Sodium Acetate Enhances Neutrophil Extracellular Trap Formation via Histone Acetylation Pathway in Neutrophil-like HL-60 Cells

Abstract

Neutrophil extracellular trap formation has been identified as a new cell death mediator, termed NETosis, which is distinct from apoptosis and necrosis. NETs capture foreign substances, such as bacteria, by releasing DNA into the extracellular environment, and have been associated with inflammatory diseases and altered immune responses. Short-chain fatty acids, such as acetate, are produced by the gut microbiota and reportedly enhance innate immune responses; however, the underlying molecular mechanisms remain unclear. Here, we investigated the effects of sodium acetate, which has the highest SCFA concentration in the blood and gastrointestinal tract, on NETosis by focusing on the mechanisms associated with histone acetylation in neutrophil-like HL-60 cells. Sodium acetate enhanced NETosis, as shown by fluorescence staining with SYTOX green, and the effect was directly proportional to the treatment duration (16-24 h). Moreover, the addition of sodium acetate significantly enhanced the acetylation of Ace-H3, H3K9ace, and H3K14ace. Sodium acetate-induced histone acetylation rapidly decreased upon stimulation with the calcium ionophore A23187, whereas histone citrullination markedly increased. These results demonstrate that sodium acetate induces NETosis via histone acetylation in neutrophil-like HL-60 cells, providing new insights into the therapeutic effects based on the innate immunity-enhancing effect of dietary fiber.

Keywords: NETosis; acetate; histone acetylation; histone citrullination; immune response; neutrophil extracellular trap; peptidyl arginine deiminase 4.

Figure 1

Sodium acetate increases A23187-induced neutrophil extracellular trap formation (NETosis) (NOX-independent NETosis). (a) After treatment with or without 10 mM sodium acetate during differentiation, NETosis levels in nHL-60 cells treated with (+) or without (−) 10 μM A23187 for 4 h were analyzed through SYTOX green assay (n = 5). Control was not treated with sodium acetate. 16 hr and 24 hr: incubation time of sodium acetate. After sodium acetate incubation, cells were stimulated by A23187. (b) After treatment with or without 10 mM sodium acetate during differentiation, NETosis images were obtained using confocal microscopy of SYTOX green (DNA)-stained nHL-60 cells treated with (+) or without (−) 10 μM A23187 for 2 h.

Figure 2

Sodium acetate treatment did not increase ROS production in nHL-60 cells after treatment with or without 10 mM sodium acetate during differentiation. Control was not treated with sodium acetate. 16 hr and 24 hr: incubation time of sodium acetate. After sodium acetate incubation, cells were stimulated by A23187. Quantitative chemiluminescence analysis of ROS production in nHL-60 cell culture incubated with or without 10 μM A23187 for 90 min was performed. Data are shown as mean ± SD (n = 3).

Figure 3

Effect of ACSS2 inhibitor on release of extracellular DNA. After treating nHL-60 cells with or without 10 μ M ACSS2 inhibitor during sodium acetate treatment, extracellular DNA was isolated and measured by performing extracellular DNA quantification assay. +: Addition of reagents, −: No treatment. Data are shown as mean ± SD (n = 3).

Figure 4

Expression of ACSS2, MCT-1, and MCT-4 in nHL-60 cells. After treatment with or without sodium acetate (1 or 10 mM) for 24 h, ACSS2, MCT-1, and MCT-4 expression levels in nHL-60 cells were analyzed using Western blotting. −: No treatment. Data are shown as mean ± SD (n = 3).

Figure 5

Sodium acetate enhanced histone acetylation. The nHL-60 cells were incubated with or without sodium acetate for 1 or 24 h. −: The control was not treated with sodium acetate. Following histone extraction, histone acetylation levels were analyzed using Western blotting. Loading of the histones was monitored using Coomassie staining (denoted as CBB). The data are shown as the mean ± SD (n = 3).

Figure 6

Histone H3 acetylation in nHL-60 cells. The nHL-60 cells were incubated with or without sodium acetate for 24 h. Following histone extraction, histone acetylation (denoted as H3K9ace, H3K14ace, H3K18ace, and H3K27ace) levels were analyzed by Western blotting using specific antibodies. −: The control was not treated with sodium acetate. +: Addition of 10 mM CH₃COONa. Loading of the histones was monitored using Coomassie staining (denoted as CBB). The data are shown as the mean ± SD (n = 3).

Figure 7

Histone acetylation levels before and after A23187 stimulation. The nHL-60 cells were incubated with or without 10 mM sodium acetate for 24 h. Then, the cells were stimulated with 10 μM A23187. Following histone extraction, histone acetylation levels were analyzed by Western blotting. The control was not treated with sodium acetate. +: Addition of 10 mM CH₃COONa, −: No treatment. Loading of the histones was monitored using Coomassie staining (denoted as CBB). The data are shown as the mean ± SD (n = 3).

Figure 8

Sodium acetate enhanced histone citrullination but not PAD4 expression. (a) nHL-60 cells (treated with or without 10 mM sodium acetate) were treated with or without 10 μM A23187 for 3 h. Following histone extraction, citrullinated histone H3 (denoted as citH3) protein levels were analyzed via Western blotting. Loading of histones was monitored using Coomassie staining (denoted as CBB). +: Addition of reagents, −: No treatment. (b) nHL-60 cells were incubated with or without 10 mM sodium acetate. After cell extraction, PAD4 expression was determined through Western blotting. Ct: Control was not treated with sodium acetate. Data are shown as mean ± SD (n = 3).