PSA Promotes Inducible Foxp3⁺ Tregs with Suppressive Activity.

We orally administered PSA to mice and monitored the CD4⁺CD25⁺Foxp3⁺ population of Tregs in the MLN. Mice treated with PSA displayed increased percentages of CD4⁺Foxp3⁺ T cells compared with control mice (Fig. 2A). One of the primary functions of Treg cells is to suppress the activation and proliferation of inflammatory effector T cells (Teffs). The suppressive properties of Tregs following PSA administration were determined by the addition of varying ratios of CD4⁺CD25⁺ T cells purified from MLNs to naive responder cells. Tregs isolated from PSA-treated animals have increased suppressive capacity compared with PBS-treated control animals (28.5% proliferating responder cells vs. 43.0% at a 1:2 Treg:Teff ratio) (Fig. 2B). These findings demonstrate that PSA induces the differentiation of functional Foxp3⁺ Tregs with enhanced suppressive activity.

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Fig. 2.

PSA is sufficient to induce functional Foxp3⁺ Tregs with an inducible phenotype. (A) Foxp3-GFP mice were orally gavaged with purified PSA, MLNs harvested, and cells stained for CD4 and CD25. Each symbol represents the percent of CD4⁺ Foxp3-GFP⁺ cells from a single mouse. Results are representative of three independent trials with four mice per group. **P < 0.01. (B) CD4⁺CD25⁺ cells were purified from the MLNs of mice treated with either PBS or PSA, as indicated in A, and incubated with CFSE-pulsed CD4⁺CD25⁻ effector T cells in an in vitro suppression assay. Numbers indicate the percentage of cells undergoing at least one cellular division at three different ratios of effector T cells (Teff) and Tregs. These data are representative of two independent trials. (C) Foxp3-GFP mice were orally treated with purified PSA. MLNs were extracted and CD4⁺Foxp3⁺ or the CD4⁺Foxp3⁻ T cells were purified based on ±GFP expression (purity >99%). RNA was extracted and used for qRT-PCR. These data are representative of three independent experiments. Light bars indicated cells derived from PBS treated mice and dark bars from PSA-treated mice. (D) TLR2^−/− mice were orally treated with PSA. MLNs were extracted and analyzed for the percentage of CD4⁺Foxp3⁺ cells. Each symbol in the bar graph represents the percentage of CD4⁺Foxp3⁺ from an individual mouse. NS, not significant. Results are representative of three independent trials with at least three mice per group. (E) CD4⁺CD25hi⁺ and CD4⁺CD25⁻ T cell populations were FACS purified from MLNs of PSA-treated TLR2^−/− mice. IL-10 levels were analyzed by qRT-PCR. Light and dark bars indicate IL-10 levels in PBS or PSA treated animals, respectively. Error bars represent the SD from samples of the same experiment run in triplicate. Results are representative of two independent trials with four mice per group.

