The Role And Mechanism Of Interlukin-38 In Concanavalin A-induced Autoimmune Hepatitis In Mice

Background:Autoimmune hepatitis(AIH)is an immune-mediated liver injury mainly characterized by the presence of specific auto-antibody against hepatocytes,the morbidity of which has gradually increased in recent years,and AIH has become one of liver diseases with the highest incidence rate worldwide.Numerous triggers can lead to the occurrence of AIH,the definite pathogenesis of which has not been fully elucidated yet.It is significant to acquire a deep understanding of the pathogenesis of AIH and seek for more potential therapeutic targets.As a member of interlukin-1 family,the anti-inflammatory effects of IL-38 have been brought into focus in recent years.Numerous evidences suggest that IL-38 exerts antiinflammatory effects by inhibiting the function of T cells and macrophages.Concanavalin A(Con A)-induced AIH is a T cell-dependent and macrophage-dependent liver injury,which is the most widely used animal model of immune-mediated liver injury in the world.However,the biological effect and mechanism of action of recombinant IL-38 protein have not been reported in Con A-induced AIH yet.Objective:Firstly,to clarify the expression pattern of IL-38 and the correlation between IL-38 levels and disease severity in Con A-induced AIH.Secondly,to determine the effects of IL-38 on liver injury phenotypes by exogenous administration of recombinant IL-38 protein for AIH mice and to verify the biological functions of IL-38 on immune cells and hepatocytes through in vitro experiments.Lastly,the specific mechanism of IL-38 in regulation of the disease progress of AIH was further explored based on high-throughput sequencing.Methods:Mice were challenged with Con A solutions through the tail vein to construct AIH model and samples were collected at different time points after Con A injection.The dynamic episode of disease progression in AIH mice at different time points were determined by detecting liver enzyme levels,hematoxylin-eosin staining for liver and quantitative polymerase chain reaction(q PCR).The enzyme-linked immunosorbent assay(ELISA)and q PCR were conducted to detect the dynamic variation of IL-38 levels in mice at different time points.The time point at which the variation of IL-38 expression levels was the most remarkable was selected,and the alteration of IL-38 expression in the liver and spleen were identified by Western-Blot and immunohistochemistry.The correlation between the IL-38 levels and liver enzymes was analyzed.According to experimental requirements,mice were divided into four groups: the normal control group,the group challenged with IL-38 alone,the group challenged with Con A alone and the group treated with a combination of IL-38 and Con A.Mice in the treatment group were transfused with recombinant IL-38 protein through the tail vein 2 h in advance,and samples were collected in all groups 12 h after modeling.The liver injury phenotypes were determined by measurement of liver enzyme levels,H&E staining and TUNEL staining.q PCR and Cytometric Bead Array(CBA)were applied to detect cytokines.The level of malondialdehyde and superoxide dismutase were detected by extracting liver tissue homogenate.The activation and differentiation of T cell and proliferation of macrophage in spleen and lymph nodes were detected by flow cytometry.The infiltration of macrophages in liver was detected by immunohistochemistry.GO,KEGG and GSEA enrichments were analyzed by transcriptome sequencing of mouse liver and the signal pathway were further validated by WB and q PCR.Primary cells of the spleen and lymph nodes in mice were cultured in vitro and flow cytometry was used to analyze the effect of pretreatment with IL-38 at different concentrations on the activation and differentiation of T cells induced by Con A.The inflammatory cytokines levels in the culture supernatants were detected by CBA.AML12 cells were stimulated by Con A in vitro to induce oxidative stress injury and pretreated with IL-38 at different concentrations and the expression levels of reactive oxygen species was detected by flow cytometry.Results:Con A-induced AIH is an acute and self-confinement liver injury,which displayed that the disease progression peaked 12 h after modeling and tended to resolve 48 h later.Con A can induce up-regulated expression of IL-38 in serum,liver and spleen of mice,and the serum Il-38 levels displayed the most significant increase at 12 h after Con A modeling.The results of Western-Blot and immunohistochemistry further confirmed that the expression of IL-38 in AIH mice was significantly higher than that in the control group at 12 h after modeling.The serum IL-38 levels were correlated with the liver enzyme levels.Pretreatment with IL-38 can significantly reduce the liver enzyme levels,improve liver and spleen pathological manifestations and reduce hepatocytes apoptosis in AIH mice.Pretreatment with IL-38 can also significantly reduce the levels of cytokines such as IL-6,IFN-γ and TNF-α etc.In addition,pretreatment with IL-38 can significantly reduce the MDA levels and upregulate the SOD levels.IL-38 inhibited the activation and differentiation of T cells in spleen and lymph nodes and infiltraation of macrophages in liver.Besides,IL-38 can inhibit the activation and differentiation of T cells induced by Con A in vitro and reduce the inflammatory levels of the culture supernatants.Pre-administration of IL-38 can also reduce the production of ROS in hepatocytes induced by Con A.Transcriptome sequencing showed that IL-38 could alleviate liver injury by down-regulating multiple inflammatory signaling pathways and oxidative stress pathway.The results of WB and q PCR further confirmed that IL-38 was involved in the regulation of AIH by interfering with the activation of NF-κB and MAPK pathways and up-regulating Nrf2 pathways.Conclusion:IL-38 may alleviate liver injury induced by Con A through blocking the activation of NF-κB and MAPK inflammatory pathways and upregulating the Nrf2 pathways.