HDAC inhibitors inhibit cytokine release by macrophages exposed to microbial products and bacteria. BMDMs were preincubated for 1 hour with or without TSA (100nM unless specified) before exposure to LPS (100 ng/mL), Pam₃CSK₄ (100 ng/mL), and heat-killed E coli or S aureus (10⁷ CFU/mL). (A-C) TNF, IL-6, and IL-12p40 mRNA (A) and protein (B-C) production by BMDMs. TNF, IL-6, and IL-12p40 mRNA levels were analyzed by RT-PCR, and results are expressed as the ratio of cytokines to GAPDH mRNA levels. Data are representative of 3 independent experiments. Cytokine were quantified in cell culture supernatants collected after 8 hours (TNF) and 18 hours (IL-6 and IL-12p40). Data are mean ± SD of triplicate samples from one experiment representative of 3 independent experiments. A.U. indicates arbitrary units. (D) BMDMs were preincubated for 1 hour with or without SAHA (4, 20, and 100nM) or VPA (4, 20, and 100μM) before exposure to Pam₃CSK₄ (100 ng/mL). TNF, IL-6, and IL-12p40 were quantified in cell culture supernatants collected after 8 hours (TNF) and 18 hours (IL-6 and IL-12p40). Data are mean ± SD of triplicate samples from 1 experiment representative of 3 independent experiments. (E-F) Human whole blood was incubated for 18 hours with VPA (E, 100μM) or TSA together with either LPS (10 ng/mL) or Pam₃CSK₄ (100 ng/mL). Cytokine and chemokine production was assessed by the Luminex technology (“Cytokine measurements”), and LPS/LPS + VPA ratios were calculated (E). TNF was quantified by bioassay. Data are mean ± SD of triplicate samples from one donor and are representative of 2 independent experiments (F).