Espectroscopia Raman aplicada ao estudo da diferenciação funcional de células: uma avaliação sobre os fenótipos M1 e M2 de macrófagos

Abstract

Macrophages are important cells of the immune system, distributed by different tissues and considered the first cells of defense against aggressive agents, playing a fundamental role in the development of the inflammatory process. Macrophages have great functional plasticity and are classified within a phenotypic spectrum that basically includes two activation profiles called phenotype M1 (classical activation) and phenotype M2 (alternative activation). It is known that each of these phenotypes is involved with different pathophysiological processes, and that their identification basically depends on the quantification of secreted mediators and/or the presence of typical surface markers of each phenotype. Therefore, in order to develop a methodology that allows the rapid identification of the macrophage phenotype without the need to use staining or labeling techniques, we aim in this study to apply the Raman spectroscopy technique as an alternative tool to distinguish M1 and M2 of macrophages. For this, the cell line J774.1 was used, submitted to stimulation with LPS/IFNγ for differentiation in the M1 profile, and with IL-4 for differentiation in the M2 profile. To certify the differentiation in each phenotype, the cytokines typical of each M1 (IL-6) or M2 (IL-10) profile were quantified by ELISA. In addition, we also determined morphological changes in each phenotypic profile using scanning electron microscopy. Then, the Raman analyzes were performed on each profile already defined, with the results of the variables being evaluated from the principal component analysis (PCA). The increased production of cytokines IL-6 and IL-10 confirmed the success of the polarization protocol for profiles M1 and M2, respectively. The morphological analysis of the groups observed a significant change only with regard to the M2 profile. The Raman spectra submitted to the analysis by the PCA technique allowed to distinguish the M1 and M2 profiles. In conclusion, the present study demonstrated that the polarization of macrophages in the M1 and M2 profile can be identified by Raman spectroscopy, opening the possibility of applying this technique to distinguish other cell types that show phenotypic differentiation.