METHODS: Macrophage polarization was investigated in bone marrow-derived macrophages (BMDMs) treated with or without Fasudil, the Rho-kinase specific inhibitor. Macrophage polarization was also studied in BMDMs transfected with dominant positive (L63), or dominant negative (L19) RhoA recombinant adenovirus. The effect of RhoA signaling inhibition was also assessed in mouse model of asthma.
RESULTS: CCL2 induced M1 macrophage polarization by upregulating IL-1β, iNOS, and IL-6 mRNA levels in alveolar macrophages and BMDMs. CCL2 synergistically enhanced LPS-induced M1 polarization. Fasudil diminished the effect of CCL2 and LPS-induced M1 polarization. M1 makers had higher expression levels in BMDMs transfected with RhoA L63 compared to RhoA N19. Vice versa, RhoA L63 downregulated, while RhoA N19 upregulated M2 markers (Arg1, Fizz1, and Chi3l3). Importantly, Fasudil treatment protected mice against cockroach allergen induced allergic inflammation by decreasing lung inflammatory cell infiltrates, goblet hyperplasia, airway hypersensitivity, BAL Th2 cytokines (IL4 and IL13), and serum IgG1 and IgE. Furthermore, inhibition of RhoA signaling also led to rise in Arg1 expression and decrease in iNOS expression in F4/80+ lung macrophages compared to vehicle controls.
CONCLUSIONS: RhoA signaling affect macrophage polarization and inhibition of RhoA signaling protects mice against allergen induced allergic inflammation. Therefore, RhoA pathway may serves as a therapeutic target for asthma.