Novel Roles for HDAC4 in Innate Immunity and Inflammasome Signaling
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Date
2020-05
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The Ohio State University
Abstract
Histone deacetylases (HDACs) are important mediators of gene expression, in which deacetylation leads to changed chromatin structure that can cause repression or activation of transcription. HDACs have also been found to modulate innate immunity through post-translational regulation; however, the mechanisms involved aren't well understood. Although pan-HDAC inhibitors have been shown to limit IL-1beta secretion in response to inflammasome activation, these broad-spectrum agents have many off-target effects. The objective of this study was two-fold: 1) to examine post-translational mechanisms that regulate cytosolic DNA sensing and inflammasome activation, two important aspects of the innate immune system, and 2) to assess the specific HDAC enzymes involved which may pave the way for more targeted therapies.
First, GFP-tagged nucleotide-binding domain (NOD)-like receptor family, pyrin domain containing 3 (NLRP3), a component of NLRP3 inflammasome, was co-transfected into HEK 293T cells along with various FLAG-tagged HDACs. NLRP3-HDAC association was then analyzed via GFP pull-down assay followed by anti-FLAG western blot, which revealed that NLRP3 associates with HDAC4. Transfection of cells with the double-stranded DNA absent in melanoma 2 (AIM2) inflammasome agonist followed by analysis by confocal microscopy revealed that HDAC4 also colocalizes with AIM2 inflammasomes. Since DNA has negative charge and HDAC inhibition increases acetylation levels, we hypothesized that HDAC4 loss of function may compromise the ability of high mobility group box (HMGB) proteins to sense cytosolic DNA. Both pharmacological inhibition of HDAC4 and knockout via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) impaired HMGB1 and HMGB2 interaction with poly dA:dT as assessed by biotin-pulldown assays and confocal microscopy. Finally, we note impaired IL-1beta secretion in HDAC4 (-/-) mouse embryonic fibroblasts relative to wild-type controls in an in vitro inflammasome reconstitution model. Impaired IL-1beta release in the HDAC4 (-/-) cells could be partially rescued via exogenous HDAC4 transfection, indicating that HDAC4 plays a novel,
likely post-transcriptional role in cytosolic DNA sensing and inflammasome function. Current studies are underway to further characterize the mechanism(s) at work, and to determine whether specific targeting of HDAC4 may be a possible, more targeted treatment option for autoimmune or inflammatory disorders.
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Keywords
Inflammasomes, Innate Immunity, HDAC4, HDAC Inhibitors