The Dynamics of Interleukin-10-Afforded Protection during Dextran Sulfate Sodium-Induced Colitis

Abstract

Inflammatory bowel disease encompasses a group of chronic-inflammatory conditions of the colon and small intestine. These conditions are characterized by exacerbated inflammation of the organ that greatly affects the quality of life of patients. Molecular mechanisms counteracting this hyperinflammatory status of the gut offer strategies for therapeutic intervention. Among these regulatory molecules is the anti-inflammatory cytokine interleukin (IL)-10, as shown in mice and humans. Indeed, IL-10 signaling, particularly in macrophages, is essential for intestinal homeostasis. We sought to investigate the temporal profile of IL-10-mediated protection during chemical colitis and which were the underlying mechanisms. Using a novel mouse model of inducible IL-10 overexpression (pMT-10), described here, we show that mice preconditioned with IL-10 for 8 days before dextran sulfate sodium (DSS) administration developed a milder colitic phenotype. In IL-10-induced colitic mice, Ly6C cells isolated from the lamina propria showed a decreased inflammatory profile. Because our mouse model leads to transcription of the IL-10 transgene in the bone marrow and elevated seric IL-10 concentration, we investigated whether IL-10 could imprint immune cells in a long-lasting way, thus conferring sustained protection to colitis. We show that this was not the case, as IL-10-afforded protection was only observed if IL-10 induction immediately preceded DSS-mediated colitis. Thus, despite the protection afforded by IL-10 in colitis, novel strategies are required, specifically to achieve long-lasting protection.

Keywords: colitis; inflammation; interleukin-10; macrophages; therapy.

Figure 1

A novel mouse model for inducible interleukin (IL)-10 expression: pMT-10 mice. (A) Schematic representation showing the targeting vector and insertion site. (B) Kinetics of IL-10 overexpression in the serum at different time points post Zn administration and Zn withdrawal. pMT-10 mice were fed with normal (pMT-10-Zn) or Zn-enriched (pMT-10 + Zn) water and at the indicated time points blood was harvested and the amount of IL-10 in serum measured by immunoassay. (C) qRT-PCR identified CD45⁻ TER119⁻ cell subsets from skin, bone marrow, and small intestine (SI) as the main producers of IL-10 in pMT-10 mice fed for 8 days with Zn-enriched water. In both (B,C), each point or bar represents the mean ± SEM for three independent mice. Data were analyzed with (B) two-way analysis of variance (Sidak’s multiple comparisons test) or (C) Student’s t-test, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2

Dextran sulfate sodium (DSS)-induced pathology is ameliorated by preexposure to interleukin (IL)-10. (A) BL/6, pMT-10, and pMT-10.IL-10Rα^−/− mice were fed for 8 days with normal (BL/6-Zn, pMT-10-Zn, and pMT-10.IL-10Rα^−/− Zn, respectively) or Zn-enriched (BL/6 + Zn, pMT-10 + Zn, and pMT-10.IL-10Rα^−/− + Zn) water, followed by 8 days of 3% DSS administration also in the drinking water. (B,F) Disease progression based on DAI parameters was registered every day for 8 days. Each point represents the mean ± SEM for three to five independent mice, in two independent experiments. (C) Colon length measurement at day 8 of DSS administration. (D) Representative H&E-stained sections of large bowel at 40× magnification (scale bar = 200 µm). (E) Colitis scores derived from evaluation of colon and cecum from either group. Each dot represents one independent animal; represented is also mean ± SEM. Data were analyzed with (B,F) two-way analysis of variance (Sidak’s multiple comparisons test) or (C,E) Student’s t-test. (B) # compare BL/6-Zn against BL/6 + Zn; ^ compare BL/6-Zn against pMT-10-Zn; • compare BL/6-Zn against pMT-10 + Zn; ∅ compare BL/6 + Zn against pMT-10-Zn; x compare BL/6 + Zn against pMT-10 + Zn, * compare pMT-10-Zn against pMT-10 + Zn. One symbol, p < 0.05; two symbols, p < 0.01; three symbols, p < 0.001.

Figure 3

Ly6C⁺ cells preexposed to interleukin (IL)-10 reveal a less inflammatory profile upon DSS-induced colitis than those preexposed to Zn. (A) pMT-10 or BL/6 mice were fed with Zn-enriched water for 8 days, followed by 4 days of 3% DSS administration. (B) At the end of the DSS treatment, Lamina propria leukocytes (LPLs) were isolated and Ly6C⁺ cells sort-purified. Shown is the gating strategy for Ly6C⁺ cells purification. (C) Sort-purified Ly6C⁺ cells (n = 25 cells) were analyzed by qRT-PCR for a total of 22 genes using the BioMark HD system. Samples were normalized for Hprt expression. Represented is the expression heatmap compiling the genes which expression was detected in either mouse group. Each heatmap rectangle represents the mean of gene expression obtained for cells isolated from five independent mice. (D) The frequency of the different leukocyte subsets was determined upon staining of LPLs for Ly6C⁺ cell sorting. Each dot represents one independent animal; represented is also mean ± SEM. Data were analyzed with Student’s t-test, *p < 0.05.

Figure 4

The interleukin-10 protection conferred against DSS-induced colitis is not long lasting. (A) pMT-10 mice were fed with control (pMT-10-Zn) or Zn-enriched (pMT-10 + Zn) water for 8 days, followed by a 7- or 21-day resting period where only normal water was available, and by 8 days of 3% DSS. (B) Disease progression based on Disease Activity Index (DAI) parameters was registered every day for 8 days. (C) Colon length measurement at day 8 of DSS administration. (D) Representative H&E-stained sections of large bowel at 40× magnification (scale bar = 200 µm). (E) Colitis scores derived from evaluation of colon and cecum from both groups. Each dot represents one independent animal, in two independent experiments; represented is also mean ± SEM. Data were analyzed with (B) two-way analysis of variance (Sidak’s multiple comparisons test) or (C,E) Student’s t-test.