Data_Sheet_1_IL-7-Treated Periodontal Ligament Cells Regulate Local Immune Homeostasis by Modulating Treg/Th17 Cell Polarization.docx

Both interleukin (IL)-7 and human periodontal ligament cells (hPDLCs) have immunomodulatory properties. However, their combined effect on CD4+T cells has never been studied. In this study, we aimed to investigate the effect of conditioned medium of hPDLCs treated with rhIL-7 on the differentiation of CD4+T cells into regulatory T cells/T helper 17 cells (Treg/Th17 cells) and observe the effect of IL-7 on the immunomodulatory properties of PDLCs. After hPDLCs were treated with different concentrations of rhIL-7 for 24 h, the collected supernatants were used to incubate CD4+T cells for 3 days. A gamma-secretase inhibitor (DAPT) was used to suppress the activation of the Notch1 signaling pathway. Cell proliferation, apoptosis, and necrosis were determined using the cell counting kit-8 (CCK-8) and flow cytometry (FCM). The expressions of forkhead box P3 (Foxp3) in CD4+T cells and transforming growth factor (TGF-β) and IL-6 in the supernatants were determined by ELISA. Reverse transcription-quantitative PCR (RT-qPCR), and the Western blot (WB) determined the mRNA levels and protein expression of various target factors. FCM was used to detect the mean fluorescence intensity of PD-L1 in hPDLCs and to analyze the differentiation of Treg/Th17 cells. Our results showed that IL-7 promoted proliferation and inhibited apoptosis in hPDLCs, promoted the expression of TGF-β, PD-L1, Notch1, Jagged1, and Hes1, and inhibited the levels of hypoxia-inducible factor (HIF)-1α and TCF7, whereas the addition of DAPT effectively reversed these effects. Importantly, we found that the conditioned medium of hPDLCs treated with rhIL-7 promoted the polarization of CD4+T cells into Treg cells but had no significant effect on the differentiation of Th17 cells. Our study indicated that treatment of PDLCs with IL-7 can promote the polarization of CD4+T cells into Treg cells by modulating the expression of inflammatory factors and signaling molecules through activating the Notch1 signaling pathway, thus participating in the regulation of immune homeostasis in the periodontal microenvironment.

白介素-7（IL-7）与人类牙周韧带细胞（hPDLCs）均具备免疫调节特性。然而，二者对CD4+T细胞的联合作用尚未得到深入研究。本研究旨在探究经重组人IL-7（rhIL-7）处理的hPDLCs条件培养基对CD4+T细胞分化为调节性T细胞/T辅助17细胞（Treg/Th17细胞）的影响，并观察IL-7对PDLCs免疫调节特性的作用。将hPDLCs用不同浓度的rhIL-7处理24小时后，收集的培养基用于与CD4+T细胞共孵育3天。采用γ-分泌酶抑制剂（DAPT）以抑制Notch1信号通路的激活。通过细胞计数试剂盒-8（CCK-8）和流式细胞术（FCM）检测细胞增殖、凋亡和坏死。利用ELISA检测CD4+T细胞中叉头框P3（Foxp3）以及培养基中转化生长因子（TGF-β）和IL-6的表达。反转录定量PCR（RT-qPCR）和蛋白质印迹（WB）检测目标因子的mRNA水平和蛋白质表达。利用FCM检测hPDLCs中PD-L1的平均荧光强度，并分析Treg/Th17细胞的分化。我们的研究结果显示，IL-7可促进hPDLCs的增殖并抑制其凋亡，促进TGF-β、PD-L1、Notch1、Jagged1和Hes1的表达，同时抑制缺氧诱导因子（HIF）-1α和TCF7的水平，而DAPT的加入能有效逆转这些效应。重要的是，我们发现经rhIL-7处理的hPDLCs条件培养基可促进CD4+T细胞向Treg细胞的极化，但对Th17细胞的分化无显著影响。本研究表明，通过激活Notch1信号通路调节炎症因子和信号分子的表达，IL-7处理PDLCs可促进CD4+T细胞向Treg细胞的极化，从而参与牙周微环境中免疫稳态的调节。