RORγt represses Th17 cell IL-10 production to maintain their pathogenicity in inducing intestinal inflammation

Abstract

The role of retinoid-related orphan receptor gamma t (RORγt) in Th17 cell differentiation has been well-established, however, how it regulates other T cell lineages is still not clearly understood. We report here that in mice while promoting Th17 cell differentiation, RORγt inhibited IL-10 production by T cells, thereby preserving the pathogenicity of Th17 cells. Treatment with RORγt-specific inhibitor suppressed Th17 cell signature cytokines, but promoted IL-10 production. RORγt inhibitor-treated Th17 cells induce less severe colitis compared to control Th17 cells. Mechanistically, the RORγt inhibitor induced T cell expression of Blimp-1 (encoded by Prdm1). Prdm1^−/− T cells produced significant less IL-10 when treated with RORγt inhibitor compared to wild-type (WT) T cells. Furthermore, RORγt inhibitor-treated Prdm1^−/− Th17 cells induce more severe colitis compared to RORγt inhibitor-treated WT Th17 cells. Collectively, our studies reveal a novel mechanism by which RORγt drives and maintains pathogenic Th17 cell development by inhibiting IL-10 production.

Introduction

It has been well established that development of different T cell subsets requires different transcription factors, such as T-bet in Th1 cells, GATA3 in Th2 cells, RORγt in Th17 cells, and Foxp3 in Treg cells (1–4). Th17 cells, which produce predominantly IL-17A (IL-17), IL-17F, IL-21, and IL-22, have been implicated in the induction of chronic inflammation and autoimmune diseases, such as psoriasis, ankylosing spondylitis, and inflammatory bowel disease (IBD) (5–9). A number of studies assessing the genome-wide transcriptome of Th17 cells have provided big data sets for discoveries of Th17 cell-relevant genes in mice and humans. However, only a few functional targets have been identified (10–13). A central role of RORγt in the differentiation of Th17 cells has been established by the fact that RORγt^−/− mice lack Th17 cells and cannot develop experimental autoimmune encephalomyelitis (3). It has been demonstrated recently that in addition to binding to Th17 cell signature genes, RORγt also binds to signature genes related with other T cell lineages, such as Il2, which induces Th1 cell but inhibits Th17 cell differentiation. Furthermore, in coordination with different transcription factors related with Th17 or other T cell lineages, RORγt could promote Th17 cell function, or inhibit expression of genes important for other T cell lineages (14). However, it is not completely clear how RORγt regulates development of other T cell lineages.

Although Th17 cells have been well established in mediating the pathogenesis of IBD, their signature cytokines IL-17 and IL-22 protect the intestines from inflammation rather than driving intestinal damage (15–17). These activities are much different from their roles in other autoimmune diseases, such as psoriasis (18). Thus, the mechanisms involved in Th17 cell induction of IBD are still not clear. IL-10 is a key pleiotropic immunosuppressive cytokine produced mainly by Treg and Tr1 cells, as well as by T effector cells, dendritic cells, macrophages and B cells. Critically, IL-10 plays a central role in the regulation of intestinal homeostasis and inhibition of IBD. IL-10 producing-Tr1 cells have been shown to inhibit pathogenic T cells and colitis development (19, 20), and T effector cell production of IL-10 serves as a self-limiting mechanism to prevent an exaggerated T cell response, which otherwise would be detrimental (21). It has been shown that polymorphisms in the IL-10 locus are associated with a risk for IBD, including both UC and CD (22, 23). Deficiency in either IL-10 or IL-10 receptor (IL-10R) in mice and humans is associated with severe intestinal inflammation (24–26). The IL-10-IL-10R signaling is crucial for suppressive function and amelioration of colitis by Tregs, as well as in T effector cells and innate immune cells for inhibiting exaggerated T cell responses in mucosal compartments and regulating intestinal homeostasis and prevention of colitis (27–30). The importance of T cell production of IL-10 in controlling the pathogenesis of IBD and maintaining intestinal homeostasis is further demonstrated by an elegant study in which CD4⁺ T cell-specific IL-10 conditional knockout mice develop spontaneous colitis closely resembling the phenotype in complete IL-10 deficient mice, despite intact IL-10 gene in other cell types, implicating T cells are the major source of IL-10 production in the intestines (31). A detailed analysis of the transcriptional network in Th17 cells indicates that RORγt could act as a repressor for IL-10 (11), and a recent study further demonstrates a reduced IL-10 gene expression in human pro-inflammatory Th17 cells in multiple sclerosis compared to healthy controls (32), suggesting that RORγt could inhibit T cell IL-10 production whereas positively regulates the expression of Th17 signature cytokines, thus to reinforce the effector function of Th17 cells.

In this report, we demonstrate that blockade of RORγt not only decreased IL-17 and IL-22, but also increased IL-10 production in Th17 cells. RORγt inhibitor treated Th17 cells demonstrated lower pathogenicity in the induction of colitis in Rag^−/− mice, which could be reversed by blockade of IL-10-IL-10R signaling. Mechanistically, inhibition of RORγt during Th17 cells development promoted Blimp-1 expression, which is essential for IL-10 production. Our study, thus, identifies a novel function of RORγt in maintaining the pathogenicity of Th17 cells in inducing colitis by inhibiting IL-10, which supports the argument that a master transcript factor for one T cell lineage may also inhibit the key cytokines of other T cell lineages, i.e. promoting one T cell lineage but inhibit other T cell lineage(s).

