The Role Of HBx In Mediating Immune Regulation Of Renal Tubular Epithelial Cells

Objective:To explore the role of TLR4 in immunity disorder of renal tubular epithelial cells mediated by hepatitis B virus X protein.Methods:(1)The 6-week old C57BL/6J-TgN mice were randomly divided into the experimental group and the intervention group.C57BL/6J mice were used as normal control group.Mice of the intervention group were injected TLR4 shRNA lenti virus from 8-week old,and injected every four weeks.Serum and urine were collected at 8,12,16,20,24-week old.Mice were sacrificed at 24 week old,to observe the mice renal function and pathological changes.HBx and TLR4 expression in renal tissue were observed by immunohistochemistry,and macrophages and T cells were also detected by immunohistochemistry.MHC-II,CD40,CD40L were detected by immunofluorescence.(2)We constructed a HBx-overexpression plasmid and a TLR4 shRNA plasmid and transfected them into human proximal tubular epithelial cell line(HK-2).Western blot was used to detected the expression of HBx and TLR4.Flow cytometry was used to investigate the expression of MHC-II,CD40 on HK-2 cells.Mixed lymphocyte reaction was used to detecte the proliferation and CD40L expression of T cells by flow cytometry.IFN-gamma and IL-4 in supernatant were determined by ELISA.Macrophages adherent capacity was detected,and MCP-1,tumor necrosis factor-a and IL-1? secretion of macrophages were detected after stimulation of HK-2 cells supernatants.(3)TLR4 downstream MyD88-dependent and MyD88-independent signal pathways were detected by Western blot.And location of p-IRF3 and NF-?B p65 were determined by confocal microscopy.After treatment of MyD88 inhibitor,the expression of MHC-II,CD40 in HK-2 cells were determined.Results:(1)Proteinuria of C57BL/6J-TgN mice increased from 16-week old,and the renal function deteriorated up to 24 week-old.Both glomerulus and renal tubule had varying degrees of damage.HBx and TLR4 expression was observed in tubular epithelial cells of C57BL/6J-TgN mice,and they had significant interstitial CD4+T cells and macrophages accumulation.In C57BL/6J-TgN mice,MHC-II antigen and CD40 co-localized in tubular epithelial cells,and MHC-? antigen and CD40 expression were higher in tubular epithelial cells of C57BL/6J-TgN mice than in wild-type C57BL/6J mice.We also stained CD4 and CD40L with immunofluorescence and CD40L could co-localize with CD4 which deposited in the interstitium in C57BL/6J-TgN mice.Mice with TLR4 shRNA lenti virus intervention appeared reduced proteinuria and remission of renal function.Mice in intervention group also had fewer CD4+T cells and macrophages infiltration and lower MHC-?,CD40 and CD40L expression.(2)After transfection of HBx gene,the expressions of MHC-II and CD40 in HK-2 cells were up-regulated,stimulation of T cell proliferation and CD40L expression were enhanced,and IFN-gamma/IL-4 ratio increased.The adhesion index of macrophages increased after HBx-transfected HK-2 cell supernatant treatment,and MCP-1 production and pro-inflammatory cytokines IL-1? and TNF-a were also increased.TLR4 shRNA transfection down-reagulated expression of MHC-II and CD40 on renal tubular epithelial cells,decreased ability of stimulating T cell proliferation and lowered ratio of IFN-gamma/IL-4.The adhesion index of macrophages decreased,and MCP-1,IL-1? and TNF-a were all decreased.(3)After transfection of HBx gene,the expression of MyD88-dependent and MyD88-independent signal pathways molecules MyD88?IRAK4?TRAF6?TRAM?TRIF?IRF3?p-IRF3 all up-regulated,and p-IRF3 and NF-?B p65 entered nuclear.After treatment of MyD88 inhibitor,the MHC-? and CD40 expression decreased in renal tubular epithelial cellsConclusion:We found that HBx could stimulate HK-2 cells MHC-II and CD40 expression,functioning as nonprofessional antigen-presenting cells,and activate inflammatory immune response to cause the release and imbalance of numerous inflammatory cytokines,which may contribute to the pathogenesis of HBV-GN.TLR4 and down-stream MyD88-dependent signal pathway may play an important role in HBx-mediated renal tubular epithelial cells immunity disorder,and inhibiton of TLR4 can depress immune function of tubular epithelial cells and have prevention and treatment effect.