ligand, poly I:C, induced LPS tolerance via down-regulation of TLR4 expression on macrophages.Preferential recruitment and activation of hepatic NKT cells in mice by the TLR9 ligandIn the study, we found that TLR9 ligand, CpG ODN, could preferentially promote the accumulation and activation of hepatic NKT cells in mice. After CpG ODN injection, the amount of hepatic NKT cells was increased about 1 fold, the cytokine release and cytotoxicity of hepatic NKT cells were largely enhanced, and the expressions of CD69 and Fas L on the hepatic NKT cells were also up-regulated. The depletion of macrophages markedly inhibited the effect of CpG ODN on hepatic NKT cells, while CpG ODN treated macrophages or the supernatant from macrophages could partially restore such phenomenon. IL-12 might be the effective component in the supernatant, which was demonstrated by the experiment with anti-IL-12 antibody. It suggested that cell-to-cell contact and the cytokine were all necessary for the effect of CpG ODN on hepatic NKT cells.Based on the specific effect of CpG ODN on hepatic NKT cells, we observed the effect of CpG ODN on the Con A-induced liver injury mediated by hepatic NKT cells in mice. We found that CpG ODN pretreatment could aggravate the Con A-induced liver injury, showing massive necrosis areas and elevated serum transaminase levels compared with PBS-pretreated group. Additionally, CpG ODN pretreatment largely enhanced the cytokine release of hepatic NKT cells.In summary, our study suggests that (1) TLR3 ligand, poly I:C, could down-regulate TLR4 (the ligand of LPS) expression on Kupffer cells, and diminished the ability of macrophages to respond to LPS-induced signaling activation, which was possibly contributed to the inhibitory effect of poly I:C on liver injury. (2) TLR9 ligand, CpG ODN-activated macrophages could increase the number of hepatic NKT cells and enhance hepatic NKT cell function by increasing its activation, cytokine release and cytotoxicity, which aggravated Con A-induced NKT cell-mediated liver injury in mice. The study strongly showed that TLR signalings are actively involved in the pathophysiology in the liver and exert their regulatory effects on autoimmune liver injury. It provides a valuable principle to the clinic treatment and medicine exploration.