肠道上皮细胞中的鞘氨醇-1-磷酸/鞘氨醇-1-磷酸受体2轴在肠道上皮细胞与CD4+T细胞相互作用过程中调节肠道屏障功能。
The Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis in Intestinal Epithelial Cells Regulates Intestinal Barrier Function During Intestinal Epithelial Cells-CD4+T-Cell Interactions.
作者信息
Chen Tanzhou, Lin Ruoyang, Jin Sisi, Chen Renpin, Xue Haibo, Ye Huajun, Huang Zhiming
出版信息
Cell Physiol Biochem. 2018;48(3):1188-1200. doi: 10.1159/000491985. Epub 2018 Jul 25.
BACKGROUND/AIMS: Epithelial cells line the intestinal mucosa and form an important barrier for maintaining host health. This study aimed to explore the mechanism of the Sphingosine-1-phosphate (S1P)/Sphingosine-1-phosphate receptor 2 (S1PR2) pathway in intestinal epithelial cells (IECs) that participate in the intestinal barrier function.
METHODS
In this study, we constructed a knockout of the S1PR2 gene in mice, and Dextra sulfate sodium (DSS) was used to induce colitis. We isolated IECs from wild type (WT) and S1PR2-/- mice, and the endogenous expression of S1PR2 and Zonula occludens 1 (ZO-1) in IEC were detected by Western blot. Next, the major histocompatibility complex II (MHC-II) expression was analyzed by reverse transcription quantitative real-time (RT-qPCR) and flow cytometry. The in vivo and in vitro intestinal permeability were evaluated by serum fluorescein isothiocyanate (FITC) concentration. The tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-γ (IFN-γ) levels in cell suspension were analyzed by enzyme-linked immuno sorbent assay (ELISA). A carboxyfluorescein diacetate succinimidyl ester (CFSE) assay was used to detect the T-cell proliferation in a co-culture system.
RESULTS
The intestinal mucosal barrier damage in S1PR2-/- mice was more severe than in the WT mice, and there were more CD4+T-cells in the colon tissue of DSS-treated S1PR2-/- mice. Either the mouse colon carcinoma cell line (CT26. WT) or the IECs upregulated MHC-II expression, which then promoted CD4+T-cell proliferation. The S1P/S1PR2 pathway controlled MHC-II expression to regulate CD4+T-cell proliferation via the extracellular signal-regulated kinase (ERK) pathway. In addition, the IFN-γ that was secreted by CD4+T-cells increased DSS-induced damage of intestinal epithelial cell barrier function. ZO-1 expression was increased by S1P in CT26.WT cells, while S1PR2 antagonist JTE-013 expression was downregulated. However, in CT26.WTsi-S1PR2 cells, S1P had no effect on ZO-1 expression.
CONCLUSIONS
The S1P/S1PR2 axis in IECs mediated CD4+T-cell activation via the ERK pathway and MHC-II expression to regulate intestinal barrier function.
背景/目的：上皮细胞排列在肠道黏膜表面，是维持宿主健康的重要屏障。本研究旨在探讨鞘氨醇-1-磷酸（S1P）/鞘氨醇-1-磷酸受体2（S1PR2）通路在参与肠道屏障功能的肠上皮细胞（IECs）中的作用机制。
方法
本研究构建了S1PR2基因敲除小鼠，并用葡聚糖硫酸钠（DSS）诱导结肠炎。我们从野生型（WT）和S1PR2基因敲除小鼠中分离出IECs，通过蛋白质免疫印迹法检测IECs中S1PR2和紧密连接蛋白1（ZO-1）的内源性表达。接下来，通过逆转录定量实时（RT-qPCR）和流式细胞术分析主要组织相容性复合体II（MHC-II）的表达。通过血清异硫氰酸荧光素（FITC）浓度评估体内和体外肠道通透性。通过酶联免疫吸附测定（ELISA）分析细胞悬液中肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和干扰素-γ（IFN-γ）水平。使用羧基荧光素二乙酸琥珀酰亚胺酯（CFSE）测定法检测共培养系统中的T细胞增殖。
结果
S1PR基因敲除小鼠的肠道黏膜屏障损伤比WT小鼠更严重，在DSS处理的S1PR2基因敲除小鼠的结肠组织中有更多的CD4+T细胞。小鼠结肠癌细胞系（CT26.WT）或IECs均上调MHC-II表达，进而促进CD4+T细胞增殖。S1P/S1PR2通路通过细胞外信号调节激酶（ERK）通路控制MHC-II表达，以调节CD4+T细胞增殖。此外，CD4+T细胞分泌的IFN-γ增加了DSS诱导的肠上皮细胞屏障功能损伤。S1P可增加CT26.WT细胞中ZO-1的表达，而S1PR2拮抗剂JTE-013的表达下调。然而，在CT26.WTsi-S1PR2细胞中，S1P对ZO-1表达没有影响。
结论
IECs中的S1P/S1PR2轴通过ERK通路和MHC-II表达介导CD4+T细胞活化，从而调节肠道屏障功能。
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