Deciphering the nervous and immune systems dialogue

提出的研究计划的总体目标是创建一个体外双报告共培养系统，以揭示驱动外周神经支配神经元亚群和粘膜驻留免疫细胞之间对话的机制。因此，虽然神经系统和免疫系统传统上被认为是两个独立的实体，但越来越多的证据表明，它们以一种综合和协调的方式起作用。因此，它们共存于粘膜屏障，共享感知外部和内部环境威胁、发现危险并协调适当防御的能力。它们还具有细胞因子、生长因子和神经肽以及细胞表面酪氨酸激酶和G蛋白偶联受体的共同语言；使它们能够对各自产生的信号做出反应。在外周，由有害刺激激活伤害感受器产生的局部去极化引发动作电位放电、反射和感觉。当动作电位到达感觉神经元分支点时，它们也被传递回外周末梢，提高细胞质钙水平，导致神经肽的局部释放。反过来，这些肽作用于内皮细胞和平滑肌细胞，产生发红、发热和神经源性水肿，并作用于组织驻留免疫细胞，调节其功能。因此，伤害感受器在解剖学上位于原发性和继发性淋巴组织、皮肤和粘膜中，在那里它们可以与免疫细胞相互作用并检测屏障功能的紊乱。伤害感受器对免疫细胞活性的影响取决于所涉及的神经元亚群、所释放的神经肽以及驱动相互作用的神经元的解剖学起源（三叉神经、腰椎或迷走神经）。我们建立了一个体外实验来模拟和系统地研究粘膜神经免疫相互作用，使用高含量的双报告系统，使用钙内流作为主要的激活读数。在对系统的一只手臂进行药理学刺激后，将分别收集相应的应答细胞及其上清，对转录组和蛋白质组进行分析，并进行计算机分析以揭示潜在的打击。接下来，基因和药理学的功能获得和功能丧失方法将用于靶标验证，而神经元敏感性（钙显微镜）和活性（电生理学），以及免疫细胞增殖（活力染料稀释试验），极化（内部细胞因子表达）和迁移（定量transwell试验）能力将作为功能读取来测量。总的来说，拥抱宿主防御的神经和免疫方面将促进我们对控制健康粘膜屏障内稳态的生物学的理解

The overarching objective of the proposed research program is to create an in vitro double reporter co-culture system to unravel the mechanisms driving the dialogue between subpopulation of peripherally innervating neurons and mucosal resident immune cells. Thus, while the nervous and immune systems are traditionally thought of as two autonomous entities, accumulating evidence suggests that they function in an integrated and coordinated manner. As such, they co-exist at mucosal barrier and share the ability to sense external and internal environmental threats, detect danger and coordinate appropriate defenses. They also possess a common language of cytokines, growth factors and neuropeptides, as well as cell surface tyrosine kinase and G protein-coupled receptors; enabling them to respond to the signals each produce. In the periphery, the local depolarization produced by noxious stimuli activation of nociceptors initiates action potential firing, reflexes and sensations. When action potentials reach the sensory neuron branch points, they are also transmitted back to the peripheral terminals, raising cytoplasmic calcium levels which lead to the local release of neuropeptides. In turn, these peptides act on the endothelial and smooth muscle cells to produce redness, heat and neurogenic edema, as well as on tissue resident immune cells to modulate their function. Thus, nociceptors are anatomically positioned in primary and secondary lymphoid tissues, skin and mucosa where they can interact with immune cells and detect disturbances in barrier function. Such influence of nociceptors on immune cell activity depends on the subset of neurons involved, the neuropeptides being released and the anatomical origin of the neurons driving the interaction (trigeminal, lumbar or vagal). We set out an in vitro assay to model and systematically study the mucosal neuro-immune interplay using a high-content double reporter system which uses calcium influx as primary activation readout. Following the pharmacological stimulation of one arm of the system, the corresponding responsive cells and their supernatant will be respectively harvested, transcriptome and proteome profiled and in silico analysis performed to reveal potential hits. Next, genetic and pharmacological gain- and loss-of-function approaches will be used for target validation while neurons sensitivity (calcium microscopy) and activity (electrophysiology), as well as immune cells proliferation (viability dye dilution assay), polarization (internal cytokine expression) and migration (quantitative transwell assay) capacities will be measured as functionality readouts. Overall, embracing the neurological and immune aspects of host defense will advance our understanding of the biology controlling homeostasis at healthy mucosal barriers