Materials and Methods

Mice.

C57BL/6 and C57BL/6 Rag-1^−/− mice were purchased from the Jackson Laboratory and maintained in the animal facilities of the University of Texas Medical Branch (UTMB). CBir1 TCR Transgenic (Tg) mice were bred in the animal facilities of UTMB. B6.129-Prdm1^tm1Clme/J (Prdm1^/fl) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and Cd4^Cre mice were from Taconic (Hudson, NY). Cd4^Cre Prdm1^fl/fl mice were generated by crossing Prdm1^fl/fl mice with Cd4^Cre mice. Cd4^Cre Prdm1^−/− mice were used as WT controls. Rorγt^−/− mice and WT mice with the same background were purchased from Jackson Laboratory. All experiments were reviewed and approved by the Institutional Animal Care and Use Committees of UTMB.

Reagents and antibodies.

The RORγt inhibitor, BMS336 (compound#25a), which is a benzothiazine and tetrahydroquinoline sulfonamide analog, was discovered at Bristol-Myers Squibb (BMS) Company as a potent and selective inverse agonist of RORγt and its detailed profile was previously described (33). RPMI 1640, HEPES, penicillin–streptomycin, FBS, 2-mecapto-ethanol, and sodium pyruvate were purchased from Life Technologies (Carlsbad, CA, USA); CellTracker™ CFSE and TRIzol reagents, from Invitrogen (Carlsbad, CA, USA); and qScript reverse transcriptase from Quanta Biosciences. Reagents for TaqMan gene expression assays were obtained from Applied Biosystems (Carlsbad, CA, USA). EDTA, and collagenase type IV were obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant IL-6, TGF-β1, IL-1β, and IL-23 were purchased from Cell Signaling, BioLegend (San Diego, CA, USA) and R&D Systems, respectively. Anti-mouse IFN-γ (XMG1.2), anti-IL-4 (11B11), and ani-IL-10R (1B1.3A) neutralizing monoclonal antibodies (mAb) were purchased from BioXCell (Lebanon, NH). Fluorochrome-conjugated anti-mouse CD4, CD45.1, IL-17, IL-10, Foxp3, and IFN-γ monoclonal antibodies were purchased from BioLegend. Foxp3 staining buffer sets were purchased from eBioscience (San Diego, CA, USA). Live/dead dye indicating cell viability was obtained from Invitrogen.

Isolation of CD4⁺ T cells and in vitro T cell cultures.

CD4⁺ T cells were isolated by using anti-mouse CD4-magnetic beads (BD Biosciences) and cultured with irradiated splenic antigen presenting cells (APCs) at 37°C in humid air with 5% CO₂ under Th17 polarization conditions with TGF-β (10 ng/mL), IL-6 (30 ng/mL), anti-IFN-γ (10 μg/mL), and anti-IL-4 (5 μg/mL).

T cell adoptive transfer colitis.

CD4⁺ T cells were isolated and cultured under Th17 polarizing conditions as described above. After 5 days of culture, 1 × 10⁶ Th17 cells were adoptively transferred to Rag^−/– mice by intravenous injection. Mice were monitored weekly for signs of disease and sacrificed 5–6 weeks post transfer.

Flow cytometry.

Cells were stimulated with PMA (50 ng/mL) and ionomycin (750 ng/mL) for 5 h, with GolgiStop added for the last 3 h. Cells were fixed and permeabilized using a Foxp3 staining buffer set. Staining was performed with live/dead dye, CD4, IL-10, Foxp3, IL-17, and IFN-γ by using fluorescence-conjugated Abs, and the cells were harvested on an LSRII Fortessa flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).

RT-PCR.

Total RNA was extracted with TRIzol reagent and followed by cDNA synthesis. Quantitative PCR reactions were performed by using TaqMan Gene Expression Assays for IL-21, Rorc, Batf, Rora, Runx1, c-Maf, Prdm1, and IRF4 on a Bio-Rad iCycler (Bio-Rad, Hercules, CA, USA), and all data were normalized to GAPDH mRNA expression.

ELISA.

Mouse IL-17, IL-22, IL-10, IFN-γ, TNF-α, and IL-6 ELISA kits were purchased from Biolegend. The detection for each cytokine was performed following manufacturer’s instructions.

T cell suppression assay.

CD45.1 CBir1 CD4⁺ T cells were labeled with CFSE (5 μM) and cultured in 24-well plates at 2 × 10⁵ cells per well with 2 × 10⁵ irradiated splenic APCs in the presence of 2 × 10⁵ cells per well of different CD45.2 CBir1 effector T cells with or without anti-IL-10R mAb (20 µg/ml). The cells were harvested 3 days later. CFSE intensity was analyzed by flow cytometry by gating on CD45.1⁺ cells.

Preparation of lamina propria lymphocytes.

Lamina propria lymphocytes were isolated as previously described (34). Briefly, the intestines were rinsed, sliced into small pieces, and incubated at 37°C for 40 min with 0.5 mM EDTA. The tissues were then digested with 0.5 mg/ml collagenase IV for 50 min at 37°C with stirring. The liberated cells were collected by passage through a stainless steel sieve and 100 μm cell strainer (BD Falcon). The isolated cells were pooled together and separated on a 40/75% discontinuous Percoll gradient (Amersham Pharmacia Biotech).

