Characterization of In vitro Generated Human Polarized Macrophages

Abstract

Objective: Contact with invading pathogens and/or tissue injury leads to the polarization of macrophages into either a M1 or a M2 state which is further divided into M2a, M2b and M2c subsets. The human macrophage subsets have been poorly characterized. The present study was undertaken to characterize macrophage polarization using a non-exhaustive panel of surface markers with respect to M1, M2a, M2b and M2c macrophages and production of pro-and anti-inflammatory cytokines in response to various toll-like receptors (TLR), ligands. Methods: We generated various macrophage subsets by treating monocyte-derived macrophages (MDMs) with IFNγ (M1), IL-4 (M2a), LPS and IL-1β (M2b) or IL-10 (M2c) followed by stimulation with toll-like receptor (TLR)-2, TLR-3 and TLR-4 agonists and analysis of surface marker and cytokines expression was carried out by flow cytometry and ELISA, respectively. Results: M2a subset was characterized by CD14 low , CD163 low and TLR4 low phenotype and produced high levels of IL-10. M2b subset was characterized by CD14 high , CD80 high and CD200R low phenotype and produced IL-6 prior to stimulation. M2c subset displayed a CD86 low , CD163 high phenotype and produced high levels of IL-10. M1 subset was characterized by CD80 high , CD86 high , CD163 low and TLR4 high phenotype and produced high levels of proinflammatory IFN-g, IL-12, TNFα and IL-23 following stimulation. Conclusion: This study characterizes all four polarization states in human macrophages. Each polarization state demonstrated a unique cell surface marker profile and cytokine profile. These phenotypic markers can be used to characterize macrophage populations in tissue inflammatory disease conditions in vivo to further understand disease pathogenesis.