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Individual intestinal symbionts induce a distinct population of RORγ+ regulatory T cells

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Science  28 Aug 2015:
Vol. 349, Issue 6251, pp. 993-997
DOI: 10.1126/science.aaa9420

Gut microbes make T cells keep the peace

Our guts harbor trillions of microbial inhabitants, some of which regulate the types of immune cells that are present in the gut. For instance, Clostridium species of bacteria induce a type of T cell that promotes tolerance between the host and its microbial contents. Ohnmacht et al. and Sefik et al. characterized a population of gut regulatory T cells in mice, which required gut microbiota to survive. Multiple bacterial species of the microbiota could induce transcription factor–expressing regulatory T cells that helped maintain immune homeostasis. Mice engineered to lack these transcription factors exhibited enhanced susceptibility to colonic inflammation and had elevated amounts of proinflammatory molecules associated with allergies (see the Perspective by Hegazy and Powrie).

Science, this issue pp. 989 and 993

Abstract

T regulatory cells that express the transcription factor Foxp3 (Foxp3+ Tregs) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct Treg population in the mouse colon, which constrains immuno-inflammatory responses. This induction—which we find to map to a broad, but specific, array of individual bacterial species—requires the transcription factor Rorγ, paradoxically, in that Rorγ is thought to antagonize FoxP3 and to promote T helper 17 (TH17) cell differentiation. Rorγ’s transcriptional footprint differs in colonic Tregs and TH17 cells and controls important effector molecules. Rorγ, and the Tregs that express it, contribute substantially to regulating colonic TH1/TH17 inflammation. Thus, the marked context-specificity of Rorγ results in very different outcomes even in closely related cell types.

FoxP3 regulatory T (Foxp3+Treg) cells are essential regulators of immunologic homeostasis and responses (1). Beyond their well-described role in regulating the activity of other immunocytes, Tregs located in parenchymal tissues control other, nonimmunological, processes. These “tissue Tregs” include those that reside in visceral adipose tissue and regulate metabolic parameters (2, 3) and those that help channel inflammatory and regenerative events in injured muscle (4). The activities, transcriptomes, and T cell receptor (TCR) repertoires of these tissue Tregs are distinct from their counterparts in secondary lymphoid organs.

Another essential and specific population of tissue Tregs resides in the lamina propria (LP) of the digestive tract, in particular in the colon, where these cells modulate responses to commensal microbes [reviewed in (5)]. Colonic Tregs are an unusual population that has provoked some contradictory observations. TCRs expressed by colonic Tregs show marked reactivity against microbial antigens that seem to be important drivers of their differentiation and/or expansion (6, 7). Many of them appear to arise by conversion from FoxP3 conventional CD4+ T cells (Tconv) (6, 7), although arguments for a thymic origin have been made (7). Many colonic Tregs express marker profiles (Helios and Nrp1) that differ from Tregs of thymic origin [reviewed in (8)], although the importance of these markers has been questioned (5, 8). Accordingly, most studies have found a decreased abundance of colonic Tregs in germ-free (GF) mice [reviewed in (5)], and colonization of GF mice by pools of microbes [Schadler’s flora (9) or Clostridia combinations (10, 11)] elicited the differentiation or expansion of HeliosNrp1 colonic Tregs. The ability of single microbes to induce colonic Tregs has been more controversial, and the need for complex combinations (10, 11) has been questioned (12).

The transcriptomes of tissue-resident Tregs adapt to their location, most strikingly in terms of transcription factors (TFs) (13), and we searched for such elements in colonic Tregs. Comparison of transcriptomes of highly purified CD4+FoxP3+ Tregs [from Foxp3ires–gfp reporter mice (14)] from colon or spleen uncovered 933 differential transcripts [at a fold change >2 and false discovery rate (FDR) <0.1] [Fig. 1A (top), fig. S1A, and table S1]. These encompassed important signaling and effector pathways (Icos, Gzmb, Lag3, Areg, and Il1rl1) [Fig. 1A (top) and table S1], shared in a patchwork manner by other tissue Tregs. Yet ~39% (at a colon-specific bias of >1.5-fold) had preferential expression in colonic Tregs (including Il10, Ctla4, Havcr2, Ccl20, Jak2, and Fosl2] [Fig. 1A (bottom) and table S2]. GeneOntology analysis revealed no enriched function or pathway, except for a high proportion of TFs, including Ahr, Epas1, Hey1, Bcl6, Npas2, Nr1d1, and Maf. To our surprise, the most differential of these TFs proved to be Rorc (encodes Rorγ) (fig. S1B). Rorγ controls many aspects of immunocyte differentiation (15) but is perhaps best known as the key regulator of interleukin-17 (IL-17)–producing CD4+ T cells (TH17), and as a reciprocal antagonist of FoxP3 during in vitro differentiation in which CD4+FoxP3+ Treg and TH17 represent alternative cell fates [reviewed in (16)].

