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Anti-microbial Functions of Group 3 Innate Lymphoid Cells in Gut-Associated Lymphoid Tissues Are Regulated by G-Protein-Coupled Receptor 183

SUMMARY The intestinal tract is constantly exposed to various stimuli. Group 3 innate lymphoid cells (ILC3s) reside in lymphoid organs and in the intestinal tract and are required for immunity to enteric bacterial infection. However, the mechanisms that regulate the ILC3s in vivo remain incompletely defined. Here, we show that GPR183, a chemotactic receptor expressed on murine and human ILC3s, regulates ILC3 migration toward its ligand 7α,25-dihydroxycholesterol (7α,25-OHC) in vitro, and GPR183 deficiency in vivo leads to a disorganized distribution of ILC3s in mesenteric lymph nodes and decreased ILC3 accumulation in the intestine. GPR183 functions intrinsically in ILC3s, and GPR183-deficient mice are more susceptible to enteric bacterial infection. Together, thes1e results reveal a role for the GPR183-7α,25-OHC pathway in regulating the accumulation, distribution, and anti-microbial and tissue-protective functions of ILC3s and define a critical role for this pathway in promoting innate immunity to enteric bacterial infection.


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 cells (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., 2013Spits et al., , 2016. ILCs are lineage-negative (Lin À ), interleukin-7 (IL-7) receptor a-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 RORgt 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 a, 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., 2013Hepworth et al., , 2015Kim 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 Gai-coupled seventransmembrane 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., , 2013Li et al., 2016;Pereira et al., 2009;Yi and Cyster, 2013;Yi et al., 2012). GPR183 ligand, 7a,25-dihydroxycholesterol (7a,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 (Emgå rd et al., 2018). However, whether GPR183 and 7a,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 7a,25-OHC in vitro. Quantitative PCR (qPCR) analysis indicated 7a,25-OHC production by gut stromal cells, and genetic deletion of GPR183 or 7a,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 GPR183-7a,25-OHC pathway in regulating ILC3-dependent immunity to enteric bacterial infection.

GPR183 Is Constitutively Expressed on ILC3s and
Mediates Migration toward 7a,25-OHC To study the pathways that regulate the distribution and accumulation of ILC3s in lymphoid and mucosal tissues in vivo, we undertook an unbiased analysis of molecules associated with these processes by RNA sequencing (RNA-seq). Among the genes listed in the gene ontology (GO) term ''lymphocyte chemotaxis,'' Gpr183 was the most abundant gene expressed in intestinal LP ILC3s ( Figure S1A). To further examine this, we sorted ILC subsets from intestinal LP cells as well as mLN cells harvested from Rorc(gt) Gfp mice and analyzed the expression of Gpr183 mRNA by quantitative PCR. Consistent with the RNAseq data, intestinal CCR6 + ILC3s and CCR6 À ILC3s and mLN ILC3s had high Gpr183 mRNA expression, and the expression in intestinal CCR6 + ILC3s was higher than in ILC1s and ILC2s ( Figure 1A). To examine GPR183 protein expression, we employed Gpr183 LacZ/+ reporter mice, which were generated by replacing the Gpr183 coding region with the LacZ gene, and we could detect LacZ expression in non-GC B cells, but not in GC B cells ( Figure S1B). We confirmed the LacZ expression in mLN and small intestinal (SI) ILC3s ( Figure 1B; gating strategy in Figure S1C). We also detected GPR183 protein staining on CD45 + Lin À CD127 + CD117 + CRTH2 À cells, which are ILC3s in human ileal LP and ILC precursors in peripheral blood mononuclear cells (PBMCs) (Lim et al., 2017) from healthy donors (Figures 1C and 1D; gating strategy in Figure S1D). Collectively, these data show that murine and human ILC3s express GPR183.

