Research Article: A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular inflammatory event
Abstract:
A significant challenge in the study of neuroimmune responses is the identification and separation of resident microglia from infiltrating bone marrow-derived macrophages. Therefore, we investigated whether dual labelling of Cx3cr1 and Ccr2 is sufficient to discriminate retinal microglia from infiltrating macrophages in mice that develop spontaneous autoimmune uveoretinitis–a condition dependent on infiltration of lymphocytes and antigen presenting cells from the circulation into the retina. Mice expressing red fluorescent protein (RFP) under control of the Ccr2 promoter and green fluorescent protein (GFP) under control of the Cx3cr1 promoter (Ccr2 RFP Cx3cr1 GFP mice) were crossed with the uveitogenic R161H mouse line. Retinas of R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 mice, including those given an optic nerve crush injury to stimulate retinal MG expansion, and R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 mice were analyzed for RFP and GFP expressing cells by fundoscopy, flow cytometry, fluorescence microscopy, and for Ccr2 expression by single cell RNA sequencing of retinal CD45 + cells. Retinas from control and optic nerve crushed R161H neg x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 mice contained a single population of CD45 + cells expressing a uniquely high level of GFP but were devoid of RFP + cells, while retinas from R161H +/- x Ccr2 RFP+/- Cx3cr1 GFP+/- F 1 mice had GFP hi cells and increasing amounts of GFP neg RFP + and GFP lo-med RFP + cells as uveoretinitis progressed. Thus, dual labelling of Ccr2 and Cx3cr1 can distinguish retinal microglia from infiltrating immune cells, and as GFP + , RFP + , and GFP + RFP + cells can be easily separated by flow cytometry, our system provides a method for their separation for downstream cellular analysis.
Introduction:
Microglia (MG) are derived from embryonic yolk sac progenitor cells, reside within the parenchyma of central nervous system (CNS) tissue, including the retina, and are essential for the development and maintenance of neuronal tissue ( 1 – 3 ). MG also act as innate immune cells functioning like macrophages derived from bone marrow monocytic precursors. Under normal physiological conditions, MG are maintained in the retina with little, if any, input from bone marrow-derived precursors ( 4 – 7 ). However, under…
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