Histopathological assessment.

At necropsy, cecum and colon were separated and Swiss rolls of each were prepared. Tissues were fixed in 10% buffered formalin. Following paraffin embedding, 5 μm sections were prepared, stained with H&E. The severity of colitis was quantified by hyperplasia; goblet cell number; crypt abscesses; ulceration; mucosa and submucosa inflammatory cell infiltration. A score of 0–3, denoting increasingly severe abnormality, was assigned for each of these parameters, and added together to calculate the overall histological score.

Statistical analysis.

Student’s t-test was used to measure the difference between two groups, and one-way ANOVA test was performed for comparison among three or more groups. The nonparametric Mann–Whitney U test was used for assessing pathology scores. All results were analyzed by Graphpad Prism 5.0 software. A p value < 0.05 was considered statistically significant and shown as asterisk (*).

Results

Inhibition of the RORγt pathway promotes IL-10 producing T cells and inhibits Th17 cell development under Th17 polarization conditions

RORγt has been identified as the master transcription factor for Th17 cells (3). To determine how the RORγt inhibitor regulates T cell development and its role in T cell induction of colitis, we cultured CD4⁺ T cells from CBir1 Tg mice, which are specific for an immunodominant microbiota antigen CBir1 flagellin (35), under Th17 polarization conditions with TGF-β and IL-6 in the presence or absence of the RORγt inhibitor (36). As expected, addition of the RORγt inhibitor suppressed production of IL-17 (Figure 1A, B). Interestingly, the frequency of IL-10 producing T cells was significantly increased in the presence of the RORγt inhibitor (Figure 1A, B). Although inhibition of RORγt increased IFN-γ expression, most of the IFN-γ⁺ T cells also expressed IL-10 (Supplementary Figure 1A). Furthermore, RORγt inhibitor did not affect Foxp3 expression under Th17 conditions (Supplementary Figure 1B). Additionally, protein levels of IL-17 and IL-22 were decreased, whereas IL-10 was increased in culture supernatants when the RORγt inhibitor was included (Figure 1C). However, addition of the RORγt inhibitor did not affect the expression of IL-21, confirming that Th17 cell production of IL-21 does not require RORγt (Figure 1D). Similar results were obtained when another well-described RORγt antagonist, GSK805 (14), was used to treat T cells under Th17 polarization conditions (Supplementary Figure 1C). We then used RORγt^−/− T cells to further confirm the roles of RORγt in regulating Th17 cell IL-10 production. We cultured WT and RORγt^−/− T cells under Th17 polarization conditions. Consistent with previous reports, deficiency of RORγt impaired T cell production of IL-17 (3), while increasing production of IL-10 (Figure 1E).

Figure 1. Treatment with RORγt inhibitor suppresses T cell IL-17 and IL-22, but promotes IL-10 production under Th17 polarization conditions.

CD4⁺ T cells from CBir1 Tg mice were cultured with APCs and CBir1 peptide in the presence or absence of RORγt inhibitor (1 μM) under Th17 polarization conditions with TGF-β (10 ng/ml) and IL-6 (30 ng/ml) for 5 days. (A) Expression of IL-17, IFN-γ, and IL-10 was examined by flow cytometric analysis. (B) Bar chart of IL-17⁺ and IL-10⁺ CD4⁺ T cells. (C) Production of IL-17, IL-22, and IL-10 in culture supernatants of day 3 was detected by ELISA. (D) Expression of IL-21 was determined by qRT-PCR and normalized to Gapdh expression. (E) WT and RORγt^−/− CD4 T cells were cultured with APC and anti-CD3 mAb (2 μg/ml) under Th17 polarization conditions with TGF-β and IL-6 for 5 days. T cell cytokine production was determined by flow cytometry. (F) CD4⁺ T cells from CBir1 Tg mice were cultured with or without IL-17 (30 ng/ml) in the presence of RORγt inhibitor (1 μM) under Th17 polarization conditions with TGF-β and IL-6 for 5 days. Cytokine production of T cells was determined by flow cytometry. qRT-PCR and ELISA results are shown as mean ± SD of three samples and are representative from one of five experiments performed. *p < 0.05, **p < 0.01, ***p < 0.001.

Since T cells do not express the IL-22 receptor, we then determined if decreased IL-17 affects IL-10 expression by T cells. To do this, CD4⁺ T cells were treated with RORγt inhibitor in the presence or absence of IL-17 under Th17 polarization condition. Addition of IL-17 did not affect RORγt inhibitor-induced IL-10 and IL-17 production (Figure 1F), indicating that RORγt inhibitor-induced IL-10 production in T cells is not due to the decreased production of IL-17. Addition of the RORγt inhibitor did not affect expression of other Th17-associated genes, including Batf, Runx1, and Rora (Supplementary Figure 2). Collectively, these data indicated that while blockade of RORγt suppresses Th17 cell differentiation, it promotes IL-10-producing T cells under Th17 polarization conditions.

