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

Abstract

Regulatory T cells (Tregs) that express the transcription factor Foxp3 are critical for regulating intestinal inflammation. Candidate microbe approaches have identified bacterial species and strain-specific molecules that can affect intestinal immune responses, including species that modulate Treg responses. Because neither all humans nor mice harbor the same bacterial strains, we posited that more prevalent factors exist that regulate the number and function of colonic Tregs. We determined that short-chain fatty acids, gut microbiota-derived bacterial fermentation products, regulate the size and function of the colonic Treg pool and protect against colitis in a Ffar2-dependent manner in mice. Our study reveals that a class of abundant microbial metabolites underlies adaptive immune microbiota coadaptation and promotes colonic homeostasis and health.

Fig. 1. SCFA restore colonic Treg populations and function in germ-free mice

(A) Colonic lamina propria (LP) lymphocytes were isolated and stained for CD4 and Foxp3. Left panel: Representative dot plots and percentage of CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population from SPF, GF, and GF mice treated with propionate (P), acetate (A), butyrate (B), or the SCFA mix in the drinking water. Right panel: Numbers of Foxp3⁺ Tregs for the left panel. (B) cTregs were isolated from in vivo propionate-treated GF mice, sorted by CD4, CD127, and CD25, and examined ex vivo for expression of Foxp3 and IL-10 by RTqPCR. (C) cTregs were isolated from GF mice and purified as in 1B, cultured for 24 hrs in the presence or absence of 0.1 mM propionate and examined for expression of Foxp3, TGFβ, and IL-10 by RTqPCR and IL-10 protein production by ELISA. For panel A, symbols represent data from individual mice. Horizontal lines show the mean and error bars the SD. For panels B and C, each symbol or bar represents pooled cTregs from 3-5 mice. All data shown are representative of at least 3 independent experiments. A Kruskal-Wallis test with a Dunn's post hoc test was performed in panel A, *** P <0.001 and * P <0.05. A Mann-Whitney U test was performed in panels B and C.

Fig. 2. SCFA augment colonic Treg population size and function in SPF mice

(A) SPF Foxp3^YFP-Cre mice were treated with water alone (-) or P, A, B, or the mix. Colonic LP lymphocytes were isolated and stained for CD4 and IL-10. Upper panel: Representative dot plots and percentage of CD4⁺ Foxp3-YFP⁺ within the CD45⁺CD3⁺ population. Lower panel: Representative dot plots and percentage of the CD4⁺Foxp3⁺IL-10⁺population. (B) Cell numbers for the data in (A) upper panel. (C) Cell numbers for the data in (A) lower panel. Symbols in B and C represent data from individual mice and represent 4 independent experiments. (D) cTregs were co-cultured with splenic effector T cells (Teff) and P, A, B, or media (sodium and pH matched) for 96 hrs. Percent suppression (y-axis), Treg:Teff ratios (x-axis). Symbols represent mean values and error bars SD for four independent experiments. * P < 0.01. (E) cTregs were isolated from the LP of in vivo propionate treated SPF Foxp3^YFP-Cre mice, sorted for CD4 and YFP, and examined for ex vivo expression of Foxp3 and IL-10. Each symbol represents pooled cTregs from 3-5 mice, horizontal lines show the mean and error bars the SD. Four independent experiments were performed. A Kruskal-Wallis test with a Dunn's post hoc test was performed in panels B, C, D, *** P <0.0001, ** P <0.01, * P <0.05. A Mann-Whitney U test was performed for experiments in panel E and P-values are shown where significant.

