The Transcription Factor RORα Preserves ILC3 Lineage Identity and Function during Chronic Intestinal Infection

Bernard C. Lo, Diana Canals Hernaez, R. Wilder Scott, Michael R. Hughes, Samuel B. Shin, T. Michael Underhill, Fumio Takei and Kelly M. McNagny
J Immunol December 15, 2019, 203 (12) 3209-3215; DOI: https://doi.org/10.4049/jimmunol.1900781

Key Points

  • RORα deficiency leads to a reduction in ILC2 and ILC3 subsets during infection.

  • Residual Rorasg/sg ILC3s have lower expression of Rorc and lineage-specific receptors.

  • RORα conserves ILC3 function by modulating integration of environmental cues.

Abstract

Innate lymphoid cells (ILCs) are critical for host defense and tissue repair but can also contribute to chronic inflammatory diseases. The transcription factor RORα is required for ILC2 development but is also highly expressed by other ILC subsets where its function remains poorly defined. We previously reported that Rorasg/sg bone marrow chimeric mice (C57BL/6J) were protected from Salmonella-induced intestinal fibrosis due to defective ILC3 responses. In this study, single-cell RNA analysis of ILCs isolated from inflamed tissues indicates that RORα perturbation led to a reduction in ILC3 lineages. Furthermore, residual Rorasg/sg ILC3s have decreased expression of key signature genes, including Rorc and activating cytokine receptors. Collectively, our data suggest that RORα plays a key role in preserving functional ILC3s by modulating their ability to integrate environmental cues to efficiently produce cytokines.

This article is featured in In This Issue, p.3089

Introduction

Innate lymphoid cells (ILCs) are common lymphoid progenitor–derived cytokine-producing cells but lack Ag-specific TCRs that are the hallmark of the adaptive immune system (1). ILCs are enriched at mucosal surfaces where they play key roles in host defense against infections, tissue homeostasis, and repair but can also contribute to pathological conditions associated with unresolved inflammatory responses (1, 2). Although distinct from the Th cell lineages, ILCs are functionally comparable, as they express a similar repertoire of cytokines and the associated lineage-maintaining transcription factors; ILC1s express T-bet and are an important source of IFN-γ, ILC2s are GATA3-dependent and produce IL-5 and IL-13, whereas ILC3s require RORγt expression and produce IL-17 and IL-22 (3). In addition to helper-like ILCs, eomesodermin (EOMES)-dependent NK cells represent a distinct developmental lineage of cytotoxic ILCs that express granzymes and perforin (3).

ILCs have been studied extensively in the context of intestinal inflammation. In multiple mouse models of colitis, activated ILC1s and ILC3s promote intestinal immunopathology through the secretion of IFN-γ and IL-17A (4–6). Similarly, inflamed intestinal tissues of patients with Crohn disease (CD) are selectively enriched for IL-23–responsive ILCs, suggesting a possible pathological role of ILC3s and IL-17A (7). However, conflicting reports have indicated that the frequencies of ILC1s are highly elevated in the intestinal lamina propria of CD patients at the expense of NCR+ ILC3s (8); furthermore, the bidirectional plasticity between ILC1s and ILC3s can be modulated by IL-12 or IL-23 exposure and the antagonistic expression of T-bet and RORγt (9). Consistent with these observations, fate mapping experiments in mice suggest that ILC3s can downregulate RORγt in response to IL-12 and IL-15 and acquire ILC1-like properties such as elevated IFN-γ production and NK cell receptor expression (10). These studies emphasize the significance of environmental cues and the stability of transcription factors in ILC lineage maintenance.

