Application of Raman spectroscopy for characterization of the functional polarization of macrophages into M1 and M2 cells

Macrophages are key cells in the immune inflammatory response that can be differentiated into M1 and M2 phenotypes. Polarization has a critical therapeutic value, especially in diseases in which an M1/M2 imbalance plays a pathophysiological role. Raman spectroscopy has proven to be a promising bioanalytical technique for discriminating different cell types. However, to our knowledge, its application to identify the functional polarization of macrophages into M1 or M2 cells is yet to be investigated. In this work, Raman spectroscopy was applied to the analysis of macrophage polarization, and the spectral datasets were analyzed using principal component analysis (PCA). In vitro, resting J774.1 macrophages were treated with LPS/IFN-γ to induce the M1 phenotype or with IL-4 to induce the M2 phenotype. The resulting Raman spectra showed sufficient biochemical information to distinguish between M1 and M2 phenotypes when analyzed by PCA, reflecting the changes in cell markers caused by differentiation. The Raman spectra collected from LPS-stimulated M1 and M2 macrophages were more intense. The functional phenotype of M1 macrophages was confirmed by IL-6 secretion and TNF-α mRNA expression, while M2 macrophages produced IL-10 and Arg-1 mRNA, as well as by the morphological changes observed by scanning electron microscopy. Taken together, the results indicate that Raman spectroscopy combined with PCA analysis is a useful tool to identify the functional phenotypes of macrophages, providing an alternative way to distinguish between cells in distinct differentiation stages.