Control of mucosal defense and innate immunity by neuroregulators

Mucosal surfaces are constantly exposed to environmental insults, being susceptible to infection, inflammation and tissue damage. At these sites, an integrated network of non-hematopoietic and hematopoietic cells is critical to maintain epithelial barrier integrity and mucosal homeostasis. In the past years, a novel family of innate effectors has been identified and termed as innate lymphoid cells (ILCs). These innate lymphocytes are important regulators of inflammation and immunity at mucosal barriers. The combined expression of lineage-specific transcription factors and their cytokine profile led to the identification of three distinct ILCs subsets, namely group 1 ILCs (ILC1s), ILC2s and ILC3s. ILC2s sense barrier disruption by integrating locally produced alarmins, including interleukin (IL)-33, IL-25 and thymic stromal lymphopoietin. In response to alarmins, activated ILC2s produce IL-5, IL-13 and amphiregulin (AREG), which contribute to anti-helminth immunity, allergic inflammation and tissue remodeling. Despite increasing progress in the understanding of ILC2 biology, how ILC2s perceive and integrate environment-derived cues remains poorly understood. Recently, pivotal studies demonstrated that enteric glial cells and neurons can regulate immune cell function and mucosal homeostasis. These studies raised the hypothesis that neurons and immune cells might interact in a bidirectional manner to form functional neuro-immune cell units (NICUs) and to ensure tissue physiology. Work in this thesis initially analyzed the ILC transcriptome, revealing that ILC2s selective express neuromedin U receptor 1 (Nmur1), which encodes the peripheral receptor of the neuropeptide Neuromedin U (NMU). NMU is preferentially expressed in the central nervous system and in the gastrointestinal tract and it was described to regulate, among others, smooth muscle contraction, inflammation, energy homeostasis and feeding. Herein, we further shown that mucosal ILC2s are found in close proximity to NMUexpressing cholinergic neurons. Furthermore, in vitro stimulation of ILC2s with NMU lead to a fast and potent production of type 2 cytokines, notably IL-5, IL-13 and AREG in a Nmur1-dependent manner. Accordingly, in vivo NMU treatment induced a selective increase of ILC2-derived type 2 cytokines, while T helper cells remain unaffected. Remarkably, NMU stimulation of ILC2s, lead to a faster and stronger production of type 2 cytokines when compared with the canonical ILC2-activating alarmins. Using pharmacological inhibitors, we further demonstrated that NMU induces type 2 cytokine production downstream of Ca2+/calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal–regulated kinase (ERK) signaling pathways. Since ILC2s are enriched at highly innervated mucosae we hypothesized that NMUNMUR1 axis might orchestrate barrier defense. Strikingly, infection with Nippostrongylus brasiliensis induced a strong Nmu upregulation in infected tissues. Interestingly, in vivo administration of NMU to N. brasiliensis infected mice resulted in a selective increase of ILC2 responses, reduced tissue hemorrhage, increased cellular infiltrates and reduced infection burden when compared to control-untreated mice. Furthermore, infected Nmur1-deficient mice or chimeric mice reconstituted with Nmur1-deficient ILC2s displayed an impaired ILC2 response and increased N. brasiliensis infection burden. Sensing molecules such as toll-like receptors (TLRs) contributes to an efficient initiation of type 2 response. To uncover whether neurons could coordinate the initiation of ILC2 responses, we have employed a neuronal organoid system. Notably, stimulation of neurosphere-derived neurons with the alarmin IL-33 or with N. brasiliensis excretory/secretory products (NES) resulted in upregulation of Nmu in a myeloid differentiation primary response 88 (MYD88)-dependent manner. Interestingly, deletion of MYD88 on cholinergic neurons impaired ILC2 responses upon helminth infection. Collectively, the data presented in this thesis uncover a novel layer of homeostatic regulation where mucosal neurons by sensing worm components or alarmins produce NMU that specifically activates ILC2s to produce type 2 cytokines, providing an immediate protection against hookworm infections. Altogether, in a context of a mucosal aggression, functional NICUs ensure a synchronized, integrated and effective tissue homeostatic and protective responses