Effect And Mechanism Of Tumor Necrosis Factor-α-induced Protein 8 On The Immune Response Of CD4~+T Lymphocytes In Immunocompromised Mice Following Acute Insult

Background and purposeSince the its inception in 1954,allograft kidney transplantation has been the treatment of choice for ESRDs.With the development of modern immunosuppression medications,renal transplantation has become the largest and most successful large organ transplantation of all kinds of solid organ transplantation.However,despite satisfactory surgical success and five-year survival,long-term use of immunosuppressive agents,can inhibit the immune response in a non-specific manner,increasing the risk of infection and subsequent development of sepsis that are the leading cause for mortality in kidney transplant recipients.Sepsis is characterized by host’s inappropriate immune response to infection or other insult,and is commonly complicated by life-threatening organ failure,which in part explains the high mortality with sepsis.Studies have shown that over-enhanced pro-inflammatory compensatory anti-inflammatory response and imbalance of immune homeostasis are the main mechanisms of the pathogenesis of sepsis,and there are different degrees of impaired immune function and immunosuppression in sepsis patients.The disorders of immune function caused by sepsis,especially the inhibition of cellular immune function,are mainly manifested as enhanced T lymphocyte apoptosis and decreased proliferation,favored polarization of T cells into Th2 cells.Other cells,including DCs,may also be affected in setting of sepsis where they exhibit impaired antigen presenting and/or cytokine secretion abilities.It has been confirmed that T lymphocyte subsets have a downward trend in the pathogenesis of sepsis,among which CD4+T cells have the most significant downward trend.Tumor necrosis factor-alpha-induced protein 8(TNFAIP8),also known as SCC-S2,GC2-1 and MDC-3.13,is the first discovered member of the TNFAIP8 family.Studies have shown that TNFAIP8 can promote cell survival and inhibit the activities of apoptotic enzymes caspase-8 and caspase-3.TNFAIP8 is highly expressed not only in tumors,but also in immune organs and lymphoid tissues.However,its function in the immune system is still unclear.Therefore,it is important to study the potential effect of TNFAIP8 on CD4+T cell immune response in sepsis for exploring the regulatory factors of imbalance and disorder of immune function in sepsis.This study focused on the role of TNFAIP8 in regulation of immune function imbalance in sepsis.We enrolled renal transplantation recipients,who were further divided into stable patients,chronic rejectors and those complicated with sepsis.We then used cecal ligation and puncture(CLP)to induce sepsis in mouse pretreated with CT,isolated mouse spleen CD4+T lymphocytes for mechanistical study.Through the in-vivo and in-vitro studies,we tried to obtain in-depth understanding of TNFAIP8 and its key regulatory links mediating CD4+T cell immune function,further clarify the contribution of T cells in the pathogenesis of sepsis,provide new clues for the searching of immunoregulatory pathway s of sepsis and finally mitigate the key factors in this vicious circle.Method:1.The kidney transplant recipients were divided into 3 groups:stable kidney transplant,chronic rejectors,sepsis group.We also chose a group of healthy people as the normal control.CD4+T lymphocytes were isolated from peripheral blood by magnetic beads.RT-qPCR was used to detect the difference expression of TNFAIP8 mRNA of CD4+T in peripheral blood of each group.Serum IL2.IFN-gamma and IL4 levels were measured using ELISA2.immunofluorescence staining of mouse spleen CD4+T lymphocytes was performed by FITC-labeled antibody,and the cellular localization of TNFAIP8 protein was observed by laser scanning confocal microscopy3.The immune compromised mouse sepsis model was established by cecal ligation and puncture(CLP)in C57BL/6 mice that were pretreated with peritoneal injection of cyclophosphamide.The spleens of normal mice and sepsis mice were taken and CD4+T lymphocytes were extracted by magnetic beads sorting method.The expression of TNFAIP8 protein in CD4+T lymphocytes of normal mice spleen and the abnormal expression of TNFAIP8 protein in CD4+T lymphocytes of sepsis mice spleen were detected by Western Blot.4.The TNFAIP8 RNA lentiviral vector was constructed and transfected into CLP mice,which were divided into four groups:sham group,CLP group,TNFAIP8 RNA-CLP group and negative-CLP(empty vector)group.TNFAIP8 expression in spleen CD4+T lymphocytes of mice in each group was detected by RT-qPCR;The proliferation of spleen CD4+T lymphocytes in septic mice and TNFAIP8 overexpressing mice was detected by MTT assay.IL2,IL4 and IFN gamma levels were measured by ELISA;Activity of nuclear factor NF-AT was detected using ELISA after extraction of NFAT from CD4+T cells.Result:1.The expression of TNFAIP8 in peripheral blood CD4+T cells of kidney transplant recipients was significantly lower than that of healthy controls,stable transplant and chronic rejectors(P<0.05),while the expression of TNFAIP8 in peripheral blood CD4+T cells of chronic rejection patients was not significantly different from health control or stable transplants.IL2 and IFN gamma were significantly lower in septic patients when compared with stable transplant and chronic rejectors.However,IL4 in septic patients was much higher.(p<0.05)2.TNFAIP8 was stained green by FITC labeled antibody,while DAPI stained the nucleus blue.TNFAIP8 was expressed abundantly in the cytoplasm of CD4+T cells from mouse spleen3.The expression of TNFAIP8 protein in CD4+T cells of sepsis mice spleen decreased significantly(P<0.05).This reduction can be effectively reversed by overexpression of TNFAIP8 via lenti-virus transduction.4.MTT assay showed that CD4+T lymphocyte proliferation in CLP sepsis mice was decreased,while TNFAIP8 overexpression could increase CD4+T lymphocyte proliferation activity;IL-2 secreted by CD4+T lymphocyte in spleen of CLP mice decreased significantly,while IL-2 level in TNFAIP8 overexpression mice increased significantly.The expression of TNFAIP8 probably promoted the secretion of IL-2 in T lymphocyte.The activity of NF-AT in spleen CD4+T lymphocyte of CLP mice decreased and can almost be fully restored by TNFAIP8 overexpression.5.The Th1/Th2 balance of CD4+T cells in sepsis shifted to Th2.After the overexpression of TNFAIP8,the Th1/Th2 balance of CD4+T cells shifted to Thl.TNFAIP8 may affect the polarization of CD4+T cells in sepsis.ConclusionTNFAIP8 is highly expressed in CD4+T lymphocytes and is suppressed in context of sepsis at the clinical level and mouse experimental model.It can enhance the proliferation activity of CD4+T lymphocytes by increasing the activity of NF-AT and help tilt the Thl/Th2 balance to Thl pole.Thus,TNFAIP8 could be affected in sepsis or engage in this process to favor the development of a full-blown picture of sepsis.Targeting this molecule may carry the promise of holding sepsis in its early stage and impro ving clinical outcome.