Fig 3. Ffar2 mediates SCFA effects on cTregs

(A) Tregs were isolated from the colon, small intestine, spleen and MLN of SPF and GF BALB/c mice, purified as in 1B and Ffar2 expression examined by qPCR. Each symbol represents data from 3-5 individual mice, horizontal lines show the mean and error bars the SD. Data reflect 3-7 independent experiments. (B) Lymphocytes were isolated from the colon, small intestine, spleen, and MLN of SPF Ffar2^−/− and littermate Ffar2^+/+ mice. Cells were stained for CD4, Foxp3, and Ffar2. Left panel depicts a representative flow cytometry histogram comparing colonic Ffar2 expression in Ffar2^−/− vs littermate Ffar2^+/+ mice. Right panel shows the MFI for Ffar2 for Tregs from the indicated sites. Bars show the mean, error bars SD, and data reflect 4 independent experiments. (C) Colonic LP lymphocytes were isolated from Ffar2^−/−and littermate Ffar2^+/+ mice exposed to propionate (P) or water alone and stained for CD4 and Foxp3. Left panel: Representative dot plots with percentage of CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population. Right panel: Foxp3⁺ Treg number for the left panel. Each symbol represents data from individual mice, horizontal lines show the mean and error bars the SD. (D) Ffar2^−/− and littermate Ffar2^+/+ cTregs were co-cultured with splenic Teff cells in media with or without propionate for 96 hrs. Percent suppression (y-axis) and Treg:Teff ratios (x-axis). Symbols represent the mean of 3 independent experiments and error bars show the SD. (E) cTregs were isolated from the LP of Ffar2^−/− and littermate Ffar2^+/+ mice, purified as in 1B, cultured in the presence of 0.1 mM propionate or media (pH and sodium matched) for 24hrs and examined for expression of HDAC 1,2,6,7 and 9 by RTqPCR. Bars show the mean and error bars the SD of 3 independent experiments (F) Whole cell extracts were generated from cTregs isolated from the LP of Ffar2^−/− and littermate Ffar2^+/+ mice, purified as in 1B, and cultured in the presence of 0.1 mM propionate or media (pH and sodium matched) for 24hrs. Samples were analyzed by western blotting for histone acetylation by examining levels of acetylated histone (H3K9), total histone levels were used as a loading control. The western blot shown is representative of two independent experiments with cTregs cell lysates pooled from 10-12 mice per group. A bar graph of densitometry ratios of acetylated Histone H3:total Histone H3 is shown. Bars represent the mean and error bars the SD. A Kruskal-Wallis test with a Dunn's post hoc test was performed for panels A and D, *** P < 0.001. The Mann-Whitney U test was performed for panels C and E. The student's t-test was performed for panel B and F.

Fig. 4. SCFA exposure ameliorates T cell transfer colitis in a Treg-intrinsic, Ffar2-dependent manner

BALB/c Rag2^−/− mice were injected with CD4⁺CD45RB^hiCD25^lo naive T cells alone or in combination with Tregs. Following injection, mice received propionate, SCFA mix, or pH and sodium-matched drinking water. (A) Weekly percentage body weight change is shown across the experimental groups from experimental d0-d42. Symbols show the mean and error bars the SD. Data reflect three independent experiments. Colonic LP lymphocytes were isolated and stained for CD4 and Foxp3 and (B) percentage and (C) number of CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population are shown. Symbols represent data from individual mice, horizontal lines show the mean and error bars the SD. (D-F) C57BL/6 Rag2^−/− mice were injected with CD4⁺CD45RB^hiCD25^lo naive T cells alone or in combination with Ffar2^+/+ or Ffar2^−/− Tregs. Following injection mice received propionate or pH and sodium-matched drinking water. (D) Histologic colitis score is shown along the y-axis, the treatment group and experimental conditions are shown along the x-axis. Colonic LP lymphocytes were isolated and (E) percentage and (F) number of CD4⁺Foxp3⁺ within the CD45⁺CD3⁺ population are shown. Symbols represent data from individual mice. Horizontal lines show the mean and error bars the SD. Panels D-F represent data from 2 independent experiments. The Kruskal-Wallis test with a Dunn's post hoc test was performed for panels A-F. ** P <0.01, * P <0.05.