Investigating the role of epigenetics in the regulation of inflammatory skin disease

Abstract

Psoriasis represents a complex interplay between genetic predisposition, the environment and inflammatory responses, and increasing evidence suggests alterations in the epigenome, including histone acetylation, plays a role. Bromodomain-containing proteins regulate gene expression by binding to acetyl-lysine residues on histones and recruiting transcription factors to gene regulatory regions. The development of small molecule inhibitors of bromodomain extraterminal (BET) proteins has enabled interrogation of this pathway. Interestingly BET inhibitors demonstrate anti-proliferative and anti-inflammatory effects in in vitro and in vivo models of cancer and inflammation. An established in-vitro keratinocyte model of cutaneous inflammation was further developed to characterise IL-6 and IL-8 (mRNA and protein) responses to TNF+ IL-17 stimulation. Chromatin immunoprecipitation (ChIP) studies showed psoriasis-relevant stimuli induced dynamic, gene-specific alterations of the epigenome, including histone hyperacetylation, with co-ordinated recruitment of BET proteins (Brd2, Brd3 and Brd4) and RNA polymerase II, to the promoter region of IL-6 and IL-8. The effects of TNF+ IL-17 stimulation in keratinocytes were validated through global gene expression array studies which showed stimulation modulated expression of keratinocyte genes known to be differentially expressed in psoriasis and involved in its pathogenesis. The hypothesis that BET proteins are involved in regulating inflammatory responses in keratinocytes was tested using a specific BET inhibitor, I-BET151; this blocked pathogenic inflammatory responses. In particular, IL-6 and IL-8 responses to TNF + IL-17 stimulation demonstrated potent sensitivity to I-BET151 treatment; this could be accounted for by the decreased binding of BET proteins and RNA polymerase II to IL-6 and IL-8 gene promoter regions in the presence of the BET inhibitor. Global gene expression array studies showed genes sensitive to BET inhibition were primarily involved in the cell cycle and inflammation, with many relevant to the pathogenesis of psoriasis. In addition, ~20% of genes identified in a previously published meta-analysis of five psoriasis transcriptomic studies were differentially modulated by I-BET151 treatment in TNF + IL-17 stimulated NHEKs. Furthermore, acetate, a principle metabolite of ethanol, a factor implicated in the development and exacerbation of psoriasis, enhanced IL-6, but not IL-8, responses to TNF + IL-17 stimulation through gene-specific epigenetic modifications at the promoter region. IFN also enhanced IL-6, but not IL-8, response to TNF + IL-17 stimulation, suggesting i) IL-6 is more sensitive to enhancement by additional disease relevant stimuli and ii) IL-6 and IL-8 are differentially regulated at the transcriptional level; ChIP studies showed increased enrichment of Brd4/p65 at the IL-6 promoter compared to the IL-8 promoter.