| BACKGROUND AND OBJECTIVE:Tumor necrosis factor-α induced protein 8(TNFAIP8)is a newly-recognized protein which is discovered from researches on head and neck squamous carcinoma,and belongs to TNFAIP8 protein family.TNFAIPS is overexpressed in a bunch of malignant cell lines and maybe involved in the processes like oncogenesis and cell survive/death.Immune organs and immune cells such as spleen,thymus and T lymphocytes steadily synthesis TNFAIP8,but it remains unclear whether TNFAIP8 is expressed and functioned in CD4+CD25+ regulatory T cells(Tregs),thereby contributing to the development of sepsis.The present study was conducted to investigate the expression and function of TNFAIP8 in rodent CD4 CD25 Tregs and to further elucidate its potential regulatory mechanisms.METHODS:CD4+CD25 Tregs from spleens of BALB/c mice were isolated with mouse CD4 CD25 Treg isolation kits containing MACS microbeads.Levels of gene expression and protein synthesis of TNFAIP8 were determined by RT-PCR and Western blot,respectively.Tregs were transinfected with recombinant lentiviruses that earned TNFAIP8-siRNA or TNFAIP8-RNA and were stimulated by lipopolysaccharide(LPS)to simulate sepsis environment in vitro.Wild type mice were injected intravenously(via caudal vein)with the same lentiviruses two weeks prior to cecal ligation and puncture(CLP).Cytotoxic T lymphocyte-associated antigen-4(CTLA-4)and forkhead/winged helix transcription factor p3(Foxp3)expressions were measured by flow cytometry,and proliferation of T lymphocytes was tested by CCK-8.The apoptosis of Tregs was determined by Annexin V detection kits.All cytokines including transforning growth factor(TGF)-β and interleukin(IL)-10 released by Tregs,and IL-2,IL-4 and interferon(IFN)-γ secreted by T cells,as well as nuclear factor of activated T cells(NF-AT)and active protein(AP)-1 were all quantified by enzyme linked immunosorbent assay(ELISA).In addition,activities of mitogen-activated protein kinase(MAPK)signaling pathway member proteins were also analyzed by Western blotting.STATISTICAL ANALYSIS:All data were represented as mean±standard deviation(SD).Data sets were examined by one-way ANOVA,and individual group means were then compared with Least Significant Difference(LSD)-t test.All statistical tests were two sided and a P value of 0.05 or less was considered to indicate statistical significance.RESULTS AND CONCLUSIONS:1.TNFAIP8 was found to be postively expressed at both gene and protein levels in mouse CD4 CD25+Tregs.2.In vitro,compared with normal splenic Tregs,Tregs transfected with TNFAIP8-siRNA lentivirus had lower expression of CTLA-4 and Foxp3,lower secretion of IL-10 and TGF-β,increased cell apoptotic rate,and co-cultured T lymphocytes exihibited higher proliferative rate,increased IL-2 release,elevated AP-1,NF-AT activity and helper T cell(Th)1 polarization in Thl/Th2 balance;while Tregs transfected with TNFAIP8-RNA lentivirus had higher expression of CTLA-4 and Foxp3,higher secretion of IL-10 and TGF-p,decreased cell apoptotic rate,and co-cultured T lymphocytes exihibited lower proliferative rate,decreased IL-2 secretion,declined AP-1,NF-AT activity and Th2 polarization in Thl/Th2 balance.3.In vivo,compared with splenic Tregs in wild type mice with CLP,Tregs in CLP mice injected with TNFAIP8-siRNA and TNFAIP8-RNA lentivirus manifested similar trends with same lentivirus transfecting Tregs in vitro,respectively.4.Compared with normal splenic Tregs,TNFAIP8-S1RNA transfecting Tregs showed significantly enhanced phosphorylation of c-Jun N-terminal kinase(p-JNK),whereas p-JNK expression in TNFAIP8-RNA transfecting Tregs was markedly down-regulated.Expression of phospho-extracellular regulated protein kinases(ERK)and p-p38 MAPK had no statistical differences between groups.These results suggest that TNFAIPS can obviously affect the immune inhibitory function of CD4+CD25+Tregs,which might be associated with JNK signal transduction. |
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