RORα (retinoic acid receptor–related orphan receptor α) is a member of the nuclear receptor transcription factor superfamily (11). A natural mutation in Rora (Rorasg/sg) results in a nonfunctional protein lacking the C-terminal ligand-binding domain (12). Rorasg/sg bone marrow (BM) transplant experiments have revealed that hematopoietic RORα is essential for ILC2 development (13, 14) but also acts synergistically with RORγt in the induction of normal Th17 responses. Intriguingly, we recently found that mice lacking hematopoietic expression of RORα are protected from intestinal fibrosis in a Salmonella-driven model of CD due to attenuated ILC3 production of IL-17A (15). In this study, we use single-cell RNA sequence analysis to further define the role of RORα in ILC responses during infection-induced gut inflammation and fibrosis. Our data suggest RORα plays an essential role in preserving ILC3 fate under inflammatory conditions and that, in its absence, these cells take on an ILC1-like fate.

Materials and Methods

Mouse models

B6.SJL-PtprcaPepcb/BoyJ (CD45.1) and B6.C3(Cg)-Rorasg/J (Rorasg/sg) mice were maintained specific pathogen-free at the Biomedical Research Centre. BM chimeras were generated by lethally irradiating (10 Gy) and reconstituting CD45.1 animals with 2 million BM cells from sex-matched wild-type (WT) or Rorasg/sg littermates. Mice were given 20 mg of streptomycin by gavage 24 h prior to oral infection with 3 × 106 Salmonella Typhimurium ΔaroA CFU (16). All experiments performed were approved by the University of British Columbia Animal Care Committee.

Cell sorting

For immune cell isolation from ceca, tissues were flushed with PBS to remove luminal contents followed by mincing and incubation with collagenase D (1.5 U/ml) and dispase II (2.4 U/ml) (Roche) for 30 min at 37°C with gentle rotation. Samples were then passed through a 70-μm mesh nylon strainer. Hematopoietic cells were enriched by Percoll (GE) separation. Mesenteric lymph nodes (mLNs) were passed through a 70-μm strainer. Single-cell suspensions were then incubated with anti-CD16/32 prior to cell surface Ab staining. Cells were sort purified using a BD Influx system.

RNA sequencing

In droplet-based 3′ end massively parallel single-cell RNA sequencing (scRNA-seq) experiments, sort-purified cells were encapsulated into droplets. Libraries were then prepared using Single Cell 3′ reagent kits according to the manufacturer’s protocol (10x Genomics). The scRNA-seq libraries were sequenced using a 75 cycle NextSeq500 with 26 bp read 1, 58 bp read 2, and 8 bp index 1 read. Samples were aligned using the Mus musculus mm10 reference transcriptome and the standard Cell Ranger count pipeline. Data analysis and visualization was performed using the R package Seurat (17). Highly variable genes for aggregate ILC libraries from cecum (995 genes) and mLNs (1079 genes) were used for principal component (PC) analysis; PCs used were highly significant as determined by Jackstraw analysis and visualization of heatmaps (Supplemental Figs. 2, 3). PCs 1:7 (cecum) and 1:8 (mLN) at a resolution of 0.3 were used for graph-based clustering. For bulk RNA-seq experiments, cells were sorted directly into TRIzol LS (Thermo Fisher Scientific) for RNA isolation. The Agilent 2100 Bioanalyzer was used to assess RNA integrity; qualifying samples were then prepared following the standard protocol for the TruSeq stranded mRNA library kit on the Neoprep Library System (Illumina). Sequencing was performed on the Illumina NextSeq 500 with Paired End 75 × 75 bp reads. Demultiplexed read sequences were then aligned to the Mus musculus mm10 reference sequence using TopHat2 splice junction mapper and differential expression assessed using Cuffdiff through bioinformatics applications on Basespace. Scatter plot and heat maps were generated using open source R and VisR applications (18).

Nuclear and intracellular Ag staining and analysis

Cell surface staining was performed using fluorescence-conjugated Abs against CD45.2 (104), CD11b (M1/70), CD11c (N418), NK1.1 (PK136), Gr1 (RB6-8C5), CD19 (1D3), B220 (RA3-6B2), CD8 (53.67), CD3e (145-2C11), CD90.2 (30-H12), CD127 (SB/199), and KLRG1 (2F1). Then cells were fixed and permeabilized using a Foxp3/Transcription Factor staining buffer set (eBiosciences) followed by transcription factor staining with fluorescence-conjugated Abs against RORγt (B2D), T-bet (4B10), and EOMES (Dan11mag). Nonviable cells were excluded using a fixable viability dye (eBiosciences). Data acquisition was performed on a BD LSR II and analyzed using FlowJo.

