Induction of Colonic Regulatory T Cells by Mesalamine by Activating the Aryl Hydrocarbon Receptor

Abstract

Background & aims: Mesalamine is a first-line drug for treatment of inflammatory bowel diseases (IBD). However, its mechanisms are not fully understood. CD4⁺ Foxp3⁺ regulatory T cells (Tregs) play a potential role in suppressing IBD. This study determined whether the anti-inflammatory activity of mesalamine is related to Treg induction in the colon.

Methods: We examined the frequencies of Tregs in the colons of wild-type mice, mice deficient for aryl hydrocarbon receptor (AhR^-/- mice), and bone marrow-chimeric mice lacking AhR in hematopoietic cells (BM-AhR^-/- mice), following oral treatment with mesalamine. We also examined the effects of mesalamine on transforming growth factor (TGF)-β expression in the colon.

Results: Treatment of wild-type mice with mesalamine increased the accumulation of Tregs in the colon and up-regulated the AhR target gene Cyp1A1, but this effect was not observed in AhR^-/- or BM-AhR^-/- mice. In addition, mesalamine promoted in vitro differentiation of naive T cells to Tregs, concomitant with AhR activation. Mice treated with mesalamine exhibited increased levels of the active form of TGF-β in the colon in an AhR-dependent manner and blockade of TGF-β signaling suppressed induction of Tregs by mesalamine in the colon. Furthermore, mice pretreated with mesalamine acquired resistance to dextran sodium sulfate-induced colitis.

Conclusions: We propose a novel anti-inflammatory mechanism of mesalamine for colitis: induction of Tregs in the colon via the AhR pathway, followed by TGF-β activation.

Keywords: AhR, aryl hydrocarbon receptor; Aryl Hydrocarbon Receptor; BM, bone marrow; DSS, dextran sodium sulfate; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; LPL, lamina propria lymphocytes; MLN, mesenteric lymph nodes; Mesalamine; PBS, phosphate-buffered saline; Q-PCR, quantitative polymerase chain reaction; RPMI, Roswell Park Memorial Institute; Regulatory T Cells; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TGF, transforming growth factor; TGF-β; TNF, tumor necrosis factor; Tregs, regulatory T cells; WT, wild-type; XRE, xenobiotic responsive element; mAb, monoclonal antibody.

Graphical abstract

Figure 1

Mesalamine increases the accumulation of Tregs in the LP of the colon. (A–E) Male 6-week-old WT mice were orally treated with mesalamine for 2 weeks. (A) Representative plots (left) and quantitative data (right) of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the colon LP. n = 4 mice per group. (B) Percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the LP of small intestine (SI) (n = 3 mice per group), MLNs (n = 4 mice per group), and spleen (n = 4 mice per group). (C) Foxp3 mRNA levels in the colon (n = 8 mice per group), SI (n = 8 mice per group), MLNs (n = 4 mice per group), and spleen (n = 4 mice per group). (D) Representative images (left) and the numbers of CD4⁺Foxp3⁺ cells in square millimeter in the lamina propria (right). Scale bar, 100 μm. Arrowheads indicate CD4⁺Foxp3⁺ cells (n = 5 mice per group). (E) The percentages of Ki67⁺ cells within the CD3⁺CD4⁺ Foxp3⁺ cell population isolated from the colon LP (n = 4 mice per group). (F) Female 6-week-old WT mice (left, n = 5 mice per group) or male aged (30 ± 4 week-old) mice (right, n = 5 mice per group) were orally treated with mesalamine for 2 weeks. Percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the colon LP. Error bars represent mean ± SEM. *P < .05, ^∗∗P < .01. 5-ASA, mesalamine.

Figure 2

Mesalamine does not increase the accumulation of Th1 cells and Th17 cells in the LP of the colon. Wild-type mice were orally treated with 50 mg/kg of mesalamine for 2 weeks. (A) Percentages of IFN-γ⁺ cells (n = 8 mice per group) or IL17⁺ cells (n = 4 mice per group) within the CD3⁺CD4⁺ cell population isolated from the colon LP. (B) T-bet and RorC mRNA levels in the colon (n = 4 mice per group). Error bars represent mean ± SEM. 5-ASA, mesalamine.

