The microbial metabolites, short chain fatty acids, regulate colonic Treg cell homeostasis

The intestinal immune system has co-evolved with the gut microbiota for the maintenance of intestinal health (1). Disruption of this homeostasis leads to intestinal inflammation and disease (2, 3). Colonic regulatory T cells (cTregs) expressing the transcription factor Foxp3 are critical for limiting intestinal inflammation and depend on microbiota-derived signals for proper development and function (4-7). Bacteroides fragilis and clostridial species induce Treg responses (6, 7); however, how the gut microbiota affect cTreg responses across mammalian hosts remains unclear. While polysaccharide A from B. fragilis modulates Treg responses (6), such effects are also likely mediated through more common factor(s) produced by many bacterial genera.

Humans and mice rely on bacteria to breakdown undigestible dietary components, such as fibers (8). Short chain fatty acids (SCFA) are bacterial fermentation products and range in concentration between 50-100mM in the colonic lumen (9). We examined SCFA concentrations in specific pathogen-free (SPF), gnotobiotic Altered Schaedler Flora (ASF)-colonized mice, and germ-free (GF) mice and found that GF mice had reduced concentrations of the three most abundant luminal SCFA, acetic acid, propionic acid and butyric acid (Table S1), as previously reported (10). The decrease of these SCFA in GF mice suggested that SCFA may contribute to their immune defects, specifically reduced cTreg numbers. We provided SCFA in the drinking water (150mM) to GF mice for three weeks and found that SCFA individually or in combination (SCFA mix) increased cTreg frequency and number (Fig. 1A) but did not increase the number or frequency of splenic, mesenteric lymph node (MLN), or thymic Tregs (fig. S1). These effects coincided with increased luminal SCFA (Table S1). SCFA increased CD4⁺ T cell frequency and number (fig. S2) but did not alter colonic Th1 or Th17 cell numbers significantly (fig. S3).

Microbiota-induced cTreg development is associated with increases in de novo generation of inducible Tregs (iTregs) and not in Tregs of thymic origin (nTregs) (7). These populations can be distinguished by expression of Helios, which in vivo is restricted to nTregs (11). We found that Helios⁺ Treg frequency was similar between GF and SCFA-treated GF mice (fig. S4) but lower in SPF mice. SCFA-treatment increased Helios⁺ Treg numbers in GF mice, indicating that Tregs already present in the colonic lamina propria (cLP) were expanding.

To test if SCFA could affect cTregs in a GF setting, we isolated cTregs from GF mice treated with propionate in vivo for three weeks and examined expression of Foxp3 and interleukin (IL)-10, a key cytokine in Treg-mediated suppression. We also isolated cTregs from GF mice and stimulated them in vitro with propionate. Both treatments significantly increased Foxp3 and IL-10 expression (Fig. 1B, C). In vitro treatment increased IL-10 production but not transforming growth factor-β (TGF-β), a Treg-mediated suppression factor, suggesting that SCFA specifically induce Foxp3⁺ IL-10-producing Tregs (Fig. 1B, C).

The antibiotic vancomycin, which targets Gram-positive bacteria and disrupts the gut microbiota (12), reduces cTregs to similar levels as observed in GF mice (7) (fig. S5). However, when SPF mice were treated with a combination of vancomycin and SCFA, the reduction in cTregs was completely restored (fig. S5). Collectively, these results suggest that SCFA, play a role in cTreg homeostasis.

We questioned whether SCFA would augment cTregs in SPF mice. SCFA treatment of SPF mice increased Foxp3⁺ and Foxp3⁺IL-10⁺ cTreg frequency and number (Fig. 2A-C) but not Foxp3⁺TGFβ⁺ cTregs (fig. S6). We did not observe changes with SCFA treatment in small intestinal Treg numbers (fig. S7). Neither colonic Th17 and Th1 nor MLN and splenic Treg frequency and number) from SPF mice were affected by SCFA (fig. S8-S11).

These results may explain the benefits of dietary fibers and bacteria, such as clostridia and bifidobacteria (13) that can increase colonic luminal SCFA production and modulate inflammation in mice and humans. We measured SCFA production of species belonging to Clostridium cluster XI (Clostridium bifermentans), XIV (ASF 356 and 492), XVII (C. ramosum), and the bacteroides species, B. fragilis as Clostridium cluster XIV members and B. fragilis affect cTregs (6,7). ASF 356 and 492 and C. ramosum generated more propionate (14-62 vs 0.05-1.1 μmol/10⁵ CFU) and acetate (118-220 vs 0.1-2 μmol/10⁵ CFU) as compared to the other strains (Table S1).

cTregs regulate intestinal homeostasis and control inflammation by limiting proliferation of effector CD4⁺ T cells (Teff). Addition of SCFA to cTreg and Teff co-cultures increased cTreg suppressive capacity (Fig. 2D and fig. S12). In SPF mice, SCFA are taken up by colonic epithelial cells but also diffuse through the epithelium into the lamina propria where they can mediate their effects directly (9, 14). To determine if SCFA directly affect cTregs, we isolated cTregs from SCFA-treated SPF mice. In vivo treatment increased cTreg Foxp3 and IL-10 expression (Fig. 2E). We also isolated cTregs from SPF mice and incubated them with SCFA in vitro. Foxp3 expression as well as IL-10 expression and protein production increased (fig. S13), whereas TGFβ levels remained unchanged (fig. S14). As enhanced suppressive activity (Fig. 2D) could be attributed not only to higher IL-10 levels per cTreg but also to increased cTreg proliferation, we examined cTreg proliferation. cTregs exhibited enhanced proliferation when cultured in the presence of propionate (fig. S15).