Statistics

Data are presented as mean ± SEM. Statistical significance was assessed by Student t test using GraphPad Prism.

Results

RORα preserves ILC2s and ILC3s during chronic inflammation and fibrosis

Although the nuclear receptor RORα is a core transcript expressed by all ILC subsets (19), its deletion does not affect ILC3 numbers in mucosal tissues of naive mice (20, 21). We previously reported that RORα and ILCs are involved in the development of fibrotic disease. To further define the role of RORα in ILC function and gene expression and how this contributes to intestinal fibrosis, we sort purified cytotoxic and helper-like ILCs (CD45.2+ Lin− CD90+) from Salmonella-infected ceca of WT or Rorasg/sg BM-transplanted (BMT) chimeric mice during peak disease and performed droplet-based scRNA-seq. Contaminating cells expressing Jchain, Cd5, Rag1, Rag2, Mcpt8, Fcer1a, Rrm2, Elane, and collagen transcripts were excluded from further analysis. Graph-based clustering of the aggregate WT and Rorasg/sg single ILC libraries was then performed, and cluster identities were assigned based on reported signature ILC subset genes (1, 19). Cytotoxic NK cells of clusters 1–3 were distinguished by high expression of Eomes, Gzma, and Sell (Fig. 1A–D, Supplemental Fig. 1). In contrast, helper-like ILC1, ILC2, and ILC3 clusters displayed enhanced Il7r and Cxcr6 expression when compared with the NK cell clusters (Fig. 1A–D). Gata3high ILC2s, represented by cluster 5, exhibited discrete expression of Il4 and Il17rb (Fig. 1A–D, Supplemental Fig. 1). ILC3s were segregated into two clusters; cluster 6 cells expressed high levels of Cd4, likely representing the LTi-like ILC3s, whereas cluster 7 constituted a subset of Ncr1+ ILC3s that are a major innate source of IL-22 and lack features of cytotoxicity and do not express Ifng (22). The separate ILC3 clusters may also reflect divergent gene expression profiles related to their distinct ontogeny (23, 24). To directly assess alterations in ILC subset identities as a result of RORα dysfunction, the ratio of relative contributions of WT:Rorasg/sg cells to each cluster was determined (Fig. 1A, 1B). Loss of functional RORα led to an increase in the abundance of cytotoxic NK cells, as Rorasg/sg single cells constituted 67–73% of Eomes+ cell clusters (numbers 1, 2, 3) (Fig. 1B). Meanwhile, the Il7r-expressing ILC1 cluster (number 4) displayed a comparable ratio of WT and Rora-deficient ILCs. This suggests that the ILC1 lineage was largely unaffected by RORα perturbation (Fig. 1B). Rora is well known to be required for ILC2 development. Accordingly, 97% of the GATA3high ILC2 cluster consisted of WT-derived ILCs indicating highly efficient ILC2 ablation in Salmonella-infected Rorasg/sg chimeric mice (Fig. 1A, 1B). Lastly, 76% of the two clusters representing intestinal LTi-like ILC3 (no. 6) and Ncr1+ ILC3 (no. 7) were derived from the WT ILC libraries (Fig. 1A, 1B). These selective perturbations in ILC lineages were further revealed by the striking alterations in ILC subset distribution in WT or Rorasg/sg cecal tissues (Fig. 1C). In the analysis of ILCs purified from infected WT BMT ceca, ILC3s were the most abundant subset (44.6%), followed by NK cells (32.2%), ILC1s (12.8%), and ILC2s (10.5%) (Fig. 1C). In contrast, RORα dysfunction resulted in a 2.3-fold increase in the abundance of NK cells, whereas the frequency of ILC3s was reduced to 13.9% (Fig. 1C). Notably, we found that cells of the NK cell clusters expressed the truncated Rora transcript, further indicating lineage plasticity due to RORα dysfunction. These data suggest that, in addition to aborted ILC2 development, RORα deficiency leads to significantly impaired generation or maintenance of ILC3s and a concomitant expansion of cytotoxic NK cells, which collectively led to attenuated fibrosis in the gut.

