Enteric Nervous System Derived IL-18 Orchestrates Mucosal Barrier Immunity [RNA-seq]

Using RNA sequencing, we report that neuron derived-IL-18 is required for intestinal antimicrobial protein expression. Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the anti-microbial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA-hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron derived IL-18 signaling controls tissue wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing. For Bulk RnaSeq experiments proximal colons were removed from: 1) Il18f/f and Il18f/fHand2Cre+, 2) Il18f/f and Il18f/fVillinCre+, and 3) Il18f/f and Il18f/fFLK1Cre+ littermate mice and RNA extracted.本研究通过RNA测序（RNA sequencing）证实，神经元来源的IL-18对于肠道抗菌蛋白的表达不可或缺。黏膜屏障免疫对于维持共生菌群稳态以及抵御侵袭性细菌感染至关重要。尽管既往认为免疫细胞与上皮细胞是维持这一复杂稳态的经典调控者，但本研究证实，肠神经系统（enteric nervous system, ENS）在调控抗菌蛋白（anti-microbial protein, AMP）应答过程中发挥着不可或缺且不可替代的作用。本研究通过共聚焦显微镜及单分子荧光原位mRNA杂交（single-molecule fluorescence in situ mRNA-hybridization, smFISH）实验，观察到肠道神经元可分泌多效性细胞因子IL-18。令人瞩目的是，仅特异性敲除肠神经元中的IL-18（而非免疫细胞或上皮细胞中的IL-18），即可使小鼠对侵袭性鼠伤寒沙门氏菌（Salmonella typhimurium, S.t.）感染易感。从机制层面分析，无偏RNA测序与单细胞测序结果显示，肠神经元来源的IL-18是稳态条件下杯状细胞产生抗菌蛋白所特异性必需的因素。综上，本研究证实神经元来源的IL-18信号通路可调控全组织肠道免疫，并对黏膜屏障功能及侵袭性细菌清除产生深远影响。针对批量RNA测序（bulk RNA sequencing）实验，本研究从以下3组同窝小鼠中摘取近端结肠：1）Il18f/f与Il18f/fHand2Cre+小鼠；2）Il18f/f与Il18f/fVillinCre+小鼠；3）Il18f/f与Il18f/fFLK1Cre+小鼠，随后提取其总RNA。