Regulation of type 2 immune responses via neuropeptides and neurotransmitters

先天淋巴样细胞(ILC)是抵抗感染的第一道防线，协调组织的免疫状态和动态平衡。ILC2参与2型免疫反应，包括蠕虫感染和慢性过敏反应。它们产生2型细胞因子(主要是IL-5和IL-13)，以响应广泛的环境刺激，如警素(IL-33、IL-25、TSLP)和神经调节剂(NE、Ach、CGRP、NMU)。这些信号中的每一个及其组合都独一无二地控制着ILC2的活动。因此，这些细胞在不同的环境和组织中具有异质性表型。血管活性肠肽(VIP)是一种神经肽，被认为是ILC2s的另一种调节因子，是昼夜节律系统的重要调节因子。众所周知，VIP的释放受到喂食的影响，ILC2以昼夜节律的方式产生IL-5。然而，与其他神经递质不同的是，VIP介导的ILC2调节的确切作用、位置和生理相关性仍未完全阐明。此外，缺乏有效和特定的遗传工具来干扰ILC2中的VIP信号，以及相互矛盾的研究限制了我们对这一潜在的神经免疫回路的了解。在这里，我们建议使用我们的新的小鼠模型，它允许在ILC2s中产生Vipr2条件基因敲除，以及其他现有的小鼠模型，来研究体内的VIP神经元(例如，完整的DREADD系统)，结合经典的(例如，流式细胞术、细胞因子分析)和创新的基因组学方法(例如，单核RNAseq和空间RNAseq)来从功能上表征VIper能神经元和ILC2s的相互作用。我们将首先确定ILC2s中的VIP信号是否参与了它们的异质性、细胞因子的产生以及随后嗜酸性粒细胞在组织中的募集。我们将使用呼吸道和口腔过敏模型进一步确定在2型免疫反应过程中是否需要这种神经免疫回路。然后我们将确定这个回路是否驱动昼夜ILC2活动，包括IL-5的产生和随后嗜酸性粒细胞的组织动力学。我们将进一步评估受肠道嗜酸性粒细胞影响的肠道昼夜吸收、运动和绒毛结构是否依赖于VIP-ILC2轴。最后，我们将剖析VIP神经元的空间和时间活动，以及它们在稳态和食物过敏期间与小肠中ILC2的接近程度。总之，这项研究将有助于破译涉及ILC2的另一个神经免疫回路的生理功能，并阐明VIP在ILC2活性的昼夜调节中的难以捉摸的作用，以及它对嗜酸性粒细胞动态平衡和健康和疾病组织功能的影响。

Innate lymphoid cells (ILC) are the first line of defense against infections and orchestrate tissue immune status and homeostasis. ILC2s are involved in type 2 immune responses, including helminth infections and chronic allergic reactions. They produce type 2 cytokines (mainly IL-5 and IL-13) in response to a wide range of environmental stimuli, such as alarmins (IL-33, IL-25, TSLP) and neuromodulators (NE, Ach, CGRP, NMU). Each of these signals and their combination uniquely controls ILC2s' activity. Hence, these cells adopt a heterogenic phenotype in different contexts and tissues. The vasoactive intestinal peptide (VIP), an essential regulator of the circadian system, is a neuropeptide that has been proposed as another regulator of ILC2s. VIP release is known to be influenced by feeding, and ILC2s produce IL-5 in a circadian fashion. However, unlike other neurotransmitters, the precise action, location, and physiological relevance of VIP-mediated regulation of ILC2s are still not fully elucidated. Besides, the lack of efficient and specific genetic tools to interfere with VIP signaling in ILC2s and conflicting studies limit our understanding of this potential neuro-immune circuit. Here, we propose to use our new mouse model that allows the generation of Vipr2 conditional knockout in ILC2s, as well as other existing mouse models, to study VIP neurons in vivo (e.g., INTACT, DREADD systems ), together with a combination of classical (e.g., flow cytometry, cytokines assays), and innovative genomics approaches (e.g., single nuclei RNAseq, and spatial RNAseq) to functionally characterize the interplay of VIPergic neurons and ILC2s. We will first determine whether VIP signaling in ILC2s participates in their heterogeneity, cytokine production, and subsequent eosinophil recruitment to tissues. We will further determine whether this neuro-immune circuit is required during the type 2 immune response using respiratory and oral allergy models. Then we will determine whether this circuit drives circadian ILC2 activity, including IL-5 production and subsequent eosinophils' tissue dynamic. We will further evaluate whether circadian intestinal absorption, motility, and villi architecture, influenced by intestinal eosinophils, rely on the VIP-ILC2 axis. Finally, we will dissect VIP neurons' spatial and temporal activity and their proximity to ILC2s in the small intestine both at steady-state and during food allergy. Altogether, this study will help decipher the physiological functions of an additional neuro-immune circuit involving ILC2s and shed light on a yet elusive role of VIP in the circadian regulation of ILC2s' activity and its impact on eosinophil homeostasis and tissue functions in health and disease.