RORγt inhibitor-treated Th17 cells exhibit regulatory function through promoting IL-10 production

As the RORγt inhibitor represses T cell production of IL-17 but promotes IL-10 production, we then sought to investigate whether RORγt inhibitor suppresses T cell proliferation. We cultured CFSE-labeled CD4⁺ T cells under Th17 polarization conditions in the presence or absence of the RORγt inhibitor. T cell proliferation was determined by CellTrace™ CFSE. As shown in Figure 2A and B, addition of the RORγt inhibitor did not affect T cell proliferation, indicating that the RORγt inhibitor specifically blocks Th17 cell commitment, without impairing T cell proliferation, which is consistent with previous studies (3). As we have shown that the RORγt inhibitor induces IL-10-producing T cells during Th17 cell differentiation, we then investigated whether the RORγt inhibitor-treated CD4⁺ T cells exhibit suppressive functions through promoting IL-10 production. We cultured CD45.2 CBir1 Tg CD4⁺ T cells under Th17 polarization conditions in the presence or absence of the RORγt inhibitor. We also cultured CD45.2 CBir1 Tg CD4⁺ T cells with TGF-β to generate Treg cells to serve as positive controls. Five days later, IL-17, IL-10, and Foxp3 expression by these cells was determined by flow cytometry (Supplementary Figure 3). We then performed a suppressive assay by co-culturing CFSE-labeled CD45.1 CBir1 Tg CD4⁺ T cells with T cells generated above, in the presence or absence of anti-IL10R mAb. As expected, Treg cells inhibited CD45.1 CD4⁺ T cell proliferation (Figure 2C, D). While Th17 cells did not affect CD45.1 CD4⁺ T cell proliferation, RORγt inhibitor-treated Th17 cells suppressed CD45.1 T cell proliferation, which was reversed by anti-IL10R mAb (Figure 2C, D). These data indicate that RORγt inhibitor-treated T cells acquire a regulatory function via upregulating IL-10 production.

Figure 2. RORγt inhibitor-treated T cells suppress T cell proliferation.

CBir1 CD4⁺ T cells were labeled by CFSE and cultured under Th17 polarization conditions with TGF-β (10 ng/ml) and IL-6 (30 ng/ml) in the presence or absence of RORγt inhibitor (1 μM) for 5 days. (A) The CFSE dilution was determined by flow cytometry. (B) CD4⁺ T cell proliferation is shown as mean ± SD of two samples and is representative from one of two experiments performed. (C) CD45.2 CBir1 CD4⁺ T cells were cultured under Th17 polarizeing conditions with TGF-β and IL-6 ± RORγt inhibitor or cultured with TGF-β (Treg) for 5 days. These T cells were then co-cultured with CFSE-labeled CD45.1 CBir1 CD4⁺ T cells at ratio of 1:1 for 3 days in the presence or absence of anti-IL-10R mAb (20 µg/ml). The CFSE dilution was determined by flow cytometry. (D) CD45.1 cell proliferation is shown as mean ± SD of two samples and is representative from one of three experiments performed. *p < 0.05.

RORγt inhibitor-treated T cells demonstrate lower pathogenicity in induction of colitis

Th17 cells have been implicated in the pathogenesis of colitis (34, 37), while IL-10 producing-Tr1 cells inhibit inflammatory responses (19, 20). As the RORγt inhibitor inhibits Th17 cell development while promoting IL-10 producing-T cells, we next sought to investigate whether these T cells have a lower capacity for inducing colitis. We cultured CBir1 CD4⁺ T cells under Th17 polarization conditions with TGF-β and IL-6 in the presence or absence of the RORγt inhibitor, then transferred them into groups of Rag^−/− mice (n = 5/group). We have shown previously that Cbir1-specific T cells induced colitis in Rag^−/− recipient mice (38). The mice were sacrificed 5 weeks post cell transfer, and histopathology of the colon and cecum were examined. Compared with control Th17 cells, RORγt inhibitor-treated Th17 cells induced less severe colitis in Rag^−/− mice (Figure 3A, B). We also assessed T cell cytokine production in the lamina propria (LP) of recipient mice by flow cytometry. In comparison with mice receiving control Th17 cells, Rag^−/− recipients reconstituted with RORγt inhibitor-treated Th17 cells exhibited a lower percentage and absolute number of IL-17⁺ CD4⁺ T cells, but a significantly higher proportion of IL-10⁺ T cells from the LP. Similar levels of Foxp3⁺ and IFN-γ⁺ CD4⁺ T cells were detected between both groups (Figure 3C, D).

Figure 3. RORγt inhibitor-treated Th17 cells induce less severe colitis.

Splenic CD4⁺ T cells from CBir1 Tg mice were cultured under Th17 polarization conditions in the presence or absence of RORγt inhibitor (1 μM) for 5 days. 1 × 10⁶ T cells were injected i.v. into groups of Rag^−/− mice (n = 5/group), respectively. Five weeks after cell transfer, the severity of intestinal inflammation was assessed by histological analysis. (A) Colonic histopathology and (B) histological scores are shown. (C) Cytokine production of CD4⁺ T cells in intestinal lamina propria of recipient mice was measured by flow cytometry. (D) Bar charts of the percentage and absolute number of cytokine-expressing T cells over live CD4⁺ T cells. Data are shown as mean ± SD of five mice samples pooled from each group. One representative of three experiments. *p < 0.05.

