br Experimental Procedures br Author Contributions br Acknow
Introduction The intestinal mucosal barrier surface is constantly exposed to food antigens, beneficial microbes, pathogens, and a multitude of other environmental stimuli (Turner, 2009). Innate lymphoid Nintedanib (ILCs) are known to contribute to innate and adaptive immune responses against these stimuli and play a critical role in maintaining barrier function and intestinal homeostasis (Artis and Spits, 2015, Diefenbach et al., 2014, Eberl et al., 2015, Klose and Artis, 2016, Spits et al., 2013, Spits et al., 2016). ILCs are lineage-negative (Lin−), interleukin-7 (IL-7) receptor α-positive (CD127+), CD90+ innate immune cells that are widely distributed throughout the body, particularly enriched at the mucosal barriers (Artis and Spits, 2015, Diefenbach et al., 2014, Eberl et al., 2015, Klose and Artis, 2016). Group 3 ILCs (ILC3s) express the transcription factor RORγt and play pivotal roles in protecting against bacterial, viral, and fungal infections in the intestine by fortifying the epithelial barrier via rapid secretion of soluble factors, such as IL-22, lymphotoxin α, and IL-17A, as well as regulating CD4+ T cell responses toward intestinal commensal bacteria (Fernandes et al., 2014, Gladiator et al., 2013, Hepworth et al., 2013, Hepworth et al., 2015, Kim et al., 2012, Klose et al., 2013, Satoh-Takayama et al., 2008). ILC3s are enriched in lymphoid tissues and at mucosal barrier surfaces, such as the intestinal tract, protecting against hazardous environmental stimuli together with other immune cells (Artis and Spits, 2015, Diefenbach et al., 2014, Eberl et al., 2015, Klose and Artis, 2016). Immune cells express various G-protein-coupled receptors (GPRs), including C-C motif chemokine receptors (CCRs), C-X-C motif chemokine receptors, and other GPRs, such as GPR183 and sphingosine-1-phosphate receptors, which regulate cell migration, accumulation, and distribution in tissues. Several chemokine receptors have been reported to control the accumulation of a subset of the ILC3s (Ivanov et al., 2006, Kim et al., 2015, Mackley et al., 2015, Satoh-Takayama et al., 2014); however, the molecular mechanisms that regulate the accumulation, distribution, and function of the entire ILC3 population in lymphoid and mucosal tissues and their effects on anti-bacterial responses and tissue protection are incompletely defined. GPR183 (also known as EBI2) is a Gαi-coupled seven-transmembrane chemotactic receptor. It is highly expressed on follicular B cells, CD4+ dendritic cells (DCs), and CD4+ T cells but is downregulated on germinal center (GC) B cells in secondary lymphoid organs and controls cell migration to achieve efficient antibody responses and CD4+ T cell responses (Gatto et al., 2009, Gatto et al., 2013, Li et al., 2016, Pereira et al., 2009, Yi and Cyster, 2013, Yi et al., 2012). GPR183 ligand, 7α,25-dihydroxycholesterol (7α,25-OHC), is produced by stromal cells residing in the interfollicular regions of lymph nodes (LNs) and the bridging channels of the spleen (Hannedouche et al., 2011, Liu et al., 2011, Yi et al., 2012). GPR183 expressed on CD4+ ILC3s (also termed as lymphoid tissue inducer cells [LTis]) controls their migration and the formation of colonic tertiary lymphoid organs (Emgård et al., 2018). However, whether GPR183 and 7α,25-OHC control the accumulation, distribution, and tissue-protective function of ILC3s in the gut-associated lymphoid tissues and in the intestinal lamina propria (LP) has not been examined. In this study, we demonstrate that ILC3s isolated from the mesenteric LNs (mLNs) and intestinal LP express GPR183 and intestinal ILC3s migrate toward 7α,25-OHC in vitro. Quantitative PCR (qPCR) analysis indicated 7α,25-OHC production by gut stromal cells, and genetic deletion of GPR183 or 7α,25-OHC resulted in a disorganized accumulation of ILC3s in the subcapsular sinus of the mLNs and reduced ILC3 accumulation in the intestine. The regulation of ILC3 accumulation in the intestine by GPR183 was ILC3 intrinsic and was required for optimal IL-22 production and protective immunity against the enteric bacterium, Citrobacter rodentium (C. rodentium). Taken together, these data reveal a previously unrecognized role of the GPR183-7α,25-OHC pathway in regulating ILC3-dependent immunity to enteric bacterial infection.