雷帕霉素靶蛋白复合物1对肠道3型固有淋巴细胞功能的影响

Effect of mTORC1 on intestinal group 3 innate lymphocytes function

目的·探究雷帕霉素靶蛋白复合物1(mechanistic target of rapamycin complex 1,mTORC1)对3型固有淋巴细胞(group 3 innate lymphoid cell,ILC3)功能的影响。方法·使用mTORC1信号通路的特异性抑制剂雷帕霉素体外刺激野生型C57BL/6小鼠肠道固有层淋巴细胞(lamina propria leukocyte,LPL),初步观察ILC3细胞数量及功能的变化;之后采用流式细胞技术分选出小鼠肠道ILC3,用白细胞介素23(interleukin 23,IL-23)刺激使其活化,再通过实时荧光定量PCR检测活化后ILC3中编码转录因子视黄酸受体相关的孤儿核受体(retinoic acid receptor related orphan receptor,RORγt)、IL-22及mTORC1关键组分Raptor(regulatory associated protein of mTOR)的基因mRNA水平;构建ILC3细胞特异性敲除Raptor的基因工程小鼠,使用苏木精-伊红染色、流式细胞技术和实时荧光定量PCR检测mTORC1功能缺失后对肠道结构、结肠ILC3细胞数量及功能的影响。结果·雷帕霉素刺激后LPL中ILC3数量无变化,但分泌IL-22减少;ILC3被IL-23激活后,检测到编码Raptor、RORγt和IL-22的基因mRNA表达水平同步上调;Raptor缺失的小鼠肠道结构无明显损伤,结肠ILC3数量和亚群比例与对照小鼠无明显差异,但ILC3活化后分泌细胞因子IL-22的水平显著下降。结论·mTORC1功能缺失显著抑制ILC3分泌IL-22的能力,对肠道结构及肠道ILC3的发育无影响,可见mTORC1信号对肠道ILC3功能具有正向调控作用。

Objective·To investigate the effect of mechanistic target of rapamycin complex 1(mTORC1)on group 3 innate lymphoid cells(ILC3)function.Methods·Intestinal lamina propria leukocytes(LPL)of C57BL/6 wild type mice were stimulated by rapamycin,the specific inhibitor of mTORC1 signaling pathway,in vitro,and then quantity and function of ILC3 were detected by flow cytometry.Next,purified ILC3 from mice intestinal LPL were sorted by flow cytometry.After the activation of ILC3 with IL-23,mRNA expression levels of Rorc(the gene encoding retinoic acid receptor related orphan receptor,i.e.RORγt),Il22 and Rptor(the gene encoding key component protein of mTORC1,i.e.Raptor)were detected by real-time qPCR.For further study,a genetically engineered mouse model specifically knocked out Raptor in ILC3 was constructed.Effects of mTORC1 loss on the quantity and function of ILC3 as well as gut structure were detected by flow cytometry,real-time qPCR and hematoxylin-eosin staining.Results·The total ILC3 number had no change,but the secretion of IL-22 by ILC3 reduced after stimulation with rapamycin.Il22,Rorc and Rptor mRNA expression levels were upregulated simultaneously in ILC3 after activation with IL-23.In addition,there was no significant difference in the numbers and proportions of total ILC3 and ILC3 subsets as well as gut structure in Raptor-deficient mice,but the cytokine IL-22 secretion level of ILC3 significantly decreased in these mice.Conclusion·Loss of mTORC1 function inhibits ILC3 from secreting IL-22 but has no effect on the intestinal structure and intestinal ILC3 development,which reveals the positive regulation of mTORC1 signaling on intestinal ILC3 function.