The Effect Of Peptidoglycan On The Regulation Of T Helper17Responses In Experimental Autoimmune Uveitis

ObjectiveUveitis is a group of diseases involving the retina, retinal vessels, optic nerve and vitreous, the diseases often result in the serious damage of the eye tissue, even a significant decrease or loss in visual function. The kind of human uveitis is diverse and the pathogenesis is complex. Although many factors, such as infection, trauma, can give rise to uveitis, the autoimmune disorders is one of the most common and important causes supported by most of the researches. And the T helper17cells are the key cells in the pathogenesis of the autoimmune uveitis. It has been reported that many TLRs were involved in the development of clinical uveitis and EAU, and only TLR2was involoved in the gene susuceptibility to uveits with Behcet’s disease. Recently, some studies have found that Toll-like receptor2plays a critical pathogenic role in the pathogenesis of autoimmune diseases. This study aimed to investigate the effect of the peptidoglycan, the ligand of the Toll-like receptor2, on secretion of pro-inflammatory cytokines by dendritic cells and the regulation of the Toll-like receptor2on the T helper17cell in experimental autoimmune uveitis, so as to further elucidate the pathogenesis of autoimmune uveitis.Methods1、Culture of dendritic cells:Bone marrow-derived dendritic were cultured in vitro. The phenotypes of cultured cells were characterized by flow cytometry.2、DCs cultured for six days were randomly divided into two groups:PGN-treated group and control group. The DCs in PGN-treated group were stimulated with PGN and the same volume of sterile phosphate buffered saline was added to the DCs as control group. The relative mRNA expression levels of IL-23, tumor necrotic factor (TNF)-a, IL-6, IL-1(3were detected by real-time reverse transcriptase polymerase chain reaction (RT-PCR).3、Induction of EAU model:To induce EAU, the Interphotoreceptor retinoid-binding protein (IRBP)1-20peptide fragment was emulsified1:1(vol/vol) sufficiently with Freund’s complete adjuvant (CFA), containing Mycobacterium tuberculosis H37RA.The antigen was injected subcutaneously into one side hind footpad and trunk to establish EAU model.4、The separation of the IRBPl-20-specific T cells and co-culture them with DCs from the two groups respectively:Interphotoreceptor retinoid-binding protein (IRBP)1-20-specific T cells,which were isolated from the spleen and draining lymph nodes of C57BL/6mice immunized with IRBP1-20peptide fragments13days earlier, were cocultured with PGN-treated or untreated DCs, respectively. Total RNA from T cells cocultured for2days were isolated and the relative expression of retinoic acid receptor-related orphan receptor (ROR) yt, IL-17, T-box expression in T cells (T-bet), interferon (IFN) y mRNA were detected by real-time RT-PCR.5、On the second day, the fifth day, the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-γ, IL-17positive cells.6、DCs cultured for six days were randomly divided into three groups:control group, PGN-treated group and PGN+SB203580group. And the DCs were cocultured with IRBPl-20-specific T cells respectively. On the seventh day, the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-γ, IL-17positive cells.7、DCs cultured for six days were stimulated with PGN. On0min,15min,30min, lhr p-p38were detected by western blot, respectively.Results1、DCs were successfully separated and identified.2、The real-time RT-PCR results revealed that the levels of IL-23, IL-1β, IL-6, TNF-α mRNA from PGN-stimulated DCs were significantly increased compared to the control group (t=-14.363,-5.627,-3.85,-28.151; P<0.05)3、The levels of RORyt, IL-17mRNA from the T cells cocultured with PGN-stimulated DCs were greatly raised in comparison with the control group (t=-5.601,-19.76; P<0.05). However, the level of T-bet, IFN-γ mRNA from the T cells cocultured with PGN-stimulated DCs had’little chang in comparison with the control group.5、Data of flow cytometry showed that two days, five days, seven days after cocultured with PGN-treated DCs, the percentages of IL-17positive T cells were increased compared to the control group (t=-2.944,-3.03,-4.81; P<0.05), and the percentages of IFN-y positive T cells had no remarkable change (t=-1.25,-0.18,-2.16; P>0.05).6、The result of flow cytometry showed that compared with PGN-treated group, the percentages of IL-17positive T cells in SB203580pretreatment group were decreased.7、The western blot results showed that stimulated with PGN, the express level of the p-p38in DCs were increased.ConclusionsIn vitro, PGN treatment can promote the mRNA and protein expression of Th17-polarizing cytokines by DCs, which favor proliferation and differentiation of Th17cells in experimental autoimmune uveitis. This effect may be mediated, at least in part, by the activation of p38siganling pathway in DCs.