A subpopulation of activated retinal macrophages selectively migrated to regions of cone photoreceptor stress, but had limited effect on cone death in a mouse model for type 2 Leber congenital amaurosis
Abstract Background Studies of antigen presentation in retina using mice that expressed green fluorescent protein (GFP) from a transgenic CD11c promoter found that retinal GFP hi cells possessed antigen presentation function. Subsequent studies found that these high GFP hi cells preferentially localized to sites of retinal injury, consistent with their APC function. Interest in the roles of macrophages in degenerative CNS diseases led us to study the GFP hi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65 −/− knockout mice induced the GFP hi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival. Methods Rpe65 −/− mice were bred to CD11c GFP mice on the B6/J background. CD11c GFP Rpe65 −/− mice were also backcrossed to CX3CR1 YFP-creER ROSA DTA mice so that CX3CR1 + mononuclear cells could be depleted by Tamoxifen. Retinas were analyzed by immunohistochemistry, confocal microscopy, fluorescence fundoscopy and flow cytometry. Results Elevated numbers of GFP hi cells were concentrated in photoreceptor cell layers of CD11c GFP Rpe65 −/− mice coinciding with the peak of cone death at 2 to 4weeks of age, and persisted for at least 14months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFP hi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFP hi cells and MG in Rpe65 −/− mice from day 13 to day 41, and from day 390 to day 420 promoted a small increase in cone survival. We found no evidence that the GFP hi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFP hi cells were well segregated in the dystrophic retina; GFP hi cells were foremost in the photoreceptor cell layer, while MG were concentrated in the inner retina. Conclusions The expression of GFP on a subset of retinal mononuclear cells in CD11c GFP mice identified a distinct population of cells performing functions previously attributed to MG. Although GFP hi cells dominated the macrophage response to cone death in the photoreceptor cell layer, their ablation led to only an incremental increase in cone survival. The ability to identify, ablate, and isolate these cells will facilitate analysis of this activated, antigen-presenting subset of MG. Highlights Retinal macrophages expressing GFP in CD11c GFP mice concentrated in the photoreceptor cell layer of RPE65 knockout mice. Elevated numbers of GFP hi cells remained in the outer retinas of knockout mice for more than a year. Generation of the GFP hi cells was found to be dependent on microglia. Tamoxifen-induced macrophage ablation gave a small increase in S-cone survival in acute and chronic phases of cone loss. GFP expression on retinal mononuclear cells in CD11c GFP mice identified cells performing functions of activated microglia.
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