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Significance T cells control many immune functions, with Th17 cells critical in regulating inflammation. Following activation, T cells undergo metabolic reprogramming and utilize glycolysis to increase the ATP availability. Epigenetic mechanisms controlling metabolic functions in T cells are currently not well-defined. Here, we establish an epigenetic link between the histone H3K27me3 demethylases KDM6A/B and the coordination of a metabolic response. Inhibition of KDM6A/B leads to global increases in the repressive H3K27me3 histone mark, resulting in down-regulation of key transcription factors, followed by metabolic reprogramming and anergy. This work suggests a critical role of H3K27 demethylase enzymes in maintaining Th17 functions by controlling metabolic switches. Short-term treatment with KDM6 enzyme inhibitors may be useful in the therapy of chronic inflammatory diseases., T helper (Th) cells are CD4+ effector T cells that play a critical role in immunity by shaping the inflammatory cytokine environment in a variety of physiological and pathological situations. Using a combined chemico-genetic approach, we identify histone H3K27 demethylases KDM6A and KDM6B as central regulators of human Th subsets. The prototypic KDM6 inhibitor GSK-J4 increases genome-wide levels of the repressive H3K27me3 chromatin mark and leads to suppression of the key transcription factor RORγt during Th17 differentiation. In mature Th17 cells, GSK-J4 induces an altered transcriptional program with a profound metabolic reprogramming and concomitant suppression of IL-17 cytokine levels and reduced proliferation. Single-cell analysis reveals a specific shift from highly inflammatory cell subsets toward a resting state upon demethylase inhibition. The root cause of the observed antiinflammatory phenotype in stimulated Th17 cells is reduced expression of key metabolic transcription factors, such as PPRC1. Overall, this leads to reduced mitochondrial biogenesis, resulting in a metabolic switch with concomitant antiinflammatory effects. These data are consistent with an effect of GSK-J4 on Th17 T cell differentiation pathways directly related to proliferation and include regulation of effector cytokine profiles. This suggests that inhibiting KDM6 demethylases may be an effective, even in the short term, therapeutic target for autoimmune diseases, including ankylosing spondylitis.

Background Alterations in epigenetic mechanisms have been implicated in the regulation of pro-inflammatory cytokine production, and bromodomain-containing proteins (the BET family) may have a specific role in the transcriptional regulation of inflammatory genes. Our objective was to examine specific epigenetic variation between the responses of macrophages derived from RA patients and healthy volunteers, and those derived from inflammatory synovial fluid (SF). Delineation of any epigenetic differences between different macrophage populations may highlight new therapeutic targets in inflammatory disease. Materials and methods A library of small molecule inhibitors, including BET protein inhibitors, was used to identify key epigenetic regulators of pro-inflammatory cytokine production from macrophages. Macrophages were differentiated from the blood monocytes of normal volunteers and RA patients. Synovial fluid derived macrophages were isolated from RA patients undergoing therapeutic arthrocentesis. Cell culture media was supplemented with a small molecule inhibitor, and lipopolysaccharide subsequently used to stimulate inflammatory cytokine production. Cell viability was confirmed using WST-1 assay, cytokine production was measured using an MSD platform and qPCR to assess gene expression. Each experimental condition was performed in triplicate, alongside positive and negative controls. The study was ethically approved and all samples were obtained with informed consent. Results Inhibitors of BET proteins (JQ1, I-BET151 and PFI-1) were the only class of inhibitor to show consistent down-regulation of pro-inflammatory cytokines in both healthy and RA-patient derived macrophages. BET protein inhibitors significantly suppressed the production of TNFα and IL-6 production from RA blood derived macrophages and IL-6 from corresponding SF-derived macrophages. However, only JQ1 achieved significant inhibition of TNFα production from SF-derived macrophages; it also abrogated LPS-induced cytokine and chemokine mRNA expression in the macrophages of patients with RA. Conclusions This data suggests that the BET proteins are essential regulators of pro-inflammatory cytokine and chemokine production from the macrophages of patients with RA. The differential response to BET inhibition by macrophages derived from blood and synovial fluid of RA patients could indicate alteration in BET protein distribution at pro-inflammatory cytokine promoters, influenced by the chronic inflammatory environment. This study highlights the therapeutic potential of BET inhibitors (particularly JQ1) in RA. Funding acknowledgements NIHR BRU, BBSRC and GlaxoSmithKline