Fig. 1 Rorγ, encoded by Rorc, is preferentially expressed in colonic Tregs.

Gene expression profiles from purified Treg cells of various origins. (A) Transcripts that are enriched in tissue and colonic Tregs. (Top) Transcripts differentially represented in tissue versus splenic Tregs (at a fold change >2). VAT, visceral adipose tissue. (Bottom) Transcripts that are most biased in colonic Tregs (fold change >1.5 versus any other tissue Treg). Means of at least two duplicates. (B) Representative flow cytometry plots of CD4+ T cells and a compilation of frequencies (bottom) of Rorγ+Helios Tregs within the FoxP3+CD4+TCRβ+ population. Each point is an individual mouse. Data are representative of more than three independent experiments. (C) Representative Rorγ versus Helios, Nrp1, IL-33R, or Gata3 plots for colon or spleen Foxp3+CD4+TCRβ+ Tregs (see fig. S2 for quantification). (D) Frequencies of Rorγ+Helios Tregs among FoxP3+CD4+TCRβ+ cells of different tissues (SI, small intestinal lamina propria; PP, Peyer’s patches; MLN, mesenteric LNs; scLN, subcutaneous LNs; spleen, Spl). Each point is an individual mouse. Data pooled from at least two independent experiments. (E) Flow cytometry analysis of human colon biopsies and frequencies of human RORγ+ Tregs within the FOXP3+CD4+CD8CD3+CD45+ population. Healthy tissue samples were endoscopically determined normal areas from chronic constipation or irritable bowel syndrome patients; inflamed tissue was from Crohn’s lesions. Each point is an individual patient. Data pooled from five independent experiments. (F) IL-17a (after phorbol 12-myristate 13-acetate + ionomycin activation and intracellular staining) or IL-17f (reporter in Il17frfp mice) expression among Foxp3+ Treg or FoxP3 Tconv mice. Each point is an individual mouse. Data are representative of three independent experiments.

Cytometry confirmed that many colonic CD4+FoxP3+ Tregs express Rorγ (40 to 60% in C57BL/6J or other inbred mouse strains) (Fig. 1B and fig. S2A), a phenotype largely absent in spleen or lymph node (LN) and among FoxP3+ cells induced in vitro. Helios and Nrp1, described as markers of thymus-derived Tregs [reviewed in (8)], were absent on colonic Rorγ+ Tregs (Fig. 1C); this absence demarcated three distinct subsets of colonic Tregs, with Rorγ+ representing the majority of Helios cells (Fig. 1C and fig. S2, B and C). Consistent with the RNA data, Rorγ+ Tregs were also detected in low proportions in the small intestine (SI) and regenerating muscle (Fig. 1D and fig. S2D). In keeping with a recent report (17), Rorγ+ Tregs were distinct from those expressing the IL-33 receptor, most of which were Helios+ (Fig. 1D and fig. S2, B, C, E, and F), and from Gata3hi Tregs (18), which also belong to the Helios+ Treg subset (Fig. 1D and fig. S2, B and C).

We asked whether RORγ is also expressed by colonic Tregs in humans, by staining cells from healthy or inflamed (Crohn’s) colon biopsies. Rorγ+ Tregs were indeed detected at comparable levels in both contexts (Fig. 1E).

Rare Tregs expressing IL-17 and Rorγ have been observed during chronic inflammation or cancer, usually being Helioshi [reviewed in (19)]. We tested IL-17 production in colonic Rorγ+ Tregs. Although IL-17–expressing Tregs could be detected in the SI LP, colonic Rorγ+ Tregs did not secrete detectable IL-17a or f (Fig. 1F).

