The effect of peptidoglycan on the secretion of pro-inflammatory cytokines by dendritic cells and the regulation of T helper 17 responses in experimental autoimmune uveitis

Abstract

Objective To investigate the effect of peptidoglycan (PGN) on the secretion of proinflammatory cytokines by dendritic cells (DCs) and the regulation of T helper 17 (Th17) responses in experimental autoimmune uveitis.Methods Bone marrow cells from naive mice were cultured with granulocyte macrophage-colony-stimulating factor and interleukin (IL)-4 to induce DCs.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 phosphate buffered saline was added to the DCs as control group.The relative mRNA expression levels of IL-23,tumor necrotic factor α (TNF-α),IL-6,IL-1βwere measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR).Peptide fragment of interphotoreceptor retinoid-binding protein (IRBP1-20)-specific T cells,which were isolated from the spleen and draining lymph nodes of C57BL/6 mice immunized with IRBP1-20 peptide fragments 13 days earlier,were co-cultured with PGN-treated or untreated DCs,respectively.Total RNA from T cells cocultured for two days were isolated and the relative expression of retinoic acid receptor-related orphan receptor γt (ROR-γt),IL-17,T-box expression in T cells (T-bet),interferon γ (IFN-γ) mRNA were detected by real-time RT-PCR.On the second,the fifth and the seventh day,the cocultured T cells were analyzed by flow cytometry to detect the percentages of IFN-γ,IL-17 positive cells.Results The real-time RT-PCR results revealed that the level 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).The level of RORγt,IL-17 mRNA from the T cells cocultured with PGN-stimulated DCs were greatly increased compared with the control group (t=-5.601,-19.76; P＜0.05).However,the level of T-bet,IFN-γmRNA from the T cells cocultured with PGN-stimulated DCs were significantly decreased compared with the control group (t=4.717,11.207; P＜0.05).Data of flow cytometry showed that at two days,five days,seven days after cocultured with PGN-treated DCs,the percentages of IL-17 positive T cells were increased compared to the control group (t=-2.944,-3.03,-4.81; P＜0.05),and the percentages of IFN-γpositive T cells had no remarkable change (t=-1.25,-0.18,-2.16; P＞0.05).Conclusion PGN can promote the secretion of Th17-related cytokines by DCs,which favors proliferation and differentiation of Th17 in experimental autoimmune uveitis. Key words: Peptidoglycan/pharmacology; Uveitis/physiopathology; Toll-like receptor 2; T-cell antigen receptor specificity; Th17 cells