Various subsets of Tregs exist within the Foxp3⁺ and Foxp3⁻ T-cell populations; therefore, we analyzed the expression of Treg-associated genes to understand how PSA affects the development of Foxp3⁺ Tregs. Foxp3-GFP mice were gavaged with purified PSA (or PBS control), and RNA was extracted from either CD4⁺Foxp3⁻ or CD4⁺Foxp3⁺ T cells from the MLNs following cell purification. As expected, gene expression in Foxp3⁻ and Foxp3⁺ T-cell subsets differed dramatically and included higher basal levels of IL-10, TGF-β2, GITR (glucocorticoid-induced TNF-related protein), ICOS, CTLA-4, and Ebi3 (subunit of IL- 35) in Foxp3⁺ T cells (Fig. 2C and Fig. S4). Remarkably, PSA induces over 8-fold increased levels of IL-10 from CD4⁺Foxp3⁺ Tregs than that expressed in PBS-treated cells, and had virtually no impact on CD4⁺Foxp3⁻ T cells. Accordingly, although TGF-β2 expression in CD4⁺Foxp3⁻ T cells was not altered, PSA elicited significant induction of TGF-β2 from Foxp3⁺ Treg cells. Although Treg subsets that do not express Foxp3 have been described based on IL-10 and TGF-β production (Tr1 and Th3 cells, respectively) (20), PSA's effects are restricted to the Foxp3⁺ Treg population. PSA treatment also significantly increases the transcription of granzyme B, perforin, and CCR6 (a chemokine receptor associated with the migration of Treg cells) from Foxp3⁺ Tregs (Fig. 2C and Fig. S4). It is important to note that PSA does not globally impact all Treg-derived cytokines, as expression of TGF-β1 and Ebi3 are not altered, demonstrating specificity for a distinct Treg profile. Furthermore, production of the natural Treg-associated surface receptors CTLA-4, GITR, and ICOS are not changed among Foxp3⁺ cells in response to PSA treatment (Fig. 2C and Fig. S4). Taken together, these data reveal that PSA activates inducible Foxp3⁺ Tregs and identifies a “PSA-specific” gene expression program within Foxp3⁺ Treg cells.

We have previously reported that colonization of germ-free animals with PSA-producing bacteria induces production of the Th1 cytokine IFN-γ among splenic CD4⁺ T cells (21). To further investigate the lineage differentiation of immune cells directed by PSA, we examined animals for IFN-γ expression among Foxp3⁺ T cells. Intriguingly, IFN-γ expression in the MLNs is found exclusively in the Foxp3⁻ subset and is not affected by microbial colonization (Fig. S5). Furthermore, splenic CD4⁺ T cells produce IFN-γ only from Foxp3⁻/IL-10⁻ T cells that is dependent on PSA expression (Fig. S6). These results demonstrate that there is a compartmental difference in the ability of PSA to induce a Th1 profile in the spleen, although promoting a tolerogenic immune environment in the gut consisting of CD4⁺Foxp3⁺IFN-γ⁻ Treg cells.

PSA Activity Requires TLR2 Signaling.

Many microbial products are sensed by pattern recognition receptors, such as TLRs. Although historically believed to induce inflammation, a series of studies now show that TLR signaling can also promote anti-inflammatory responses (reviewed in ref. 22). PSA has been shown to coordinate cytokine production from innate immune cells through TLR2 signaling (23); however, a role for TLR2 in Treg development remains uncertain. To understand the mechanism by which PSA promotes Tregs, TLR2-deficient animals were orally treated with PSA and analyzed for CD4⁺Foxp3⁺ T-cell development. In contrast to the Treg expansion seen in WT animals (see Fig. 2A), we observed no difference in the percentage of CD4⁺Foxp3⁺ T cells in TLR2^−/− mice treated with PSA compared with PBS controls (Fig. 2D). Additionally, induction of IL-10 by Tregs (CD4⁺CD25⁺) in response to PSA is lost in the absence of TLR2 expression (Fig. 2E). Our findings are entirely consistent with reports that TLR2^−/− mice have defects in Foxp3⁺ Treg cells (24, 25). Although further work is needed to fully understand how innate immune signaling contributes to Treg lineage differentiation, PSA-mediated Treg development is a TLR2-dependent mechanism.

PSA Expands Functional Foxp3⁺ Tregs During Protection from Experimental Colitis.

