B Lymphocyte–Induced Maturation Protein-1 Contributes to Intestinal Mucosa Homeostasis by Limiting the Number of IL-17–Producing CD4+ T Cells

Soofia Salehi, Rashmi Bankoti, Luciana Benevides, Jessica Willen, Michael Couse, Joao S. Silva, Deepti Dhall, Eric Meffre, Stephan Targan and Gislâine A. Martins
J Immunol December 15, 2012, 189 (12) 5682-5693; DOI: https://doi.org/10.4049/jimmunol.1201966

Abstract

The transcription factor B lymphocyte–induced maturation protein-1 (Blimp-1) plays important roles in embryonic development and immunity. Blimp-1 is required for the differentiation of plasma cells, and mice with T cell–specific deletion of Blimp-1 (Blimp-1CKO mice) develop a fatal inflammatory response in the colon. Previous work demonstrated that lack of Blimp-1 in CD4+ and CD8+ T cells leads to intrinsic functional defects, but little is known about the functional role of Blimp-1 in regulating differentiation of Th cells in vivo and their contribution to the chronic intestinal inflammation observed in the Blimp1CKO mice. In this study, we show that Blimp-1 is required to restrain the production of the inflammatory cytokine IL-17 by Th cells in vivo. Blimp-1CKO mice have greater numbers of IL-17–producing TCRβ+CD4+cells in lymphoid organs and in the intestinal mucosa. The increase in IL-17–producing cells was not restored to normal levels in wild-type and Blimp-1CKO–mixed bone marrow chimeric mice, suggesting an intrinsic role for Blimp-1 in constraining the production of IL-17 in vivo. The observation that Blimp-1–deficient CD4+ T cells are more prone to differentiate into IL-17+/IFN-γ+ cells and cause severe colitis when transferred to Rag1-deficient mice provides further evidence that Blimp-1 represses IL-17 production. Analysis of Blimp-1 expression at the single cell level during Th differentiation reveals that Blimp-1 expression is induced in Th1 and Th2 but repressed by TGF-β in Th17 cells. Collectively, the results described here establish a new role for Blimp-1 in regulating IL-17 production in vivo.

Introduction

Interleukin-17 is an inflammatory cytokine produced by Th17 cells (1), a Th subset that develops independently (2, 3) of the transcription factors T-bet and GATA-3, which are required for the development of Th1 and Th2 cells, respectively (4). Th17 cells are associated with protection against bacterial and fungal infections encountered at mucosal surfaces (5) but also with several autoinflammatory disorders, including rheumatoid arthritis, psoriasis, multiple sclerosis, corticosteroid-resistant asthma, and inflammatory bowel disease (6, 7).

Differentiation of Th17 cells is induced by the cytokines TGF-β, IL-6, and IL-1β (8–10), and it is dependent on the expression of the transcription factors RORγt and RORα (11, 12), which induce transcription of the Il17a gene (11). ROR-γt acts in cooperation with RORα and other transcription factors, including STAT3, IFN regulatory factor-4, BATF, and Runx1, to induce full commitment of precursors to the Th17 subset (13–15). Activation of RORγt also promotes expression of the receptor for IL-23 (11), which is thought to maintain the expansion and pathogenesis of mature Th17 cells (16).

Although Th17 cells are clearly a distinct Th subset, recent data demonstrate that similar to other Th subpopulations, Th17 cells are considerably plastic and can acquire features and carry on effector functions characteristics of other Th subsets (17, 18). Th17 cells can express the regulatory T (Treg) cell specifying factor Foxp3 under certain conditions, and in the context of infectious and autoimmune inflammation, Th17 cells can also produce IFN-γ and IL-17 simultaneously (17, 19). IL-17/IFN-γ double-positive (IL-17+/IFN-γ+) T cells are found in elevated numbers in inflamed tissues of both humans and mice (20–22), and similar populations are observed in Th17 cells differentiated in vitro from human and murine naive CD4+ T cells (21–23). Recent studies indicate that IL-23 signaling is important for the differentiation of IFN-γ+/IL-17+ CD4+ T cells both in vivo and in vitro (23, 24), but the transcriptional mechanisms underlying the differentiation and/or conversion of these cells from non-IFN-γ–producing Th17 cells are poorly understood.

B lymphocyte–induced maturation protein-1 (Blimp-1; also called PRDI-BF1 in humans) is a transcription factor (encoded by the PRDM-1 gene) that plays crucial roles in regulating B and T lymphocyte function. Blimp-1 is expressed in both Treg and conventional T cells (25–27). Previous studies show that in Foxp3+ Treg cells Blimp-1 interacts with IFN regulatory factor-4 to induce IL-10 production (26). However, little is known about Blimp-1’s role in non Treg cell function and how lack of Blimp-1 contributes to the development of chronic mucosal inflammation. Despite their documented deficiency in producing IL-10, Blimp-1–deficient Treg cells can suppress T cell–mediated colitis (25, 28) suggesting that defects in other T cell subsets may underlie the chronic intestinal inflammation developed in the Blimp-1CKO mice. The studies described in this paper address this possibility and identify a new, Treg-independent role for Blimp-1 in controlling intestinal mucosal homeostasis by limiting the numbers of IL-17–producing Th cells in vivo.

Materials and Methods

Mice

C57BL/6 Prdm1flox/floxCD4-Cre+ (Blimp-1 CKO) and Prdm1+/+ CD4-Cre (control) mice were generated as described previously (25, 29). Previously described (30) C57BL/6 Prdm1flox/floxCD19-Cre+ mice were obtained from Dr. M. McHeyzer-Williams (Department of Immunology and Microbial Science, Scripps Research Institute, La Jolla, CA). Mice bearing a bacterial artificial chromosome transgene encoding yellow fluorescent protein (YFP) under the control of Blimp-1 regulatory elements (Blimp-1 YFP reporter mice) (31) were also described previously (32). In these mice, YFP expression closely recapitulates Blimp-1 mRNA expression (32). Foxp3-IRES-GFP knockin (33) (Foxp3 reporter) mice were obtained from V. Kuchroo (Brigham and Women’s Hospital, Boston, MA) and crossed to C57BL/6 Prdm1flox/floxCD4-Cre+ mice to generate Ctrl and Blimp-1CKO Foxp3 reporter mice. All mice were maintained in a specific pathogen-free animal facility at the Cedars-Sinai Medical Center (CSMC) and handled in accordance with the institutional guidelines.

