Grant: Discovery Grants Program - Individual

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