Activation of innate and adaptive immune cells and secretion of inflammatory cytokines are not found under steady state conditions within the intestine. We next wished to examine Foxp3⁺ Treg cell development during intestinal disease. TNBS (2,4,6-trinitrobenzene sulfonic acid) treatment of animals results in gut inflammation, which activates T-cell responses; animals lose a significant amount of weight and display marked thickening of the colon, with lymphocyte infiltration and concomitant epithelial hyperplasia (26). As previously reported, disease was not evident in TNBS-treated animals that were treated with purified PSA (15). Vehicle treated (PBS; −TNBS) and PBS treated TNBS animals (TNBS+PBS) had a similar percentage of Foxp3⁺ Treg cells within the CD4⁺CD25⁺ T cells of the MLN (Fig. 3A). Consistent with PSA's anti-inflammatory properties, mice given PSA reproducibly had a 5 to 10% increase in the percentage of Foxp3⁺ cells within the CD4⁺CD25⁺ compartment of the MLNs (Fig. 3A). Additionally, the absolute number of CD4⁺CD25⁺Foxp3⁺ cells in the MLNs was significantly higher in PSA-treated mice compared with PBS or noncolitic animals (Fig. 3B). PSA expansion of the Foxp3⁺ Treg population is specific, as the percentage of B cells in the MLNs did not differ between PBS and PSA fed mice (Fig. S7). Consistent with an increase in the percentage of Foxp3⁺ cells in PSA-treated mice, there was an increase in the expression of the foxp3 transcript in MLNs (Fig. 3C). Furthermore, we also found that the Foxp3 expression was increased on a per cell basis in CD4⁺CD25⁺ cells during PSA-mediated protection from colitis (Fig. S8), demonstrating that PSA up-regulates proportional and cell-intrinsic Foxp3 expression. The suppressive capacity of Tregs during PSA-mediated protection from intestinal inflammation was determined by in vitro suppression assays. As expected, proliferation was partially suppressed (proliferation of effector cells in the absence of Tregs was > than 90%) when Tregs from vehicle (PBS; −TNBS) or PBS-treated colitic mice (TNBS+PBS) were added to the culture (Fig. 3D). Notably however, Tregs isolated from the MLNs of PSA fed mice (TNBS+PSA) suppressed T-cell proliferation to a significantly higher level than cells from untreated animals (43.5% proliferating responder cells vs. 63.5% at a 1:2 Treg:Teff ratio), demonstrating Tregs from animals protected from colitis by PSA have increased functional suppressive activity.

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Fig. 3.

PSA expands functional Foxp3⁺ Tregs during protection from experimental colitis. (A) BALB/c mice were treated with PSA or PBS during TNBS induced colitis and analyzed for the percentage of CD25⁺Foxp3⁺ cells within the CD4⁺ population of the MLN. These data are representative of three independent trials with four mice per group. (B) Mice were treated as in A, MLN cells were counted, and absolute numbers of CD4⁺CD25⁺Foxp3⁺ cells determined (×10³). Numbers represent the average of four mice with error bars indicting SD, and are representative of three independent trials. (C) RNA was extracted from the MLNs of mice treated with PSA and foxp3 transcript expression is shown, normalized to β-actin expression in the total lymph node. Numbers represent the average of four mice, with error bars indicating SD, and are representative of three independent trials. (D) CD4⁺CD25⁺ cells were purified from the MLNs of colitic PBS or PSA-treated mice and incubated with CFSE-pulsed CD4⁺CD25⁻ responder cells in an in vitro suppression assay. Numbers indicate the percentage of cells undergoing at least one cellular division at two different ratios of Teffs and Tregs. These data are representative of two independent trials. (E) C57BL/6 WT or TLR2^−/− animals were gavaged with purified PSA (or PBS) before TNBS administration. Colons were fixed, sectioned, and H&E stained. Representative histological sections are shown. (F) Colitis scores show that PSA protects WT but not TLR2^−/− animals from experimental colitis. Each symbol represents a separate animal, and data are from two independent trails. *P < 0.05. NS, not significant. (G) RNA was extracted from the MLNs of indicated mice either treated with PBS or PSA, and the expression levels of IL-10 and IL-17A were assayed by qRT-PCR. Relative units are represented as transcript levels relative to expression of a housekeeping gene (L32). Results are representative of three independent trials with four mice per group; error bars indicate SD. (H) Percentages of CD4⁺CD25⁺Foxp3⁺ T cells in the MLNs of TLR2^−/− mice treated with PBS or PSA during TNBS induced colitis. Plots are gated on CD4⁺ T cells. Results are representative of three independent trials with four mice per group.