FIGURE 1.
FIGURE 1.

RORα preserves IL-7R+ intestinal ILC2 and ILC3 during chronic Salmonella infection. Sort-purified ILCs (CD45.2+ Lin− CD90+) from ceca were analyzed by droplet-based scRNA-seq. (A) Graph-based clustering of single WT and Rorasg/sg ILC libraries visualized by t-distributed stochastic neighbor embedding (tSNE). (B) Normalized contribution of WT or Rorasg/sg cells for each ILC subset cluster. (C) Relative abundance of ILC subsets in WT or RORα-deficient samples. (D) Expression of ILC signature genes by cluster. Dot plot representation of canonical marker genes per cluster. Color intensity indicates log2-scaled mean gene expression level. Dot size indicates the fraction of cells in the cluster expressing each gene. ILCs were pooled from the cecal tissues of five WT or Rorasg/sg BMT animals.

Lymphoid tissue–dependent effects of RORα in ILC lineage maintenance during chronic infection

To explore tissue-specific effects of RORα in ILC responses, we performed analogous single-cell transcriptome analysis on sort-purified ILCs from the mLNs of Salmonella-infected animals. After cell exclusion on the basis of Mzb1, Cd5, Rag1, Rag2, Mcpt8, Nusap1, Rrm2, Arpp21, and Elane expression, graph-based clustering of aggregate WT and Rorasg/sg ILCs was performed. Similar to the intestinal ILC analyses, mLN NK cells of clusters 1 and 2 were defined by elevated expression of Eomes, Gzma, and Sell, whereas helper-like ILC subsets (clusters 3–6) expressed high levels of Il7r and Cxcr6 (Fig. 2A–D, Supplemental Fig. 1). GATA3high ILC2s were defined by cluster 4, and ILC3s were represented in clusters 5 and 6 (Fig. 2A–D, Supplemental Fig. 1). Comparison of WT versus Rorasg/sg ratios for each cluster revealed differential requirements of RORα in the preservation of ILC lineages in lymphoid tissues when compared with the cecum (Fig. 2A, 2B). The major NK cell cluster (number 1) and ILC1 cluster (number 3) were overrepresented by Rorasg/sg single-cell libraries (Fig. 2A, 2B). This was further reflected by their respective 1.6-fold (NK cell) and 1.5-fold (ILC1) increase in relative abundance in the Rorasg/sg BMT mLNs when compared with their WT controls (Fig. 2C). Surprisingly, deletion of RORα had an attenuated effect on ILC2 ablation in the mLNs when compared with the inflamed gut; the Gata3high ILC2 cluster consisted of 27.4% Rorasg/sg cells, indicating compensatory regulatory programs independent of RORα are involved in ILC2 maintenance in the mLNs (Fig. 2B, 2C). This magnitude of cell depletion was similar to the effect of RORα dysfunction in ILC3s; 21.8% of cluster 5 (Ncr1+, Il22+) cells and 25.8% of cluster 6 (Cd4+, Ifng+) cells were derived from Rorasg/sg libraries (Fig. 2B). Consistent with the scRNA-seq analyses of cecal ILCs, RORα deletion resulted in the enrichment of NK cell subsets at the expense of Il7rhigh ILCs (Fig. 2C). In summary, as with the inflamed cecum, RORα is required for the maintenance of ILC2s and ILC3s, although these effects were milder in the mLNs.

FIGURE 2.
FIGURE 2.

