Keratinocyte-derived extracellular vesicles induce macrophage polarization toward an M1-like phenotype

Multiple reports have shown an effect of keratinocyte-derived extracellular vesicles (EVs) on keratinocytes and other cell types. However, the contribution of keratinocyte-derived EVs under physiological and pathological conditions is not fully elucidated. Therefore, whether there is an effect of EVs on macrophages in cervical cancer (CC) is also unknown. Here, we evaluated the effect of tumor and non-tumor keratinocyte-derived EVs on the polarization of peripheral blood mononuclear cells (PBMCs)-derived macrophages and THP-1 cell line. Flow cytometric evaluation of macrophages cultured in the presence of keratinocyte-derived EVs mainly indicated an increase in classical activation markers CD80 and CD86 (M1 phenotype) and little or no modification of alternative activation markers (M2 phenotype). ELISA evaluation of macrophage supernatants revealed an increase in the secretion of proinflammatory cytokines such as IL-1 beta and IL-6. On the other hand, TGF-beta was not significantly modified and only EVs derived from non-cancerous keratinocytes induced a significant increase in IL-10. The expression levels of transcripts associated with the M1 phenotype were also evaluated by qRT-PCR with similar results to ELISA for TGF-beta and IL-10; but also an increase in the expression of HLA-DR alpha and TNF-alpha was observed, and no statistically significant changes in ARG1. The ROS production was also evaluated and this increase mainly in macrophages treated with CC keratinocytes-derived EVs. So, our results suggest that the uptake of EVs derived from released by non-tumor and cervical cancer keratinocytes promotes in macrophages their polarization to an M1-like phenotype.