Cutting edge: IFN-gamma is a negative regulator of IL-23 in murine macrophages and experimental colitis

Abstract

IL-23 regulation is a central event in the pathogenesis of the inflammatory bowel diseases. We demonstrate that IFN-gamma has anti-inflammatory properties in the initiation phase of IL-23-mediated experimental colitis. IFN-gamma attenuates LPS-mediated IL-23 expression in murine macrophages. Mechanistically, IFN-gamma inhibits Il23a promoter activation through altering NF-kappaB binding and histone modification. Moreover, intestinal inflammation is inhibited by IFN-gamma signaling through attenuation of Il23a gene expression. In germ-free wild-type mice colonized with enteric microbiota, inhibition of colonic Il23a temporally correlates with induction of IFN-gamma. IFN-gammaR1/IL-10 double-deficient mice demonstrate markedly increased colonic inflammation and IL23a expression compared with those of IL-10(-/-) mice. Colonic CD11b(+) cells are the primary source of IL-23 and a target for IFN-gamma. This study describes an important anti-inflammatory role for IFN-gamma through inhibition of IL-23. Converging genetic and functional findings suggest that IL-23 and IFN-gamma are important pathogenic molecules in human inflammatory bowel disease.

FIGURE 1

IFN-γ negatively regulates LPS-mediated IL-23 expression in macrophages. A, BMMs from WT and IFN-γR1^−/− mice were cultured in the presence of LPS (100 ng/ml, black bars) with or without IFN-γ (10 ng/ml, white bars). Supernatants were analyzed for IL-23 by ELISA. Results are expressed as mean ± SEM of three independent experiments. *p < 0.05 versus LPS-stimulated BMMs. B, WT BMMs were stimulated with LPS (100 ng/ml, solid line) and IFN-γ (10 ng/ml, dash line). Il23a mRNA expression was quantified by real-time RT-PCR. Results are expressed as fold induction normalized to β-actin. Error bars represent mean ± SEM of three independent experiments. *p < 0.05 versus LPS-stimulated BMMs. C, BMMs from WT, IL-10^−/−, and IFN-γR1/IL-10/^−/− mice were cultured with LPS (black bars, 100 ng/ml) plus IFN-γ (gray bars, 10 ng/ml). Supernatants were analyzed for IL-23 by ELISA. Results are expressed as mean ± SEM of three independent experiments. *p < 0.05 versus LPS-stimulated BMMs. D, WT BMMs were incubated for 30 min with DMSO or CHX (5 μg/ml) and then stimulated with LPS (100 ng/ml, black bars) ± IFN-γ (10 ng/ml, grey bars). Il23a (upper panel) and Il12b (bottom panel) mRNA was analyzed by real-time RT-PCR after 1 h. Results are expressed as fold induction normalized to β-actin. Error bars represent mean ± SEM of three independent experiments. *p < 0.05 versus LPS-stimulated BMMs.

FIGURE 2

IFN-γ inhibits Il23a promoter activity. A, BMMs were transfected with an Il23a promoter luciferase reporter plasmid and cultured with LPS (100 ng/ml) ± IFN-γ (10 ng/ml) for 18 h. Reporter activity is represented as luciferase units normalized to heat shock protein promoter β-galactosidase activity. Data represent mean ± SEM of three independent experiments. *p < 0.05 versus LPS-stimulated Il23a promoter. B, Binding of RelA, p50, and H4Act to the distal NF-κB site on the endogenous WT Il23a promoter and H4Act at the nucleosome 1 position of the Il12b promoter was assessed by ChIP 1 h after incubation with LPS (100 ng/ml) ± IFN-γ (10 ng/ml). Real-time PCR was performed on anti-RelA, anti-p50, and anti-H4Act precipitated DNA samples, respectively. Results are presented as enrichment (percentage input) of RelA, p50, or H4Act DNA binding. Error bars represent mean ± SEM of three independent chromatin preparations from three independent experiments. *p < 0.05 versus LPS-stimulated BMMs

FIGURE 3

Enteric microbiota induce colonic IL-23 expression in experimental colitis. WT and IL-10^−/− mice raised in GF conditions were colonized with enteric microbiota from CNV mice at 8 wk of age. A, IL-23 secretion at day 14 postcolonization in colonic explant cultures was determined by ELISA. B, Colonic Il23a (upper panel) and ifng mRNA (bottom panel) expression was detected by real time RT-PCR in GF WT mice (day 0) and at days 3 and 14 postcolonization. Each time point includes three individual mouse colons and is representative of three independent experiments. C, CD11b⁺ LPMCs were isolated from WT and IL-10^−/− mouse colons. CD11b⁺ LPMCs were activated with heat-killed E. coli ± IFN-γ (10 ng/ml). Il23a and β-actin mRNA expression was detected by real-time RT-PCR. Results are expressed as fold induction normalized to β-actin. Error bars represent mean ± SEM of three independent experiments. *p < 0.05 versus heat-killed E. coli-stimulated IL-10^−/− CD11b⁺ LPMCs.

FIGURE 4

Increased colonic inflammation and expression of IL-23 in IFNγR1/IL-10^−/− mice. A, Colitis scores of WT, IFN-γR1^−/−, IL-10^−/−, and IFN-γR1/IL-10^−/− mice at 8 wk of age. B, Colonic Il23a mRNA was examined by real-time RT-PCR. C, IL-23 protein in supernatants from colon explants cultures were analyzed using cytokine-specific ELISA and from WT, IFN-γR1^−/−, IL-10^−/−, and IFN-γR1/IL-10^−/− mice. Results are expressed as mean ± SEM from four to six mice per group. *p < 0.05 versus IL-10^−/− mice. D, CD11b⁺ LPMCs isolated from WT, IFN-γR1^−/−, IL-10^−/−, and IFN-γR1/IL-10^−/− mice at 8 wk of age were activated with heat-killed E. coli, multiplicity of infection (10:1). Il23a mRNA expression was detected by realtime RT-PCR. Results are expressed as fold induction relative to unstimulated CD11b⁺ LPMCs normalized to β-actin and represent mean ± SEM of three independent experiments. *p < 0.05 versus heat-killed E. coli-stimulated IL-10^−/− CD11b⁺ LPMCs.