Figure 2: α-tubulin acetylation activates LPS-induced IL-10 production in macrophages.

(a) Macrophages (RAW264.7) were pretreated with a HDAC6 inhibitor, Tubastatin A (TBSA), at indicated doses for 12 h and followed by LPS (1 μg ml⁻¹) treatment for 18 h. Medium was subjected to ELISA for IL-10 production. TBSA treatment markedly increased LPS-induced IL-10 production in a dose-dependent manner. (b) Macrophages were stained with an acetylated α-tubulin-specific antibody after LPS treatment at the indicated time. Immuno-staining showed that LPS treatment increased acetylated microtubules (green: Ac-tubulin; blue: DAPI; scale bar, 10 μM). (c) Macrophages were treated with LPS and analysed for acetylated tubulin, MEC17 and HDAC6 protein levels at indicated time. (d) MEC17 KD and control macrophages were pretreated with or without TBSA (10 μM) for 4 h and followed by LPS treatment for 18 h. Medium was harvested for IL-10 assay. Note that MEC17 KD strongly inhibited IL-10 production. (e) MEC17 KD macrophages were electroporated with pcDNA -EGFP, -EGFP-α-tubulin WT, K40→R and K40→Q mutant plasmids. Twenty four hours after transfection, cells were treated with LPS (1 μg ml⁻¹) for 18 h and medium was subjected to IL-10 analysis. The graph shows the means with s.e.m. (error bars) from three experiments (Student’s t-test). (f) HDAC6 KD and control macrophages were pretreated with nocodazole (NOC) for 4 h followed by another 18-h LPS treatment. NOC treatment reduced α-tubulin acetylation (bottom panel: immuno-blotting) and significantly blunted LPS-induced IL-10 levels in control and HDAC6 KD macrophages (upper panel: ELISA). (g) Macrophages were pretreated with microtubule-stabilizing agent, Taxol, for 4 h and then followed by another 18-hours LPS treatment. Taxol treatment led to a dose-dependent increase in α-tubulin acetylation (bottom panel: immuno-blotting) and a higher induction of IL-10 (upper panel: ELISA). (h) MEC17 KD macrophages were pretreated with Taxol for 4 h and followed by 18 h of LPS treatment. MEC17 KD significantly reduced Taxol-induced IL-10 production. For all ELISA assay, the graph shows the means with s.e.m. (error bars) from three experiments. Asterisks indicate statistical significance (**P<0.01 and ***P<0.001, Student’s t-test).