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 GFPhi cells possessed antigen presentation function. Subsequent studies found that these high GFPhi 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 GFPhi cells in a retinal model of neurodegeneration. We asked if apoptotic cone photoreceptor cell death in Rpe65−/− knockout mice induced the GFPhi cells, explored their relationship to resident microglia (MG), and tested their role in cone survival. Methods Rpe65−/− mice were bred to CD11cGFP mice on the B6/J background. CD11cGFPRpe65−/− mice were also backcrossed to CX3CR1YFP-creERROSADTA 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 GFPhi cells were concentrated in photoreceptor cell layers of CD11cGFPRpe65−/− mice coinciding with the peak of cone death at 2 to 4 weeks of age, and persisted for at least 14 months. After the initial wave of cone loss, a slow progressive loss of cones was found that continued to retain GFPhi cells in the outer retina. Sustained, four-week Tamoxifen depletions of the GFPhi 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 GFPhi cells were recruited from the circulation; all data pointed to a MG origin. MG and GFPhi cells were well segregated in the dystrophic retina; GFPhi 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 CD11cGFP mice identifies a distinct population of cells performing functions previously attributed to MG. Although GFPhi 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.

Optic nerve as a source of activated retinal microglia post-injury

“…The CX3CR1 YFP-CreER :R26 RFP mice were crossed with CD11c GFP mice for these experiments. Tam was given twice on alternate days at the 3 mg/dose as previously described [ 62 ] so that CX3CR1 + cells upregulated expression of RFP. At 70 days post-Tam, mice were given an optic nerve crush.…”

“…Mice were euthanized, perfused, and the retinas removed as described [ 19 , 33 , 46 , 62 ]. Retinas were suspended in 0.5 mg/ml Liberase/TM (Roche) and 0.05% DNase in DPBS and gently dispersed by trituration.…”

“…To reduce non-specific binding, 10–40 μm thick sections were blocked with 10% normal donkey serum for 1 h followed by staining for CD11b (BDPharmingen), CD11c-GFP (Invitrogen), Ki67 (Abcam), SMI-31 (Covance) and IsolectinB 4 (Invitrogen). Sections were incubated as described [ 54 , 62 ] with antibodies overnight at 4 °C. After three rinses in PBS, appropriate secondary antibodies were applied (Alexa Fluor 350, 488 and 594; Molecular Probes, CA).…”

“…Since dendritic cells are the classic “professional” APC and express CD11c, we tested the CD11c GFP reporter mice [ 27 ] in our search for retinal APC. GFP reporter expression in retina revealed a population of microglia-like cells (CD45 med CD11b hi Iba1 + F4/80 + Ly6C lo CX3CR1 hi ) that expressed GFP (GFP hi cells) from the transgenic CD11c promoter [ 33 , 62 ]. We found by in vitro and in vivo studies that GFP expression in these retinal cells correlated with APC function [ 17 , 18 , 46 ].…”

“…Recently we reported that the GFP hi myeloid cells in CD11c GFP mouse retina were major responders in the outer retina during cone photoreceptor degeneration in RPE65 knockout mice. The evidence suggested that they were an activated form of microglia [ 62 ], and no evidence of recruited myeloid cells was found in this degeneration model.…”