Figure 3

Treatment of mice with mesalamine for at least 3 consecutive days can promote the accumulation of Tregs in the colon. (A–C) WT mice were orally treated with 50 mg/kg of mesalamine for 1 (n = 5 mice per group) or 3 days (n = 4 mice per group). The percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from colon LP (A) and Foxp3 (B) or Cyp1A1 (C) mRNA levels in the colons. (D) WT mice were orally treated with 5 mg/kg of mesalamine for 2 weeks, and the percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the colon LP are shown (n = 4 mice per group). Error bars represent mean ± SEM. *P < .05, **P < .01. 5-ASA, mesalamine.

Figure 4

Mesalamine activates the AhR pathway, thereby inducing colon LP Tregs. (A) Cyp1A1 mRNA levels in the colon, small intestine (SI), MLNs, and liver isolated from WT mice orally treated with 50 mg/kg of mesalamine for 2 weeks (n = 8 mice per group). (B) Cyp1A1 and Foxp3 mRNA levels of the CD4⁺ cells in the LP of the colon isolated from WT mice orally treated with 50 mg/kg of mesalamine for 3 days (n = 5 mice per group). (C) Kinetics of serum SEAP in AhR-reporter DRESSA mice following orally treated once with 5 μg/kg TCDD (n = 3 mice per group) or orally treated with 50 mg/kg mesalamine for 4 days (n = 8 mice per group). (D, E) Percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population in the colon LP (D) (representative plots [left] and quantitative data [right]) and Foxp3 mRNA levels in the colon (E) isolated from AhR^-/- mice orally treated with mesalamine for 2 weeks (n = 4 mice per group). (F) AhR mRNA levels in the MLNs from WT mice reconstituted with WT mice BM (BM-AhR^+/+) or with AhR^-/- mice BM (BM-AhR^-/-) (n = 5 mice per group). (G) The percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell populations in the colon LP isolated from WT mice reconstituted with WT mice BM (BM-AhR^+/+) or WT mice reconstituted with AhR^-/- mice BM (BM- AhR^-/-) orally treated with mesalamine for 2 weeks (n = 5 mice per group). Representative plots (left) and quantitative data (right). Error bars represent mean ± SEM. *P < .05, **P < .01. 5-ASA, mesalamine.

Figure 5

TCDD, but not aspirin induces Tregs in the LP of the colon, concomitant with AhR activation. (A, B) WT mice were orally treated once with 5 μg/kg of TCDD. Three days later, the percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the colon LP of mice were analyzed by flow cytometry. (A) Representative plots (left) and quantitative data (right). (B) Cyp1A1 mRNA levels in the colon. (C, D) WT mice were orally treated with aspirin added to drinking water for 2 weeks, the percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the colon LP of mice were analyzed by flow cytometry. (C) Representative plots (left) and quantitative data (right). (D) Cyp1A1 mRNA levels in the colon. Error bars represent mean ± SEM, n = 4 mice per group. *P < .05, **P < .01.

Figure 6

Mesalamine promotes Treg differentiation, concomitant with AhR activation. (A) Naive CD4⁺ T cells isolated from the spleen of WT mice or AhR^-/- mice were cultured in Treg differentiation conditions with the indicated concentrations of mesalamine for 3 days, and the percentages of Foxp3⁺ cells within CD4⁺ cells were analyzed by flow cytometry (n = 3). (B) Naive CD4⁺ T cells isolated from the spleen in WT mice were stimulated in Th0, Th1, or Th17 differentiation conditions with the indicated concentrations of mesalamine for 3 days, and the supernatants of the cultures were collected. The concentrations of IFN-γ or IL17A were measured by ELISA (n = 2). (C) Chromatin immunoprecipitation assay analysis of the interaction of AHR with the nonconserved AhR binding site (NCABS) and conserved AhR binding site (CABS) in Foxp3 or XRE sequence of Cyp1A1 in naive CD4⁺ T cells under Treg differentiation conditions supplemented with PBS, mesalamine (1 μM), or TCDD (1 nM) (n = 3). Error bars represent mean ± SEM. *P < .05, **P < .01. Similar results were obtained from at least 3 independent experiments. 5-ASA, mesalamine.

