Interleukin-10 Inhibits Interleukin-12 p40 Gene Transcription by Targeting a Late Event in the Activation Pathway

Abstract

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/EBPβ, as monitored by genomic footprinting and chromatin immunoprecipitation. It also had little effect on NF-κB 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.

We thank Michelle Bradley, Bradley Cobb, and Vladimir Ramirez-Carrozzi for critical reading of the manuscript. We are also grateful to Valerie Schuman and Dana Russell for performing the thioglycolate injections.

A.A.N. was supported by the Ingram Research Endowment. S.T.S. is an Investigator with the Howard Hughes Medical Institute.