Interleukin (IL)-23 suppresses IL-10 in inflammatory bowel disease

Abstract

Interleukin (IL)-10 plays an important role in immune regulation in the intestine. Immune deregulation is suggested in the pathogenesis of inflammatory bowel disease (IBD). This study aims to elucidate the role of IL-23 in the suppression of IL-10 in the IBD intestinal mucosa. Surgically removed colon specimens were obtained from 16 IBD patients. The expressions of IL-10, IL-23, and IgA in the specimens were examined at the protein and gene transcriptional levels. The gene transcription of IL-10 was assessed by chromatin immunoprecipitation assay and promoter accessibility assay. The levels of IgA and IL-10 were significantly lower, whereas the levels of IL-23 were higher, in IBD specimens than in normal controls. The levels of IgA and IL-10 were negatively correlated with the infiltration of inflammatory cells in the IBD mucosa. The production of IL-10 by lamina propria mononuclear cells was lower in the IBD group than in the control group, and these levels could be enhanced by blocking IL-23. The gene transcription of IL-10 was significantly suppressed in CD4(+) T cells of IBD mucosa; this phenomenon could be replicated in vitro by adding IL-23 in the culture of polarized Th2 cells. Overexpression of IL-23 in the intestinal mucosa suppresses the production of IL-10, which weakens the defensive barrier by reducing the production of IgA in the gut.

FIGURE 1.

Expression of IL-10, IL-23, and IgA in IBD colon mucosa. Surgically removed colon tissues were obtained from IBD patients (nine UC patients; seven CD patients) and nine colon cancer patients (controls (Con)); the marginal normal tissue (proved by pathologists) was used in the experiments. The samples were analyzed by ELISA, quantitative RT-PCR, Western blotting, and flow cytometry individually. A, B, and E, the bar graphs indicate the levels of mRNA (A) and proteins (B) of IL-10 and IL-23 and the levels of total IgA (E). Data are presented as means ± S.D. *, p < 0.05, as compared with the control group. In C1, D1, and F1, the dot plots show CD4⁺ T cells (C1), CD4⁺ (C1), macrophages (D1), and plasma cells (F1), respectively; the gated cells in the panels were further analyzed for the expression of IL-10 (C2–C4), IL-23 (E2–E4), and IgA (F2–F4). The tissue types are annotated above histograms. Isotype controls are shown in C5, D5, and F5. Samples obtained from patients were analyzed individually.

FIGURE 2.

Frequencies of inflammatory cells and MPO are increased in IBD colon mucosa. All samples used in Fig. 1 were also analyzed by H&E staining. A, the bars indicate the frequencies of PMN and mononuclear cells that were counted under a light microscope (counted in 20 fields per sample at ×400) in colon tissue. B, the bars indicate the levels of MPO in colon tissue extracts. The data were presented as means ± S.D. *, p < 0.01, as compared with controls (Con).

FIGURE 3.

In vitro production of IL-10 and IgA by isolated LPMCs. LPMCs were isolated from the samples in Fig. 1. The cells were cultured in the presence or absence of phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) (Medium group) and ionomycin (1 μg/ml) with or without anti-IL-23 antibody (αIL-23; 100 ng/ml) for 3 days. A and B, the bars indicate the levels of IL-10 (A) or IgA (B) in the supernatants as determined by ELISA. Specimens from different patients were assessed individually. *, p < 0.01, as compared with the control group (Con). #, p < 0.01, as compared with the medium group. Inactive, cells were not activated by phorbol 12-myristate 13-acetate/ionomycin. Isotype, cells were treated with isotype IgG instead of αIL-23. αIL-23/αIL-10, Cells were treated with antibodies of both IL-23 and IL-10.

FIGURE 4.

IL-10 gene transcription is compromised in IBD mucosa. CD4⁺ cells were isolated from the samples in Fig. 1. Cells were analyzed by a ChIP assay. A, the gel graphs show acetylated histone H3 and H4. B, the bars indicate the mRNA levels of IL-10 in activated CD4⁺ T cells. C, the bars indicate the levels of activated promoters of IL-10. D, micrococcal nuclease accessibility by chromatin accessibility by real-time PCR was performed on the nuclei of the CD4⁺ T cells. The y axis indicates the amount of PCR products, and the x axis indicates the sources of the samples. *, p < 0.01, as compared with the control group (Con).

FIGURE 5.

IL-23 suppresses IL-10 expression in polarized Th2 cells. Polarized IL-10-producing T cells were generated from human peripheral CD4⁺ CD25⁻ T cells. The cells were treated with IL-23 in the culture at graded doses for 3 days. The cells were processed for determining the expression of IL-10 in the same procedures as described in the legend for Fig. 1. A, the gel shows acetylated histone H3 and H4. B–E, the bars indicate the levels of IL-10 promoter accessibility (B), activated promoter of IL-10 (C), IL-10 mRNA expression (D), and IL-10 protein in the culture supernatant (E), respectively. The data were presented as the means ± S.D. of triplicate samples resulting from three experiments. *, p < 0.01, as compared with the control group.