Fig. 2
M1 to M2a conversion of microglia alters secretory profiles, occurs at the single cell level, and requires PKA, but not EPAC, signaling in the presence of IL-4. Treatment of M1 (LPS; 100 ng/ml or TNF-α stimulated 10 ng/ml) BV2 microglia with db-cyclic AMP (1 mM) and IL-4 (10 ng/ml) beginning at 15 min prior to activation produced maximal abrogation of cytokines, TNF-α (a) and IP-10 (b) at 24 h post-stimulus. Levels of IL-1β remained statistically indifferent between M0, M1, and M2a phenotypes (c), with only db-cyclic AMP alone producing a significant increase over TNF-α stimulated controls. Examination of primary cortical microglia at the single cell level showed, compared to untreated M0 microglia (d, e), a significant shift in the ratio of Arginase-1 (red) to iNOS (green) expression, favoring the latter (g–i), occurred when the microglia were induced to the M1 form with TNF-α. The exposure of M1 microglia to either IL-4 (j–l) or db-cyclic AMP (m–o) independently failed to alter this ratio. In contrast, concurrent use of db-cyclic AMP and IL-4 significantly shifted the ratio of Arginase-1 to iNOS expression to one favoring the former (p–r), indicative of M1 to M2a phenotypic conversion. Statistical significance indicated at ***p < 0.001 or **p < 0.01 versus iNOS FL intensity/cell. Scale bar = 15 μm. s Immunoblotting for Arginase-1 in BV2 microglia showed little expression in M0-untreated cells or M1 cells induced with LPS and either untreated or exposed to IL-4. Concurrent delivery of IL-4 with cyclic AMP analogs showed that those that selectively activated PKA (db-cyclic AMP, 1 mM; dioctanoyl-cyclic AMP, 100 μM; phenyl-cyclic AMP, 100 μM), but not EPAC (CPT-2′O methyl-cyclic AMP, 100 μM and 8-bromo-2′O methyl-cyclic AMP, 100 μM), induced robust Arginase-1 expression, indicative of M1 to M2a phenotypic conversion. t Densitometry values for Arginase-1 in untreated and treatment groups normalized to β-actin. The expression of Arginase-1 (red) and iNOS (green) in primary microglia treated with LPS alone (u1–u2) or in conjunction with IL-4 and cyclic AMP analogs that activate PKA and/or EPAC (v1–x2), shows the importance of PKA in mediating conversion towards an Arg1+/iNOS- phenotype in the presence of IL-4. Statistical significance indicated at ***p < 0.001 or *p < 0.05 versus untreated controls or ### p < 0.001 or # p < 0.05 versus LPS-only-treated controls. c–j Immunocytochemical staining of primary microglia for Arginase-1 (red) and macrosialin (ED1; green) in control and treated microglia showed robust induction of Arginase-1 with PKA-selective, but not EPAC-selective, cyclic AMP analogs in the presence of LPS and IL-4. Cell nuclei were counterstained with Hoechst (blue). Scale bar = 15 μm. Results shown from three independent culture replicates