The Regulation Of CD4+T Cell Senescence By Mi R-181a And Its Signaling Pathway In Hcv Infected Patients

T cells play a crucial role for viral clearance or persistence; however, the precise mechanisms that control their responses during viral infection remain largely unknown. micro RNAs(mi R) have been implicated as key regulators controlling biological processes through posttranscriptional repression. Evidence has suggested that some mi RNAs are able to regulate hepatitis C virus(HCV) replication and its related liver diseases by directly interacting with the HCV genome or indirectly controlling virus-associated host pathways. The thesis studied the regulation of CD4+T cell senescence by mi R-181 a in HCV infected patients.Methods: Separate CD4+T cells from human peripheral blood mononuclear cells(PBMCs) of HCV infected patients or healthy subjects(HS), mi Script mi RNA array to assay the expression levels of mi RNAs in the CD4+ T cells of HCV patients compared to HS. Flow cytometry and Real-time PCR quantification of DUSP6 and IL-2 on CD4+T cell. Transfect human primary CD4+ T cells with mi R-181 a precursors or block DUSP6 of CD4+T cells with DUSP6 inhibitor, analysis of CD69, CD25 and IL-2 expression by flow cytometer; DUSP6 expression by real-time PCR.Separate CD4+T cells from human peripheral blood mononuclear cells(PBMCs) of HCV infected patients or healthy subjects(HS), assay the telomere length by Flow-FISH and stain senescence-associated β-galactosidaese(SA-β-gal) expression. Flow cytometry of SIRT1 on CD4+T cells, Western Blot of ΔNp63 on CD4+T cells. Purified CD4+ T cells from HCV-infected individuals were transfected with Sirt1 si RNA or p63 si RNA and negative control or mi R181 a precursors and negative control. After transfection, Flow-FISH to assay telomere length of CD4+T cell, PCR-ELISA to assay relative telomerase activity(RTA), stain SA-β-gal expression, Flow cytometry of Ed U.Results:hepatitis C virus(HCV)-mediated decline of mi R-181 a expression impairs CD4+ T cell function via over-expression of dual specific phosphatase 6(DUSP6). Specifically, a significant decline of mi R-181 a expression, along with over-expression of DUSP6, were observed in CD4+ T cells from chronically HCV-infected individuals compared to healthy subjects; and the levels of mi R-181 a loss were found to be negatively associated with the levels of DUSP6 over-expression in these cells. Importantly, reconstitution of mi R-181 a or blockade of DUSP6 expression in CD4+ T cells led to improved T cell responses, including enhanced CD25 and CD69 expressions, increased IL-2 expression, and improved proliferation of CD4+ T cells derived from chronically HCV-infected individuals.HCV-induced T cell senescence is counter-regulated by a ΔNp63-mi R181a-Sirt1 pathway. Chronically HCV-infected individuals show signs of accelerated T cell aging compared to age-matched healthy subjects. Mechanistic studies revealed that up-regulation of transcription factor ΔNp63 leads to the decline of mi R181 a expression, resulting in the over-expression of Sirt1-- an anti-aging molecule-- in CD4+ T cells from HCV-infected individuals versus healthy subjects. Silencing ΔNp63 or Sirt1 and reconstitution of mi R181 a expression in CD4+ T cells from HCV patients led to an accelerated T cell senescence, as evidenced by a remarkably reduced telomerase activity, shortened telomere length, increased SA-β-galexpression, and decreased Ed U incorporation. Paradoxically, an increased IL-2 expression was observed in CD4+ T cells by these treatments, primarily due to a reduced frequencies of Foxp3+ regulatory T cells(Tregs) and increased or unchanged numbers of Foxp3- effector T cells(Teffs) along with manipulating the ΔNp63-mi R181a-Sirt1 pathway.These findings provide novel insights into how HCV utilizes different pathways to balance T cell senescence/exhaustion and reveal new targets for therapeutic rejuvenation of impaired T cell responses during chronic viral infection, and provide new model of how chronic viral infection cause cell senescence.