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HDAC Inhibitor Sodium Butyrate-Mediated Epigenetic Regulation Enhances Neuroprotective Function of Microglia During Ischemic Stroke

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Abstract

Cerebral ischemia leads to neuroinflammation and activation of microglia which further contribute to stroke pathology. Understanding regulation of microglial activation will aid in the development of therapeutic strategies that mitigate microglia-mediated neurotoxicity in neuropathologies, including ischemia. In this study, we investigated the epigenetic regulation of microglial activation by studying histone modification histone 3-lysine 9-acetylation (H3K9ac) and its regulation by histone deacetylase (HDAC) inhibitors. In vitro analysis of activated microglia showed that HDAC inhibitor, sodium butyrate (SB), alters H3K9ac enrichment and transcription at the promoters of pro-inflammatory (Tnf-α, Nos2, Stat1, Il6) and anti-inflammatory (Il10) genes while inducing the expression of genes downstream of the IL10/STAT3 anti-inflammatory pathway. In an experimental mouse (C57BL/6NTac) model of middle cerebral artery occlusion (MCAO), we observed that SB mediates neuroprotection by epigenetically regulating the microglial inflammatory response, via downregulating the expression of pro-inflammatory mediators, TNF-α and NOS2, and upregulating the expression of anti-inflammatory mediator IL10, in activated microglia. Interestingly, H3K9ac levels were found to be upregulated in activated microglia distributed in the cortex, striatum, and hippocampus of MCAO mice. A similar upregulation of H3K9ac was detected in lipopolysaccharide (LPS)-activated microglia in the Wistar rat brain, indicating that H3K9ac upregulation is consistently associated with microglial activation in vivo. Altogether, these results show evidence of HDAC inhibition being a promising molecular switch to epigenetically modify microglial behavior from pro-inflammatory to anti-inflammatory which could mitigate microglia-mediated neuroinflammation.

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Acknowledgments

This research was funded by the NUHS seed fund for basic science research (Grant No. T1-BSRG 2014-02; WBS No. R181-000-166-112).

Authors’ Contributions

RP, graduate student conceived the study, designed and performed experiments, and wrote the manuscript. TVA performed tMCAO and in vivo SB treatment. NG performed in vitro experiments relating to pSTAT3 targets and analyzed data. TVA, NG, and STD provided intellectual contribution and edited the manuscript. STD is the principal investigator of the study.

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  1. Department of Anatomy, The Yong Loo Lin School of Medicine, National University of Singapore, MD10, 4 Medical Drive, Singapore, 117594, Singapore

    Radhika Patnala, Neelima Gupta & S Thameem Dheen

  2. Department of Physiology, The Yong Loo Lin School of Medicine, National University of Singapore, MD9, 2 Medical Drive, Singapore, 117597, Singapore

    Thiruma V Arumugam

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  1. Radhika Patnala
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Ethics Approval and Consent to Participate

All animal procedures were carried out in accordance with the National University of Singapore Institutional Animal Care and Use committee (IACUC) guidelines (NUS/IACUC/R15-0051). All efforts were made to minimize pain and number of animals used.

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Online resource 1

Table 1: Antibodies. The following table lists the antibodies used in the study and their optimal dilutions and the secondary antibody pairing respective to the experiment. Table 2: Primer sets used for qPCR gene expression analysis. Table 3: Primer sets used for CHIP-qPCR analysis (PDF 135 kb)

Online resource 2

Expression of H3K9ac, pSTAT1, and IL10 in primary microglia subject to SB treatment. a Immunofluorescence staining displayed an upregulation of H3K9ac (red) levels in primary microglia in response to SB treatment. b Immunofluorescence staining displayed an upregulation of pSTAT1 in response to LPS-mediated microglial activation in primary microglial cultures. The pSTAT1 (red) expression was suppressed in the presence of SB treatment. c Immunofluorescence staining displayed an upregulation of IL10 (red) in response to SB treatment. Primary microglial cells stained with CD11b (green) used as microglial marker. DAPI (blue) staining nuclei; n = 3. Scale bars (white) denote 20 μm. (PDF 344 kb)

Online resource 3

Enrichment of total H3 indicates nucleosome density, from cell cultures consisting of untreated control, 1 h, and 6 h LPS treatment, with and without pre-treatment with SB (2.5 mM) analyzed by chromatin immunoprecipitation assay. Primers flank promoter approx. 100 bp regions near and downstream of transcription start sites (TSS) of gene promoters Il10 (a), Fcrlb (b), Tnf-α (c), Il6 (d), Nos2 (e), and Stat1 (f), respectively. Data represented as mean + SEM; n = 3 cultures; One-way ANOVA, Tukey’s post hoc test; *p value <0.05, **<0.01; ***<0.001. (PDF 211 kb)

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Patnala, R., Arumugam, T.V., Gupta, N. et al. HDAC Inhibitor Sodium Butyrate-Mediated Epigenetic Regulation Enhances Neuroprotective Function of Microglia During Ischemic Stroke. Mol Neurobiol 54, 6391–6411 (2017). https://doi.org/10.1007/s12035-016-0149-z

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