Induction of colonic regulatory T cells by indigenous Clostridium species

Abstract

CD4(+) T regulatory cells (T(regs)), which express the Foxp3 transcription factor, play a critical role in the maintenance of immune homeostasis. Here, we show that in mice, T(regs) were most abundant in the colonic mucosa. The spore-forming component of indigenous intestinal microbiota, particularly clusters IV and XIVa of the genus Clostridium, promoted T(reg) cell accumulation. Colonization of mice by a defined mix of Clostridium strains provided an environment rich in transforming growth factor-β and affected Foxp3(+) T(reg) number and function in the colon. Oral inoculation of Clostridium during the early life of conventionally reared mice resulted in resistance to colitis and systemic immunoglobulin E responses in adult mice, suggesting a new therapeutic approach to autoimmunity and allergy.

Fig. 1

Indigenous intestinal bacteria-dependent accumulation of colonic T_regs. (A) The percentage of Foxp3⁺ cells within the CD4⁺ cell population isolated from iLNs or LP of colon or SI of SPF BALB/c mice at the indicated age was analyzed by flow cytometry. (B) Lymphocytes from SI, colon, and iLN of 8-week-old BALB/c, IQI and C57BL/6 (B6) GF, and SPF mice were analyzed for CD4 and Foxp3 expression. (C) GF IQI mice were conventionalized (Conv) by oral administration of the fecal suspension from B6 SPF mice purchased from Jackson Laboratory. Colonic LP lymphocytes were isolated 3 weeks later and analyzed for Foxp3 expression. (D) Four-week-old SPF B6 mice were treated with polymyxin B (poly B) or vancomycin (Vanco) for 4 weeks and analyzed for the percentage of Foxp3⁺ cells within the CD4⁺ cell population. (E) GF mice were gavaged with chloroform-treated feces from SPF mice (+chloro) and analyzed for the percentage of Foxp3⁺ cells within the CD4⁺ cell population. Each circle in (B) to (E) represents an individual mouse, and error bars indicate the SD. Data were obtained from more than two independent experiments with similar results (n ≥ 4 mice per group). *P < 0.01; **P < 0.001, unpaired t test.

Fig. 2

Clostridia induce T_reg accumulation in colonic LP. (A) GF BALB/c or IQI mice were colonized with segmented filamentous bacteria (SFB), 16 strains of Bacteroides (Bactero.), 3 strains of Lactobacillus (Lacto.), or 46 strains of Clostridium (Clost.) for 3 weeks. The percentage of Foxp3⁺ cells within the CD4⁺ cell population in the colon and SI of individual mice was analyzed by flow cytometry (n ≥ 5 mice per group). (B) Electron micrograph showing the proximal colon of Clost.-colonized B6 mice. (C) Lymphocytes from the colonic LP of indicated mice were analyzed for the expression of CD4, Foxp3, and Helios by flow cytometry. The percentage of Helios⁻ cells within Foxp3⁺CD4⁺ lymphocytes is shown (n = 4 mice per group). (D and E) Whole colons (D) or IECs (E) from GF, Lacto.-, or Clost.-colonized mice were cultured for 24 hours. The concentration of TGF-β1 in the supernatant was determined by enzyme-linked immunosorbent assay (ELISA). (F) Splenic CD4⁺ T cells were cultured with antibodies against CD3 and CD28 and 50% conditioned medium from cultured IECs isolated from GF or Clost.-colonized mice in the presence or absence of anti–TGF-β. After 5 days, T cells were collected and assayed for Foxp3 expression by real-time reverse transcription–polymerase chain reaction. Each circle represents a mouse or a sample, and error bars indicate the SD (n ≥ 3 per group). *P < 0.02; **P < 0.001, unpaired t test. Data are representative of at least two independent experiments with similar results.

Fig. 3

Clostridia induce IL-10–expressing T_regs in the colon. (A to D) Splenic and colonic LP lymphocytes from SPF, GF, or Clostridium-colonized Il10^Venus mice were analyzed for expression of CD4, Foxp3, Venus, and CTLA4 by flow cytometry. Representative dot-plots and histograms are shown in (A) and (D), and the percentage and absolute number of Venus⁺ cells within the CD4⁺Foxp3⁺ cell population in individual mice are shown in (B) and (C). Each symbol represents a mouse, and error bars indicate the SD. Data are representative of at least two independent experiments with similar results (n ≥ 5 mice per group). **P < 0.001 versus GF, unpaired t test. (E) Purified CD4⁺ Venus⁺ cells from the colonic LP of SPF or Clostridium-colonized Il10^Venus mice were cultured with splenic CD4⁺CD25⁻ T cells (T_eff) in the presence of irradiated splenic CD11c⁺ cells and anti-CD3 for 72 hours at the indicated ratios. Proliferation was measured by [³H]thymidine uptake. Data represent the mean ± SD of triplicate cultures.

Fig. 4

Effect of Clostridium abundance on regulation of colitis and IgE response. (A) Two-week-old SPF mice were orally inoculated with Clostridium (SPF+Clost.) or untreated (SPF). After 6 weeks, the percentage of Foxp3⁺ cells within CD4⁺ cells in colonic LP was analyzed. The experiment was repeated more than three times with similar results. Each circle represents a mouse, and error bars represent the SD (n = 7 mice per group). (B to D) SPF and SPF+Clost. mice were treated with 2% DSS and monitored and scored for body weight loss, stool consistency, and bleeding for 6 days (n = 7 mice per group). (B) On day 6, the colons were collected (C) and dissected for histological analysis by hematoxylin and eosin staining (D). (E and F) SPF and SPF+Clost. mice were immunized with OVA + alum twice at a 2-week interval. Sera were collected and examined for OVA-specific IgE levels by ELISA (E). Splenocytes were collected from mice in each group and examined for IL-4 and IL-10 production upon restimulation with OVA in vitro (F). Error bars represent the SD (n = 5 mice per group). *P < 0.02; **P < 0.001, unpaired t test.