In addition, we analyzed the expression patterns of Treg trafficking molecules. Although levels of the chemokine receptor CCR9 or α₄β₇ integrin were not altered in propionate-treated GF and SPF mice, levels of the cTreg homing receptor, GPR15 (15), did increase (fig. S16). Taken together, these data indicate that SCFA may have a beneficial effect in SPF mice through their ability to increase Foxp3⁺IL-10 producing cTregs and cTreg proliferative capacity, as well as alter cTreg GPR15 expression.

Considering that SCFA can influence cTregs directly, we asked if this was a receptor-mediated process. G coupled protein receptor (GPR) 43 (Ffar2 is the gene that encodes GPR43) binds SCFA and through its expression on innate immune cells mediates resolution of inflammatory responses (3, 16). We found that intestinal Tregs and, particularly cTregs, had significantly higher levels of Ffar2 than Tregs isolated from spleen or MLN (Fig. 3A, B), which was largely dependent upon microbiota-derived signals. As a reference, we compared cTreg Ffar2 expression levels to colonic myeloid (CD11b⁺) cells, which are known to express Ffar2 (3), and found that on average CD11b⁺ cells expressed 1.6 fold more Ffar2 than cTregs (fig. S17).

To determine if Ffar2 contributes to cTreg homeostasis, we treated Ffar2^−/− mice and Ffar2^+/+ littermates with propionate, which has the highest affinity for Ffar2 (17), and measured cTreg numbers. Propionate enhanced cTreg frequency and number in Ffar2^+/+ but not Ffar2^−/−mice (Fig. 3C). SCFA-mediated, enhanced cTreg suppressive capacity was also dependent on Ffar2 (Fig. 3D). In addition, propionate enhanced Foxp3 and IL-10 expression and IL-10 protein levels in Ffar2^+/+, but not in Ffar2^−/−, cTregs (fig. S18). Furthermore, we examined whether propionate could restore cTreg populations and numbers in the setting of vancomycin treatment and found that Ffar2 was necessary (fig. S19).

One mechanism by which SCFA mediate their effects is histone deacetylase (HDAC) inhibition (18). The HDAC inhibitor trichostatin-A (TSA) increases Treg gene expression and suppressive capacity (19); and HDAC6 and 9 down-regulate nTreg function (20). Given that SCFA promote cTreg homeostasis in our studies, we hypothesized that SCFA mediate their effect through HDAC inhibition. We treated Ffar2^+/+ and Ffar2^−/− mice with propionate and measured HDAC expression. Propionate treatment of Ffar2^+/+ mice reduced cTreg HDAC6 and HDAC9 (class IIb and IIa, respectively) expression, but did not reduce HDAC1 and HDAC2 (class I) or HDAC7 (class IIa) expression (Fig. 3E). Western blot analysis demonstrated that propionate treatment of cTregs enhanced histone acetylation, all of which were dependent on Ffar2 expression (Fig. 3F). These results suggest that SCFA via Ffar2 may affect cTregs through HDAC inhibition.

To further evaluate the effects of SCFA on cTregs, we utilized the T cell transfer model of colitis (21). In this model, lymphopenic mice (e.g. Rag2^−/−) are injected either with naïve T cells, which results in severe colitis, or injected with naïve T cells in combination with Tregs, which reduces colitis severity. Propionate or SCFA mix-treated mice injected with naïve T cells and Tregs had less severe weight loss, reduced disease score, and lower levels of colonic pro-inflammatory cytokines than mice that received water alone (Fig. 4A and fig. S20). SFCA mix or propionate did not affect colitis severity in mice that received only naïve T cells (fig. S20). The frequency and number of cLP Foxp3⁺ Tregs in mice receiving propionate and SCFA mix increased (Fig. 4B, C). SCFA, however, did not result in conversion of naïve T cells to cTregs (Fig. 4B, C: group 1 vs 2). To evaluate whether these effects were cTreg intrinsic and dependent upon Ffar2, we performed the T cell transfer colitis model using Rag2^−/− recipients, wild type naïve T cells, and Ffar2^+/+ or Ffar2^−/− Tregs with or without propionate in the drinking water (Fig. 4D, fig. S21). The effect of propionate on intestinal inflammation was dependent upon Ffar2 expression in Tregs as indicated by the colitis scores (Fig. 4D). Treg cell populations and cell numbers further substantiated that the propionate effects on cTregs were dependent on Ffar2 (Fig. 4E, F).

Gut microbiota-host immune misadaptation has been implicated in the rising incidence of IBD, other inflammatory diseases, and obesity (22). The western dietary pattern, specifically reduced ingestion of plant-based fibers, may be a critical factor that links the gut microbiome to disease (23). While the gut microbiota composition is divergent across individuals, functional gene profiles are quite similar (24, 25), and alterations to common gut microbial metabolic pathways may affect production of symbiotic factors, such as SCFA, which regulate intestinal adaptive immune responses and promote health.