Lymphoid tissue–specific effects of RORα in ILC lineage maintenance following chronic Salmonella infection. Sort-purified ILCs (CD45.2+ Lin− CD90+) from mLNs of infected mice were analyzed by droplet-based scRNA-seq. (A) Graph-based clustering of single WT and Rorasg/sg ILC libraries visualized by t-distributed stochastic neighbor embedding (tSNE). (B) Normalized contribution of WT or Rorasg/sg cells for each ILC subset cluster. (C) Relative abundance of ILC subsets in WT or RORα-deficient libraries. (D) Expression of ILC signature genes by cluster. Dot plot representation of canonical marker genes per cluster. Color intensity indicates log2-scaled mean gene expression level. Dot size indicates the fraction of cells in the cluster expressing each gene. ILCs were pooled from the mLNs of five WT or Rorasg/sg BMT animals.

RORα is required for ILC3 lineage maintenance

Our scRNA-seq analysis of ILC subsets suggests that RORα deletion results in partial depletion of ILC3 subtypes during chronic Salmonella infection. To confirm these findings by flow cytometry, we performed transcription factor staining of CD127+ ILCs purified from infected tissues (Fig. 3A). RORγt expression by cecal Rorasg/sg ILCs was highly attenuated, whereas T-bet expression was enhanced; moreover, EOMES expression by ILCs was unaltered due to RORα deletion (Fig. 3B). The frequency of RORγt+ ILC3s is reduced in the cecum and mLNs of Rorasg/sg BMT animals (Fig. 3C). This depletion was proportional in both T-bet+ and T-bet- ILC3 subsets, whereas the total numbers of T-bet+ and EOMES+ ILCs were unchanged. These data suggest that RORα dysfunction results in a selective loss in the frequency of RORγt+ ILC3s under chronic inflammatory conditions.

FIGURE 3.
FIGURE 3.

RORγt+ ILC3s are depleted in Rorasg/sg BMT following Salmonella infection. (A) Gating strategy for the analysis of transcription factor expression by ILCs isolated from infected tissues. (B) Percent expression of RORγt, T-bet, and EOMES by WT and Rorasg/sg ILCs. (C) Total numbers of RORγt+, T-bet+, EOMES+ ILC subsets from the cecum and mLN of WT or Rorasg/sg BMT animals infected with Salmonella. Significance determined by Student t test (n = 6, 7). Representative data from two independent experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001.

To gain further mechanistic insights into the role of RORα in ILC3 gene regulation, we isolated bulk populations of ILC3s from WT and Rorasg/sg BMT animals 21 d after Salmonella infection and performed whole-transcriptome sequence analyses. We found a striking reduction in the expression of a number of ILC3-defining transcripts, including the transcription factor Rorc, cytokine receptors Il1r, Il23r, Il17re, and the cytokine Il22 (Fig. 4A) (19). It has been proposed that ILC3 behavior can be modulated by the contrasting expression of RORγt and T-bet (10, 25). In response to environmental cues, namely IL-12 and IL-15, ILC3s downregulate RORγt expression; these “ex-RORγt” ILC3s have a reduced capacity to secrete IL-17A and IL-22 and acquire ILC1-like properties, including increased expression of T-bet and IFN-γ (10, 25). Consistent with this model, we found that the downregulation of Rorc in Rorasg/sg ILC3s led to an increase in expression of transcripts uniquely associated with the ex-RORγt ILC3 subset and several ILC1 signature genes (Fig. 4A) (19). Interestingly, RORα deficiency in ILC3s resulted in the enhanced expression of several genes involved in cytotoxic responses, including Gzma, Gzmb, and Prf1 (Fig. 4A). This suggests that RORα plays a key role in preserving functional ILC3s in a chronic inflammatory setting and that, in its absence, cells rapidly adopt an ex-RORγt phenotype. In addition, we found a substantial decrease in the expression of several important cytokine and chemokine receptor genes that would allow ILC3s to respond to their local inflammatory milieu, including receptors for IL-2, IL-17, IL-18, TNF, CCL16, CCL20, MIP-3, and IGF-1 (Fig. 4A, 4B). These data suggest that loss of RORα leads to a lesion upstream of cytokine production in ILC3s.