Large intestines lamina propria lymphocyte isolation

Large intestines (LI) (cecum, colon, and rectum) were removed, opened longitudinally, cleaned and cut into strips 1 cm in length. Tissues were washed in ice-cold PBS and subjected to enzymatic digestion as described previously (34). Lamina propria (LP) mononuclear cells were purified on a 45/72% Percoll gradient by centrifugation for 20 min at 25°C and 600 × g with no brake.

Abs and flow cytometry

Abs used for cell surface staining were Alexa 700–conjugated TCR-β, Pacific Blue–conjugated anti-CD4, PE-conjugated anti-CD25, allophycocyanin-conjugated anti-CD44 (all from BioLegend), PE-conjugated anti–IL-17A, Alexa 647–conjugated anti–IL-17F, PE-conjugated anti–IL-4, and allophycocyanin-conjugated IFN-γ (eBioscience). Cell surface staining was performed as described previously (25). Samples were analyzed on a LSRII analyzer (BD Biosciences, San Jose, CA).

Determination of cytokine production

Total cell suspensions obtained from the LI-LP, spleen (SP), and mesenteric lymph nodes (mLN) were stimulated with plate-bound anti-CD3 (5 μg/ml) plus anti-CD28 (2.5 μg/ml) for 24 h with addition of brefeldin A (BFA; 1 mg/ml) for the last 6 h. The production of IL-17 and IFN-γ was determined by ELISA on supernatants collected at 18 h (before BFA addition) using eBioscience ELISA kits, following the manufacturer’s instructions. Where indicated, IL-17, IL-4, and IFN-γ were also detected by intracellular staining, as previously described (29). Cells were analyzed on a LSRII analyzer. Cytokine staining was evaluated in live CD4+TCRβ+-gated lymphocytes.

Quantitative real-time PCR

Total mRNA was isolated using RNAeasy kits (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed on equal amounts of RNA (as determined by Nanodrop measurements) for each sample using Superscript III (Invitrogen). SYBR Green incorporation quantitative real-time PCR (qRT-PCR) was performed using a FastStart SYBR Green Master mix (Roche) in the Realplex2 Mastercycler ep gradient S (Eppendorf). Primers used are described on Supplemental Table I.

Mixed bone marrow chimera

Recipient mice (CD45.1+ wild-type) were irradiated (10 Gy) for depletion of hematopoietic cells. Donor Ctrl (CD45.1+) or Blimp-1CKO (CD45.2+) Lin− bone marrow cells, enriched for hematopoietic progenitors by magnetic bead depletion (StemCell Technologies), were injected i.v. and chimeras were analyzed 11 wk later.

T cell transfer model of colitis

Induction of colitis by transfer of naive CD4+ T cells was performed as previously described (24, 35), with the following modifications: C57BL/6 Rag1−/− mice were injected i.p. (4 × 105 cells in 200 μl PBS) with naive CD4+CD25−CD45RBHigh or CD4+CD25−CD44low sorted from SP and LN from 4- to 6-wk-old Ctrl or Blimp-1CKO mice or CD4+CD25−CD44lowFoxp3− cells sorted from Ctrl or Blimp-1CKO-Foxp3 reporter mice. Mice were weighed weekly and inspected for clinical signs of disease (including weight loss, hunched appearance, pilo-erection of fur coat, and loose stool). Mice presenting clinically severe disease were sacrificed according to the CSMC Animal Care and Use Committee guidelines. At 7–12 wk posttransfer, recipient mice were sacrificed and colons were removed, cleaned in ice-cold PBS, and pieces of ∼0.5 cm in length were obtained from the proximal, middle, and distal portion of the colon and fixed immediately in 10% formalin. Fixed tissue was later embedded in paraffin, and 3-μm sections were cut and stained with H&E. Samples were coded and scored by a pathologist in a blinded fashion as described previously (35).

T cell isolation and in vitro Th differentiation

Naive CD4+ (CD25−CD44low) or effector (CD25−CD44High) T cells were sorted from SP and lymph nodes cell suspensions, using a FACSAria III fluorescent cell sorter (BD Biosciences, San Jose, CA). Purity of sorted cells preparations were >98%. For in vitro Th differentiation, sorted naive CD4+ T cells were stimulated in Iscove’s DMEM (Cellgro, Manassas, VA) supplemented with 10% FBS (Omega Scientific, Tarzana, CA) and penicillin/streptomycin (Cellgro) with plate-bound anti-CD3 (5 μg/ml), anti-CD28 (2.5 μg/ml) (both from BioXCell, West Lebanon, NH), and rHuIL-2 (25 U/ml; Roche) (neutral conditions). Experimental Th1 conditions also included rMuIL12 (5 ng/ml) plus anti–IL-4 (10 μg/ml) in addition to IL-2. Th2 conditions included rMuIL-4 (10 ng/ml) and anti–IFN-γ (10 ng/ml). Th17 conditions excluded IL-2 and included IL-1β (20 ng/ml), IL23 (50 ng/ml), IL-6 (10 ng/ml), TGF-β (5 ng/ml) (Promega), anti–IL-4, and anti–IFN-γ. With the exception of TGF-β, all recombinant cytokines were obtained from eBioscience. Neutralizing Abs anti–IFN-γ and anti–IL-4 were obtained from BioXCell. Cells were split every 3 d, and fresh medium (with the appropriate recombinant cytokines) added each time cells were split. For experiments in which TGF-β was used to repress Blimp-1 expression, recombinant TGF-β was only added at the beginning of the cultures and not replaced upon splitting of the cells. Restimulation was performed with PMA (50 ng/ml) and ionomycin (500 ng/ml) (both from Sigma-Aldrich) for 4 h; BFA was added in the last 2 h or with plate-bound anti-CD3 for 6 h (BFA added in the last 2 h).