To confirm the roles of RORγt inhibition in Th17 cells during colitis induction, we cultured RORγt^−/− and WT T cells under Th17 polarization conditions, and then transferred them into Rag^−/− mice. The mice were sacrificed 6 weeks post cell transfer, and disease severity was measured by histopathology. As shown in Figure 4A-B, the recipients of RORγt^−/− T cells cultured under Th17 conditions developed much less severe colitis than the mice receiving WT Th17 cells. Additionally, lower percentages of IL-17⁺ T cells, as well as IFN-γ⁺ T cells, but higher levels of IL-10⁺ T cells, were observed in colonic LP from Rag^−/− mice reconstituted with RORγt^−/− T cells cultured under Th17 conditions, compared with mice reconstituted with WT Th17 cells. Additionally, there was no difference in Foxp3 expression between these two groups (Figure 4C-D). Moreover, IL-17 was decreased, and IL-10 was increased in the supernatants of colonic organ cultures from the mice reconstituted with RORγt^−/− T cells cultured under Th17 conditions (Figure 4E). The colonic IFN-γ and IL-6 production from mice reconstituted with RORγt^−/− T cells cultured under Th17 conditions was similar to those that received WT Th17 cells (Figure 4E).

Figure 4. RORγt^−/− T cells cultured under Th17 conditions induce less severe colitis.

Splenic CD4⁺ T cells from WT and RORγt^−/− mice were cultured with APCs and anti-CD3 mAb (2 μg/ml) under Th17 polarization conditions for 5 days. 1 × 10⁶ T cells were injected intravenously. into groups of Rag^−/− mice (n = 4/group), respectively. Six weeks after cell transfer, the severity of intestinal inflammation was assessed by histological analysis. (A) Colonic histopathology and (B) histological scores are shown. (C) Cytokine production of T cells in lamina propria was determined by flow cytometry. (D) Bar charts of the percentage of cytokine-expressing CD4⁺ T cells. (E) Colonic tissues were cultured in the medium for 24 h, and cytokine production in supernatants was assessed by ELISA. Data are shown as mean ± SD of four mice samples pooled from each group. One representative of two experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Increased IL-10 production in RORγt inhibitor-treated Th17 cells contributes to less severe colitis

To determine if the increased IL-10 production of RORγt inhibitor-treated Th17 cells plays a role in controlling the excessive inflammation during Th17-induction of colitis, we transferred control Th17 cells or RORγt inhibitor-treated Th17 cells into Rag^−/− mice. One group of mice that received the RORγt inhibitor-treated Th17 cells were administered an anti-IL-10R antibody by intraperitoneal route weekly. The other two groups of mice, which received either RORγt inhibitor-treated or control Th17 cells were given control anti-IgG antibody. Consistent with previous results, mice receiving RORγt inhibitor-treated Th17 cells exhibited mild colitis as compared with the mice transferred with control Th17 cells (Figure 5A, B). Administration of anti-IL-10R antibody exacerbated disease induced by RORγt inhibitor-treated Th17 cells (Figure 5A, B). In addition, we examined cytokine production in colonic tissue cultures using ELISA. The mice reconstituted with RORγt inhibitor-treated Th17 cells produced lower levels of pro-inflammatory cytokines, including IL-17, IFN-γ, and TNFα, but increased IL-10 production compared to the mice reconstituted with control Th17 cells. Administration of anti-IL-10R antibody significantly increased production of IL-17, IFN-γ and TNFα. Of note, there was no difference in IL-6 production among the groups (Figure 5C).

Figure 5. Treatment with anti-IL-10R mAb increases severity of colitis induced by RORγt inhibitor-treated Th17 cells.

Splenic CD4⁺ T cells from CBir1 Tg mice were cultured under Th17 polarization conditions ± RORγt inhibitor (1 μM) for 5 days, and 1 × 10⁶ T cells were injected into groups of Rag^−/− mice (n = 5–6/group) i.v., respectively. A group of mice transferred with RORγt inhibitor-treated Th17 were administered with anti-IL-10R mAb (25mg/kg) i.p. weekly. The other two groups of mice, which received control or RORγt inhibitor-treated Th17 cells, were given with anti-IgG antibody as controls. Five weeks after cell transfer, the severity of intestinal inflammation was assessed. (A) Colonic histopathology and (B) histological scores are shown. (C) Colonic tissues were cultured in the medium for 24 h, and cytokine production in supernatants was assessed by ELISA. Data are shown as mean ± SD of twelve mice samples pooled from two experiments. *p < 0.05, **p < 0.01.