Figure 7

Mesalamine increases the level of the active form of TGF-β1, which is necessary for Treg induction. (A) Levels of the active form of TGF-β1 (acid-) or total TGF-β1 (acid+) in colons isolated from WT mice orally treated with mesalamine for 2 weeks (n = 5 mice per group). (B) Colon tissue samples (1.6 mg) from WT mice orally treated with mesalamine for 2 weeks were added into the culture of TGF-β reporter cells (MFB-F11) for 24 hours with or without HTS466284 (a chemical inhibitor of TGF-β signaling); then, SEAP activities of the supernatants were measured. 10 ng/mL human TGF-β1 was used as a positive control (n = 5). (C) TGF-β1 mRNA levels in the colon isolated from WT mice orally treated with mesalamine for 2 weeks (n = 8 mice per group). (D) Levels of active TGF-β1 (acid-) or of total TGF-β1 (acid+) in the colon isolated from AhR^-/- mice treated orally with mesalamine for 2 weeks (n = 4 mice per group). (E) Levels of active TGF-β1 (acid-) or of total TGF-β1 (acid+) in the colon isolated from bone marrow chimeric mice (BM-AhR^+/+ mice and BM-AhR^-/- mice) treated orally with mesalamine for 2 weeks (n = 5 mice per group). (F, G) TSP-1 and Integrin αV mRNA levels in the colon isolated from WT mice or AhR^-/- mice orally treated with mesalamine for 2 weeks (n = 8 mice per group). (H) WT mice were orally treated with mesalamine with or without GW788388, an inhibitor of TGF-β type I receptor kinase, in drinking water for 2 weeks, and the percentages of Foxp3⁺ cells within the CD3⁺CD4⁺ cell population isolated from the colon LP were determined (n = 8 mice per group). Error bars represent mean ± SEM. *P < .05, **P < .01. 5-ASA, mesalamine.

Figure 8

Mesalamine increases fecal IgA production. (A) WT (n = 6 mice per group) and AhR^-/- mice (n = 4 mice per group), or (B) bone marrow chimeric mice (BM-AhR^+/+ mice and BM-AhR^-/- mice) (n = 10 mice per group) were orally treated with 50 mg/kg of mesalamine for 2 weeks. Concentrations of IgA of stool samples were measured by ELISA. Error bars represent mean ± SEM. *P < .05. 5-ASA, mesalamine.

Figure 9

Mice pretreated with mesalamine acquire the resistance to DSS-induced colitis. (A) Experimental protocol. WT or AhR^-/- mice (see Figure 10) were orally treated with mesalamine for 2 weeks; 24 hours after the last administration of mesalamine, the mice were treated with 3% DSS to induce colitis. (B) Body weight changes in mice after administration of DSS (n = 7 mice per group). (C) Stool score changes in mice after administration of DSS (n = 7 mice per group). (D) Colon length in mice after administration of DSS (n = 7 mice per group). (E) Colon histopathology. Representative pictures (left) and quantitative data (right). Scale bar indicates 100 μm (n = 3 per group). (F) TNF-α, IL1β, IL6, MPO, and HO-1 mRNA levels in the colon after administration of DSS were analyzed by Q-PCR (n = 7 mice per group). Error bars represent mean ± SEM. *P < .05, **P < .01. 5-ASA, mesalamine.

Figure 10

AhR^-/-mice pretreated with mesalamine do not acquire the resistance to DSS-induced colitis. AhR^-/- mice were orally treated with mesalamine for 2 weeks; 24 hours after the last administration of mesalamine, the mice were treated with 3% DSS to induce colitis. (A) Body weight changes in mice after administration of DSS (n = 5 mice per group). (B) Stool score changes in mice after administration of DSS (n = 5 mice per group). (C) Colon length in mice after administration of DSS (n = 5 per group). (D) TNF-α, IL1β, IL6, MPO, and HO-1 mRNA levels in the colon after administration of DSS were analyzed by Q-PCR (n = 5 per group). Error bars represent mean ± SEM. 5-ASA, mesalamine.

Figure 11

Proposed model. 5-ASA, mesalamine.