Western blots

Whole-cell lysates (30 ng/sample) were subjected to SDS-PAGE transferred to Invitrolon polyvinylidene difluoride membranes (Invitrogen) and immunoblotted with monoclonal anti-mouse Blimp-1 (3H2E8) or monoclonal anti-mouse β-actin (Sigma-Aldrich).

Chromatin immunoprecipitation

Chromatin immunoprecipitation assays were performed as described previously (29). Briefly, naive CD4+ T cells were sort-purified from cell suspensions of lymph nodes and SPs from wild type mice and stimulated with plate bound anti-CD3, plus anti-CD28 under Th2 polarizing conditions. After seven to eight days cells were restimulated with PMA and ionomycin for 4 h before crosslinking by fixation with 1.1% paraformaldehyde for 10 min at room temperature. Sonicated chromatin from 4 to 5 × 107 cells was immunoprecipitated with 25 μl of either rabbit anti–Blimp-1 polyclonal Ab (clone 267, recognizing the C terminal of Blimp-1) (36) or preimmune serum as a control. qRT-PCR using SYBR Green incorporation was performed in DNA recovered from IP and input samples (see Supplemental Table II for primers sequences). Fold enrichment for each sample was calculated by dividing the percentage of input values obtained with anti-Blimp by the values obtained with control Ab, followed by normalization to the values obtained from the input chromatin. Analysis of sequence homology and identification of putative Blimp-1 consensus sites were performed using Evolutionary Conserved Regions browser (http://ecrbrowser.dcode.org) and rVista 2.0 software. All genomic sequences were obtained from Ensembl.

Retroviral gene transduction

Recombinant retroviruses encoding Blimp-1 (in the MigR1 vector, containing an internal ribosomal entry site–GFP cassette) were produced from transfected Plat-E packaging cells (37) and were used to transduce naive CD4+ T cells stimulated under Th17-polarizing conditions or CD3−B220+ cells sorted from the SPs of Prdm1Flox/FloxCD19-CRE+/− and Prdm1Flox/ FloxCD19 cre−/− mice and stimulated for 24 h with 2 μg/ml LPS (Sigma-Aldrich). Transduction was performed by mixing the B cells (24 h after stimulation) or the T cells (48–56 h after stimulation) with supernatant-containing virus and Polybrene (8 μg/ml; Sigma-Aldrich) and then centrifuged at room temperature for 90 min (10,000 × g). After transduction B cells were replated in regular media and T cells were replated under Th17-polarizing conditions, B cells were restimulated with LPS (10 μg/ml) 2 d after transduction and analyzed 36 h after LPS restimulation. T cells were analyzed 3 and 4 d after transduction and upon restimulation with PMA and ionomycin.

Statistics

Student t test with two-tailed distribution of equal variances was used to calculate p values, using the JMP software (SAS Institute, NC).

Results

Lack of Blimp-1 is associated with accumulation of CD4+ T cells in the colon and increased production of IL-17 in vivo

To identify the cellular mechanisms leading to the inflammatory response observed in the intestinal mucosa of the Blimp-1CKO mice, we first investigated the presence of T cells in the inflammatory infiltrate observed in 8- to 16-wk-old mice with signs of colitis development. We found that the absolute numbers of mononuclear cells recovered from the LI (cecum, colon, and rectum)-LP were significantly higher in the Blimp-1CKO mice (Fig. 1A). Both the percentages and absolute numbers of TCRβ+CD4+ were elevated in the LI-LP of Blimp-1CKO mice; CD4+ T cells percentages were on average 2-fold higher in the Blimp-1CKO mice (Fig. 1B), whereas the absolute numbers were 3- to 4-fold higher in the Blimp-1CKO mice (Fig. 1B). Thus, Blimp-1–deficient CD4+ T cells accumulate in the colonic LP.

FIGURE 1.
FIGURE 1.

Accumulation of CD4+TCRβ+ cells in the colon and increased amounts of IFN-γ– and IL-17–producing cells in Blimp-1 CKO mice. (A) Total numbers of mononuclear cells in the LI (cecum, colon, and rectum) LP in 8- to 16-wk-old Prdm1+/+CD4-CRE+/− (Ctrl) and Prdm1F/FCD4-CRE+/− (CKO) mice. Each symbol represents one animal (●, Ctrl; ▲, CKO), and bars represent average for each group. Results shown are representative of five independent experiments. (B) Percentages of TCRβ+CD4+ T cells (FACS plots) and total numbers of TCRβ+CD4+ T cells (chart) in LI-LP from Ctrl and Blimp-1CKO mice. (C) Production of and IL-17A (left) and IFN-γ (right) measured by ELISA on supernatants of LI-LP cells stimulated with plate-bound anti-CD3 plus anti-CD28 for 24 h. (D) ICC for IL-17A and IFN-γ (D, left plots) or IL-17A and IL-17F (D, right plots) and FACS analysis of CD4+TCRβ+ cells in mLN (top row) and SP (SP, bottom row) from Ctrl and CKO mice. SP and mLN total cell suspensions were stimulated as in C) before analysis (BFA was added in the last 6 h). Data shown are from gated TCRβ+CD4+ cells. (E and F) combined results of the percentage (E) or absolute numbers (F) of IL-17A or IFN-γ+CD4+ T cells in SP or mLN in five to six independent experiments for a total of six to eight mice per group. (G) Production of IL-17A (ELISA) by cells from SP or mLN after 18 h stimulation with plate-bound anti-CD3 plus anti-CD28. Data shown are the average and SEM from three different experiments with three to seven mice per group.