CH25H and CYP7B1 Are Expressed in Intestinal Stromal Cells
Cholesterol 25-hydroxylase (CH25H) and oxysterol 7a-hydroxylase (CYP7B1), which are enzymes required for the biosynthesis of GPR183 ligand 7a,25-OHC, are highly expressed in spleen and LN stromal cells (Hannedouche et al., 2011;Liu et al., 2011;Yi et al., 2012). To determine whether CH25H and CYP7B1 are also expressed in intestinal stromal cells, we fractionated mouse SI into epithelial, LP leukocyte (LPL), and stromal compartments and measured the relative expression of Ch25h and Cyp7b1 mRNA in each compartment by qPCR. Expression of Ch25h and Cyp7b1 was higher in the stromal compartment compared to epithelial and LPL compartments of the SI (Figure 1F), suggesting 7a,25-OHC production by intestinal stromal cells. Together with previous reports (Emgå rd et al., 2018;Yi et al., 2012), these data indicate that the interaction of GPR183 expressed on ILC3s and 7a,25-OHC produced by stromal cells in mLNs and the intestine may control the accumulation of ILC3s in these organs.
Previous studies identified that the localization of GPR183expressing T cells, B cells, and DCs within lymphoid tissues is regulated by 7a,25-OHC produced from tissue stromal cells (Gatto et al., , 2013Hannedouche et al., 2011;Li et al., 2016;Liu et al., 2011;Pereira et al., 2009;Yi and Cyster, 2013;Yi et al., 2012). To examine whether GPR183 regulates the localization of ILC3s in mLNs, we stained mLN sections from WT mice and Gpr183 À/À mice with anti-RORgt, CD3ε, and CD127 antibodies to visualize ILC3s ( Figure 2E). Serial sections were stained for B220 or MAdCAM-1 to examine the position of follicles and the subcapsular sinuses (Figures 2F and 2G). There was no overt difference in the structure of follicles and T cell areas in mLNs from Gpr183 À/À mice compared to WT mice, which is consistent with the previous report (Pereira et al., 2009). RORgt + CD3ε À CD127 + ILC3s were found predominantly in the interfollicular areas of WT mLN sections, which is consistent with published results Mackley et al., 2015). A significantly higher proportion of the Gpr183 À/À ILC3s were found in the outer regions of the interfollicular areas rather than their normal localization in mLNs compared to that of the WT ILC3s (Figures 2E-2H; refer to Figure S2F for the definition of inner and outer regions). Similarly, staining of mLN sections from WT mice and Ch25h À/À mice showed more ILC3s localized in the outer regions of the interfollicular areas of Ch25h À/À mLNs compared to WT mLNs (Figures 2I,2J,and S2F). Consistent with the flow cytometry data (Figures 2A and 2B), more ILC3s were found in the interfollicular areas in the Gpr183 À/À mLNs compared to WT ( Figure 2K). We also examined the Peyer's patches (PPs) and found increased ILC3s in the Gpr183 À/À PPs compared to WT (Figure S2G). Immunofluorescent staining of PP sections revealed accumulation of ILC3s in between the follicle and the T cell area in the luminal side of Gpr183 À/À mice ( Figure S2H). Collec- Data are representative of 2 independent experiments. **p < 0.01. (F) qPCR analysis of 7a,25-OHC-producing enzymes Ch25h and Cyp7b1 transcript abundances in fractionated SI samples from WT mice, presented relative to Hprt1. LPL, lamina propria leukocyte. Each symbol represents one mouse unless specifically indicated. Data are mean ± SEM. See also Figure S1. tively, these results suggest that GPR183 and 7a,25-OHC control the distribution and accumulation of ILC3s in the mLNs and PPs. Notably, ILC3s from Gpr183 À/À mice had comparable expression of CCR7 and CCR9 but significantly lower expression of integrin a4b7, which mediates cell trafficking to the intestine (Kim et al., 2015), compared to ILC3s from WT mice ( Figures 2L and S2I).
(H) Percentages of ILC3s localized in the outer regions over ILC3s localized in the interfollicular areas between follicles in mLN sections of WT and Gpr183 À/À mice. Twenty-three interfollicular areas in 15 mLNs from 3 WT mice and 21 interfollicular areas in 15 mLNs from 3 Gpr183 À/À mice were examined. ***p < 0.001. (I and J) Representative images of ILC3 distribution (I) and percentages of ILC3s localized in the outer regions (J) in mLN sections of WT and Ch25h À/À mice. Ten interfollicular areas in 10 mLNs from 2 WT mice and 14 interfollicular areas in 10 mLNs from 2 Ch25h À/À mice were examined. The scale bars represent 100 mm. In (J), *p < 0.05. between WT and Gpr183 À/À mice or Ch25h À/À mice ( Figures  S3A-S3D), but Gpr183 À/À mice had fewer CPs and isolated lymphoid follicles in the colon compared to WT mice ( Figures  S3E and S3F). Taken together, these results suggest that, through binding to 7a,25-OHC produced by intestinal stromal cells, GPR183 promotes the accumulation of ILC3s in the intestine.
The expression of the proliferation marker Ki67 was comparable in CD45.1 + CD45.2 + ILC3s (WT) and CD45.2 + ILC3s (WT or Gpr183 À/À ) in both chimeras ( Figure 3L). Therefore, GPR183 appears to regulate the accumulation, but not the proliferation, of ILC3s in the intestine and mLNs in a cell-intrinsic manner. Moreover, Gpr183 À/À :WT mice exhibited increased ratios of CD45.2 + cells in the ILC1 gate and comparable ratios of ILC2s compared to WT:WT mice, suggesting that the reduced ILC1s and ILC2s in Gpr183 À/À mLNs was secondary to the cell-intrinsic effect on ILC3s ( Figure S3I).

