Development of molecular sensors based on fluorescent proteins for polarized macrophages identification

Macrophages are a type of white blood cells that play key roles in innate immune responses as a part of cellular immunity for host defence and tissue homeostasis. To perform diverse functions, macrophages show high plasticity by transforming to polarized states. They are mainly identified as unpolarized, pro-inflammatory and antiinflammatory states and termed as M0, M1 and M2 macrophages respectively. Discriminating polarized states is important due to strict implication with inflammatory conditions resulting in many diseases as chronic inflammation, neurodegeneration, and cancer etc. Many polarization protein markers have been identified and applied to investigate expression profiles through PCR and other techniques with antibodies. However, they are time and cost consuming and sometimes show insufficient performances. We focused on the mannose receptor (CD206) as representative marker of M2 macrophage recognising terminal mannose. We developed dose dependent mannosylated fluorescent proteins (FPs) by conjugations with mannose derivative for around 20 modifiable sites on FPs surfaces. Maximum modifications did not spoil various features of FPs. We found further sensitive and specific discriminations among M2, M1 and M0 macrophages after treating polarized macrophages with adequately conditioned FPs compared to already established approaches using anti CD206 antibody through flow cytometric analysis. These results might be derived from direct ligand utilizations and increased avidity due to multivalent bindings with abundantly modified multimeric FPs. Our strategy is simple but addresses disadvantages of preceding methods. Moreover, this strategy is applicable to detect other cell surface receptors as FPs can be modified with ligands or recognizable aptamer like molecules.