Acetate reduces microglia inflammatory signaling in vitro

Abstract

Acetate supplementation increases brain acetyl-CoA and histone acetylation and reduces lipopolysaccharide (LPS)-induced neuroglial activation and interleukin (IL)-1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen-activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor-kappa B (NF-κB) in primary and BV-2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS-induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL-6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor-β1 (TGF-β1) and both IL-4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2-4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS-induced elevation of NF-κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non-histone protein acetylation.

Figure 1

Time-dependent acetate-induced H3K9 hyperacetylation and dose-response study showing the effects of different LPS concentrations (0–25 ng/ml, 4 hr) on H3K9 acetylation and the expression of pro-inflammatory cytokines in BV-2 microglia. Panels A and C show representative images of the Western blots. Panel B shows the averaged proportion of H3K9 normalized to total H3 (n = 3) after 1, 2 and 4 hr of treatment with 12 mM sodium acetate. Panels D, E, F and G show the optical densities of H3K9 normalized to total H3 and the pro-inflammatory cytokines pro-IL-1β, IL-6, and TNF-α normalized to the loading control α-tubulin (n = 3). The graphs represent the means ± SD where statistical significance (* = compared to LPS 0 ng/ml) was set at p ≤ 0.05, as determined by One Way ANOVA followed by Tukey’s post-hoc test.

Figure 2

Changes in histone acetylation in primary mouse microglial cell culture stimulated for 4 hr with LPS 6.25 ng/ml, and the reversal of these effects upon treatment with 12 mM sodium acetate. Panel A shows representative images of the Western blots. Panels B and C show the optical densities of acetyl-CoA synthetase enzyme normalized to the loading control α-tubulin (n = 3) and H3K9 normalized to total H3 (n = 5), respectively. Panel D shows the quantification of the ratio of secreted LDH in the media to total cellular LDH (n = 5). Bars represent means ± SD where statistical significance (a = compared to NaCl, and b = compared to LPS + NaCl) was set at p ≤ 0.05, as determined by a one way ANOVA followed by Tukey’s post-hoc test.

Figure 3

Changes in the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in primary mouse microglial cell culture stimulated for 4 hr with LPS 6.25 ng/ml with and without 12 mM sodium acetate. Panel A shows representative images of the Western blots. Panels B, D and F show the optical densities of the pro-inflammatory cytokines pro-IL-1β, IL-6 and TNF-α, respectively, normalized to the loading control α-tubulin (n = 5). Panels C, E and G show the changes in the mRNA levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, quantified by qrt-PCR and normalized to β-actin (n = 5). Bars represent means ± SD where statistical significance (a = compared to NaCl, and b = compared to LPS + NaCl) was set at p ≤ 0.05, as determined by a one way ANOVA followed by Tukey’s post-hoc test.

Figure 4

Changes in histone acetylation in BV-2 microglial cell culture stimulated for 4 hr with LPS 6.25 ng/ml, and the reversal of these effects upon treatment with 12 mM sodium acetate. Panel A shows representative images of the Western blots. Panels B and C show the optical densities of acetyl-CoA synthetase enzyme normalized to the loading control α-tubulin and H3K9 normalized to total H3, respectively (n = 6). Panel D shows the quantification of the ratio of secreted LDH in the media to total cellular LDH (n = 6). Bars represent means ± SD where statistical significance (a = compared to NaCl, and b = compared to LPS + NaCl) was set at p ≤ 0.05 (n = 6, per group), as determined by a one way ANOVA followed by Tukey’s post-hoc test.

Figure 5

Changes in the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in BV-2 microglial cell culture stimulated for 4 hr with LPS 6.25 ng/ml with and without 12 mM sodium acetate. Panel A shows representative images of the Western blots. Panels B, D and F show the optical densities of the pro-inflammatory cytokines pro-IL-1β, IL-6 and TNF-α, respectively, normalized to the loading control α-tubulin. Panels C, E and G show the changes in the mRNA levels of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α, quantified by qrt-PCR and normalized to β-actin. Bars represent means ± SD where statistical significance (a = compared to NaCl, and b = compared to LPS + NaCl) was set at p ≤ 0.05 (n = 6, except pro-IL-1β where n = 5), as determined by a one way ANOVA followed by Tukey’s post-hoc test.

Figure 6

Changes in the anti-inflammatory cytokines TGF-β1, IL-4, and IL-10 in BV-2 microglial cell culture stimulated for 4 hr with LPS 6.25 ng/ml with and without treatment with 12 mM sodium acetate. Panel A shows representative images of the Western blots. Panels B, D and F show the optical densities of the anti-inflammatory cytokines TGF-β1, IL-4, and IL-10, respectively, normalized to the loading control α-tubulin (n = 6). Panels C, E and G show the changes in the mRNA levels of the anti-inflammatory cytokines TGF-β1, IL-4, and IL-10, quantified by qrt-PCR and normalized to β-actin (n = 6). Bars represent means ± SD where statistical significance (a = compared to NaCl, and b = compared to LPS + NaCl) was set at p ≤ 0.05, as determined by a one way ANOVA followed by Tukey’s post-hoc test.

Figure 7

Changes in the phosphorylation state of MAPK p38, JNK and ERK1/2 in BV-2 microglia stimulated for 0.5, 1, 2 and 4 hr with LPS 6.25 ng/ml with and without 12 mM sodium acetate. Panel A shows representative images of the Western blots from the 4 hr experiment. Panels B, C and D show the optical densities of phosphorylated MAPK p38, JNK and ERK1/2 normalized to the loading controls MAPK p38, JNK and ERK1/2, respectively (n = 6). The data represent the means ± SD where statistical significance (* = compared to NaCl) was set at p ≤ 0.05, as determined by One Way ANOVA followed by Tukey’s post-hoc test.

Figure 8

Changes in the protein level, phosphorylation and acetylation states of NF-κB p65 in BV-2 microglia cell culture stimulated for 4 hr with LPS 6.25 ng/ml with and without 12 mM sodium acetate. Panel A shows representative images of the Western blots. Panel B shows the optical density of total NF-κB p65 normalized to the loading control α-tubulin. Panels C, D and E show the optical densities of phosphorylated p65 at S536, phosphorylated p65 at serine 468 and acetylated p65 at lysine 310 normalized to total p65, respectively (n = 6). The data represent the means ± SD where statistical significance (a = compared to NaCl and b = compared to LPS) was set at p ≤ 0.05, as determined by One Way ANOVA followed by Tukey’s post-hoc test. Abbreviations are: S536, serine 536; S468; serine 468: and K310, lysine 310.