PSA Protection from Intestinal Inflammation Requires TLR2.

Multiple studies have implicated TLR signaling by innate immune cells during inflammatory disease (27). Furthermore, TLR2 signaling has been shown to have significant effects on Treg expansion and function (24, 25). We are therefore in a unique position to examine how TLR2 responses to a natural microbial ligand affect intestinal inflammatory disease. WT or TLR2^−/− animals were orally treated with PSA or PBS before induction of TNBS colitis. WT animals had ulcerative lesions, lymphocyte infiltration, and thickening of the intestinal mucosa following administration of TNBS (PBS treatment); WT animals treated with PSA were protected from disease (Fig. 3E). In contrast, TLR2^−/− mice treated with PSA had marked inflammation including thickening of the mucosa, infiltrating leukocytes, and crypt loss (Fig. 3E). Scoring by a blinded pathologist revealed that TLR2^−/− animals are resistant to PSA-mediated protection, as no differences in colitis scores were observed for TLR2-deficient animals (Fig. 3F). As previously reported, WT animals were protected from colitis by PSA (Fig. 3F) (15). We next determined the impact of TLR2 signaling on cytokine induction by PSA in animals. IL-10 expression was induced in response to PSA during protection from colitis in WT animals (Fig. 3G); however, in the absence of TLR2 signaling, IL-10 was no longer up-regulated in PSA-treated animals. Suppression of the proinflammatory cytokine IL-17 by PSA was evident in WT animals; intriguingly, IL-17 levels were actually increased in TLR2^−/− mice given PSA compared with PBS consistent with no protection from colitis in the absence of TLR2 (Fig. 3G). To examine a role for TLR2 in coordination of Treg responses by PSA, we determined the proportions of CD4⁺Foxp3⁺ Tregs in TLR2^−/− animals treated with PSA during TNBS colitis. As shown in Fig. 3H, there are no differences in the percentage of Tregs in TLR2^−/− animals in response to PSA. These results demonstrate that TLR2 signaling by PSA is required for Treg induction and provide a molecular mechanism for protection from experimental colitis.

In Vitro Suppression Assay.

Either CD4⁺CD25⁺ or CD4⁺Foxp3⁺ cells were used as a source of Tregs. CD4⁺CD25⁻ cells were pulsed with 1 μL of a 5 μM CFSE stock for 10 min at 37 °C. CFSE labeled cells were washed in PBS twice and immediately used. 1 × 10⁵ mitomycin C (Sigma)-treated CD4-depleted splenocytes were mixed with CFSE-pulsed CD4⁺CD25⁻ (or Foxp3⁻) responder cells. Indicated dilutions of CD4⁺CD25⁺ Treg cells were titrated in and 1 μg/mL of anti-CD3 was added in a round bottom 96-well plate. Cultures were incubated for 3 to 5 d and then analyzed by flow cytometry.

Experimental Colitis.

Eight-week-old BALB/c mice were purchased from Taconic Farms. For prophylactic studies, animals were pretreated with 50 μg of PSA every other day for 6 d before administration of TNBS and every other day after TNBS induction until necropsy. For curing studies, animals were treated with a single dose of 50 μg of PSA 1 or 2 d following TNBS and every other day until necropsy. See SI Materials and Methods for details.

We thank members of the Mazmanian laboratory for their critical review of the manuscript and Dr. Talal Chatila (University of California Los Angeles) for the kind gift of the Foxp3-GFP mice, Rochelle A. Diamond for help with cell sorting, and Dr. Gregory W. Lawson (University of California Los Angeles) for providing expert pathology analysis. J.L.R. is a Merck Fellow of the Jane Coffin Child's Memorial Fund. S.K.M. is a Searle Scholar. Work in the laboratory of the authors is supported by funding from the National Institutes of Health (DK 078938), the Damon Runyon Cancer Research Foundation, and the Crohn's and Colitis Foundation of America (to S.K.M.).