*2.4. HDAC Inhibition Promotes an M1-to-M2 Phenotypic Transition and Skews the M2 Macrophage Phenotype Towards an M2c Feature*

To explore the role of HDAC inhibition on macrophage plasticity, we analyzed the phenotypes of interstitial macrophages in UUO after the administration of TSA. Upon treatment with TSA, immunohistochemistry showed that the infiltration of iNOS-positive M1 macrophages significantly decreased as compared to the vehicle group. Conversely, Arg1-positive macrophages were significantly upregulated in the TSA-treated kidneys following 14 days of obstruction (Figure 4A,B). Remarkably, TSA increased the number of SLAM-positive M2c macrophages but decreased the number of M2a macrophages. Immunofluorescent staining showed that both CLEC7A and SLAM colocalized with F4/80+CD206+ cells in obstructed kidneys, supporting that CLEC7A-positive and SLAM-positive cells represented M2a and M2c macrophages, respectively. Immunoblot confirmed that TSA downregulated the expression of iNOS and CLEC7A, as well as upregulated the expression of Arg1 and CLEC7A (Figure 4C–F). These data indicated that TSA suppressed the accumulation of proinflammatory M1 macrophages and profibrotic M2a macrophages in UUO. Instead, TSA increased the anti-inflammatory M2c macrophage infiltration, which limited the extent of inflammation and inhibited the activation of myofibroblasts and deposition of the extracellular matrix.

**Figure 4.** Trichostatin A (TSA) suppresses M1 and M2a macrophage infiltration but promotes M2c macrophage infiltration in unilateral ureteral obstruction (UUO). (**A**) Representative immunohistochemical images of inducible nitric oxide synthase (iNOS, M1 macrophage marker), arginase-1 (Arg1, pan-M2 macrophage marker), C-type lectin domain family 7 member A (CLEC7A, M2a macrophage marker) and signaling lymphocytic activation molecule (SLAM, M2c macrophage marker) in day 7 and day 14 obstructed kidneys either treated with the vehicle or TSA. Scale bar = 25 μm. (**B**) Quantification of the iNOS-, Arg1-, CLEC7A- and SLAM-positive interstitial cells in day 7 and day 14 obstructed kidneys treated with the vehicle or TSA. \*\* *p* < 0.01 and \*\*\* *p* < 0.001 by the unpaired Student's *t*-test; *n* = 5 for each group. (**C**) Western blots of iNOS, Arg1, CLEC7A and SLAM in day 7 and day 14 obstructed kidneys either treated with the vehicle or TSA. β-actin served as the loading control. (**D**) Quantification of iNOS, Arg1, CLEC7A and SLAM expression levels in obstructed kidneys. \* *p* < 0.05 and \*\* *p* < 0.01 by the unpaired Student's *t*-test; *n* = 5 for each group. (**E**) Immunofluorescent staining of CLEC7A, CD206 and F4/80 in obstructed kidneys. Scale bar = 25 μm. (**F**) Immunofluorescent staining of SLAM, CD206 and F4/80 in obstructed kidneys. Scale bar = 25 μm. White color indicates colocalization in the merged panels.
