RESUMO
Inflammasomes are cytoplasmic protein complexes that regulate the secretion of pro-inflammatory cytokines including IL-1ß and IL18, thereby playing a crucial role in inflammatory and chronic diseases. Plant compound Withaferin A (WFA) has been demonstrated to possess numerous biological activities including anti-inflammatory and anti-cancer effects. However, the effect of WFA on macrophage polarization and inflammasome expression in polarized macrophages has not been documented. In this study, cultured THP-1 macrophages were polarized into M1/M2 phenotypes. Subsequently, macrophage characterization was tested for M1 markers (CXCL10 and CXCL9) and M2 markers (CCL20 and CCL13). NOD-like receptor protein 3 (NLRP3) and Absent in melanoma (AIM2) inflammasome gene and protein expressions were measured by RTqPCR and Western blot respectively. Colocalization of both proteins in polarized macrophages was analyzed by immunofluorescence. Our results show that M1 polarized macrophages express elevated NLRP3 and AIM2 gene expressions. Furthermore, WFA treatment stimulated AIM2 and caspase-1 protein expression in M2W macrophages in comparison to M2 cells. ELISA analysis of the cell culture supernatant showed that WFA treatment of M2 macrophages inhibited the secretion of TGF-ß in comparison to M1. Immunofluorescence studies showed NLRP3/ASC colocalized in the cytoplasm in M1 macrophages, which was not the case in M2 and M2W cells. AIM2/ASC were found colocalized in M1 and M2W cells, indicating an activation of inflammasome. These results provide basis for better understanding the effect of WFA in inflammatory diseases and some cancers by modulating macrophage polarization and inflammasome activation.
