Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells

Abstract

Natural killer (NK) cells are innate lymphocytes, important in immune surveillance and elimination of stressed, transformed, or virus-infected cells. They critically shape the inflammatory cytokine environment to orchestrate interactions of cells of the innate and adaptive immune systems. Some studies have reported that NK cell activation and cytokine secretion are controlled epigenetically but have yielded only limited insight into the mechanisms. Using chemical screening with small-molecule inhibitors of chromatin methylation and acetylation, further validated by knockdown approaches, we here identified Jumonji-type histone H3K27 demethylases as key regulators of cytokine production in human NK cell subsets. The prototypic JMJD3/UTX (Jumonji domain–containing protein 3) H3K27 demethylase inhibitor GSK-J4 increased global levels of the repressive H3K27me3 mark around transcription start sites of effector cytokine genes. Moreover, GSK-J4 reduced IFN-γ, TNFα, granulocyte–macrophage colony-stimulating factor (GM-CSF), and interleukin-10 levels in cytokine-stimulated NK cells while sparing their cytotoxic killing activity against cancer cells. The anti-inflammatory effect of GSK-J4 in NK cell subsets, isolated from peripheral blood or tissue from individuals with rheumatoid arthritis (RA), coupled with an inhibitory effect on formation of bone-resorbing osteoclasts, suggested that histone demethylase inhibition has broad utility for modulating immune and inflammatory responses. Overall, our results indicate that H3K27me3 is a dynamic and important epigenetic modification during NK cell activation and that JMJD3/UTX-driven H3K27 demethylation is critical for NK cell function.

Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism

“…It was of interest to validate the earlier anti-inflammatory observations within a context of chronic inflammation. Consistent with our earlier work on chronic inflammation in NK cells (36), we observe IFN-γ reduction in GSK-J4 treated CD3 + T cells isolated from Rheumatoid Arthritis (RA) patients ( Figure 2A ), and also of IL-17 in AS patients ( Figure 2B ). A significant reduction in the proportion of cells in G2/M phase ( Figure 2C ), associated with reduced Th17 proliferation following treatment with GSK-J4 ( Figure 2D ) was observed.…”

“…A conspicuous transcriptional response to GSK-J4 treatment, corroborated by knockdown experiments, is a change in metabolic gene expression. Although we observe a characteristic ATF4 signature in NK cells following GSK-J4 treatment (36), we only observe a robust DDIT3 upregulation in this study with a consistent lack of upregulated metabolic targets, which are otherwise associated with an ATF4-DDIT3 mediated stress response. Importantly, DDIT3 has been shown to directly repress critical Tcell transcription factors like T-bet, leading to reduced cytokine output in tumour-infiltrating Tcells and thus might contribute to the observed anti-inflammatory phenotype (42).…”

“…Single cell profiling using mass cytometry (CyTOF) allowed deep immune phenotyping and analysis of cell cycle progression of Th cells enriched from AS patients in response to GSK-J4 treatment. t-distributed stochastic neighbour embedding (t-SNE) plots ( Figure S4A ) confirm that GSK-J4 has a broad anti-inflammatory effect (36, 37, 40), highlighting a reduction in TNFα, IL-22 and IL-17 cytokines ( Figure S4B ). Activation markers such as HLA-DR and CD25 were found to be reduced following treatment with GSK-J4, in addition to a reduction in cell proliferation marker Ki-67 ( Figure S4C ).…”

“…A consistent downregulation of cytokine expression following treatment with inhibitors against the JmjC-domain containing histone demethylases (KDM6A/KDM6B), bromodomains and histone deacetylases (HDACs) was observed in all effector subsets ( Figure 1A, Figure S1A ), suggesting a common epigenetic susceptibility. Given the importance of H3K27 (KDM6) demethylases in cellular development (34, 35) and inflammation (36), we focused this study towards the prototypic KDM6 inhibitor GSK-J4 (37), displaying an EC 50 of 2μM ( Figure S1B) , and its inactive regional isomer GSK-J5 as a control. Using two different Th17 differentiation models, in which either CD4 + or CD45RA + cells are cultured in a cocktail of IL-6, IL-23 and TGF-β cytokines for 7 days, followed by treatment with DMSO, GSK-J4 or GSK-J5 for 48 hours, we observed a significant reduction in IL-17 and IFN-γ ( Figure 1B, Figure S2A ) by GSK-J4, with constant levels of RORC expression ( Figure S2A ).…”

Epigenetic inactivation of oncogenic brachyury (TBXT) by H3K27 histone demethylase controls chordoma cell survival

“…To identify epigenetic processes of biological importance in chordoma, we screened a library of tool compounds (Dataset S1) (25) targeting proteins involved in chromatin biology including several components of epigenetic readers, writers and erasers of a 'histone' or 'chromatin code', against five well characterised chordoma cell lines (www.chordomafoundation.com). A resazurin-based viability assay was used as the primary anti-proliferative readout (Fig.…”