Opposite role of tumor necrosis factor receptors in dextran sulfate sodium-induced colitis in mice

Abstract

Tumor necrosis factor-α (TNF-α) is a key factor for the pathogenesis of inflammatory bowel diseases (IBD), whose function is known to be mediated by TNF receptor 1 (TNFR1) or 2. However, the precise role of the two receptors in IBD remains poorly understood. Herein, acute colitis was induced by dextran sulfate sodium (DSS) instillation in TNFR1 or 2-/- mice. TNFR1 ablation led to exacerbation of signs of colitis, including more weight loss, increased mortality, colon shortening and oedema, severe intestinal damage, and higher levels of myeloperoxidase compared to wild-type counterparts. While, TNFR2 deficiency had opposite effects. This discrepancy was reflected by alteration of proinflammatory cytokine and chemokine production in the colons. Importantly, TNFR1 ablation rendered enhanced apoptosis of colonic epithelial cells and TNFR2 deficiency conferred pro-apoptotic effects of lamina propria (LP)-immune cells, as shown by the decreased ratio of Bcl-2/Bax and enhanced nuclear factor (NF)-κB activity.

Figure 1. TNF-R1 or 2−/− mice have more or less weight loss and mortality after DSS.

DSS colitis was established as described in Materials and methods. (A) Weight in each group was monitored daily at 8-day intervals. Weight was presented as percentage of the initial weight at day 0. (B) Survival rate was calculated at the end of experiments. Data are pooled from three independent experiments (n = 10–30 mice per group). *P<0.05, **P<0.01 vs WT mice after DSS.

Figure 2. TNF-R1 or 2 deficiency exacerbates or ameliorates mucosal damage after DSS.

Colons from each group were collected at day 8 after DSS instillation. (A) Macroscopic images of the colons are shown. (B) Colon edema was determined by wet-to-dry weight ratios. (C) Microscopic images of the colons are shown by haematoxylin and eosin staining. Magnification: ×200. (D) Colitis score was determined as described in Materials and methods. Data represent the mean±SD of eight to ten mice per group. *P<0.05, **P<0.01 vs WT control mice; #P<0.05, ##P<0.01 vs WT mice after DSS.

Figure 3. TNF-R1 or 2−/− mice have increased or reduced infiltrates of neutrophils into the colons after DSS.

Levels of myeloperoxidase (MPO) in the colons of mice in each group were measured at day 8 after colitis induction. Data represent the mean±SD of four to six mice per group. **P<0.01 vs WT control mice; #P<0.05, ##P<0.01 vs WT mice after DSS.

Figure 4. Lack of TNF-R1 or 2 increases or reduces proinflammatory mediator production in the colons after DSS.

Colons were isolated and homogenized at day 8 after colitis induction. (A) IL-6, IL-1β, MCP-1, and IL-17A concentrations in the supernatants of homogenates were determined by ELISA. (B) mRNA were isolated from homogenates and CXCL1/2, CCL3 expression was examined by quantitative RT-PCR. Data represent the mean±SD of six to eight mice per group from two independent experiments. **P<0.05 vs WT control mice; #P<0.05, ##P<0.01 vs WT mice after DSS.

Figure 5. Systemic inflammatory response in TNF-R−/− and WT mice after DSS.

(A) Spleen was isolated and weighed at day 8 after colitis induction. The percentage of granulocytes (Gr-1⁺) (B) or monocytes (F4/80⁺) (C) in the spleens were examined by flow cytometry. Representative plots were shown. Data represent the mean±SD of four to six mice per group from three independent experiments. *P<0.05 vs WT control mice; #P<0.05, ##P<0.01 vs WT mice after DSS.

Figure 6. TNF-R1 or 2 ablation affects apoptosis of colonic epithelial cells or infiltrating immune cells differently after DSS.

Colons were dissected at day 8 after colitis induction. (A) Colonic epithelial cells and LP-immune cells were isolated as described in Materials and methods. TNF-R1 (brown line) and 2 (red line) expression in these cells was examined by flow cytometry. (B) Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining (brown nuclei). Magnification: ×400. Histology is representative for more than 10 mice per group. (C) Colonic epithelial cells and LP-immune cells were stained with annexin V and PI. Their apoptosis was detected by flow cytometry. (D,E) Colonic epithelial cells (D) and lamina propria-immune cells (E) were isolated respectively and the proteins were extracted. Bcl-2, Bax, and IκBα expression was examined by western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Changes in quantity of expression of these factors were determined by densitometric assays. Data are representative of three independent experiments. *P<0.05 vs WT control mice; #P<0.05, ##P<0.01 vs WT mice after DSS.