IL-33/ST2L Pathway Is Involved In Con A-induced Hepatitis And Its Mechanism

Objective:Concanavalin A (Con A)-induced hepatitis (CIH) is a well-established experimental murine model that resembles human autoimmune hepatitis. The purpose of our experiment is to investigate the role of the IL-33/ST2L pathway in Con A-induced hepatitis, and the underlying mechanism of its involvement.Methods:1. To investigate whether IL-33 play a role in Con A induced hepatitis, real-time PCR was used to measure IL-33 expression in hepatic tissues of mouse after Con A administration. Additionally, immunohistochemistry analysis was also explored to determine IL-33 expression of liver tissue from mice with Con A injection.2. An exogenous recombinant IL-33 was administrated together with Con A into animals, or rabbit anti-mouse IL-33 antibody/rabbit IgG was injected intraperitoneally into mice 1 h before Con A treatment. Then liver injury was assessed by detecting serum levels of ALT and AST. Additionally, HE and TUNEL staining were done to analyze necrosis and apoptosis of hepatocytes.3. The effect of a-IL-33 on accumulation and late stage activation of lymphocytes (mainly T cells, NKT cells, NK cells) were analyzed by using FCM. In addition, the production of proinflammatory cytokines by these cells were measured with ELISA and FCM, and data were treated with stastical analysis.4. Based on observations above, to further confirm the central effect of NKT cells and IFN-y, IFN-y deficient mice and NKT cells depletion antibody were used to be investigated. The protective effect of IL-33 blocking antibody in Con A-treated IFN-y deficient mice and the hepatodetrimental effect of IL-33 to Con A-treated NKT depletion animals were measured by serum level of ALT and AST, together with HE staining.Results:1. IL-33 is involved in mouse hepatitis induced by Con AReal-time PCR of hepatic mRNA indicated that IL-33 expression was significantly elevated at 12 h after Con A injection, and decreased at 24 h. Similarly, liver tissue from mice with Con A injection showed a significantly up-regulated expression of IL-33 by immunohistochemistry analysis when compared with PBS injection. Additionally, compared with administration of Con A, mice injected with both IL-33 and Con A displayed a markedly elevated serum ALT and AST levels. HE staining revealed a massive necrosis in liver from IL-33 and Con A treated animals, but a slightly necrotic area in liver of animals treated with Con A.2. Pretreatment of IL-33 antibody protects mouse from Con A-induced hepatitisIn a-IL-33 pretreated mice, serum ALT and AST levels and HE staining revealed a significant amelioration of liver injury compared with IgG controls. Additionally, TUNEL staining revealed massive apoptosis in IgG control group, whereas it was markedly decreased in anti-IL-33 antibody pretreated animals. For mice with delayed treatment of neutralizing antibodies to IL-33, it also showed significant protection against Con A-induced liver injury. Otherwise, in mice treated with lethal dose of Con A (25 mg/kg body weight). IL-33 neutralizing antibody showed a significant protection against lethal dose of Con A as compared with IgG group.3. Neutralizing of IL-33 inhibits the late stage of T cells and NKT cells activation and decreases IFN-y secretion in the liverNeutralizing of IL-33 significantly suppressed activation of T and NKT cells but not NK cells, yet it failed to prevent accumulation of T and NKT cells into the injured liver. Flow cytometry analysis showed that administration of a-IL-33 inhibited IFN-y expression of T and NKT cells, but no conspicuous effect on NK cells. Moreover, levels of IL-4 and IL-17 produced by cells above exhibited no conspicuous difference between a-IL-33 pretreated groups and IgG controls. Consistently, as compared with IgG controls, a significantly decreased production of IFN-y in sera and liver tissues were observed by ELISA and western blot analysis, while levels of IL-4, IL-17 and TNF-a still presented no perceptible difference.4. ST2L expression was up-regulated in the liver of Con A-treated miceST2L expression on T cells and NKT cells peaked at 12h to 24h after Con A challenge, while decreased at 48h. Moreover, no perceptible up-regulation of ST2L was found in NK cells. The expression of ST2L in isolated MNCs of liver tissue cocultured with Con A or IL-33 demonstrated that up-expression of ST2L on T and NKT cells was induced directly by Con A but not IL-33.5. The protective effect of a-IL-33 for Con A-induced hepatitis is NKT cells and IFN-γ dependentIn NK- and NKT- depleted mice, serum levels of ALT and AST were significantly elevated in Con A and IL-33 co-treatment when compared with Con A treatment alone. Interestingly, this effect was not observed in NKT-depleted only animals. Moreover, in IFN-γdeficient mice,α-IL-33 pretreatment failed to protect the animals from Con A-induced hepatitis.6. Pretreatment of sST2 attenuates Con A-induced hepatitisSoluble ST2 (sST2), the specific antagonist of IL-33, was explored to investigate the effect of IL-33 on Con A-induced liver injury. As a result, psST2-Fc deeply decreased serum levels of ALT and AST, and significantly ameliorated liver injury analyzed by HE staining. Moreover, psST2-Fc considerably prolonged the survival of mice challenged with lethal dose of Con A as compared with pcDNA3.1 control group.Conclusions:1. IL-33/ST2L pathway is involved in Con A-induced hepatitis in mice.2. Pretreatment of IL-33 blocking antibody ameliorates Con A-induced hepatitis, while exogenous injection of recombinant IL-33 excerbates the liver injury.3. The protective effect of IL-33 blocking antibody is associated with impaired activation of T cells and NKT cells and reduced production of IFN-γ. This is predominantly depdent on NKT cells and IFN-γ.