Characterization of M1 and M2 polarization phenotypes in peritoneal macrophages after treatment with graphene oxide nanosheets

Macrophages play a key role in nanoparticle removal and are primarily responsible for their uptake and trafficking in vivo. Due to their functional plasticity, macrophages display a spectrum of phenotypes between two extremes indentified as pro-inflammatory M1 and reparative M2 macrophages, characterized by the expression of specific cell surface markers and the secretion of different cytokines. The influence of graphene oxide (GO) nanosheets functionalized with poly(ethylene glycol-amine) and labelled with fluorescein isothiocyanate (FITC-PEG-GO) on polarization of murine peritoneal macrophages towards M1 and M2 phenotypes was evaluated in basal and stimulated conditions by flow cytometry and confocal microscopy through the expression of different cell markers: CD80 and iNOS as M1 markers, and CD206 and CD163 as M2 markers. Although FITC-PEG-GO did not induce M1 or M2 macrophage polarization after 24 and 48 h in basal conditions, this nanomaterial decreased the percentage of M2 reparative macrophages. We have also compared control macrophages with macrophages that have or have not taken up FITC-PEG-GO after treatment with these nanosheets (GO(+) and GO(-) cells, respectively). The CD80 expression diminished in GO(+) macrophages after 48 h of GO treatment but the CD206 expression in GO(+) population showed higher values than in both GO- population and control macrophages. In the presence of pro-inflammatory stimuli (LPS and IFN-gamma), a significant decrease of CD80(+) cells was observed after treatment with GO. This nanomaterial also induced significant decreases of CD206(+) and CD163(+) cells in the presence of reparative stimulus (IL-4). The CD80, iNOS and CD206 expression was lower in both GO(-) and GO(+) cells than in control macrophages. However, higher CD163 expression was obtained in both GO(-) and GO(+) cells in comparison with control macrophages. All these facts suggest that FITC-PEG-GO uptake did not induce the macrophage polarization towards the M1 pro-inflammatory phenotype, promoting the control of the M1/M2 balance with a slight shift towards M2 reparative phenotype involved in tissue repair, ensuring an appropriate immune response to these nanosheets.