Abstract
Microglia exert diverse functions by responding in diverse ways to different stimuli, yet little is known about the plasticity of various phenotypes that microglia display. We used interferon (IFN)-γ, interleukin (IL)-4 and IL-10 to induce different phenotypes in mouse primary microglia. RNA sequencing was used to identify genes differentially expressed in response to stimulation, and the different stimulated populations were compared in terms of morphology, proliferative capacity, phagocytic ability and neurotoxicity. IFN-γ induced an “immunodefensive” phenotype characterizing both induction of filopodia and upregulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor α. Microglia with this phenotype mediated an acute inflammatory response accompanied by excellent proliferative capacity and neurotoxicity, and remained susceptible to remodeling for up to 48 h after initial stimulation. IL-4 induced an enduring “neuroimmunoregulatory” phenotype involving induction of lamellipodium and persistent upregulation of arginase (Arg)-1 and YM-1 expression. Microglia with this phenotype remained susceptible to remodeling for up to 24 h after initial stimulation. IL-10 induced an “immunosuppressive” phenotype involving induction of ameba-like morphology and upregulation of transforming growth factor β and IL-10 as well as inhibition of inflammation. This phenotype was accompanied by inhibition of self-proliferation, while its morphology, molecular properties and function were the least susceptible to remodeling. IFN-γ, IL-4, or IL-10 appear to induce substantially different phenotypes in microglia. The immunodefensive microglia induced by IFN-γ showed remarkable plasticity, which may help repair CNS inflammation damage under pathological condition. Chronic activation with IL-10 decreases microglial plasticity, which may help protect the brain form the immune response. Our research justifies and guides further studies into the molecular pathways that operate in each phenotype to help multitasking microglia regulate homeostasis in the brain.
Highlights
MATERIALS AND METHODSMicroglia play crucial roles in the development and homeostasis of the central nervous system, as well as in its acute responses to infection or injury (Tambuyzer et al, 2009)
Transcriptional Profiling of Primary Microglia Stimulated by IFN-γ, IL-4, or IL-10
IL-10 promoted phagocytic ability of microglia at 8–24 h, but inhibited phagocytic ability of microglia at 68–72 h (Figures 6B,C). These findings suggest that IFN-γ, IL-4, and IL-10 differentially regulate phagocytic ability of primary microglia, which depends on exposure time in the stimulating molecule
Summary
Microglia play crucial roles in the development and homeostasis of the central nervous system, as well as in its acute responses to infection or injury (Tambuyzer et al, 2009). At 2–72 h later, microglia were fixed for 30 min in 4% PFA, blocked with 10% bovine serum albumin, labeled overnight using the same goat anti-Iba antibody as in Section “Immunofluorescence” and mouse anti-BrdU antibody (1:400; Cell Signaling Technology, United States), followed by the antimouse and anti-goat secondary antibodies mentioned in Section “Immunofluorescence”. Microglia were plated at a density of 5 × 105 cells/cm and treated with each of the cytokines as described in Section “Stimulation of Primary Microglia,” followed by washing with PBS twice, and addition of DMEM-F12 + GlutaMax medium for another 24 h. Apoptosis of NSPCs were labeled overnight using the mouse anti-cleaved caspase antibody (1:300; Cell Signaling Technology, United States), followed by the anti-mouse secondary antibodies mentioned in Section “Immunofluorescence.” cells were stained for 5 min with DAPI (1:10,000) and imaged using a fluorescence microscope (Olympus BX51, Japan). Levels of significance are marked in figures as ∗p < 0.05, significant; ∗∗p < 0.01, very significant; and ∗∗∗p < 0.001, highly significant
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
