Transcriptional regulation of IL10 gene in human macrophages

Abstract

Abstract IL-10 is an anti-inflammatory cytokine with a crucial role in immune homeostasis. Dysregulated production of IL-10 has been linked to certain autoimmune and inflammatory diseases. IL-10 is strongly induced by TLR ligands and suppressed by IFN-γ as part of the synergistic activation of inflammatory genes. However, the molecular mechanisms that regulate expression of the IL10 gene, especially in human macrophages, are incompletely understood. By using epigenomic analysis of ATAC-seq and ChIP-seq analysis we have identified human macrophage-specific enhancers in the IL10 gene locus. In these putative enhancers, we observed that chromatin accessibility and histone acetylation are increased by LPS stimulation. We also found that p300 was frequently recruited to these macrophage-specific cis-regulatory elements (CREs) in LPS-stimulated macrophages. One of these potential LPS-induced enhancers, CRE+6, is located 6kb upstream of the IL10 transcriptional start site and contains transcription factor binding motifs and autoimmune disease-associated SNPs. CRE+6 showed constitutive binding of the macrophage-lineage-determining factor PU.1 and LPS-inducible binding of AP-1 and STAT3. LPS stimulation increased eRNA transcription from CRE+6. The functional relevance of CRE+6 eRNA was confirmed by eRNA-targeted knockdown experiments. Strikingly, the mechanism by which IFN-γ suppresses IL10 expression appeared to be decommissioning of enhancers, as IFN-γ strongly suppressed TLR-induced positive enhancer marks and eRNA transcription. Our study implicates that crucial enhancer elements cooperate with the core promoter in LPS- and IFN-γ-mediated regulation of IL-10 gene transcription in human macrophages.