DISCUSSION
Mucosal barriers are constitutively challenged by various stimuli, and the homeostasis of mucosal barriers both at steady state and upon challenge are maintained by tissue-resident immune cells (Kurashima et al., 2013;Okumura and Takeda, 2016). ILC3s are found in lymphoid tissues and are enriched in the intestine, where they play critical roles in regulating adaptive immune responses against commensal bacteria, as well as in innate immunity against enteric bacterial infections Rankin et al., 2016;Satoh-Takayama et al., 2008;Sawa et al., 2011;Song et al., 2015;Sonnenberg et al., 2011). Although the mechanisms ILC3s employ to control infections and promote tissue repair continue to be defined (Satoh-Takayama et al., 2008;Sawa et al., 2011;Sonnenberg et al., 2011), our understanding of how the accumulation, distribution, and tissue-protective function of ILC3s in the intestine and its associated lymphoid organs are controlled remained limited. Emgå rd et al. (2018) recently reported that CD4 + LTi-like ILC3s express GPR183 that controls cell migration and formation of solitary intestinal lymphoid tissues in the colon and enhances IL-22 production by ILC3s in the colon at steady state. In the current study, we demonstrate that GPR183 is expressed on murine and human ILC3s and that GPR183 and its ligand 7a,25-OHC regulate the accumulation and distribution of ILC3s in lymphoid tissues and the intestine, and consequently, GPR183 controls ILC3-dependent innate immunity and tissue protection following enteric bacterial infection. We also identify GPR183-dependent accumulation of IL-22-producing ILC3s in the intestine following C. rodentium infection. Of note, enhanced IL-22 production by ILC3s was not detectable, possibly due to heightened inflammation elicited by the bacterial infection.
ILC3s reside in the interfollicular areas of the mLNs, where they present commensal bacterial antigen through major histocompatibility complex class II and prevent CD4 + T cellinduced chronic intestinal inflammation toward commensal bacteria . In this study, we show that GPR183 controls the distribution of ILC3s in mLNs. GPR183deficient ILC3s accumulated in the outer regions of the interfollicular areas, which are close to the subcapsular sinuses. DCs migrate into the LNs via the lymph through subcapsular sinuses and then move to the paracortex where they interact with helper T cells (Lian and Luster, 2015). This pathway is (G-J) Colon length (G), changes in body weight (H), Lcn2 mRNA expression (I), and survival (J) of infected Rag1 À/À and Rag1 À/À Gpr183 À/À mice. Body weight is presented relative to initial weight, set as 100%. Survival data are pooled from 2 experiments (8 mice per group). *p < 0.05, **p < 0.01. All data are representative of 2 independent experiments unless stated. Each symbol represents one mouse. Data are mean ± SEM. See also Figure S4. regulated by CCR7, a molecule that also controls the accumulation of ILC3s to LNs (Lian and Luster, 2015;Mackley et al., 2015). Similarly, ILC3s migrate from other organs, such as the intestine (Mackley et al., 2015), and enter the LNs through subcapsular sinuses. In the context of GPR183 deficiency, ILC3s cannot migrate into the interfollicular areas because they fail to respond to the ligand expressed in the inner regions of the interfollicular areas and hence are sequestered in the subcapsular sinuses. As GPR183 plays such important roles in regulating the distribution and function of ILC3s in both lymphoid and non-lymphoid tissues, GPR183 itself and its oxysterol ligandproducing pathway could be potential therapeutic targets for controlling and regulating ILC3 functions in multiple infectious and inflammatory diseases.

EXPERIMENTAL PROCEDURES
Further details and an outline of resources used in this work can be found in Supplemental Experimental Procedures.
Breeding of Gpr183 LacZ/+ to homozygosity resulted in Gpr183-deficient (Gpr183 LacZ/LacZ ) mice referred to as Gpr183 À/À throughout the manuscript. Sex-and age-matched WT and transgenic mice between 6 and 16 weeks of age were co-housed and used for experiments. All mice were maintained under specific pathogen-free conditions and were used in accordance with the Institutional Animal Care and Use Committee guidelines at Weill Cornell Medical College.

Quantification and Statistical Analysis
Statistical tests were performed with Prism (GraphPad). Unless specifically indicated otherwise, Student's t tests were used to compare endpoint means of different groups. Error bars depict the SEM. For the comparison of Kaplan-Meier survival curves, log rank (mantel-Cox) test was used. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures and four figures and can be found with this article online at https://doi.org/ 10.1016/j.celrep.2018.05.099.

ACKNOWLEDGMENTS
We thank the Artis lab members and the Sonnenberg lab members for discussion and critical reading of the manuscript. We also thank Dr. G. Eberl for Rorc(gt) Gfp