Figure 6.

Carbonic anhydrase inhibition suppresses M1 and M2 activation of bone marrow–derived macrophages (BMDMs). (A) Rat bone marrow was harvested, and cells were grown in the presence of macrophage colony-stimulating factor for 7 days. BMDMs were polarized toward M1 with LPS and IFN-γ or M2 with IL-4 and IL-13 for 24 hours. Untreated BMDMs were considered M0. (B) Expression of different carbonic anhydrase isoforms was assessed by qPCR (Car2 expression is set as 1). Carbonic anhydrase 2 (Car2) was the most abundant isoform in M0, M1, and M2 BMDMs (indicated by #). (C and D) Car2 is significantly upregulated in both M1- and M2-polarized BMDMs compared with M0 at both mRNA (C) and protein levels (representative Western blot shown in D). Quantification of four independent experiments in Figure E3A. (E) ACTZ suppresses M1 activation (Tnf, Il6, and Ccl2) of BMDMs in a dose-dependent manner. (F) Ethoxzolamide (EZA) suppresses M1 activation (Tnf, Il6, and Ccl2) of BMDMs in a dose-dependent manner. (G) ACTZ and EZA significantly reduce TNF-α and IL-6 secretion by M1-polarized BMDMs, measured by ELISA in cell culture supernatants. No detectable TNF-α or IL-6 in M0 control macrophage supernatants. (H) ACTZ and EZA suppress M2 activation (Ccl17, Arg1, and Cd163) of BMDMs. (B–H) Data represent mean ± SEM from three or four independent experiments. Statistical analysis by one-way ANOVA and Tukey’s post hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001).