To further confirm whether increased IL-10 production by RORγt inhibitor-treated WT Th17 cells contributes to induction of less severe colitis, we cultured WT or IL-10^−/− T cells under Th17 conditions with or without RORγt inhibitor, and transferred them into Rag^−/− mice. Mice were sacrificed 6 weeks post cell transfer. Consistently, RORγt inhibitor-treated WT Th17 cells induced less severe colitis compared with control Th17 cells. Compared to RORγt inhibitor-treated WT Th17 cells, RORγt inhibitor-treated IL-10^−/− Th17 cells induced severe colitis at the levels similar to that of control IL-10^−/− Th17 cells (Figure 6A, B). Furthermore, IL-17 production was decreased in the supernatants of colonic tissue culture from mice receiving WT or IL-10^−/− RORγt inhibitor-treated Th17 cells, compared with mice receiving WT or IL-10^−/− control Th17 cells (Figure 6C). However, there was no statistically significant difference of IL-17 production in colon between mice received RORγt inhibitor-treated WT Th17 cells and RORγt inhibitor-treated IL-10^−/− Th17 cells (Figure 6C), whereby administration with anti-IL-10R antibody significantly increased production of IL-17 in colon from mice receiving RORγt inhibitor-treated WT Th17 cells (Figure 5C). This discrepancy could be due to that only T cell-produced IL-10 is deficient when IL-10^−/− Th17 cells were used, while anti-IL-10R antibody treatment blocked IL-10 signaling in all different types of cells. The level of IFN-γ in colonic tissues of the Rag^−/− recipient mice of RORγt inhibitor-treated WT Th17 cells were decreased compared with those from WT Th17 cells-reconstituted controls, while there was no difference in IFN-γ production in the colon from mice receiving RORγt inhibitor-treated IL-10^−/− Th17 cells and IL-10^−/− control Th17 cells (Figure 6C). TNFα levels in colonic tissues were comparable between mice receiving IL-10^−/− Th17 cells and IL-10^−/− control Th17 cells, which were significantly higher than those in mice reconstituted with WT Th17 cells (Figure 6C). Additionally, colonic IL-6 production was similar in all groups. Taken together, these results demonstrated that increased IL-10 production in RORγt inhibitor-treated Th17 cells, but not decreased IL-17, contributes to protection from inflammation.

Figure 6. Inhibition of RORγt in IL-10^−/− Th17 cells does not affect their potential in induction of colitis.

Splenic CD4⁺ T cells from WT and IL-10^−/− mice were cultured with APCs and anti-CD3 mAb (2 μg/ml) under Th17 polarization conditions in the presence of RORγt inhibitor (1 μM) for 5 days. Then 1 × 10⁶ T cells were injected intravenously into groups of Rag^−/− mice (n = 4/group), respectively. Six weeks post cell transfer, the severity of intestinal inflammation was assessed. (A) Colonic histopathology and (B) histological scores are shown. (C) Colonic tissues were cultured in the medium for 24 h, and cytokine production in supernatants was assessed by ELISA. Data are shown as mean ± SD of each group of mice. One representative of two experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

RORγt inhibits IL-10 production of Th17 cells through repressing Blimp-1

These findings prompted us to investigate the mechanisms by which RORγt regulates T cell IL-10 production. Several pathways have been identified recently in mediating IL-10 production by T cells and the generation of Tr1 cells. Transcription factors c-Maf and AhR contribute to IL-10 production by Tr1 cells (39), and Blimp-1 (encoded by Prdm1) is required for the IL-10 production by CD8⁺ T cells and Foxp3⁺ Treg cells (40, 41), as well as IL-10 production of Tr1 cells (42, 43). Furthermore, interferon regulatory factor 4 (IRF4) is also implicated in regulating expression of IL-10 by T cells in combination with other transcription factors (41, 44, 45). Therefore, we next investigated whether the RORγt inhibitor skews Th17 cells into an IL-10 producing phenotype through the regulation of these transcription factors. First, we measured expression of c-Maf, AhR, Blimp-1 and IRF4 in CD4⁺ T cells cultured under Th17 polarization conditions in the presence and absence of the RORγt inhibitor. As shown in Figure 7A, treatment with the RORγt inhibitor significantly increased the expression of Blimp-1, but not that of c-Maf, AhR, and IRF4, which was confirmed by using RORγt^−/− T cells (Figure 7B, and Supplementary Figure 4A). We then determined whether increased Blimp-1 mediates IL-10 production induced by the RORγt inhibitor. To do this, we cultured WT and Prdm1^−/− CD4⁺ T cells under Th17 conditions in the presence or absence of the RORγt inhibitor. While RORγt inhibitor treatment inhibited IL-17 in both WT and Prdm1^−/− T cells, it only increased IL-10 production in WT T cells but not in Prdm1^−/− T cells (Figures 7C and 7D), suggesting that RORγt inhibition leads to IL-10 production via promoting Blimp-1 in T cells.

Figure 7. RORγt inhibits IL-10 production under Th17 polarization conditions through repressing Blimp-1.

(A) CD4⁺ T cells from CBir1 Tg mice were cultured under Th17 polarization conditions with TGF-β (10 ng/ml) and IL-6 (20 ng/ml) in the presence or absence of RORγt inhibitor (1 μM). Expression of c-Maf, AhR, IRF4, and Blimp-1 was determined by qRT-PCR at day 2. Gene expression values were normalized to Gapdh expression. (B) CD4⁺ T cells from WT mice and RORγt^−/− mice were cultured under Th17 polarization conditions with TGF-β (10 ng/ml) and IL-6 (20 ng/ml) in the presence or absence of RORγt inhibitor (1 μM). Expression of Blimp-1 was determined by qRT-PCR at day 2 and normalized to Gapdh expression. (C) CD4⁺ T cells from WT (Cd4^CrePrdm1^fl/+) and CD4 conditional Prdm1^−/− (CKO, Cd4^CrePrdm1^fl/fl) mice were cultured with APCs and anti-CD3 mAb in the presence or absence of RORγt inhibitor under Th17 polarization conditions with TGF-β and IL-6. Cytokine production of T cells was determined by flow cytometry. (D) IL-17 and IL-10 production in culture supernatants was determined by ELISA. The results are shown as mean ± SD of three samples and are representative from one of 2–3 experiments performed. *p < 0.05, **p < 0.01.

