Single cell RNA-Sequencing of adipose innate T cells for investigation of circadian rhythm.

The circadian rhythm of the immune system helps to protect against pathogens; however, the role of circadian rhythms in immune homeostasis is less well understood. Innate T cells are tissue-resident lymphocytes with key roles in tissue homeostasis47. Here we use single-cell RNA sequencing, a molecular-clock reporter and genetic manipulations to show that innate IL-17-producing T cellsincluding T cells, invariant natural killer T cells and mucosal-associated invariant T cellsare enriched for molecular-clock genes, compared with their IFN-producing counterparts. We reveal that IL-17-producing T (17 T) cells, in particular, rely on the molecular clock to maintain adipose tissue homeostasis, and exhibit a robust circadian rhythm for RORt and IL-17A across adipose depots, which peaks at night. In mice, loss of the molecular clock in the CD45 compartment (Bmal1Vav1) affects the production of IL-17 by adipose 17 T cells, but not that of cytokines by T or by IFN-producing T (IFN T) cells. Circadian IL-17 is essential for de novo lipogenesis in adipose tissue, and mice with an adipocyte-specific deficiency in IL-17 receptor C (IL-17RC) have defects in de novo lipogenesis. Whole-body metabolic analysis in vivo shows that Il17a/f/ mice (which lack the expression of IL-17A and IL-17F) have defects in their circadian rhythm for de novo lipogenesis, which results in disruptions to their whole-body metabolic rhythm and core-body temperature rhythm. This study identifies a crucial role for IL-17 in whole-body metabolic homeostasis, and shows that de novo lipogenesis is a major target of IL-17. We performed 10x single cell RNA-Sequencing of T cells and iNKT cells from murine epididymal white adipose tissue. Both innate T cell populations were sorted using FACS and then combined and sequenced as sample. A total of five visceral adipose tissue deposits from five independent biological replicates were pooled for sequencing.免疫系统的昼夜节律有助于抵御病原体，但学界对昼夜节律在免疫稳态中的作用仍未完全明晰。固有T细胞（innate T cells）是定居于组织的淋巴细胞，在组织稳态中发挥关键作用47。本研究借助单细胞RNA测序（single-cell RNA sequencing）、分子钟报告因子及遗传操纵技术，证实相较于产生干扰素（IFN）的对应细胞群，产生IL-17的固有T细胞——包括γδ T细胞、恒定自然杀伤T细胞（invariant natural killer T cells）及黏膜相关恒定T细胞（mucosal-associated invariant T cells）——的分子钟基因表达更为富集。研究发现，IL-17分泌型T（17 T）细胞尤其依赖分子钟维持脂肪组织稳态，并在不同脂肪库中呈现RORγt与IL-17A的显著昼夜节律，其表达峰值于夜间出现。在小鼠模型中，CD45+细胞群的分子钟缺失（Bmal1Vav1小鼠）会影响脂肪组织中IL-17分泌型T细胞的IL-17分泌，但不会改变γδ T细胞或产生IFN的T细胞（IFN+ T细胞）的细胞因子产生水平。昼夜节律性IL-17对脂肪组织的从头脂肪生成至关重要，而脂肪细胞特异性缺失IL-17受体C（IL-17RC）的小鼠会出现从头脂肪生成缺陷。体内全身代谢分析显示，Il17a/f缺陷小鼠（缺乏IL-17A与IL-17F的表达）的从头脂肪生成昼夜节律存在异常，进而引发全身代谢节律及核心体温节律紊乱。本研究阐明了IL-17在全身代谢稳态中的关键作用，并证实从头脂肪生成是IL-17的主要作用靶点。本研究对小鼠附睾白色脂肪组织中的固有T细胞与恒定自然杀伤T细胞开展了10x单细胞RNA测序：通过荧光激活细胞分选术（fluorescence-activated cell sorting，FACS）分离两类固有T细胞群后，将其合并为一个样本进行测序；共将5份独立生物学重复的内脏脂肪组织样本混合后完成测序。