A method for distinguishing and separating retinal microglia from infiltrating macrophages during an ocular...

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^RFPCx3cr1^GFP mice) were crossed with the uveitogenic R161H mouse line. Retinas of R161H^neg x Ccr2^RFP+/-Cx3cr1^GFP+/- F₁ mice, including those given an optic nerve crush injury to stimulate retinal MG expansion, and R161H^+/- x Ccr2^RFP+/-Cx3cr1^GFP+/- F₁ 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+/- F₁ 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+/- F₁ mice had GFP^hi cells and increasing amounts of GFP^negRFP⁺ and GFP^lo-medRFP⁺ 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.