The Effect And Mechanism Of Anti-inflammatory Cytrokines On The Immune Response To Chronic Hepatitis Infection

Objective1. To observe the relationship between virus infection and anti-inflammatory cytokines among the liver biopsy samples.2. To study whether the anti-inflammatory cytokines affect the antivirus activity and the production of the antivirus mediator. Research on the effect of the anti-inflammatory cytokines on the expression of the TLR3 and TLR3 signaling pathway. To learn the function and possible mechanism of the anti-inflammatory cytokines against the liver non-parenchymal cells mediated adaptable immunity response.3. To learn the sequence of the woodchuck IL-10R and the expression of extra cellular protein. To produce the polyclonal antibodies and study its function among the WHV infected woodchuck.Methods1. Collect the patients'liver biopsy samples, extract the total RNA, and detect the expression of the HCV RNA, IL-10 and TGF-βby RT-PCR.2. Isolate murine non-parenchymal cells and culture in vitro. Poly I:C stimulates the anti-inflammatory cytokines pretreated and untreated cells. Measure the expression of the IP-10, IFI-T1, ISG15, TNF-a in the cells by RT-PCR. Virus protection experiment detects the antivirus function of the supernatant. 3. Pretreated non-parenchymal cells by the anti-inflammatory cytokine, then stimulated them with poly I:C, detect the level of TLR3 RNA and the expression of the protein by real time PCR and Western blot. Utilize NF-κB antibody to detect the translocation and phosphorylation. IRF3 report gene transfect the non-parenchymal cells, then pretreated by the anti-inflammatory cytokine. Stimulate cells by Poly I:C before detecting the IRF3 activation. The cells pretreated and stimulated by the anti-inflammatory cytokine and Poly I:C were cultured together with allogeneic T cell. Detect the production of the supernatant IFN-y and the multiplication of T cell by ELISA. Measure the effect of anti-inflammatory cytokine on TLR3-induced expression of co-stimulating molecules (CD80, CD86, CD40, MHCⅡ) induced by FACS.4. Clone IL-10R Sequence from the healthy woodchuck spleen tissue. Sequence and analyze the homology with other species at RNA level and protein level. Prokaryotic expresses the extracellular of the IL-10R. Immunize the rabbits with purified protein to produce polyclonal antibody. And detect the titer of the antibody with ELISA and immuno fluorescence. Utilize antibody to block IL-10R in vitro. Detect the perliferation of the antigen-specific T-cell.Results1. In the liver biopsy specimens, there is a positive correlation between the positive HCV and the TGF-βexpression level.2. TLR3 stimulation induced strong antiviral activities and ISG expression in NPC. The effects were potently suppressed by pretreatment with IL-10 or TGF-βin a dose-dependent manner. The antiviral activity of the supernatant is weaker than the untreated group too.3. Compared with the untreated group, the TLR3 RNA and protein expression level of the non-parenchymal cells in the anti-inflammatory cytokine-treated group were significantly reduced. The anti-inflammatory cytokine inhibits the translocation and phosphorylation of the NF-κB and IRF3, then suppress the NF-κB and IRF3 activation, interfere TLR3 signal pathway and inhibit TLR3-mediated antiviral effects and immune response. 4. TLR3 activated non-parenchymal cells can activate the allogeneic T cells, result in the perliferation of the T cell and the production of IFN-γ, while the anti-inflammatory cytokine could inhibit this effect. FACS result suggests the anti-inflammatory cytokine can reduce the expression of CD80 and CD86 induced by TLR3.5. The whole length of the woodchuck IL-10R sequence is 1728bp, which could encode 575 amino acids. Human and mouse IL-10R have 80.11% and 70.60% homology at nucleotides level respectively with the woodchuck. Human and mouse IL-10R have 71.53% and 56.42% homology at protein level respectively with the woodchuck.6. The extracellular segment of the woodchuck IL-10R contains 621 nucleotides; insert them into the prokaryotic expression vector pET30a, then transform to BL21 expressing bacteria. After inducing by IPTG, it gets prokaryotic protein weight 27kDa.7. Immunize the rabbits with purified protein to produce polyclonal antibody. The ELISA titers of anti-serum is about 1:5000000, the immunofluorescence titer is 1:200. In the chronic infected woodchuck, blocking the IL-10R can raise the perliferation of the antigen-specific T-cell.Conclusions1. There is a relationship between the TGF-βexpression and the virus infection in the liver biopsy specimens2. In murine non-parenchymal cells, exogenous IL-10 and TGF-βmay has significantly dose-dependent effect on inhibiting the TLR3 mediated antiviral and the expression of the responsive genes. This correlated with suppression of TLR3 receptor expression and inhibition of TLR-induced activation of NF-κB and IRF-3 in these cells.3. T cell activation induced by TLR3-stimulated NPC could be repressed by IL-10 or TGF-β. The data also show that IL-10 or TGF-βsuppress the TLR3-mediated up-regulation of cell surface molecules (CD80, CD86, CD40, MHC-Ⅱ), which may explain in part the inhibition of T cell activation. 4. Obtain the whole length of woodchuck IL-10R, the truncated recombinant IL-10R developed in this study had strong immunogenicity. Immunized with the purpose protein expressed, rabbit generated polyclonal antibody against IL-10R successfully. The polyclonal antibody had both high sensitivity and high specificity. The anti-IL-10 polyclonal antibody developed can be applied in detecting IL-10 expression. Blocking the IL-10R in vitro, could up-regulation the perliferation of the antigen-specific T-cell.