To characterize the CD4+ T cells that accumulated in the intestinal mucosa of Blimp-1CKO mice, we next investigated the production of the cytokines IFN-γ and IL-17A by these cells, because these two cytokines have been shown to play effectors roles in intestinal inflammation. LI-LP were isolated from Ctrl and Blimp-1CKO mice and stimulated with plate-bound anti-CD3 plus anti-CD28 for 24 h, and IFN-γ and IL-17A production were measured in the supernatants (Fig. 1C). The amount of IFN-γ detected in the supernatant of these cultures was similar in Ctrl and Blimp-1CKO mice (Fig. 1C, right), but LI-LP from Blimp-1CKO mice produced significantly more IL-17A than cells from Ctrl mice (Fig. 1C, left).

To determine whether the increased production of IL-17A and IFN-γ was restricted to the LI mucosa, we measured production of these cytokines in SP and mLN of Blimp-1CKO mice after in vitro TCR stimulation. Intracellular cytokine staining (ICC) and FACS analysis showed increased percentages (Fig. 1D, 1E) and absolute numbers (Fig. 1F) of IL-17A–producing cells in both SP and mLN of Blimp-1CKO mice. In addition, we also observed an increase in the percentage of IL-17F+ cells in the mLN of Blimp-1CKO mice (Fig. 1D). In both Ctrl and Blimp-1CKO mice, ∼50% of the IL-17A+ cells were also IL-17F+, but CKO mice had 2- to 3-fold more IL-17–producing cells (Fig. 1D). Different from the observed for IL-17 production, the percentages of IFN-γ–producing cells were elevated in the mLNs but not in the SP of Blimp-1CKO mice (Fig. 1D, 1E), and no significant differences were observed in the absolute numbers of IFN-γ–producing cells in Ctrl and Blimp-1CKO mice (Fig. 1F). Secretion of IL-17A was also significantly increased in the SP and mLNs of the CKO mice (Fig. 1G). Thus, lack of Blimp-1 is associated with increased production of IL-17 and, to a lesser extent, IFN-γ in vivo.

Blimp-1–deficient effector/memory CD4+ T cells have increased expression of genes associated with the Th17 differentiation program

Characterization of the IL-17–producing cells present in the Blimp-1CKO mice revealed that they were all Ag-experienced cells, which expressed high levels of CD44 (data not shown). Sort-purified CD44highCD25−CD4+ cells from pooled mLN and SP from Blimp-1CKO had higher frequency of IL-17 producers and secreted significantly more IL-17A protein than Ctrl cells upon in vitro restimulation (Fig. 2A, 2B). They also expressed significantly higher amounts of Il17a, Rorc, and Il23r but not Batf mRNA than Ctrl cells when stimulated in vitro (Fig. 2C). Thus, the expression of Th17 signature genes is upregulated in Blimp-1–deficient Ag-experienced CD4+ T cells, suggesting that Blimp-1 is required to control the differentiation and/or accumulation of Th17 cells in vivo.

FIGURE 2.
FIGURE 2.

Increased expression of il17a and Th17-signature genes in Blimp-1–deficient effector/memory cells. CD4+ Ag-experienced (CD44High) T cells were sorted from pooled SP and mLN from Ctrl or Blimp-1 CKO mice and stimulated in vitro with plate-bound anti-CD3 plus anti-CD28 and IL-17 production was measured by ICC staining and FACS analysis at 6 h (A) or in the supernatants by ELISA at 48 h (B). Il17a, Il23r, RORC, and BATF steady-state mRNA levels (C) were measured by qRT-PCR in Ctrl and Blimp-1CKO stimulated as in (A). Results shown in (A)–(C) are representative of two to three independent experiments with three to five mice per group. Bars show average, and error bars represent SEM. Results in (C) are presented relative to 18S expression.

Regulation of IL-17 production by Blimp-1 is intrinsic to CD4+ effector T cells

Increased numbers of IL-17–producing CD4+ T cells in Blimp-1CKO mice could result from intrinsic effects of Blimp-1 in regulating the differentiation/accumulation of Th17-cells in vivo or could result from the previously described impaired Treg cell function in these mice (25, 26), because defective Treg cell responses can facilitate priming and development of IL-17– and IFN-γ–producing cells (38–40). Alternatively, the increased numbers of IL-17–producing cells could be a secondary effect of the ongoing inflammation in the colon of these mice (25). To distinguish these possibilities, we next analyzed the expression of IL-17 in CD4+ T cells from chimeric mice generated with a mixture of wild-type and Blimp-1CKO bone marrow cells. To delay the development of severe inflammation in the chimeric mice, we designed our experiments such that the hematopoietic compartment in the reconstituted mice would contain significantly more Ctrl than Blimp-1–deficient cells. Thus, in all sites analyzed, the majority of TCRβ+CD4+ cells were Blimp-1 sufficient (Fig. 3A).

FIGURE 3.
FIGURE 3.

Increased production of IL-17 by Blimp-1–deficient CD4+ T cells in mixed-bone marrow chimeric mice. Wild-type CD45.1-irradiated mice received a total of ∼4 × 106 HSC-enriched mixed BM cells from Ctrl (CD451.1) and Blimp-1CKO (CD451.2) mice. Eleven weeks after bone marrow cells transfer, mLN, SP, and LI-LP cells were isolated from chimeric mice, stimulated as described in Fig. 1D and IL-17 (A) production was analyzed in TCRβ+CD4+ cells in the CD45.2− (Ctrl) or CD45.2+ (Blimp1CKO) gates. (B) Frequency of IFN-γ–producing TCRβ+CD4+ cells from the mLN and SP from chimeric mice [stimulation and analysis done as in (A)]. (C) Frequency of Foxp3-expressing cells in the Ctrl and Blimp-1CKO compartments in the SP and mLN from chimeric mice [gated as shown in (A)]. Plots (A)–(C) (bottom) show compiled results from four to nine different chimeric mice.

In both compartments, Blimp-1–sufficient and Blimp-1–deficient, the frequency of Foxp3+ Treg cells was similar (Fig. 3C). In the Blimp-1–deficient compartment; however, the frequency of IL-17A–producing (but not of IFN-γ–producing) cells was significantly increased in comparison with the wild-type compartment in all sites evaluated, including the LI-LP (Fig. 3). Thus, Blimp-1–deficient CD4+ T cells are intrinsically more prone to differentiate into IL-17–producing cells.

