GITRL On Regulation The Inflammation By Inhibiting PDL1in Kupffer Cells

Objection: GITRLsiRNA and pEGFP-N1-GITRL recombinedplasmid was transfected into Kupffer cells in order to interfere the basicexpression of the GITRL in KCs. KC was co-cultured with T cells in vitro.Analyzing the PDL1expression in LPS-stimulated KCs and the activity ofT cells. Exploring the relationship between GITRL and the PDL^-1regulation of the inflammatory response mechanism in KCs.Method:1Kupffer cells were isolated by discontinuous densitygradient centrifugation and adherent selection and T cells were isolated byperfusion of nylon column.2、KCs were randomly divided into4groupsto be transfected. GITRLsiRNA and pEGFP-N1-GITRL recombinedplasmid, the siRNA of control group and no-load plasmid were transferredinto KCs separately.The effect of transfection was identified byfluorescence microscopy and preliminarily compared the efficiency oftransfection since transfected after24hours. Rt-PCR and Westernbolt werefurther adopted to analyze the gene and protein expression levels in eachgroup of transfected cells., Cells of each group and the normal KCs cellswere co-cultured with T cells in vitro After transfection （1:10）. Co-culturedcells were divided into6groups including control group, untransfectedgroup, T cell co-culture system with only medium, LPS-stimulating group,co-culture system of untransfected KC and T cell plus LPS （1ug／m1）,GITRL siRNA group, Control siRNA group, pEGFP-N1-GITRL group and pEGFP-N1control group. Each group was respectively co-cultured withtransfected KCs and T cells which was treated as same as LPS group.GITRL and PDL1expression of KCs was determined byimmunofluorescence and Westernboltafter24hours. MTT method andAnnexin-V/FITC flow analysis were adopted to analyze the activity of Tcell. The secretion levels of tumor necrosis factor （TNFα） fromsupernatants was determined by ELISA. The distribution of cytoplasm andnuclear in KCsP65was observed by laser scanning confocal.Result:1、Cell survival rate and purity were determined byswallowing ink staining experiments and F4/80immunofluorescence of93%and94%respectively. The purity of T cell analyzed by flowcytometric analysis after CD3/CD4double-antibody staining was75%.2、Transfected effects of GITRLsiRNA and pEGFP-N1-GITRL observed byfluorescence microscope since transfected with siRNA and plasmid after24hours were80%and85%respectively. There were significant differencesdetermined by Rt-PCR and Westernbolt tests, which showed in genesilencing group, over-expression group compared with control group（p<0.05）.3、GITRL on KCs,the activity of T-cell and tumor necrosisfactor TNFα secretion of supernatant is upregulated in LPS group, LPS+Control siRNA group and the pEGFP-N1control group of co-culture systemsince LPS stimulating after24hours （p＜0.05）. GITRL, the activity ofT-cell and tumor necrosis factor TNFα secretion of supernatant wereshowed more significant up-regulation otherwise the expression ofPDL1were showed more down-regulation in pEGFP-N1-GITRL group.The T cell proliferation and the expression of tumor necrosis factor TNFαsecretion were significantly decreased after LPS stimulation in GITRLsiRNA group （p＜0.05） and the degree of inhibition to PDL1was alsoshowed significantly weakened. Conclusion: The up-regulation of GITRL on KCs induced theinhibition of PD-L1could affect the corresponding receptor on T cells,activat T cell and promote the inflammation. Interference GITRLexpression on KCs could restore the immune balance and inhibit theinflammatory reaction.