HDAC8-Mediated Epigenetic Reprogramming Plays a Key Role in Resistance to Anthrax Lethal Toxin–Induced Pyroptosis in Macrophages

Abstract

Macrophages pre-exposed to a sublethal dose of anthrax lethal toxin (LeTx) are refractory to subsequent high cytolytic doses of LeTx, termed toxin-induced resistance (TIR). A small population of TIR cells (2–4%) retains TIR characteristics for up to 5–6 wk. Through studying these long-term TIR cells, we found that a high level of histone deacetylase (HDAC)8 expression was crucial for TIR. Knocking down or inhibition of HDAC8 by small interfering RNAs or the HDAC8-specific inhibitor PCI-34051, respectively, induced expression of the mitochondrial death genes Bcl2 adenovirus E1B 19 kDa–interacting protein 3 (BNIP3), BNIP3-like and metastatic lymph node 64, and resensitized TIR cells to LeTx. Among multiple histone acetylations, histone H3 lysine 27 (H3K27) acetylation was most significantly decreased in TIR cells in an HDAC8-dependent manner, and the association of H3K27 acetylation with the genomic regions of BNIP3 and metastatic lymph node 64, where HDAC8 was recruited to, was diminished in TIR cells. Furthermore, overexpression of HDAC8 or knocking down the histone acetyltransferase CREB-binding protein/p300, known to target H3K27, rendered wild-type cells resistant to LeTx. As in RAW264.7 cells, primary bone marrow–derived macrophages exposed to a sublethal dose of LeTx were resistant to LeTx in an HDAC8-dependent manner. Collectively, this study demonstrates that epigenetic reprogramming mediated by HDAC8 plays a key role in determining the susceptibility of LeTx-induced pyroptosis in macrophages.

Inhibition of Interleukin 1β (IL-1β) Expression by Anthrax Lethal Toxin (LeTx) Is Reversed by Histone Deacetylase 8 (HDAC8) Inhibition in Murine Macrophages

“…Primary bone marrow-derived macrophages (BMDMs) were prepared from 129/Svj mice or C57BL/6j mice as described previously (54). RAW264.7 cells stably transfected with pEGFP or pEGFP-HDAC8 were prepared and cultured as described previously (37). Transfections of RAW264.7 cells and BMDMs with siRNAs and ASOs were performed using the Lipofectamine RNAi Max kit (Invitrogen), according to the manufacturer's instructions.…”

“…Western Blotting-Preparation of total cell lysates and immunoblotting were performed as reported previously (37).…”

“…Chromatin Immunoprecipitation (ChIP) Analysis -ChIP analysis was conducted as described previously (37), using H3K27Ac antibody (Active Motif), anti-NF-B (p65; eBioscience), or rabbit IgG antibody (Sigma) as a control. Purified DNAs were subjected to quantitative real time PCR analysis using Power SYBR Green PCR Master Mix (Applied Biosystems) and primers targeting up to ϳ10 kb upstream of the IL-1␤ transcription starting site.…”

“…Quantitative Real Time PCR-eRNA or mRNA expression was quantified by quantitative real time PCR (qPCR) as reported previously (37). Briefly, total cellular RNAs were isolated using TRIzol (Life Technologies, Inc.) according to the manufacturer's instructions and reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (New England Biotechnology) with random hexamers (for eRNA) or oligo(dT) primers (for mRNA).…”

“…Levels of histone acetylation are regulated by two families of enzymes (36), the histone acetyltransferases and histone deacetylases (HDACs). We previously showed that LeTx up-regulates HDAC8 expression, which is involved in silencing the mitochondrial death genes, likely through targeting acetylated histone H3 lysine 27 (H3K27Ac) (37). HDAC8 was shown to target the core histones H2A/H2B, H3, and H4 in vitro (38 -40); however, in vivo activity of HDAC8 toward histones and specific histone residues is unknown (41).…”