Interleukin-10 inhibits interleukin-12 p40 gene transcription by targeting a late event in the activation pathway

Interleukin-10 (IL-10) is a potent anti-inflammatory cytokine that suppresses the induction of proinflammatory cytokine genes, including the IL-12 p40 gene. Despite considerable effort examining the effect of IL-10 on specific transcription factors and signaling molecules, the mechanism by which IL-10 inhibits gene transcription has remained elusive. To provide a different perspective to this problem, we examined the effect of IL-10 on molecular events occurring at the endogenous IL-12 p40 locus in lipopolysaccharide-stimulated peritoneal macrophages. IL-10 abolished recruitment of RNA polymerase II to the p40 promoter. However, it only modestly reduced binding of C/EBPbeta, as monitored by genomic footprinting and chromatin immunoprecipitation. It also had little effect on NF-kappaB complexes that are critical for p40 induction. A substantial reduction in nucleosome remodeling at the p40 promoter was observed, but the magnitude of this reduction appeared insufficient to account for the strong inhibition of transcription. Finally, a lipopolysaccharide-inducible DNase I hypersensitive site identified 10 kb upstream of the start site was unaffected by IL-10. Thus, despite a dramatic reduction in p40 transcription, several events required for activation of the endogenous p40 gene occurred relatively normally. These findings suggest that IL-10 blocks one or more events that occur after p40 locus decondensation and nucleosome remodeling and after, or in parallel with, the binding of a subset of p40 transcriptional activators.