METHODS: hDPSCs were isolated from human dental pulp tissues. Cells were treated with 25 μM IWP-2 for 24 h, and subsequently, the gene expression profile was examined using high-throughput RNA sequencing. The mRNA expression was analysed using qPCR. The effect of IWP-2 was investigated in both normal and LPS-induced hDPSCs (inflamed hDPSCs). CD4+ T cells and CD14+ monocyte-derived macrophages were cultured with conditioned media of IWP-2 treated hDPSCs to observe the immunosuppressive property.
RESULTS: RNA sequencing indicated that IWP-2 significantly downregulated several KEGG pathways, including cytokine-cytokine receptor interaction, IL-17 signalling pathway, and TNF signalling pathway. In both normal and inflamed conditions, IWP-2 markedly upregulated TGFB1 mRNA expression while the mRNA expression of pro-inflammatory cytokines, TNFA, IL1B, IFNG, and IL6, was inhibited. In the inhibition experiment, the pretreatment with p38, MAPK, or PI3K inhibitors abolished the effects of IWP-2 in LPS-induced inflammation. In terms of immune cells, IWP-2-treated-inflamed hDPSCs conditioned media attenuated T cell proliferation and regulated regulatory T cell differentiation. In addition, the migratory property of macrophage was decreased after being exposed to IWP-2-treated inflamed hDPSCs conditioned media.
CONCLUSIONS: IWP-2 suppressed inflammatory cytokine expression in both normal and inflamed hDPSCs. Moreover, hDPSCs exerted the immunosuppressive property after IWP-2 treatment. These results suggest the role of Wnt in inflammatory responses and immunomodulation in dental pulp tissues.
方法:从人牙髓组织中分离hDPSC。细胞用25μMIWP-2处理24小时,随后,使用高通量RNA测序检查基因表达谱.使用qPCR分析mRNA表达。在正常和LPS诱导的hDPSC(发炎的hDPSC)中研究了IWP-2的作用。用IWP-2处理的hDPSC的条件培养基培养CD4+T细胞和CD14+单核细胞来源的巨噬细胞以观察免疫抑制特性。
结果:RNA测序表明IWP-2显著下调几个KEGG通路,包括细胞因子-细胞因子受体相互作用,IL-17信号通路,和TNF信号通路。在正常和发炎的情况下,IWP-2显著上调TGFB1mRNA的表达,而促炎细胞因子的mRNA表达,TNFA,IL1B,IFNG,和IL6,被抑制。在抑制实验中,p38,MAPK预处理,或PI3K抑制剂消除了IWP-2在LPS诱导的炎症中的作用。在免疫细胞方面,IWP-2处理的发炎的hDPSC条件培养基减弱T细胞增殖并调节调节性T细胞分化。此外,暴露于IWP-2处理的发炎hDPSC条件培养基后,巨噬细胞的迁移特性降低。
结论:IWP-2抑制了正常和发炎hDPSC的炎性细胞因子表达。此外,hDPSC在IWP-2处理后发挥免疫抑制特性。这些结果表明Wnt在牙髓组织的炎症反应和免疫调节中的作用。