To investigate whether Blimp-1 mediates RORγt inhibitor-treated Th17 cell function in vivo, we generated WT and Prdm1^−/− Th17 cells with or without the RORγt inhibitor in vitro, and transferred them into groups of Rag^−/− mice. The Rag^−/− recipient mice of RORγt-treated WT Th17 cells developed less severe colitis compared with Rag^−/− mice reconstituted with WT control Th17 cells (Figures 8A and B). Furthermore, Rag^−/− mice which received control Prdm1^−/− Th17 cells or RORγt-treated Prdm1^−/− Th17 cells developed colitis at similar levels, but both developed more severe colitis than the Rag^−/− mice reconstituted with RORγt-treated and control WT Th17 cells (Figures 8A and B). The levels of IL-17, IFN-γ, and TNFα in colonic tissues of the Rag^−/− recipient mice of RORγt-treated WT Th17 cells were decreased, while the levels of IL-10 were increased, compared with those from WT Th17 cells-reconstituted controls (Figure 8C). IL-17 production in colonic tissues of mice reconstituted with RORγt-treated Prdm1^−/− Th17 cells was also decreased compared with those from the mice received control Prdm1^−/− Th17 cells (Figure 8C). IL-10, IFN-γ, and TNFα production in colonic tissues of Rag^−/− mice reconstituted with RORγt-treated Prdm1^−/− Th17 cells was similar to mice that received control Prdm1^−/− Th17 cells. Colonic IL-6 production was similar in all groups (Figure 8C). Additionally, we isolated intestinal lamina propria CD4⁺ T cells and analyzed IL-10 mRNA expressions by qRT-PCR. IL-10 expression was higher in CD4⁺ T cells from Rag^−/− recipient mice of RORγt-treated WT Th17 cells compared with those from Rag^−/− recipient mice of WT Th17 cells. Additionally, IL-10 expressions in T cells from Rag^−/− mice that received control Prdm1^−/− Th17 cells were similar with those from mice that received RORγt-treated Prdm1^−/− Th17 cells, but lower than those from Rag^−/− recipient mice of WT Th17 cells (Figure 8D). These data indicate that decreased IL-10 production contributes in part to the excessive colitis induced by RORγt inhibitor-treated and control Prdm1^−/− Th17 cells. Taken together, these data suggest that RORγt serves as a repressor of Blimp-1 expression to restrain IL-10 production in Th17 cells, thus maintaining their pathogenicity in the induction of colitis.

Figure 8. RORγt inhibitor-treated Prdm1^−/− Th17 cells induce severe colitis.

Splenic CD4⁺ T cells from WT and Prdm1 CKO mice were cultured with APCs and anti-CD3 mAb (2 μg/ml) under Th17 polarization conditions with TGF-β (10 ng/ml) and IL-6 (30 ng/ml) in the absence or presence of RORγt inhibitor (1 μM) for 5 days. After 5 days, 1 × 10⁶ T cells were injected intravenously. into groups of Rag^−/− mice (n = 4/group), respectively. Six weeks after cell transfer, the severity of intestinal inflammation was assessed. (A) Colonic histopathology and (B) histological scores are shown. (C) Colonic tissues were cultured in the medium for 24 h, and cytokine production in supernatants was assessed by ELISA. (D) IL-10 expression in lamina propria CD4⁺ T cells was analyzed by qRT-PCR, and normalized to Gapdh expression. Data are shown as mean ± SD of each group of mice. One representative of two experiments. *p < 0.05, **p < 0.01.

Discussion

Multiple mechanisms are involved in generating Th17 cells and maintaining their pathogenicity in inducing chronic inflammatory diseases. In addition to its well-established role in driving Th17 cell development, we have demonstrated in this report that RORγt inhibits Th17 cell production of IL-10 through downregulating Blimp-1, thus preventing self-regulation and the development of IL-10-producing T cells, and maintaining their pathogenicity in inducing colitis. Our findings reveal a novel mechanism of action for RORγt inhibition in potentially ameliorating Th17 cell-driven intestinal inflammation.

Th17 cells have been implicated in the pathogenesis of IBD (34, 37, 46). It is widely believed that upon microbiota antigen stimulation, Th17 cells produce several pro-inflammatory cytokines that mediate inflammatory cell infiltration and tissue destruction in the intestines, thus leading to chronic inflammation. However, among Th17 cell signature cytokines, IL-17 has been shown to have dual functions during intestinal inflammation: 1) by recruiting myeloid cells (especially neutrophils) to the inflamed intestines (47), and 2) by protecting the intestines from inflammation through the promotion of intestinal IgA production (48) and regulation of epithelial permeability (49). Perhaps not surprisingly, administration of anti-IL-17 antibody has been ineffective in clinic trials for Crohn’s disease (50, 51). IL-22 is crucial in protecting intestinal epithelial barrier function as well as in preventing gut microbiota contact with the host. These studies indicate that other mechanisms are involved in driving intestinal inflammation by Th17 cells. A recent study has suggested that T cell-derived IL-22 binding protein plays a pathogenic role in IBD (52), which could partially contribute to the pathogenic role of Th17 cells in IBD. Our current data indicated that RORγt inhibits Th17 cell production of IL-10 while mediating IL-17 production. As IL-10 is crucial for inhibiting IBD both in experimental colitis models and patients with IBD, we speculate that failure to produce IL-10 by Th17 cells could also contribute to their pathogenic role in IBD. Importantly, our findings demonstrate that inhibiting RORγt is differentiated from blocking IL-17 in that the former promotes production of IL-10 production in addition to reducing IL-17.

