*2.5. HDAC Inhibition Suppresses Proinflammatory and Profibrotic Phenotypes in Cultured M2 Macrophages*

To further clarify the effect of HDAC inhibition on the proinflammatory and profibrotic phenotypes of macrophages, J774A.1 macrophages were stimulated with IL-4/IL-13 or IL-10/TGF-b1 in the presence or absence of TSA for 24 h. IL-4/IL-13 induced the expression of Arg1 and CLEC7A in J774A.1 macrophages and polarized the cells towards an M2a phenotype. M2a J774A.1 macrophages increased the expression of profibrotic α-SMA and fibronectin, which were suppressed by TSA treatment. Furthermore, the amounts of proinflammatory tumor necrosis factor-α and iNOS in the M2a macrophages were also downregulated by TSA (Figure 5A,B). In contrast, IL-10/TGF-β1 induced the SLAM expression in J774A.1 macrophages and skewed the cells towards an M2c phenotype. The TSA treatment enhanced the SLAM expression in M2c J774A.1 macrophages and also further suppressed the amounts of TNF-α, iNOS, α-SMA and fibronectin in a dose-dependent manner (Figure 5C,D).

**Figure 5.** The effect of trichostatin A (TSA) on proinflammatory and profibrotic phenotypes of M2a and M2c macrophages. (**A**) Western blots of C-type lectin domain family 7 member A (CLEC7A), proinflammatory (tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS)) and profibrotic (α-smooth muscle actin (α-SMA) and fibronectin) expressions in J774A.1 macrophages treated with the indicated conditions for 24 h. Concentrations of interleukin (IL)-4 and IL-13 were both 20 ng/mL. β-actin served as the loading control. (**B**) Quantification for the levels of indicated proteins. \* *p* < 0.05 and \*\* *p* < 0.01 by ANOVA, followed by Tukey's post hoc multiple comparison test; *n* = 3 for each group. (**C**) Western blots of SLAM, proinflammatory (TNF-α and iNOS) and profibrotic (α-SMA and fibronectin) expressions in J774A.1 macrophages treated with the indicated conditions for 24 h. Concentrations of IL-10 and transforming growth factor (TGF)-β1 were both 20 ng/mL. β-actin served as the loading control. (**D**) Quantification for the levels of indicated proteins. \* *p* < 0.05 and \*\* *p* < 0.01 by ANOVA, followed by Tukey's post hoc multiple comparison test; *n* = 3 for each group.
