IL-21/IL-21R signaling suppresses intestinal inflammation induced by DSS through regulation of Th responses in lamina propria in mice

Abstract

Serum level of IL-21 is increased in patients with inflammatory bowel diseases (IBD), suggesting that IL-21/IL-21 receptor (IL-21R) signaling may be involved in the pathogenesis of IBD. However, the role of IL-21/IL-21 receptor signaling plays in the pathogenesis of IBD is not very clear. In this study, using IL-21R.KO mice, we tested the role of IL-21/IL-21R signaling in the regulation of T helper cell responses during intestinal inflammation. Here we found that IL-21R.KO mice were more susceptible to DSS-induced colitis as compared with C57BL/6 mice. The spontaneous inflammatory cytokines released by macrophages in LP of colon were significantly increased, and Th2, Th17 and Treg responses were down-regulated markedly. However, Th1 responses were significantly up-regulated in IL-21R.KO mice. Meanwhile, the population of CD8(+)CD44(+)IFN-γ(+) T cells was markedly elevated in LP of inflammatory intestine of IL-21RKO mice. In vivo, after disease onset, DSS-induced intestinal inflammation was ameliorated in C57BL/6 mice treated with rIL-21. Our results demonstrate that IL-21/IL-21R signaling contributes to protection against DSS-induced acute colitis through suppression of Th1 and activation of Th2, Th17 and Treg responses in mice. Therefore, therapeutic manipulation of IL-21/IL-21R activity may allow improved immunotherapy for IBD and other inflammatory diseases associated with Th cell responses.

Figure 1. Susceptibility of IL-21RKO mice to DSS-induced colitis.

Mice were oral treated with 3% DSS in the drinking water to induce colitis as described in Methods. On day 5, (A) macroscopic changes, colon length and (B) histological score (original magnification, ×200) were analyzed. (C) body weight, (D) survival rate and (E) disease activity index were daily observed. Data indicate mean ± SD of 10~15 mice of each group obtained from a representative of three independent experiments. Statistically significant differences from the value for DSS-treated C57BL/6 mice are shown (*P < 0.05, **P < 0.01 or ***P < 0.001).

Figure 2. Flow cytometry analysis of the populations of LPL in the colon of mice with DSS-induced colitis.

LPL were isolated from inflammatory intestinal tissues of mice on day 5 after DSS-induced colitis and measured by flow cytometry. (A) The total number of LPL in the colon in IL-21RKO and C57BL/6 mice on day 5 after DSS-induced colitis. The proportion and absolute number of (B) macrophage (F4/80⁺CD11b⁺), (C) NK (NK1.1⁺CD3⁻), γδ T (γδTCR⁺CD3⁺) or NKT (NK1.1⁺CD3⁺) cell and (D) CD4⁺, CD8⁺, CD4⁺CD44⁺, CD8⁺CD44⁺ T cells. Data indicate mean ± SD of 5 mice of each group obtained from a representative of three independent experiments. Statistically significant differences are shown (*P < 0.05).

Figure 3. Spontaneous inflammatory cytokine production by LP cells in mice with DSS-inducted colitis.

LPL from colon of DSS-treated IL-21RKO or C57BL/6 mice was isolated and cultured for 24 h without any stimulation. The level of cytokine production in culture supernatant was measured using ELISA kits. Data indicate mean ± SD of 8 mice of each group obtained from a representative of three independent experiments. Statistically significant differences from the value for DSS-treated C57BL/6 mice are shown (*P < 0.05).

Figure 4. Cytokine-producing T cells in the LPL of colon in IL-21RKO mice with DSS-induced colitis.

(A) The proportions and absolute numbers of IFN-γ⁺CD4⁺, IFN-γ⁺CD8⁺, IL-4⁺CD4⁺ T in LPL of colon; (B) The percentage of CD44⁺IFN-γ⁺ in CD4⁺ or CD8⁺ T cells in LPL of colon; (C) The proportion and absolute number of IL-5⁺CD4⁺ T cells in LPL of colon; (D) The proportion and absolute number of IL-17A⁺CD4⁺ T cells in LPL of colon. Data indicate mean ± SD of 3 mice of each group obtained from a representative of three independent experiments. Statistically significant differences are shown (*P < 0.05 or **P < 0.01).

Figure 5. Th cell-associated cytokines productions in LP cells of colon after DSS-inducted colitis.

LPL from colon of DSS-treated IL-21RKO or C57BL/6 mice was isolated and cultured for 48 hours with or without anti-CD3/anti-CD28 mAbs, and then cytokine production in the cultured supernatant was measured using ELISA kits. Data indicate mean ± SD of 5 mice of each group obtained from a representative of three independent experiments. Statistically significant differences are shown (**P < 0.01 or ***P < 0.001).

Figure 6. Effects of IL-21R deficiency on regulation of Treg responses in inflammatory intestine in mice.

(A) The percentage of Foxp3⁺, CD25⁺, CD25⁺Foxp3⁺ or CD25⁺Foxp3⁻ in CD4⁺ T cell in LPL of mice with DSS-induced colitis; (B) The cell numbers of CD4⁺CD25⁺, CD4⁺Foxp3⁺, CD4⁺CD25⁺Foxp3⁺ or CD4⁺CD25⁺Foxp3⁻ cells in LPL; (C) The percentage and the absolute numbers of of CD4⁺CD25⁺IL-10⁺ T cells in LPL of colon. Data indicate mean ± SD of 3 mice of either group obtained from a representative of three independent experiments. (D) LPL was cultured for 48 h under the TCR stimulations, and then IL-10 production by T-LPL in the cultured supernatant was measured using ELISA kit. Data indicate mean ± SD of 5 mice of each group obtained from a representative of three independent experiments. Statistically significant differences are shown (*P < 0.05, **P < 0.01 or ***P < 0.001).

Figure 7. Effects of in vivo treatment with rIL-21 on DSS-induced colitis in mice.

(A) The experimental design of in vivo treatment with rIL-21 on DSS-induced colitis in mice; (B) colon length (cm); (C) histological analysis; (D) body weight change, survival rate and DAI. Data indicate mean ± SD of 8~10 mice of each group obtained from a representative of two independent experiments. Statistically significant differences are shown (*P < 0.05, **P < 0.01 or ***P < 0.001).

Figure 8. rIL-21-treatment improved intestinal inflammation induced by DSS in C57BL/6 mice.

(A) IL-21 production by LP-T cell of colon from control or rIL-21-treated mice was analyzed on day 2, 4 and 6 after DSS-administration. (B) The percentage and absolute number of IFN-γ-, IL-17A-, IL-4- or IL-10-producing CD4⁺ LP-T cells from colon of control or rIL-21-treated mice were analyzed by cytokine FACS on day 6 after DSS-treatment. Data indicate mean ± SD of 3 mice of each group obtained from a representative of tow independent experiments. Statistically significant differences are shown (*P < 0.05 or **P < 0.01).