Blimp-1–deficient CD4+ T cells differentiate into IL-17+/IFN-γ+ cells and cause severe colitis in Rag1-deficient mice

To determine whether the absence of Blimp-1 can lead to the preferential differentiation of IL-17– and/or IFN-γ–producing cells in vivo, we transferred CD4+ naive (CD44low/CD25−) Ctrl or Blimp-1CKO cells into Rag1-deficient (Rag1−/−) mice and monitored colitis development in the recipient mice. Rag1−/− mice receiving Ctrl cells developed colitis and wasting disease as indicated by histological analysis and total body weight loss. Rag1−/− mice receiving Blimp-1–deficient cells showed signs of disease earlier than mice receiving Blimp-1–sufficient cells (Fig. 4A) and also developed more severe colitis, with more prominent inflammation in the colon (Fig. 4B) and significantly increased colitis scores (average score = 3 ± 0.9) than in those receiving Ctrl cells (average score = 1 ± 0.7) (Fig. 4C).

FIGURE 4.
FIGURE 4.

Blimp-1 CKO CD4+ T cells differentiate into IL-17 and IFN-γ double producer cells in vivo and cause severe colitis in Rag1−/− mice. Rag1−/− mice were injected i.p. with 4 × 105 naive (CD44lowCD25−) CD4+ T cells sorted from Ctrl or Blimp-1CKO mice and monitored for symptoms of colitis development and wasting disease, including body weight loss daily (A). Six to 7 wk after T cell transfer, recipient mice were euthanized and colons were collected and processed for H&E staining [(B), ×20 magnification] and histological analysis. Slides were analyzed blindly. Colitis score (C) was graded semiquantitatively from 0 to 4, as previously described (35) each symbol represent one animal (the circles and triangles represent Ctrl and CKO, respectively). (D) ICC staining and FACS analysis of cells from mLN from recipient mice 7 wk after adoptive T cell transfer of Ctrl (●) or Blimp1CKO (▲) cells. mLN cells were stimulated in vitro for 24 h with plate-bound anti-CD3 plus αCD28 before staining. FACS plots show cells in the TCRβ+CD4+ gate. (E) Combined results (each symbol represent one animal, and bars represent average of five to six different experiments) of the percentage of IL-17A+, IFN-γ+, or IL-17A+/IFN-γ+ CD4+ T cells in the mLN (left panel), or LI-LP (right panel) for a total of six to seven mice per group. (F) Absolute numbers of IL-17A+, IFN-γ+, or IL-17A+/ IFN-γ+ CD4+ T cells in the LI-LP based on the percentages shown in (E). (G) Percentages of TCRβ+ CD4+Foxp3+ cells in the mLN (left) or LI-LP (right) of Rag−/− mice injected with Ctrl or Blimp-1CKO 4 × 105 naive [CD44lowCD25−GFP− (Foxp3−)] CD4+ T cells and analyzed six to seven weeks later. Data are representative of five [(A)–(C)] or three (E, F) different experiments, with a total of four to seven mice per group.

Evaluation of cytokine production upon ex vivo TCR stimulation of cells from mLN and LI-LP revealed that while the percentages of IFN-γ or IL-17–single producer cells were only marginally increased in the Blimp-1CKO mice, the percentage of IFN-γ and IL-17 double producer cells were significantly increased in the mLN of mice transferred with Blimp-1-deficient cells (Fig. 4D, 4E). However, the absolute numbers of IL-17+, IFN-γ+, and IL-17+/IFN-γ+ cells were all significantly increased in the LI-LP of mice injected with Blimp-1–deficient cells in comparison with mice injected with Ctrl cells (Fig. 4F). To determine whether the differences we observed between Ctrl and Blimp-1CKO cells-mediated colitis was due to differential induction Foxp3+ Treg cells from the injected naive cells, we repeated these experiments using naive CD4+ T cells isolated from Ctrl or Blimp-1CKO mice that had been previously bred with Foxp3 reporter mice. This strategy allowed us to exclude Foxp3+ cells from the pool of naive cells adoptively transferred to the Rag1−/− mice and subsequently monitor the development of Foxp3-expressing cells in the recipient mice. Rag1−/− mice injected with CD4+ naive (CD44lowCD25−GFP− [Foxp3−] cells) from Blimp-1CKO mice developed more severe colitis than mice injected with Blimp-1–sufficient cells (data not shown), similarly to that described above, indicating that the increased severity of colitis caused by Blimp-1–deficient cells was not due to differential contamination of the transferred cells with Foxp3+ Treg cells. Moreover, analysis of GFP expression in cells from the recipient mice showed that Foxp3+ cells developed in greater numbers from Blimp-1–deficient than from Ctrl cells (Fig. 4G), indicating that increased inflammation in mice injected with Blimp-1–deficient was not due to impaired induction of Foxp3+ cells.

Blimp-1 expression is selectively regulated during Th differentiation

The results described above indicated that expression of Blimp-1 suppresses the differentiation of Th17 cells in vivo and that in the absence of Blimp-1 more cells tend to differentiate into IL-17+ or IL-17+/IFN-γ+ cells. We reasoned that if Blimp-1 antagonizes Th17 differentiation, its expression should be downregulated in cells differentiated under Th17 conditions, as opposed to cells that usually do not produce IL-17, such as Th1 and Th2 cells. Previous studies have shown that Blimp-1 is expressed in CD4+ T cells differentiating into either Th1 or Th2 conditions, but whereas one group showed increased expression of Blimp-1 in Th2 cells (41), another showed similar expression in Th1 and Th2 cells (28). Neither of these studies investigated Blimp-1 expression in Th17 cells. To clarify whether Blimp-1 expression is, in fact, differentially regulated during Th differentiation, we used cells from previously described Blimp-1-YFP reporter mice (29, 32) to analyze Blimp-1 expression at the single cell level during Th differentiation in vitro.

