RAW264.7 cells were cultured in the existence or absence of SB (5C15 m) PCI (20 nm) for 20 h and then treated with LPS (100 ng/ml) for 3 h. main virulence factor that causes immune paralysis and mortality (1). LeTx is known to cause immune suppression by targeting the MAPK pathway that is involved in all-trans-4-Oxoretinoic acid many aspects of immune responses (2,C5). It includes two proteins, protective antigen (PA)2 and lethal factor (LF) (6,C10). PA is a carrier protein that incorporates LF into the cytoplasm; LF is a metalloprotease that cleaves all MAPK kinases (MEK1C7), except MEK5 (11) and MEK7 (12). Inactivation of these MEKs results in almost complete inactivation of MAPKs, including the ERKs and p38 MAPK (p38), but partial or no effects on JNK/SAPKs in macrophages (13, 14) and other cell types (12, 15). Inhibition of ERKs and p38 by LeTx suppresses expression of various inflammatory cytokines in macrophages (16,C18). Particularly, IL-1, which is expressed as a pro-IL-1 form and proteolytically matured by caspase-1, plays an important role in mounting early immune responses to germinating (19), as well as multiple other bacterial pathogens (19,C26). Severe immune suppression caused by LeTx is the main all-trans-4-Oxoretinoic acid culprit for unrestricted proliferation of inside the host (3, 5, 27, 28). Epigenetics is a cellular mechanism that inheritably regulates expression of genes without altering genomic DNA sequences in response to developmental and environmental cues. There are three distinct but inter-related mechanisms in epigenetics as follows: DNA methylation, chromatin structure modification, and non-coding all-trans-4-Oxoretinoic acid RNAs. Among them, modifications of the N-terminal regions of histones by phosphorylation, methylation, and acetylation dynamically orchestrate chromatin structures and regulate gene expression. In macrophages, these epigenetic mechanisms are involved in activation (29), differentiation (30,C32), and endotoxin tolerance (33,C35). Various bacterial and viral microbes also epigenetically manipulate host immune responses to render environments suitable for their survival and proliferation. LeTx was shown to inhibit histone H3 serine 10 (H3S10) phosphorylation by targeting the ERK and p38 and to suppress recruitment of NF-B to gene-specific promoters, including IL-6 and IL-8 (15). Acetylation on lysine residues is another key histone modification, mainly associated with transcriptional activation of genes. 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 (38,C40); however, activity of HDAC8 toward histones and specific histone residues is unknown (41). Enhancers are O111:B4 LPS were purchased from the List Biological Laboratories. Epigenetic chemical inhibitors used in this study are the following: apicidin (Santa Cruz Biotechnology); aza-2-deoxycytidine (azacitidine; Sigma); CAY10603 (Cayman Chemical); mocetinostat (MGCD0103; Selleck); MC1568 (APExBio Technology); PCI-34051 (Cayman Chemical); and panobinostat (LBH-589; Selleck). The ERKs, p38 MAPK inhibitors, U0126, SB203580, and NF-B activation inhibitor, respectively, were purchased from Calbiochem. Anti-GFP and antibody raised against the N terminus of MEK1 (MEK1-NT) were obtained from Life Technologies, Inc., and StressGen Bioreagents, respectively. Antibodies for phospho-p38, phospho-ATF-2, phospho-IB, and IB were obtained from Cell Signaling. Monoclonal SMO antibody for HDAC8 was purchased from Epigentek (catalog all-trans-4-Oxoretinoic acid no. A-4008). Antibodies for H3K4Ac, H3K14Ac, H3K18Ac, H3K23Ac, H3K27Ac, H3K36Ac, H3K56Ac, H3K79Ac, and H3K27me3 were from Active Motif; pan-histone H3 was from BioVision; -actin was from Rockland Inc.; and anti-NF-B (p65) was from eBioscience. The pro-IL-1 antibody was a kind gift from Dr. Aurigemma (NCI-Frederick Cancer Research and Development Center, Frederick, MD). Predesigned siRNAs targeting HDAC8 (catalog no. SI1063902) and antisense oligonucleotides (ASO; LNATM GapmeRs, Exiqon) targeting pro-IL-1 eRNA (CAATCCTGGTTGATGA) were purchased from Qiagen and Exiqon, respectively. Cell Culture and Transfection RAW264.7 macrophages were cultured in Dulbecco’s modified Eagle’s medium as described previously.