FIGURE 4.
FIGURE 4.

RORα conserves ILC3 signature gene expression. Bulk RNA sequencing of ILC3s (CD45.2+ Lineage− CD90+ KLRG1−) isolated from the mLN of Salmonella-infected WT BMT or Rorasg/sg BMT mice (n = 4, 5). (A) Heat map ranking altered (p < 0.05) core ILC3, ex-RORγt ILC3, and ILC1 gene signatures (19). (B) Altered gene expression of key chemokine or cytokine receptors due to RORα dysfunction (p < 0.05).

Discussion

Numerous studies have implicated ILCs in inflammation-driven pathological conditions, including those associated with inflammatory bowel disease (26). However, less is known about their direct contributions to the development of intestinal fibrosis, a severe and largely untreatable sequela in a subset of CD patients (27). Previously, we found that mice lacking hematopoietic RORα expression were protected from Salmonella-induced intestinal fibrosis due to defective IL-17A production by ILC3s (15). In this study, we employed scRNA-seq to profile perturbations in frequencies of ILC subsets during chronic inflammation as a result of RORα dysfunction. In our single-cell transcriptome analyses, we found that 94% of ILC2s were ablated in the inflamed cecum but with reduced efficiency in the mLNs (45%). This confirms previous reports indicating that RORα is required for ILC2 development, but residual ILC2s in the lymphoid tissues can persist through compensatory programs mediated by GATA3 (14, 20, 21). Although RORα expression is a shared feature of all ILC subsets (19), its deletion does not appear to alter the frequency of ILC3s in peripheral tissues of naive mice (14, 21), possibly due to the expression of the closely related transcription factor, RORγt in this lineage. More recently, however, we found that Rorasg/sg ILC3s displayed defective cytokine expression in mice chronically infected with Salmonella. In this study, we find that the abundance of ILC3s identified by single-cell transcript sequencing is reduced by more than 3-fold in the cecum and mLNs because of RORα dysfuntion. This is further supported by flow cytometric experiments indicating a reduction in the frequency of RORγt+ (T-bet+ or −) ILC3s in the Salmonella-infected cecum and mLN.

The classification of NK cells and helper-like ILC1s has been challenging; although developmentally distinct, they share many phenotypic markers under inflammatory conditions (28). We find that graph-based clustering segregated helper-like ILC1s from NK cells on the basis of elevated expression of Il7r and Cxcr6 and low expression of Eomes. Notably, we find in the cecum and lymph nodes of Rorasg/sg BMT mice postinfection that Eomes+ NK cell and ILC1 subsets remain intact. During lymphoid cell development, RORα is expressed by nearly all committed ILC progenitors shortly after the induction of PLZF as well as by precursors in the divergent LTi lineage (29). Given that peripheral IL-7R+ ILCs are reduced under chronic inflammation, RORα perturbation may have upstream effects on BM or tissue-resident precursors of helper ILCs during persistent infections. Although RORα deletion is known to effectively ablate ILC2Ps in the BM, additional experiments would be necessary to delineate the role of RORα in the developmental hierarchies of other ILC lineages.

The regulation of ILC identity in mucosal tissues by transcriptional programs is critical for context-appropriate immunity. For instance, Gfi1 has a dual role in ILC2 progenitor development and in ILC2 lineage identity in the periphery; the deletion of Gfi1 in ILC2s leads to the derepression of ILC3 genes and hybrid ILC2s expressing IL-17–related transcripts, and this renders mice more susceptible to parasitic worm infections (30). Similarly, the histone methyltransferase G9a promotes ILC2 responses by silencing ILC3-associated genes (31). In response to Salmonella infection, ILC3s downregulate RORγt expression and display enhanced expression of T-bet and NK cell receptors. Termed ex-RORγt ILC3s, these cells are similar to archetypal ILC1s and promote Salmonella and IFN-γ–mediated enterocolitis (25). Following chronic Salmonella infection, we find that RORα is required for conserving functional ILC3s in peripheral tissues and lineage specification of residual ILC3s. Bulk-cell RNA sequencing analyses indicate that Rorasg/sg ILC3s have reduced expression of RORγt compared with WT ILC3s and as a consequence upregulate gene signatures associated with ex-RORγt ILC3s and ILC1s.