Sorted naive (CD44lowCD25−) CD4+T cells from Blimp-1-YFP reporter mice were stimulated under neutral, Th1, Th2, or Th17 conditions (Supplemental Fig. 1) and Blimp-1 mRNA expression (as reported by YFP) was analyzed at different time points. As expected from previous reports (41, 42), stimulation of naive CD4+ T with plate-bound anti-CD3 plus anti-CD28 induced expression of Blimp-1 in a small percentage of cells; addition of IL-2 to these cultures (neutral conditions) increased expression, especially at later time points (Fig. 5A). Cells cultured under Th1 or Th2 conditions began to express Blimp-1 sooner than cells cultured in neutral conditions, but Th1 cells expressed significantly more than Th2 cells at earlier time points (Fig. 5A). However, at day 7.5, >70% of Th1 and Th2 cells expressed high levels of Blimp-1 (Fig. 5A-B). In contrast to cells stimulated under neutral, Th1 or Th2 conditions, cells cultured under Th17 conditions did not upregulate Blimp-1 significantly throughout the experiment, and <5% of the cells expressed Blimp-1 at day 7.5 poststimulation (Fig. 5B). Analysis of Blimp-1 steady-state mRNA (Fig. 5C) and protein levels (Fig. 5D) in these cells confirmed the differential expression of Blimp-1 during Th differentiation. Thus, Blimp-1 is expressed in Th1 and Th2 cells but not in Th17 cells.

FIGURE 5.
FIGURE 5.

Blimp-1 is expressed in Th1 and Th2 but not Th17 cells. (A) Blimp-1 expression as reported by YFP in naive CD4+ T cells sorted from YFP transgenic Blimp-1 reporter mice and stimulated under neutral (with IL2), Th1,Th2 or Th17 conditions for 3.5 (top row) or 7.5 (bottom row) days. FACS Plots shown are from the gated live, CD4+ cells in the well. (B) Average percentages (and SD) of YFP-positive cells shown in (A) for five independent experiments for a total of seven different mice. (C) Steady-state Blimp-1 mRNA at day 7.5 in cells from experiment shown in (A). (D) Immunoblotting of total cell lysates obtained from cells stimulated as in (A) for 5.5 d (Ntrl = Neutral), using Abs to Blimp-1 or β-actin.

We next investigated the regulation of Blimp-1 expression by different Th-polarizing cytokines. Previous studies (28, 41, 42) have reported contradictory results on the induction of Blimp-1 by Th1- and Th2-inducing cytokines. We found that IL-2, IL-4, IL-6, and IL-12 were each capable of enhancing expression of Blimp-1 by anti-CD3 plus anti-CD28 stimulation (which alone induced little expression of Blimp-1) (Fig. 6A), but at different magnitudes, with IL-12 being the best inducer, followed by IL-2 and then IL-4. Among Th17-inducing cytokines, IL-6 was the best inducer of Blimp-1 expression, although in comparison with IL-2, IL-4, and IL-12, IL-6 was a poor inducer. Neither IL-1β nor IL-23 induced Blimp-1 expression; in fact, these cytokines partially inhibited Blimp-1 expression induced by IL6 (Fig. 6A). Because the expression of Blimp-1 in cells stimulated with a combination of IL-6, IL-23, and IL-1β was higher than in cells stimulated under Th17 conditions (Fig. 6A) (which included TGF-β in addition to IL-6, IL-23, and IL-1β),we suspected that TGF-β could have an inhibitory effect on Blimp-1 expression. We therefore compared Blimp-1 expression induced by the combination of TCR (and costimulation) and IL-2, IL-12, or IL-4 in the absence or presence of TGF-β. We found that TGF-β led to significant inhibition of Blimp-1 expression in these cultures (Fig. 6A). Evaluation of Blimp-1 steady-state mRNA levels confirmed these results (Fig. 6B).

FIGURE 6.
FIGURE 6.

Blimp-1 expression is induced by Th1- and Th2-inducing cytokines but repressed by TGF-β in Th17-developing cells. (A) Naive CD4+ T cells from Blimp-1 reporter mice were stimulated with plate-bound anti-CD3 plus anti-CD28 alone or in the presence of the indicted cytokines and YFP (Blimp-1) expression was analyzed by FACS at day 5. (B) qRT-PCR analysis of Blimp-1 mRNA expression in the same cells from experiment shown in (A). (C) Dose-dependent inhibition of Blimp-1 expression by TGF-β in Blimp-1 reporter naive CD4+ T cells stimulated as indicated in the presence of different amounts of TGF-β for 5 d and then analyzed by FACS. (D) qRT-PCR analysis of Blimp-1 mRNA expression in the same cells from experiment shown in (C). Data shown are from one experiment with pooled cells from three mice; similar results were obtained in two independent experiments.

We next sought to determine whether TGF-β could inhibit expression of Blimp-1 induced by the combination of IL-2 and IL-12. TGF-β inhibited Blimp-1 expression induced by TCR and costimulation in combination with IL-2 and IL-12 in a dose-dependent manner (Fig. 6C). Even at concentrations lower than the required amounts used to induce Th17 differentiation in vitro, TGF-β led to significant inhibition Blimp-1 expression in cells cultured simultaneously with IL-12 and IL-2. These results were also confirmed by mRNA expression measured by qRT-PCR (Fig. 6D). Together, these results establish TGFβ as a potent suppressor of TCR and cytokine-induced Blimp-1 expression during Th17 differentiation.

Blimp-1 binds to the Il17a gene in vivo but is not sufficient to repress Th17 differentiation

The observation that lack of Blimp-1 is associated with increased expression of Il17a mRNA and protein as well as several other Th17 genes (Fig. 2) and that Blimp-1 expression is selectively downregulated during Th17 differentiation (Fig. 5) suggested that Blimp-1 could function as a repressor of the Th17 program. Blimp-1 has potent transcriptional repression capabilities and directly regulates the transcription of several cytokine genes (26, 29, 41). Thus, we first sought to determine whether Blimp-1 response elements could be identified in the Il17a gene. We identified many putative Blimp-1 binding sites at the Il17a promoter region as well as in regions downstream of the transcription start site (data not shown). We then tested whether Blimp-1 could bind to three of the sites we identified. Using wild-type Th2-polarized cells, we found significant enrichment for Blimp-1 at least one of these sites (located at ∼3.3 kb upstream of the transcription start site [TSS]). We also detected some enrichment for Blimp-1 binding on a site located 7.2 kb upstream of the TSS, but in this case, there was no significant difference in comparison with nonspecific binding on a control gene (Fig. 7A). The third site investigated is located further downstream (∼44.9 kb from the TSS) showed no enrichment for Blimp-1 binding (Fig. 7A). Thus, in Th2 cells, Blimp-1 can bind to at least one site in the Il17a gene.

