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^GFPRpe65^−/− mice were also backcrossed to CX3CR1^YFP-creERROSA^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^GFPRpe65^−/− 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 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.

中文翻译：

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激活的视网膜巨噬细胞亚群选择性迁移到视锥细胞感光应力区域，但在2型Leber先天性黑ama病小鼠模型中对视锥细胞死亡的影响有限

背景

使用表达转基因CD11c启动子的绿色荧光蛋白（GFP）的小鼠对视网膜​​中抗原呈递的研究发现，视网膜GFP ^hi细胞具有抗原呈递功能。随后的研究发现，这些高GFP ^hi细胞优先定位于视网膜损伤部位，与其APC功能一致。对巨噬细胞在中枢神经系统退行性疾病中的作用的兴趣使我们研究了神经变性视网膜模型中的GFP ^hi细胞。我们询问Rpe65 ^-/-敲除小鼠中的凋亡视锥细胞感光细胞死亡是否诱导了GFP ^hi细胞，探索了它们与常驻小胶质细胞（MG）的关系，并测试了它们在视锥细胞存活中的作用。

方法

Rpe65 ^-/-小鼠在B6 / J背景下繁殖为CD11c ^GFP小鼠。CD11c ^GFP Rpe65 ^-/-小鼠也与CX3CR1 ^YFP-creER ROSA ^DTA小鼠回交，因此他莫昔芬可以耗尽CX3CR1 ⁺单核细胞。通过免疫组织化学，共聚焦显微镜，荧光眼底镜检查和流式细胞术分析视网膜。

结果

GFP ^hi细胞的数量增加，集中在CD11c ^GFP Rpe65 ^-/-小鼠的感光细胞层中，与2至4周龄视锥细胞死亡的高峰相吻合，并持续至少14个月。在视锥细胞丧失的最初一波之后，发现视锥细胞的缓慢进行性丧失，其继续将GFP ^hi细胞保留在外视网膜中。从第13天到第41天以及从第390天到第420天，Rpe65 ^-/-小鼠持续四周的Tamoxifen GFP ^hi细胞和MG消耗促进视锥细胞存活率的小幅增加。我们没有发现证据表明GFP ^hi细胞是从循环中募集的。所有数据均指向MG起源。MG和GFP^hi细胞在营养不良的视网膜中很好地隔离；GFP ^hi细胞在感光细胞层中最重要，而MG则集中在视网膜内。

结论

GFP在CD11c ^GFP小鼠的视网膜单核细胞的一个子集上的表达确定了不同的细胞群，这些细胞具有先前归因于MG的功能。尽管GFP ^hi细胞在感光细胞层中主导了巨噬细胞对视锥细胞死亡的反应，但它们的消融仅导致视锥细胞存活的增量增加。鉴定，消融和分离这些细胞的能力将有助于分析该激活的MG抗原呈递子集。