The mechanisms by which RORα governs ILC3 core gene transcription remains unclear. In Th17 effector cell responses, RORα is known to act synergistically with RORγt by binding to ROR response elements (ROREs) in the regulatory regions of Th17 signature genes (Il17a, Il17f, Il23r) and thereby promote their expression (13). Interestingly, RORα-specific binding motifs were found to be preferentially enriched in ROREs associated with these Th17 cell signature genes in addition to those targeted by therapeutic RORγt suppression (32). This suggests that regulatory regions containing RORα-binding motifs may be more pertinent to Th17-driven immunopathologies (32). A similar RORE consensus sequence was found to be highly enriched in the enhancer regions of ILC3s signature genes, arguing that parallel ROR-mediated regulatory programs may exist within Th17 cells and ILC3s (33). A survey of transcriptional regulatory networks further confirmed Rora as a canonical ILC3 gene that was predicted to repress type 1 signature genes in ILCs (34).

The current study highlights RORα as a regulator of cytokine and chemokine receptor gene expression that endows ILC3s with an ability to sense the inflammatory milieu and respond by activating the appropriate downstream cytokine expression. Both RORα and RORγt act as ligand-activating transcription factors; many cholesterol metabolites can serve as agonists or inverse agonists by interacting with the ligand-binding domain in the C terminus of RORs (35). Endogenous sterol RORγt ligands are important for ILC3 activation and the development of lymphoid structures (36). In addition, the therapeutic potential of ROR modulation recently led to the development of several synthetic high-affinity RORα and RORγt inverse agonists that have been used to alter ILC function (37, 38). For instance, the RORα/RORγt inverse agonist SR1001 can inhibit the differentiation of tonsillar ILC1 to ILC3, whereas the selective RORα inverse agonist SR3335 can protect mice from rhinovirus-induced type 2 immunopathology by attenuating ILC2 function in the lung (9, 39). Modulation of ILC3 gene expression to suppress proinflammatory responses offers an attractive therapeutic strategy for chronic inflammatory diseases. Previous efforts have focused on targeting RORγt suppression, as it has long been known to be essential to Th17 cell and ILC3 function, but this strategy has been linked to severe immunosuppression and an increased incidence of T cell leukemia (40). Pharmacological inhibition of RORγt (or its inducible deletion) reduced the pathological features of colitis by attenuating pathogenic Th17 responses without altering ILC3 functions (41). Intriguingly, this report revealed divergent requirements of RORγt in the preservation of Th17 cells and ILC3s and suggests that there are compensatory mechanisms in ILC3s that conserve their function after RORγt ablation (41). Our data now define a previously unrecognized role of RORα in maintaining the ILC3 lineage and mediating ILC3 cytokine production. These insights offer new strategies for ILC3 modulation in therapeutic applications, either through targeting RORα activity selectively or suppressing RORα-regulated ILC3 cell-surface receptors.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank the Biomedical Research Centre core members R. Vander Werff (RNA-seq), J. Wong and A. Johnson (cell sorting), T. Murakami (genotyping), I. Barta (histology), and M. Williams (Abs). We are grateful to B. Vallance for providing the Salmonella Typhimurium ΔaroA strain.

Footnotes

  • This work was supported by Canadian Institutes of Health Research Grants PJT-148681 and PJT-156235 (to K.M.M.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BM
    bone marrow
    BMT
    BM-transplanted
    CD
    Crohn disease
    EOMES
    eomesodermin
    ILC
    innate lymphoid cell
    PC
    principal component
    RORα
    retinoic acid receptor–related orphan receptor α
    RORE
    ROR response element
    scRNA-seq
    single-cell RNA sequencing
    WT
    wild-type.

  • Received July 15, 2019.
  • Accepted September 27, 2019.

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