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Fig. 1 | Journal of Neuroinflammation

Fig. 1

From: Cyclic AMP is a key regulator of M1 to M2a phenotypic conversion of microglia in the presence of Th2 cytokines

Fig. 1

Synergistic action of cyclic AMP and IL-4 in M1 to M2a phenotypic conversion of microglial cells. In LPS (100 ng/ml)-stimulated BV2 microglia, combined treatment with db-cyclic AMP (1 mM) and IL-4 (10 ng/ml) beginning at 15 min prior to M1 activation produced dramatic reductions in the M1 markers (red) iNOS (a, f) and COX-2 (b, g) at 24 h post-stimulus while concurrently increasing M2a markers (red) Arginase-1 (c, h), transglutaminase-2 (d, i), and RELM-α (e, j). Scale bar = 12 μm. Similarly, conversion of M0 BV2 microglia (k, l) to M1 with TNF-α produced robust iNOS expression (m) without immunoreactivity for Arginase-1 (n). IL-4 (10 ng/ml) alone produced some expressions of Arginase-1 (p) but did not alter iNOS production (o), while db-cyclic AMP (1 mM) reduced iNOS and induced little Arginase-1 expression in TNF-α-stimulated microglia (r). Only the simultaneous addition of IL-4 and db-cyclic AMP to TNF-α-induced M1 microglia produced profound M2a conversion as evidenced by a loss of iNOS (s) and strong Arginase-1 expression (t). Microglial cells are stained with phalloidin-488 (green) to demark the cell morphology, and the nuclei of the cells have been counterstained with Hoechst (blue). Scale bar = 20 μm. Immunoblotting showed that only the combination of db-cyclic AMP and IL-4 produced a robust induction of Arginase-1 in M0 or M1 microglia (stimulated with either LPS or TNF-α; u, v). Immunoblotting for Arginase-1 and iNOS in LPS-stimulated BV2 microglia following their exposure to increasing concentrations of IL-4 (fixed db-cyclic AMP concentration, w, x) or with increasing concentrations of db-cyclic AMP (fixed IL-4 concentration, y, z) shows the concentrations at which synergistic action is observed in their conversion of microglia from M1 to M2a. Densitometry values for Arginase-1 and iNOS were normalized to β-actin. Statistical significance indicated at ***p < 0.001, **p < 0.01, or *p < 0.05 versus LPS-only-treated controls. Results are shown from three independent culture replicates

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