FIGURE 7.
FIGURE 7.

Blimp-1 binds to the murine Il17a gene in Th2 cells, but it is not sufficient to repress IL-17 production in Th17 cells. (A) Chromatin immunoprecipitation analysis of wild-type Th2-polarized cells restimulated with PMA and ionomycin, followed by immunoprecipitation of chromatin with anti–Blimp-1 or control Ab and quantitative PCR analysis of binding of Blimp-1 at different regions upstream (−3.3, −7.2 kb) or downstream (+44.9 kb) of the transcription start site of the murine Il17a gene or a negative control region in the SNAIL3 gene; results were normalized to those of the input chromatin, followed by the ratio of results obtained with anti–Blimp-1 Ab and control Ab (nonspecific background). Data are representative of three to four independent experiments (mean and SEM). (B and C) Production of IL-17 upon enforced expression of Blimp-1 in Th17 cells. Naive CD4+ T cells were stimulated under Th17 conditions and transduced with retrovirus expressing GFP only (Ctrl RV) or GFP and Blimp-1 as a bicistronic message (Blimp-1 RV); cells were restimulated 3 d (B) or 4 d (C) after transduction and analyzed for IL-17A (top plots) or IL-17F (bottom plots) production. Plots shown are from CD4+ (B) or CD4+GFP+ (C) cells. Chart in (C) shows the average and SEM of three independent experiments.

To determine whether Blimp-1 could suppress Il17a activity in Th17 cells, we used retrovirus transduction to enforce Blimp-1 expression in cells stimulated under Th17-polarizing conditions and measured IL-17 production. Expression of Blimp-1 (as indicated by a GFP reporter) and confirmed by qRTPCR (data not shown) did not result in significant decrease of IL-17A or IL-17F production (Fig. 7B, 7C). However, transduction with the same retroviral construct was able to partially restore plasma cell formation in Blimp-1–deficient B cells stimulated in vitro (Supplemental Fig. 2), indicating that functional Blimp-1 protein is expressed from this construct. Therefore, although Blimp-1 is bound to the Il17a gene in Th2 cells, it is not sufficient to repress Il17a transcription in cells stimulated under Th17 conditions.

Discussion

The results of this study provide evidence for a nonredundant role for the transcriptional regulator Blimp-1 in constraining the numbers of IL-17–producing Th cells in vivo. We showed that spontaneous development of chronic intestinal inflammation in mice with T cell-specific deletion of Blimp-1 is associated with accumulation of IL-17–producing Th cells in vivo. Furthermore, Blimp-1–deficient naive CD4+ T cells preferentially differentiated into IL-17A and IFN-γ double-producing cells and caused severe colitis when transferred to Rag1−/− mice. Consistent with a role for Blimp-1 in keeping the numbers of Th17-producing cells under control, we demonstrated that Blimp-1 was selectively expressed in Th1 and Th2 but not in Th17 cells.

Our observation that IL-17A–producing cells selectively accumulate in the Blimp-1–deficient CD4+ T cell compartment of Ctrl and Blimp-1CKO mixed-bone marrow chimeric mice (Fig. 3) indicate a Treg-independent, effector T cell-intrinsic role for Blimp-1 in repressing the production of IL-17A in vivo. This is further supported by our findings that Blimp1-deficient naive CD4+ T cells generated more IL-17A–producing cells than control cells and caused severe colitis upon adoptive transfer to Rag1−/−mice. These results cannot be attributed to a defective Treg response in the absence of Blimp-1, because Blimp-1–deficient CD4+ T cells led to the generation of Foxp3+ Treg cells in greater numbers than control cells. Although IL-10 production is defective in a small subset of Foxp3+ Blimp-1–deficient Tregs (25, 28, 41), IL-10–deficient Treg cells can block adoptive T cell transfer–induced colitis (43, 44) and similar findings have been reported for Blimp-1–deficient Tregs (28).

The mechanisms underlying repression of IL-17 production/Th17 differentiation by Blimp-1 remains to be fully elucidated. Our observation that Blimp-1–deficient CD4+ T cells had increased amounts of Il17a mRNA, suggested that Blimp-1 could regulate IL-17A production at the transcriptional level. In addition, Blimp-1–deficient CD4+ effector T cells had increased amounts of Il23r and RORC mRNA, suggesting that Blimp-1 might directly repress these genes. Consistent with this possibility Blimp-1 consensus binding sites can be found in conserved, putatively regulatory regions in both loci (data not shown). Alternatively, because expression of Il23r and Il17a are both positively regulated by Rorc (encoding RORγt), Blimp-1 might also regulate Il23r and Il17a indirectly by acting as a repressor of Rorc.

Our observation that Blimp-1 can bind to at least one site on the Il17a promoter in Th2 cells suggests that Blimp-1 might function to directly repress the Il17a gene during Th2 differentiation. In fact, Il17a site -3.2, which shown significant enrichment for Blimp-1 binding is located ∼2 kb downstream of conserved non coding sequence 2 (CNS2, also called CNS5), an important regulatory region in the il17a promoter (45, 46). Consistent with the idea that Blimp-1 could repress Il17a in Th2 cells, deficiency of the histone lysine methyltransferase G9a, which can function as a cofactor for Blimp-1–mediated repression (47), leads to increased production of IL-17A in Th2-polarized cells (48). However, forced expression of Blimp-1 in cells differentiating under Th17 conditions did not result in a significant decrease of IL-17A or IL-17F production (Fig. 7B, 7C). Expression of Prdm1 mRNA in transduced Th17 cells that expressed Blimp-1 was similar to that of developing Th2 cells (data not shown). Nevertheless, it is possible that repression of Il17a in Th17 cells requires higher amounts of Blimp-1 protein than achieved in our experiments. Alternatively, repression of the Il17a by Blimp-1 might require corepressors that are selectively expressed in Th2 (and potentially Th1) but not in Th17 cells. Such a mechanism could play a role in preserving Il17a activity in Th17 cells that are exposed to conditions where Blimp-1 expression can be induced.

