Rho-specific Guanine nucleotide exchange factors (Rho-GEFs) inhibition affects macrophage phenotype and disrupts Golgi complex.

Abstract

Macrophages play crucial role in tissue homeostasis and the innate and adaptive immune response. Depending on the state of activation macrophages acquire distinct phenotypes that depend on actin, which is regulated by small GTPase RhoA. The naive M0 macrophages are slightly elongated, pro-inflammatory M1 are round and M2 anti-inflammatory macrophages are elongated. We showed previously that interference with RhoA pathway (RhoA deletion or RhoA/ROCK kinase inhibition) disrupted actin, produced extremely elongated (hummingbird) macrophage phenotype and inhibited macrophage movement toward transplanted hearts. The RhoA function depends on the family of guanine-nucleotide exchange factors (GEFs), which catalyze the exchange of GDP for GTP and activate RhoA that reorganizes actin cytoskeleton. Using actin staining, immunostaining, Western blotting, flow cytometry and transmission electron microscopy we studied how a direct inhibition of Rho-GEFs with Rhosin (Rho GEF-binding domain blocker) and Y16 (Rho GEF DH-PH domain blocker) affects M0, M1 and M2 macrophage phenotypes. We also studied how Rho-GEFs inhibition and RhoA deletion affects organization of Golgi complex that is crucial for normal macrophage functions such as phagocytosis, antigen presentation and receptor recycling. We found that GEFs inhibition differently affected M0, M1 and M2 macrophages phenotype and that GEFs inhibition and RhoA deletion both caused changes in the ultrastructure of the Golgi complex. These results suggest that actin/RhoA- dependent shaping of macrophage phenotype has different requirements for activity of RhoA/GEFs pathway in M0, M1 and M2 macrophages, and that RhoA and Rho-GEFs functions are necessary for the maintenance of actin-dependent organization of Golgi complex.