The properties of this dominant colonic Rorγ+Helios Treg population suggested a link to the gut microbiota. Indeed, GF mice had a lower proportion of Rorγ+ Tregs than their conventionally raised specific pathogen–free (SPF) counterparts (Fig. 2A). During normal maturation in the mouse, Rorγ+ Tregs appeared between 15 and 25 days of age (Fig. 2B), coincident with the changes in the gut microbiota that accompany the transition to solid food. Note that Rorγ+ Tregs appeared a few days after RorγHelios Tregs. Antibiotic treatment strongly affected Rorγ+ Tregs (Fig. 2C), a large reduction followed a broad-spectrum antibiotic combination, whereas individual antibiotics had less or no effect, which suggested the contribution of several microbes. As the reported impacts of various microbial species on total colonic Tregs have differed (10, 12), we took advantage of a panel of mice generated in a large-scale screen in which GF mice were colonized with a single species from a panel of 22 bacterial species from the human gastrointestinal tract (table S3). A number of microbes elicited colonic Rorγ+ Tregs, with a gradient of responses and, for some, at frequencies comparable with those of SPF mice (Fig. 2D). This restoration of Rorγ+ Tregs was independent of bacterial load and not accompanied by inflammation (fig. S3). Bacteria able to induce Rorγ+ Treg (and colonic FoxP3+ Tregs more generally) belonged to several phyla and genera and were not restricted to Clostridiae (10, 11). Segmented filamentous bacteria (SFB)—which are classic inducers of Rorγ-dependent TH17 cells (20) and which elicit IL-17–producing Tregs in the SI (21)—were only mediocre inducers of colonic Rorγ+ Tregs, which reinforced the distinction between the cell populations. We noticed diversity within the Bacteroides genus and assessed a wider Bacteroides panel (fig. S4A and table S3). Here again, a range of colonic Rorγ+ Tregs was observed. This distribution did not relate to the Bacteroides phylogeny for these strains, and there was no unique correlation between Treg-inducing ability and gene content (fig. S4B). Colonic Rorγ+ Tregs did not appear immediately after GF colonization but only after a few days, again after RorγHelios cells (fig. S4C).

Fig. 2 Rorγ+Helios Tregs can be induced by several bacterial species.

(A) Frequency of Rorγ+Helios within colon FoxP3+CD4+TCRβ+ Tregs of SPF and GF mice, P < 0.0001 as determined by Student’s t test. Each point is an individual mouse. Data pooled from more than three experiments. (B) Induction of Rorγ in colonic Tregs during postnatal development in SPF mice (left). Representative FACS plots (right); frequencies across ages of FoxP3+ Tregs within CD4+TCRβ+ cells, as well as Rorγ+Helios (red) and RorγHelios (black) cells within Tregs. Each point is an individual mouse. Data pooled from four or more experiments. (C) SPF mice were treated with single antibiotics (abbreviations for neomycin, vancomycin, ampicillin, metronidazole) or all four (VMNA) antibiotics for 4 weeks. Frequency of colonic Rorγ+Helios Tregs within the FoxP3+CD4+TCRβ+ population. P = 0.0004, Bonferroni-corrected Student’s t test. Each point is an individual mouse. Data pooled from two experiments. (D) GF mice were colonized with single bacterial species, and colonic Tregs were analyzed after 2 weeks (top). Representative plots and frequencies of Rorγ+Helios within FoxP3+CD4+TCRβ+ Tregs, color-coded per phyla (bottom). Each point is an individual mouse. Data are representative one to three experiments for each microbe. *Different from GF at an FDR of <0.05.

Several reports have suggested that short-chain fatty acids (SCFAs) promote increased colonic Tregs (2224). To test their relevance to Rorγ+ Tregs, SCFAs were quantified by liquid chromatography–mass spectrometry (LC-MS) in cecal content of monocolonized mice. No significant correlation was observed between any SCFA and Rorγ+ Treg frequency or to other Treg parameters (fig. S5, A and B, and table S4). In addition, we could not reproduce previously reported effects of oral or rectal SCFA administration (fig. S5, C and D). Although SCFA combinatorial effects or intercolony variation cannot be ruled out, SCFAs cannot alone explain the microbial impact on colonic Tregs observed here.

To integrate our observations with intercellular pathways that influence intestinal T cells, we measured the relative abundance of Rorγ+ Tregs in mice lacking receptors for key cytokines and alarmins. Signaling through IL-23, IL-1, or IL-33 receptors was not required to sustain Rorγ+ Tregs, nor was IL-10 (fig. S6, A to D). In fact, only the Helios+ population expanded after IL-33 administration (fig. S6E).

We then asked what transcripts Rorγ controls in Rorγ+ Tregs and whether Rorγ is necessary to specify this particular Treg lineage. We compared transcriptomes of Rorγ+ and Rorγ colonic Tregs (sorted from Foxp3Thy1.1 × Rorcgfp intercrossed mice). Rorγ+ cells were enriched in some, but not all, transcripts of the colonic Treg signature, notably Il23r, Cxcr3, Tbx21, and Havcr2 (Fig. 3A), as validated at the protein level, including the unexpected CXCR3 (Fig. 3B). Conversely, Il1rl1 (encodes IL-33R), Nrp1, and Ikzf2 were underrepresented in Rorγ+ Tregs.

