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 CD14low, CD163low and TLR4low phenotype and produced high levels of IL-10. M2b subset was characterized by CD14high, CD80high and CD200Rlow phenotype and produced IL-6 prior to stimulation. M2c subset displayed a CD86low, CD163high phenotype and produced high levels of IL-10. M1 subset was characterized by CD80high, CD86high, CD163low and TLR4high 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.
Highlights
Monocytes released from the bone marrow circulate in the bloodstream for 3 days before infiltrating tissues to maintain tissue resident macrophage populations [1] such as osteoclasts in the bone or microglia in the brain [2]
Macrophages subsets were characterized by a differential expression of the cell surface molecules that are generally present on macrophages, including CD80, CD86, HLA-DR, CD14, TLR4, CD163 and CD200R
M1 macrophages were found to have significantly higher CD80 and CD86 expression compared to monocyte-derived macrophages (MDMs)
Summary
Monocytes released from the bone marrow circulate in the bloodstream for 3 days before infiltrating tissues to maintain tissue resident macrophage populations [1] such as osteoclasts in the bone or microglia in the brain [2]. Macrophages operate at the interphase of the innate and adaptive immunity and through their plasticity can display polarized states. These polarization states are acquired due to a change in type, timing or amount of micro-environmental signals [3]. Activated macrophages or M2 polarized macrophages denote a more heterogeneous polarization state that includes several subtypes: M2a, M2b and M2c [4]. This classification of M1 and M2 has persisted despite evidence demonstrating biochemical and physiological differences between M2a, M2b and M2c polarized subtypes [6]. M2b and M2c are regulatory macrophages that are generated in the later stages of the adaptive immune response, serve to limit inflammation and dampen the immune response [8]
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