Deciphering the nervous and immune systems dialogue

拟议研究计划的总体目标是创建一个体外双报告共培养系统，以揭示驱动周围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自主的实体，但越来越多的证据表明，它们以一种综合和协调的方式发挥作用。因此，它们共存于粘膜屏障，具有感知外部和内部环境威胁、检测危险和协调适当防御的能力。它们还拥有一种共同语言，包括细胞因子、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；使它们能够对各自产生的信号做出反应。在外周，伤害性刺激激活伤害性感受器产生的局部去极化启动动作电位激发、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终末，提高胞浆钙水平，导致神经肽的局部释放。反过来，这些多肽作用于内皮细胞和平滑肌细胞，产生红肿、发热和神经源性水肿，并作用于组织驻留的免疫细胞，以调节其功能。因此，在解剖学上，伤害性感受器位于初级和次级淋巴组织、皮肤和粘膜中，在那里它们可以与免疫细胞相互作用，并检测屏障功能的障碍。伤害性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、释放的神经肽以及驱动相互作用的神经元的解剖来源(三叉神经、腰椎或迷走神经)。*我们建立了一种体外实验，以钙内流为主要激活读数的高含量双报告系统来模拟和系统地研究粘膜神经免疫相互作用。在对系统的一只手臂进行药物刺激后，将分别采集相应的反应细胞及其上清，绘制转录组和蛋白质组图，并进行计算机分析，以揭示潜在的命中。接下来，将使用遗传学和药理学获得和丧失功能的方法进行靶标验证，同时将测量神经元的敏感性(钙显微镜)和活性(电生理学)，以及免疫细胞的增殖(活性染料稀释试验)、极化(内部细胞因子表达)和迁移(定量Transwell分析)能力作为功能读数。总体而言，接受宿主防御的神经学和免疫学方面将促进我们对在健康粘膜屏障上控制动态平衡的生物学的理解**

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**