Abstract
Interleukin (IL)-23, a new member of the IL-12 family, plays a central role in the Th17 immune response and in autoimmune diseases. It is clear that activated macrophages and dendritic cells produce IL-23, but the molecular mechanisms whereby inflammatory signals stimulate IL-23 expression are not fully understood. We demonstrate that induction of IL-23 p19 gene expression by LPS depends on the TLR4 and MyD88 pathways. All three MAPK pathways (ERK, JNK, and p38) that are activated by lipopolysaccharide (LPS) stimulation were shown to exert a positive effect on p19 expression. We cloned a 1.3-kb putative p19 promoter and defined its transcription initiation sites by the 5'-rapid amplification of cDNA ends method. By analyzing IL-23 p19 promoter mutants, we have identified a promoter region (-413 to +10) that contains several important elements, including NF-kappaB and AP-1. In addition to NF-kappaB, we have demonstrated that the proximal AP-1 site is important for p19 promoter activation. Mutation of the AP-1 site resulted in the loss of p19 promoter activation. Electrophoretic mobility shift assay (EMSA) analysis showed that c-Jun and c-Fos bind to the AP-1 site, which was confirmed by a chromatin immunoprecipitation assay. Furthermore, co-transfection of c-Jun and ATF2 synergistically induced p19 promoter activation, and c-Jun and ATF2 formed a protein complex, demonstrated by co-immunoprecipitation. Finally, LPS-stimulated peritoneal macrophages from IL-10-deficient mice expressed significantly higher IL-23 p19 than macrophages from wild type mice, and the addition of recombinant IL-10 strongly inhibited LPS-induced p19 expression. Thus, this study suggests that MyD88-dependent Toll-like receptor signaling induces IL-23 p19 gene expression through both MAPKs and NF-kappaB.
Highlights
IL-23 p19 Expression Is Dependent on TLR4 and MyD88—To evaluate the importance of Tolllike receptor (TLR) signaling pathways for IL-23 p19 expression, thioglycollate-elicited peritoneal macrophages from wild type (WT), TLR4Ϫ/Ϫ, and MyD88Ϫ/Ϫ mice were activated with LPS (1 g/ml) for either 4 h or 24 h
LPS strongly induced IL-23 p19 mRNA expression in macrophages from WT mice, but IL-23 p19 mRNA expression was significantly impaired in macrophages from TLR4Ϫ/Ϫ and MyD88Ϫ/Ϫ mice (Fig. 1A), as was LPS-stimulated expression of IL-12 p35 (Fig. 1A)
IL-23 and IL-12 protein production induced by incubation with LPS plus IFN-␥ was significantly reduced in TLR4Ϫ/Ϫ and MyD88Ϫ/Ϫ macrophages (Fig. 1D), LPS itself did not induce detectable amounts of IL-23
Summary
Mice lacking IL-23 p19 were resistant to collagen-induced arthritis, experimental autoimmune encephalomyelitis, and inflammatory bowel disease, because the generation of Th17 cells is impaired in the absence of IL-23 [6, 10, 11]. IL-12-deficient mice were susceptible to collagen-induced arthritis, experimental autoimmune encephalomyelitis, and inflammatory bowel disease. The regulation of IL-23 p19 at the molecular level is not fully understood, two recent studies suggest that the NF-B subunit c-Rel is important for p19 gene expression in dendritic cells [22, 23]. AP-1 Control of p19 Gene Expression include c-Jun, ATF2, and Elk-1. In contrast to IL-12/IL-23 p40, which is negatively regulated by ERK, the TLR-activated ERK pathway is essential for IL-23 p19 gene expression. The clearer understanding of factors controlling IL-23 expression may assist efforts to control the induction and progression of autoimmune disease
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