Our results presented here show that TGF-β signaling is a potent repressor of Blimp-1 expression and might function to repress Blimp-1 expression during Th17 differentiation. Interestingly, a recent study using in vitro-generated Th17 cells to induce colitis upon transfer into Rag1−/− mice demonstrate that the extinction of IL-17 production in Th17 cells that become IFN-γ producers in the presence of IL-12 and IL-23 is contingent upon limited amounts or complete absence of TGF-β (23). The presence of TGF-β resulted in maintenance of IL-17 production in these cells (23). Using experimental autoimmune encephalomyelitis as a model of inflammation, Ghoreschi et al. (49) made similar observations, showing that Th17 cells generated in the presence of IL23 and absence of TGF-β retained the capacity to make IL-17 while simultaneously turning on IFN-γ production (49). On the basis of these observations, our results showing that Blimp-1–deficient T cells generate more IL-17+/IFN-γ+ cells under inflammatory conditions suggest a model whereby induction of Blimp-1 expression could be required to repress IL-17 production in Th17 cells converting into IFN-γ–producing cells. According to this model, under highly inflammatory conditions where IL-12 is abundant and TGF-β scarce, IL-12 would lead to induction of Blimp-1 expression in Th17 cells, in addition to inducing expression of T-bet and IFN-γ. Blimp-1 expression under these conditions would result in the repression of both IL-17 and IL23R, thus restraining the formation and/or maintenance of the IL-17+/IFN-γ+ cells. In the absence of Blimp-1, IL-12 would lead to IFN-γ production, but would fail to repress IL-17 and favor the generation/maintenance of the IL-17+/IFN-γ+ cells.

One caveat of this model is the assumption that IL12 could induce Blimp-1 and IFN-γ simultaneously, which is at odds with our previous observation that in CD4+ T cells stimulated under neutral conditions, Blimp-1 binds to and suppresses transcription of both Tbx21 and Ifng (41). It is possible, however, that the Ifng gene regions bound by Blimp-1 are not equally accessible in T cells stimulated under neutral conditions and in cells responding to IL-12 and transitioning into Th1 cells (or “ex Th17 cells”) developing during mucosal inflammation. In addition to differential accessibility of Blimp-1 binding regions at the Ifng locus, these different Th subpopulations may also diverge in the expression of coactivators and corepressors, which could interact with Blimp-1 to counteract or favor repression of Ifng. The same arguments can be made to explain the fact that Th1 cells express high levels of Blimp-1 while making high amounts of IFN-γ. Evaluation of Blimp-1 binding sites occupancy in cells stimulated under neutral conditions side by side with cell transitioning from Th17 to Th1 will be required to clarify this.

In line with our observation that IL-17–producing cells accumulate in the Blimp-1CKO mice and that Blimp-1–deficient T cells fail to turn off the Il17 gene under inflammatory conditions, recent genome-wide association studies reveal strong association between the gene encoding Blimp-1 (PRDM1) and inflammatory bowel disease (50, 51). One of the forms of inflammatory bowel disease, Crohn’s disease, is also associated with the accumulation of IL-17+/IFN-γ+ cells in the intestinal lesions (21, 22). Although the functional implications of the PRDM1 polymorphisms associated with IBD remain unknown, one intriguing possibility is that these polymorphisms cause decreased expression or partial loss of function of Blimp-1 resulting in the accumulation of IL-17+/IFN-γ+ cells in the intestinal lesions of CD patients.

Plasticity of the CD4+ T cells is now a recognized trait of immune responses, especially under conditions associated with inflammatory disorders. Understanding the mechanisms regulating Th cell plasticity in these conditions will be essential in considering new therapeutic approaches to treat these diseases. The involvement of Blimp-1 in counteracting the differentiation of the highly inflammatory IL-17+/IFN-γ+ producing cells in vivo as well as its potential role in repressing Il17a expression in Th1 and Th2 cells that we report in this paper might represent initial steps toward the understanding the mechanisms regulating effector T cell response under chronic inflammatory conditions.

Disclosures

The authors have no conflicting financial interests.

Acknowledgments

We thank Dr. Kathryn Calame for invaluable support of these studies and Dr. Jonathan Kaye for helpful discussions and for critical reading of the manuscript. We also thank Brian de la Torre and Asha Kadavallore for helping with mice irradiation and retrovirus supernatant production, respectively, and the CSMC Flow Cytometry core, especially Gillian Hultin, for sorting. We also thank Carol Landers and Richard Deem for technical help and Loren Karp for editorial assistance.

Footnotes

  • This work was supported by National Institutes of Health‑National Institute of Allergy and Infectious Diseases Grant AI083948-01 (to G.A.M.), F. Widjaja Foundation Inflammatory Bowel and Immunobiology Institute funds, and a predoctoral scholarship from Fundacao de Amparo a Pesquisa e ao Ensino do Estado de Sao Paulo (Brazil) (FAPESP#2008/04606-2) (to L.B.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BFA
    brefeldin A
    Blimp-1
    B lymphocyte–induced maturation protein-1
    CSMC
    Cedars–Sinai Medical Center
    ICC
    intracellular cytokine staining
    LI
    large intestine
    LP
    lamina propria
    mLN
    mesenteric lymph node
    qRT-PCR
    quantitative real-time PCR
    SP
    spleen
    Treg
    regulatory T
    TSS
    transcription start site
    YFP
    yellow fluorescent protein.

  • Received July 18, 2012.
  • Accepted October 13, 2012.

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