Studies On The Role And Mechanisms Of Il-17 In Con A-induced Liver Injury

Acute and chronic liver diseases are still a major health problem and caused by various etiologies. Immune-mediated mechanisms play a central role in autoimmune and viral hepatitis and determine disease outcomes. Despite the advanced treatment, a high percentage of individuals fail to respond to the conventional methods and liver transplantation is ultimately required. Therefore, a better understanding of immune mechanism underlying the haptitis is needed for the generation of more effective therapeutic strategies against the disorder. Current evidence suggests that inhibiting the over-heated immune response or directly preventing liver cell damage may have beneficial effect in autoimmune heptitis.Con A-induced hepatitis is a well-defined experimental animal model of acute hepatitis. It mimics human autoimmune hepatitis (AIH) in many aspects. Con A-induced hepatitis is accompanied by an increase in the serum concentration of several cytokines, including TNF-α, IL-6, IFN-y, and IL-1. TNF-α, appeared to be critical for tissue damage in acute liver inflammation. During the early stages, TNF-αdirectly mediates liver cell damage. The importance of TNF-a as a distal mediator in this model is substantiated by the observation that passive immunization against TNF-αprotected mice from Con A-induced liver injury. IL-6, a pleiotropic cytokine with a wide range of biological activities, produced by monocytes/macrophages/T cells and B cells, plays a central role in acute phase reaction.IL-17 expression has been found to be increased in the serum of AIH patients. However, the role of IL-17 in Con A-induced liver injury remains unclear. As such, with the controversy of the reports of IL-17 in Con A-induced hepatitis, we aim to investigate the expression of IL-17 and its effect on the production of TNF-αand IL-6 by kupffer cells isolated from Con A-induced liver injury mice. In summery, IL-17 is a proinflammatory factor for AIH and may be a novel therapeutic target in the liver disease.1. Elevated IL-17 secretion in liver is associated with Con A induced acute hepatitisAcute hepatitis was induced in immucompetent mice after Con A injection. High dose of Con A (20mg/kg) was injected intravenously into BALB/c mice.12 h after the injection, serum ALT level was greatly increased and massive necrosis in the livers of mice. More importantly, as the critical factors for tissue damage in acute inflammation, TNF-αand IL-6 cytokine secretion were significantly increased in Con A injected mice compared to the control. These data indicate that Con A-induced liver injury was successfully conducted. Elevated IL-17 secretion in liver is associated with Con A induced acute hepatitis. The expression pattern and distribution of IL-17 was detected in this model by real-time quantitative PCR, ELISA and immunohistochemistry, further the relationship of expression level of IL-17 and extend of liver injury was also evaluated. The results demonstrated that IL-17 was strongly increased in injured liver tissue. The expression level of IL-17 might closely be associated with extent of liver injury.2. The role of Trl regulatory T cells in ConA-induced tolerance modelAdministration of anti-IL-17 mAb before Con A challenge protected the mice from acute liver damage, and accompanied by a significant reduction of both ALT level and number of circulating lymphocytes infiltration. Moreover, both proinflammatory factors such as TNF-αand IL-6 expression in the liver were decreased after anti-IL-17 mAb treatment. These findings suggested that intrahepatic recruitment of IL-17+lymphocytes (dominant T cells) to IL-17 might play an important role in massive hepatic necrosis in Con A-induced liver injury, but the details mechanism need to be further investigated. To confirm whether endogenous IL-17 plays a role in Con A-induced liver inflammation, the mice were treated by AdIL-17R:Fc. Treatment with AdIL-17R:Fc, the ALT level and the liver necrosis were significantly reduced. Consequently, the levels of TNF-a and IL-6 were markedly decreased. These data indicate that IL-17 plays an essential role in Con A-induced hepatitis.Construction, expression and functions of plasmids encoded with IL-17. The eukaryotic expression plasmids, pcDNA3.1-IL-17 (pIL-17), were constructed. The plasmid DNA was then transferred to Hun7 cells and the expression of IL-17 proteins in Hun7 cell culture supernatant were measured after 48 hours. ELISA was applied to analyze protein levels. The results of ELISA indicated that the successful expression of IL-17 in vitro. In vivo level of IL-17 was detected by ELISA. All results above suggested that IL-17 proteins could be effectively expressed in vitro and in vivo by the recombinant plasmid DNA we constructed. Over-expression of IL-17 by HTV injection of plasmid pcDNA3.1-IL-17 greatly enhanced the severity of liver injury, increased serum ALT and more hepatocyte necrosis were observed. More importantly, the production of TNF-a and IL-6 were markedly increased in Con A-induced liver inflammation. These data suggest that IL-17 is critically involved in Con A-induced liver hepatitis.3. IL-17-expression Th17 and NKT cells increased in Con A-induced acute hepatitisWe examined IL-17-producing cells in hepatic MNCs after Con A injection. We found a marked increase of NKT and CD4+ T cells in liver, both in absolutely cell numbers and percentages. A higher percentage of IL-17-producing CD4+ T and NKT cells, but not CD8+ T cells were detected by the intracellular staining of IL-17. These data implicated that CD4+ T and NKT cells were the main source of IL-17 in inflamed liver.4. Effect of IL-17 on Production of TNF-a and IL-6 by Kupffer CellsWe then investigated the IL-17R expression on hepatic kupffer cells by real-time PCR with regards to its interaction with IL-17. IL-17R expression on hepatic kupffer cells was elevated 12 h after Con A injection compared to the control mice. Addition of IL-17 to kupffer cells isolated from either normal or Con A-induced hepatitis mice, resulted in a increased secretion of inflammatory cytokines TNF-a and IL-6 only in Con A-induced kupffer cells. This indicated that the interaction between IL-17 and IL-17R directly promoted the activation of kupffer cells. Additionally, inhibition of the function of kupffer cells by pretreatment with GdCl3 resulted in a 90% decrease of ALT level, and reduced the secretion of TNF-a and IL-6. These data collectively shows that the engagement of IL-17 and IL-17R promoted the production of TNF-a and IL-6 in kupffer cells, which may subsequently led to liver injury.In summary, our study clearly demonstrated that IL-17 produced by CD4+ T and NKT cells plays a critically proinflammatory role in Con A-induced hepatitis, our results also suggest that the pathogenic effects of T cell-derived IL-17 is mainly mediated via the IL-6 and TNF-a productions in kupffer cells. Therefore, we propos that the T cell-IL-17-kupffer cells pathway may be a new target in prevention and treatment of AIH.