In our study, IL-10 producing T cells induced by inhibition of RORγt displayed immune suppressive function, inhibited T cell proliferation in vitro, and induced less severe colitis as compared with control Th17 cells. Furthermore, blockade of IL-10 signaling led to an exacerbation of colitis induced by RORγt inhibitor-treated Th17 cells, indicating that the increased IL-10 production in RORγt inhibitor-treated Th17 cells plays a central role in the suppression of Th17-driven colitis. Therefore, our studies suggest the reduced pathogenic ability of RORγt inhibitor-treated Th17 in provoking colitis relies on both decreased Th17 cell function and increased production of IL-10 by those cells. In supporting this argument, a recent study demonstrated reduced IL-10 gene expression in human pro-inflammatory Th17 cells in multiple sclerosis compared to healthy controls (32), indicating that such mechanism could also function in humans.

It has been shown that effector CD4⁺ T cells produce IL-10 in addition to Treg cells. This supports the concept that IL-10 functions as a negative regulator following immune cell activation, which prevents the development of excessive inflammation. Several pathways have been identified in mediating IL-10 production in effector T cells, including STAT4, Blimp-1, c-Maf, and Notch in Th1 cells, STAT6 and GATA3 in Th2 cells, and STAT3 in Th17 cells (53). However, homeostatic mechanisms should exist to restrain IL-10 production in effector T cells to allow maximal driving of the inflammatory response when needed. c-Maf, AhR, Blimp-1 and IRF4 are transcription factors that have been shown to drive IL-10 expression of T cells. c-Maf is critical for Tr1 cell differentiation and IL-10 production by directly binding the IL-10 promoter (39). Blimp-1 is essential for effector T cells to produce IL-10, including Treg cells, Th1 cells and Th17 cells (41, 43, 54, 55), and it has been proposed to be a unique IL-10-promoted transcription factor in Tr1 cells (42, 43). IRF4 and AP-1 complexes promote the IL-10 expression by Th17 cells (45), and IRF4 synergizes with Blimp-1 to directly regulate IL-10 production by Treg cells (41). In addition, a recent study demonstrated that activin-activated IRF4, along with AhR, shape the differentiation of human Tr1 cells (44). Therefore, the effector T cells can acquire the Tr1 cell phenotype after activation of these IL-10 associated transcription factors (39, 43, 44). In our studies, the RORγt inhibitor did not affect T cell expression of c-Maf, Ahr, and IRF4, but significantly increased Blimp-1 expression under Th17 polarization conditions with TGF-β and IL-6. Furthermore, Prdm1^−/− T cells produced significantly less IL-10 when treated with the RORγt inhibitor under Th17 conditions compared to WT T cells. Transfer of RORγt inhibitor-treated Prdm1^−/− Th17 cells induced more severe colitis than that of RORγt inhibitor-treated WT Th17 cells, indicating that RORγt inhibits IL-10 production at least partially through inhibition of Blimp-1.

Our data suggested that targeting RORγt, the master transcription factor for Th17 development, could be an effective choice to treat IBD as it not only can suppress Th17 cell function but also promote IL-10 production to resolve intestinal inflammation. Successful utility of small molecular RORγt inhibitors in treatment of mice EAE and other autoimmune models (14) provides an opportunity for the development of a RORγt inhibitor that selectively suppresses the pathogenic functions of Th17 in intestinal inflammation. Indeed, we demonstrated that RORγt inhibitor-treated Th17 cells induced significantly less severe colitis, accompanied by diminished Th17 responses in the LP, which is consistent with previous reports (14, 56, 57). Interestingly, our studies also demonstrated that the RORγt inhibitor not only inhibited the production of IL-17 in Th17 cells, but also increases the production of IL-10 by those T cells, thus, providing a novel mechanism by which RORγt inhibitor downregulates colitis development. In supporting this notion, a recent study demonstrated that RORγt inhibitors not only suppressed IL-17 but also promoted IL-10 production by CD4⁺ T cells in inflammatory arthritis and IBD (58).

In summary, our study revealed a novel function of RORγt in Th17 cell development, in that RORγt promotes Th17 cells but inhibits IL-10 production, thus, maintaining their pathogenicity in inducing colitis. Our data thus supports the argument that a master transcript factor for one T cell lineage may also inhibit the key cytokines of other T cell lineages, i.e. promoting one T cell lineage but inhibiting other T cell lineage(s).

Abbreviations:

IL

interleukin

RORγt

retinoid-related orphan receptor gamma t

Th

T helper

IBD

inflammatory bowel disease