CLOTRIMAZOLE IMPACT ON MACROPHAGE M2 POLARIZATION

INTRODUCTION AND OBJECTIVES: Macrophages are cells of the innate immune system, present in all tissues where function as sentinels. They are extremely specialized cells capable of detecting and responding to pathogens invasion, tissue damage and maintaining homeostasis. Due to their plasticity, these cells are extremely responsive to microenvironment stimuli and can change their phenotype and functions. In addition to their important role in tissue homeostasis, macrophages are also involved in several pathologies. Macrophage activation is conventionally defined in a classically activated state (M1) in which these cells assume pro-inflammatory characteristics (stimulated by LPS and IFNγ) and the alternative activation state (M2) with opposite characteristics assuming an anti-inflammatory profile (activated by IL-4). Tumor-associated macrophages (TAM) are macrophages present in the tumor microenvironment with M2-like characteristics due to the immunomodulatory profile. Its tumorigenic role is highlighted in the literature, where it is described for playing important roles in tumor progression, migration, metastasis, angiogenesis and immunomodulation. Investigate the mechanisms of M2 polarization is essential to understand tumor biology and its microenvironment, generating the possibility of new therapeutic strategies. The objective of this work is to evaluate the effect of clotrimazole, an important PI3K pathway inhibitor, on M2 polarization. MATERIAL AND METHODS: Murine macrophage cell line J774 and bone marrow-derived macrophages were polarized with IL-4 and then treated with 5µM clotrimazole for 24h. Subsequently, immunofluorescence, Western blotting and qPCR assays were performed to evaluate the effects on the phenotype, modulated pathways and the gene expression of M2 markers. RESULTS AND CONCLUSION: After treatment with CTZ, a modulation in the PI3K pathway was observed, characterized by the decrease in the phosphorylation of the downstream effectors. Furthermore, a modulation on M2 activation phenotype was observed when these macrophages are treated with clotrimazole, observed by immunofluorescence and Western blotting of classic M2 activation markers. In addition, the effects of CTZ on the MAPK and NFkB pathways were analyzed, which corroborate with our hypothesis that there is a modulation in these pathways that are linked to phenotypic changes in these cells. These results support the hypothesis that CTZ modulates M2 polarization, inhibiting important pathways for M2 phenotype. These data confirm previous data and support our hypothesis that CTZ can modify the macrophage phenotype and directly impact its tumorigenic effects in the tumor microenvironment.