Figure 1: LPS induced hyper-induction of IL-10 in HDAC6-deficient macrophages and mice.

(a) HDAC6 was stably knocked down in macrophages (RAW264.7 cells) by two different HDAC6 shRNAs (no. 321323 and no. 321357). The levels of HDAC6 protein and α-tubulin acetylation were determined by immunoblotting. (b,c) Control and HDAC6 KD (shRNA 321323) macrophages were treated with LPS (1 μg ml⁻¹) for 18 h and medium was subjected to ELISA for TNF-alpha and IL-1beta (b) and IL-10 production (c).HDAC6 KD by either shRNA significantly enhanced IL-10 production. 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). (d) Bone marrow-derived primary macrophages (BMDM) isolated from WT and HDAC6 KO mice were treated with LPS (1 μg ml⁻¹) for indicated time and medium was assayed for IL-10 levels by ELISA. HDAC6 KO BMDM produced significantly more IL-10 production compared to WT macrophages. 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). (e) WT and HDAC6 KO mice were challenged by LPS (50 μg per mouse) through intraperitoneal (i.p.) injection and serum was analysed for IL-10 at 4 h post injection. Without LPS injection (PBS injection), IL-10 was not detectible. The graph shows the means with s.e.m. (error bars). Asterisks indicate statistical significance (**P<0.01, Student’s t-test).