Fig. 3 Rorγ determines a specific signature and function in colonic Tregs.

(A) Rorγ+ or Rorγ Tregs were sorted from the colon of Foxp3thy1.1x Rorcgfp intercross male mice, and gene expression profiles were determined. Expression values (triplicate averaged) are compared and highlighted according to the colon Treg signature of fig. S1. (B) Flow cytometric validation of some of the Rorγ+/Rorγ Tregs differential genes (Havcr2, which encodes Tim3, and Cxcr3) that represent two experiments. (C) Comparison by gene expression profiling of the Rorγ signature in different contexts (all mean of triplicates). Fold change between colonic Nrp1 Tregs from WT or Foxp3-cre × Rorcfl/fl mice is shown on the x axis; fold change between SI CD4+ T cells sorted from GF mice monocolonized with TH17-inducing SFB or from unmanipulated GF is shown on the y axis. Shared or specific signature genes are color-coded. (D) SPF mice were treated with Rorγ antagonist TMP778 or control dimethyl sulfoxide (DMSO) for 3 weeks. Representative cytometry plots of colonic Tregs (left) or compiled frequencies of FoxP3+ Tregs (middle) and of Rorγ+Helios (right) within FoxP3+CD4+TCRβ+ Tregs (right); P = 0.009 as determined by Student’s t test. Each point is an individual mouse. Data are representative of two or more independent experiments. (E) Analysis of Rorγ–deficient Tregs from Foxp3-cre × Rorcfl/fl mice or control (Foxp3-creRorc+/+) littermates. Cytometry plots of colonic Tregs (left) or compiled frequencies of FoxP3+ Tregs (middle) and of Rorγ+Helios (right) within FoxP3+CD4+TCRβ+ Tregs (right); P values were determined by paired Student’s t test. Each point is an individual mouse. Data are representative of more than three independent experiments.

To further delineate the transcriptional signature of Rorγ in Treg cells, RNA sequencing profiles were generated from Nrp1 cells of Foxp3-cre.Rorcfl/fl mice, which have a Treg-selective deletion of Rorc (fig. S7A), or paired wild-type (WT) littermates. Differentially expressed genes were related to the Rorγ-dependent signature in conventional TH17 cells (defined from a comparison of SI CD4+ T cells of mice colonized, or not, with SFB) (Fig. 3C and table S5). Part of the classic TH17 signature was unrelated to Rorγ in colonic Tregs (blue in Fig. 3C) or Il1r1 or the canonical TH17 cytokines Il17a/f and Il22; some were shared (Rorc itself, Il23r); and a third segment was controlled by Rorγ in Nrp1 colonic Tregs but not in TH17 cells (Havrc2, Irak3, and Il1rn). Thus, the transcriptional footprint of Rorγ is context-dependent in different T cells.

Next, we explored whether Rorγ contributes to colonic Treg homeostasis. First, mice were treated for 3 weeks with a pharmacologic Rorγ antagonist (25), which reduces SI TH17 levels. This treatment partially decreased both the total frequency of colonic FoxP3+ cells and their Rorγ+ component (Fig. 3D). Second, Foxp3-cre.Rorcfl/fl mice—which have no systemic Treg deficiency or scurfy-like pathology nor any change in FoxP3 intensity—showed a reduced frequency of colonic Tregs, and, more specifically, of Helios Tregs; the proportion of Helios+Gata3+ Tregs was correspondingly increased (Fig. 3E and fig. S7B).

We noted that the loss of Rorγ+ Tregs in Foxp3-cre.Rorcfl/fl mice led to increased production of IL-17 and interferon-γ (IFN-γ) but not TH2 cytokines like IL-5 or IL-13, by Tconv cells in colons of otherwise unchallenged mice (Fig. 4A), which suggested a decreased ability of colonic Tregs lacking Rorγ to regulate inflammatory responses. We thus assessed Foxp3-cre.Rorcfl/fl mice in the trinitrobenzenesulfonic acid (TNBS)–induced colitis model and found an exacerbation of disease severity, in colitis score and histopathology (Fig. 4, B and C). Furthermore , after TNBS challenge of GF mice monocolonized with different microbes, the frequency of Rorγ+ Tregs correlated with the colitis score (Fig. 4D). These results imply a nonredundant role for Rorγ and Rorγ+ Tregs in colonic homeostasis.

Fig. 4 Rorγ+ Tregs control gut inflammation.

