Metformin-Inducible Small Heterodimer Partner Interacting Leucine Zipper Protein Ameliorates Intestinal Inflammation

Abstract

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. We investigated the mechanism by which SMILE suppressed the development of inflammatory bowel disease (IBD) using a DSS-induced colitis mouse model and peripheral blood mononuclear cells (PBMCs) from patients with ulcerative colitis (UC). Metformin, an antidiabetic drug and an inducer of AMPK, upregulated the level of SMILE in human intestinal epithelial cells and the number of SMILE-expressing cells in colon tissues from DSS-induced colitis mice compared to control mice. Overexpression of SMILE using a DNA vector reduced the severity of DSS-induced colitis and colitis-associated intestinal fibrosis compared to mock vector. Furthermore, SMILE transgenic mice showed ameliorated DSS-induced colitis compared with wild-type mice. The mRNA levels of SMILE and Foxp3 were downregulated and SMILE expression was positively correlated with Foxp3 in PBMCs from patients with UC and an inflamed mucosa. Metformin increased the levels of SMILE, AMPK, and Foxp3 but decreased the number of interleukin (IL)-17-producing T cells among PBMCs from patients with UC. These data suggest that SMILE exerts a therapeutic effect on IBD by modulating IL-17 production.

Keywords: AMPK; Foxp3; IL-17; SMILE; fibrosis.

Figure 1

Metformin upregulates SMILE in DSS-induced colitis. (A) Western blots of STAT3, p-STAT3, and SMILE expression in metformin (5 mM)-treated and untreated HT-29 cells for 48 h, followed by densitometric analysis. (B) Metformin (50 mg/kg) was administered with DSS. Body weight is expressed as a percentage (n = 5). (C) Colon length of mice with DSS-induced colitis on day 11. Macroscopic images of the colon are shown, and the colon length was measured. (D) Hematoxylin and eosin (H&E) staining and IHC with anti-mTOR, -AMPK, and -STAT3 antibodies of colon sections from the DSS vehicle and metformin groups. The score and positive area were determined. Data are means ± SEM of three replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

SMILE overexpression vector reduced the susceptibility of mice to DSS-induced colitis. (A) Vector map showing restriction sites and the SMILE site. (B) Lysates of HT-29 cells transfected with MOCK and SMILE overexpression vectors were analyzed for SMILE protein by western blotting with an anti-SMILE antibody. (C) Changes in body weight (n = 5 per group). (D) Macroscopic images of the colon and colon lengths. (E) Colon tissue stained with an anti-SMILE antibody. Representative images are shown. Bars are percentage positive areas per field. (F) Th17 and Treg cells and (G) IL-17- and IL-10-producing B cells in MLNs by flow cytometry. Data are means ± SEM of two replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

SMILE decreases inflammation and fibrosis in DSS-induced colitis. H&E and IHC staining of the colon of C57BL/6 mice with DSS-induced colitis and administered SMILE vector (100 μg/mL) (n = 5 per group). Representative images are shown. (A) H&E staining and IHC with anti-mTOR, -AMPK, and -STAT3 antibodies. (B) IHC with anti-IL-1β, -IL-6, -IL-17, -α-SMA, and -collagen-1 (Col1) antibodies. Bars are percentages of positive areas per field. Data are means ± SEM of two replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Therapeutic effect of SMILE in transgenic mice. (A) Transgenic mice overexpressing SMILE were generated on a C57BL/6 background. (B) WT and SMILE TG mice were treated with DSS and body weight was measured as a percentage of that at baseline (n = 5 per group). (C) Representative macroscopic images and colon length of WT and SMILE TG mice. (D) MLN cells analyzed by flow cytometry. (E) Colon tissue of WT and SMILE TG mice was subjected to H&E and IHC with anti-SMILE, -mTOR, -AMPK, and -STAT3 antibodies. Data are means ± SEM of two replicates. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Regulation of SMILE expression in the colon of patients with UC. (A) Colon tissues were evaluated by H&E staining and Mayo scoring. Representative images of IHC with anti-mTOR, -STAT3, -AMPK, and -SMILE antibodies. Bars are percentages of positive area per field. (B) Fold changes in the SMILE and FOXP3 mRNA levels in UC PBMCs by real-time PCR (n = 9 per group). Correlation between SMILE and FOXP3 levels. (C) mRNA levels of SMILE, PRKAA, and FOXP3 in UC PBMCs treated with metformin (1 mM) for 48 h by real-time PCR. (D) Th17 and Treg cells in the PBMCs of metformin (1 mM)-treated patients with UC under Th17-polarizing condition for 72 h. Data are means ± SEM. **P < 0.01, ***P < 0.001.