Light-entrained and brain-tuned circadian circuits regulate ILC3 and gut homeostasis

Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation, infection, microbiota composition and metabolism. ILC3 and neuronal cells were shown to interact at discrete mucosal locations to steer mucosal defence. Nevertheless, whether neuroimmune circuits operate at an organismal level, integrating extrinsic environmental signals to orchestrate ILC3 responses remains elusive. Here we show that light-entrained and brain-tuned circadian circuits regulate enteric ILC3, intestinal homeostasis and the host lipid metabolism. We found that enteric ILC3 display circadian expression of clock genes and ILC3-related transcription factors. ILC3-autonomous ablation of the circadian regulator Arntl led to disrupted gut ILC3 homeostasis, impaired epithelial reactivity, deregulated microbiome, increased susceptibility to bowel infection and disrupted lipid metabolism. Loss of ILC3-intrinsic Arntl shaped the gut postcode receptors of ILC3. Strikingly, light-dark cycles, feeding rhythms and microbial cues differentially regulated ILC3 clocks, with light signals as major entraining cues of ILC3. Accordingly, surgical- and genetically-induced deregulation of brain rhythmicity led to disrupted circadian ILC3 oscillations, deregulated microbiome and altered lipid metabolism. Our work reveals a circadian circuitry that translates environmental light cues into enteric ILC3, shaping intestinal health and organismal homeostasis. Overall design: Small intestine lamina propria ILC3 (CD45.2+ Lin- Thy1.2hi KLRG1-) were sort-purified from the small intestine of 8-14 weeks old WT (Arntlwt/fl) and KO (Rorc-Cre.Arntlfl/fl) mice at two different timepoints: ZT05 (WT n=3; KO n=3) and ZT23 (WT n=3; KO n=3). Total RNA was extracted (Qiagen Micro Kit) and quality was assessed using an Agilent 2100 Bioanalyzer. SMART-SeqII (ultra-low input RNA) libraries were prepared using Nextera XT DNA sample preparation kit (Illumina). Sequencing was performed on an Illumina HiSeq4000 platform, PE100.

3型先天淋巴细胞（Group 3 innate lymphoid cells, ILC3）是炎症、感染、微生物群组成及代谢的核心调控因子。已有研究证实，3型先天淋巴细胞与神经元细胞可在特定黏膜部位相互作用，调控黏膜防御功能。然而，神经免疫环路是否能够在机体层面运作，整合外在环境信号以调控3型先天淋巴细胞的应答，目前仍未明确。
本研究证实，受光调控且受大脑调节的昼夜节律环路可调控肠道3型先天淋巴细胞、肠道稳态以及宿主脂质代谢。我们发现，肠道3型先天淋巴细胞的时钟基因及3型先天淋巴细胞相关转录因子呈现昼夜节律性表达。3型先天淋巴细胞自主性敲除昼夜节律调控因子Arntl后，会导致肠道3型先天淋巴细胞稳态紊乱、上皮反应性受损、微生物群失调、肠道感染易感性升高以及脂质代谢异常。3型先天淋巴细胞内源性Arntl的缺失会重塑其肠道定位受体。值得注意的是，光暗周期、进食节律与微生物信号可差异化调控3型先天淋巴细胞的生物钟，其中光信号是3型先天淋巴细胞的主要授时因子。相应地，通过手术及遗传手段诱导的大脑节律紊乱，会导致3型先天淋巴细胞的昼夜节律振荡异常、微生物群失调以及脂质代谢改变。本研究揭示了一条昼夜节律环路，可将环境光信号转化为对肠道3型先天淋巴细胞的调控信号，进而塑造肠道健康与机体稳态。
实验设计概况：从8~14周龄野生型（Arntl^wt/fl）及敲除型（Rorc-Cre.Arntl^fl/fl）小鼠的小肠中，于两个不同时间点（ZT05：野生型组n=3，敲除型组n=3；ZT23：野生型组n=3，敲除型组n=3）分选纯化小肠固有层3型先天淋巴细胞（CD45.2⁺ Lin^- Thy1.2^hi KLRG1^-）。采用Qiagen微量试剂盒提取总RNA，并通过Agilent 2100生物分析仪检测RNA质量。使用Nextera XT DNA样本制备试剂盒（Illumina）构建SMART-SeqII（超低起始量RNA）文库。在Illumina HiSeq4000平台上进行PE100双端测序。