(A) Frequency of IL-17a and IFN-γ expression in Foxp3CD4+ Tconv cells from Foxp3-cre × Rorcfl/fl mice and control Foxp3-cre × Rorc+/+ littermates at steady state; P values determined by paired Student’s t test. Each point is an individual mouse. Data are representative of three or more independent experiments. (B and C) Colitis score (B) and histology (C) of Foxp3-cre × Rorcfl/fl mice and control Foxp3-cre × Rorc+/+ littermates challenged with TNBS, calculated on the basis of weight loss, histologic score, and other physical parameters; P value as determined by paired Student’s t test. Each point is an individual mouse. Data representative of more than three independent experiments. (D) Correlation between TNBS-colitis score (x axis) with frequency of Rorγ+Helios within colonic Tregs in GF mice monocolonized for 2 weeks with bacteria that elicit different levels of Rorγ+Helios Tregs before TNBS colitis induction. B. theta, B. thetaiotaomicron. Pearson’s correlation coefficient r = 0.82, P < 0.0001. Each point is an individual mouse. Data pooled from four experiments.

Thus, Rorγ contributes unexpectedly but in an important way to the Treg response to commensal microbes. This role contrasts with the accepted dichotomy between FoxP3 and Rorγ, a notion stemming mainly from their antagonism in vitro (14, 2628); perhaps this relation has been overinterpreted. There had been indications that the two TFs are not incompatible (19), but the present data suggest a collaborative transcriptional impact, consistent with the overlap between their chromatin-binding sites (29). The context-specificity of Rorγ’s transcriptional footprint is in line with its broad involvement in many immunological and nonimmunological processes (organogenesis, circadian rhythm, and lipid metabolism) (15, 30). Rorγ-dependent Il23r expression in Tregs also raises the intriguing speculation that human IL23R genetic variants associated with inflammatory bowel disease (31) might involve balancing effects in effector and regulatory T cells.

Rorγ+ Tregs form the majority of the Helios Tregs that differentiate locally in response to antigens of commensal microbes in the gut (6) and do not respond to the alarmin IL-33, in contrast to Gata3+Helios+ cells that expand during tissue damage (17, 18). Mutually exclusive expression of Gata3 and Rorγ in colonic Tregs suggests that they may distinguish Treg responses to symbiotic (Rorγ) versus aggressive (Gata3) microbes. Contrary to expectations, many individual microbes proved able to elicit Rorγ+ and Helios Tregs, a property not restricted to Clostridiae (10). The graded range suggests that several mechanisms may be involved. The molecular mediator of Rorγ+ Treg induction remains elusive but is unlikely to be SCFAs alone. Rorγ+ induction must follow different routes in TH17 versus colonic Tregs, because the best Rorγ+ Treg inducers do not affect SI TH17 and vice versa.

In conclusion, these studies show Rorγ as a uniquely microbe-responsive factor induced in two different cellular contexts, in response to different microbes, with distinct transcriptional consequences, and with diametrically opposite functional outcomes.

Supplementary Materials

www.sciencemag.org/content/349/6251/993/suppl/DC1

Materials and Methods

Figs. S1 to S6

Tables S1 to S5

References (3258)

REFERENCES AND NOTES

  1. Acknowledgments: We thank A. Onderdonk, C. Dong, A. Rudensky, R. Lee, V. Kuchroo, and L. Bry for microbial and mouse strains and S. Edwards, A. T. Sherpa, K. Hattori, K. Rothamel, and R. Cruse for help with mice or profiling. TMP778 is available to academic investigators from G.S.K. under a material transfer agreement that does not unduly affect or prohibit publication. ⁠The data are tabulated in supplementary materials and deposited at the National Center for Biotechnology Information, NIH, Gene Expression Omnibus (GSE68009). E.S., N.G.-Z., D.K., D.M., C.B., and Harvard Medical School have filed a provisional patent application related to work presented in this paper. This work was supported by NIH R01-AI51530 and R56-AI110630 and the J.P.B. Foundation (D.M. and C.B.); a Sponsored Research Agreement from UCB Pharma (D.M., C.B., D.K., and A.E.); the Helmsley Charitable Trust and the Wolpow Family Chair in the Center for Inflammatory Bowel Disease Treatment and Research (S.B.S.). E.S. and D.Z. were supported by fellowships from the Boehringer Ingelheim Fonds, N.G.-Z. by the Human Frontier Science Program and European Molecular Biology Organization (ALTF 251-2011) fellowships and the Weizmann—National Postdoctoral Award for Advancing Women in Science.
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