CD8~+T Cell/IL-33/ILC2 Axis Exacerbates The Liver Injury In Con A-Induced Hepatitis In T Cell-Transferred Rag2-Deficient Mice

Background:Previously,we reported that the damage-associated molecular pattern molecules?DAMPs?alarmin high-mobility group box 1?HMGB1?and interleukin?IL?-33 play important roles in the pathogenesis of Con A-induced murine hepatitis,and injured hepatocytes are major sources of these DAMPs.Moreover,IL-33 is a key cytokine that promotes the development and responses of type 2 innate lymphoid cells?ILC2s?.However,the immune cells participating in the injury of hepatocytes in the setting of Con A challenge are not fully understood.Thus,we hypothesize that there exists a CD8⁺T cell/IL-33/ILC2 axis that mediates Con A-triggered hepatocyte injury.Objective:IL-33 is a pivotal and noval alarmin in ConA-induced hepatitis,released from injured hepatocytes.IL-33 is involved in variety inflammatory diseases,including immune-induced hepatitis,and plays a balancing role in the pathogenesis.ST2 receptor is the downstream of IL-33,which is expressed on the subset of ILC2 cells.Thus,we hypothesize that there exists a CD8⁺T cell/IL-33/ILC2 axis that mediates Con A-triggered hepatocyte injury.Methods:1.Two different mouse models of Con A-induced hepatitis?1?T cell-transferred Rag2^-/-mice:CD4⁺T cells and CD8⁺T cells were isolated and transferred intravenously 1 hour before injection of ConA at a dose of 15mg/kg body weight.10 hours later,livers were isolated and plasma was harvest;?2?Wild-type C57BL/6 mice:Mice were treated with ConA at 15mg/kg body weight. Tissues and sera were collected after 10 hours.2.Real-time PCR were applied to visualize IL-33 expression of hepatocytes and HMCs;3.ELISA:The levels of IFN-?,IL-6,and IL-10 and IL-33 were determined by ELISA kits.Blood samples were harvested and kept at room temperature for 30min before centrifuging.Plasma was collected and stored at-80°C until analysis.For in vitro experiments,the supernatants were collected after mixed cell co-culture for 10 h;4.HE staining and TUNEL staining were used to assess hepatitis liver injury induced by Con A in both of two mouse models;5.Immunofluorescence?IF?:IL-33 and perforin were detected using anti-perforin antibody and anti-IL-33 antibody,respectively;6.Apoptosis detection:Propidium iodide?PI?/Annexin V was determined using an Annexin V-FITC Apoptosis Detection Kit I;7.Furthermore,ST2⁺ILC2 and CD4⁺T cells cytokine production,including IL-4,IL-5 and IL-13were detected using intracellular staining by flow cytometry;8.Finally,ST2⁺ILC2s were sorted by flow cytometry and adoptively transferred into C57BL/6 mice to verify the function of the cells in mouse liver.Results:Transferred CD8⁺T cells play an important role in the pathogenesis of Con A-induced liver injury in T cell-transferred Rag2-deficient mice.Hepatocytes injured by perforin-based CD8⁺T cell cytotoxicity release the alarmin IL-33.This cytokine promotes ST2⁺ILC2 development and the secretion of cytokines IL-5 and IL-13 to mediate liver inflammation triggered by Con A challenge.In addition,these type 2 cytokines,including those originated from CD4⁺T cells,result in eosinophil accumulation in the liver after Con A administration.Conclusion:Our data revealed that CD8⁺T cells play an indispensable role in the pathogenesis of Con A-induced liver injury in T cell-transferred Rag2-deficient mice.The IL-33-mediated innate ST2⁺ILC2 response exacerbates liver tissue damage downstream of CD8⁺T cell cytotoxicity.Therefore,the CD8⁺T cells/IL-33/ILC2 axis is a potential therapeutic target for acute immune-mediated liver injury.