Glial cell-derived neuroregulators control type 3 innate lymphoid cells and gut defence

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2017In this thesis we demonstrate that three distinct players form a novel multi-tissue defence unit in the intestinal wall: group 3 of innate lymphoid cells (ILC3s), intestinal epithelial cells and enteric glial cells (EGCs). This interplay reveals a neuro-immune interaction unit that regulates epithelial homeostasis and mucosal defense. ILC3s are major regulators at mucosal surfaces being critical in tissue repair and in the maintenance of gut homeostasis. Intestinal ILC3 – that mainly aggregate into cryptopatches - integrate environmental signals leading to the production of the proinflammatory cytokines IL-22 and IL-17. IL-22 in turn induces intestinal epithelial cells to produce antimicrobial peptides and mucus. We found that ILC3s express high levels of RET, a neuroregulatory receptor for GDNF family ligands (GFLs). In order to address the effect of RET in ILC3s development and function RET-deficient mice foetal liver chimeras were analyzed. Interestingly a decrease of IL-22 expressing ILC3s was observed when compared to WT controls. In addition a RET gain of function model (RetMEN2B) resulted in increased IL-22 expressing ILC3s. In line with these experiments, cell-autonomous ablation of RET in Rorγt expressing cells was performed. RorγtCreRetfl/fl, (RetΔ) mice had decrease IL-22 expressing ILC3s and a reduction of epithelial reactivity genes such as mucins and defensins comparing with their littermate controls. Upon infection with the attaching and effacing bacteria Citrobacter rodentium, RetΔ mice had marked gut inflammation, reduced IL-22 producing ILC3, increased C. rodentium infection and translocation, reduced epithelial reactivity genes, increased weight loss and reduced survival. All these data together, suggest that RET cell autonomous ILC3 signals regulate IL-22 production. Signals downstream of Ret were regulated via GFLs which directly controled rapid phosphorylation of the p38 MAPK/ERK-AKT cascade and STAT3 activation in ILC3s. In turn, STAT3 bound to the Il22 promoter to induce transcription. Finally, we found that enteric glial cells integrated commensal and environmental signals to produce GFLs that control IL-22 production. Physical localization of glial cells in the vicinity of ILC3 was observed taking advantage of double reporter mice for GFAP (glial fibrillary acidic protein) and RET. Enteric glial cells had a stellate shape morphology, projecting into cryptopatches. In vitro co-culture studies showed EGCs capacity to produce GFLs in response to TLR2 and TLR4 activation and IL-1β and IL-33 stimulation, promoting IL-22 production by ILC3s. In vivo studies with DSS induced colitis in glial specific Myd88 deficient mice (Gfap-Cre.Myd88Δ) showed an increase of gut inflammation and weight loss along with a reduced expression of intestinal GFLs and ILC3-derived IL-22 levels compared with their littermate controls. When infected with Citrobacter Rodentium Myd88 deficient mice exhibited a pronounced susceptibility to bowel inflammation and infection. In summary, we were able to show that the enteric glial cells sense environmental cues through MYD88 to produce GFLs that in turn activate RET expressing ILC3s and via MAP kinase and STAT3 induce the transcription of Il22. The production of IL-22 promotes the expression of defence and repair genes. Thus, this novel glial-ILC3 epithelial unit is critical in the maintenance of intestinal homeostasis providing protection and repairing the epithelial barrier after injury

Neuronal regulation of type 2 innate lymphoid cells via neuromedin U

Group 2 innate lymphoid cells (ILC2s) regulate inflammation, tissue repair and metabolic homeostasis1, and are activated by host-derived cytokines and alarmins1. Discrete subsets of immune cells integrate nervous system cues2, 3, 4, but it remains unclear whether neuron-derived signals control ILC2s. Here we show that neuromedin U (NMU) in mice is a fast and potent regulator of type 2 innate immunity in the context of a functional neuron–ILC2 unit. We found that ILC2s selectively express neuromedin U receptor 1 (Nmur1), and mucosal neurons express NMU. Cell-autonomous activation of ILC2s with NMU resulted in immediate and strong NMUR1-dependent production of innate inflammatory and tissue repair cytokines. NMU controls ILC2s downstream of extracellular signal-regulated kinase and calcium-influx-dependent activation of both calcineurin and nuclear factor of activated T cells (NFAT). NMU treatment in vivo resulted in immediate protective type 2 responses. Accordingly, ILC2-autonomous ablation of Nmur1 led to impaired type 2 responses and poor control of worm infection. Notably, mucosal neurons were found adjacent to ILC2s, and these neurons directly sensed worm products and alarmins to induce NMU and to control innate type 2 cytokines. Our work reveals that neuron–ILC2 cell units confer immediate tissue protection through coordinated neuroimmune sensory responses

IFN-γ-dependent interactions between tissue-intrinsic γδ T cells and tissue-infiltrating CD8 T cells limit allergic contact dermatitis

BACKGROUND: Elicitation of allergic contact dermatitis (ACD), an inflammatory type 4 hypersensitivity disease, induces skin infiltration by polyclonal effector CD8 αβ T cells and precursors of tissue-resident memory T (TRM) cells. Because TRM have long-term potential to contribute to body-surface immunoprotection and immunopathology, their local regulation needs a fuller understanding.OBJECTIVE: We sought to investigate how TRM-cell maturation might be influenced by innate-like T cells pre-existing within many epithelia.METHODS: This study examined CD8+ TRM-cell maturation following hapten-induced ACD in wild-type mice and in strains harboring altered compartments of dendritic intraepidermal γδ T cells (DETCs), a prototypic tissue-intrinsic, innate-like T-cell compartment that reportedly regulates ACD, but by no elucidated mechanism.RESULTS: In addition to eliciting CD8 TRM, ACD induced DETC activation and an intimate coregulatory association of the 2 cell types. This depended on DETC sensing IFN-γ produced by CD8 cells and involved programmed death-ligand 1 (PD-L1). Thus, in mice lacking DETC or lacking IFN-γ receptor solely on γδ cells, ACD-elicited CD8 T cells showed enhanced proliferative and effector potentials and reduced motility, collectively associated with exaggerated ACD pathology. Comparable dysregulation was elicited by PD-L1 blockade in vitro, and IFN-γ-regulated PD-L1 expression was a trait of human skin-homing and intraepithelial γδ T cells.CONCLUSIONS: The size and quality of the tissue-infiltrating CD8 T-cell response during ACD can be profoundly regulated by local innate-like T cells responding to IFN-γ and involving PD-L1. Thus, interindividual and tissue-specific variations in tissue-intrinsic lymphocytes may influence responses to allergens and other challenges and may underpin inflammatory pathologies such as those repeatedly observed in γδ T-cell-deficient settings.</p

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation

International audienceGroup 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis