Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

BACKGROUND:Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD. METHODOLOGY/PRINCIPAL FINDINGS:In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space. CONCLUSIONS/SIGNIFICANCE:These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD

Microglial Morphology and Dynamic Behavior Is Regulated by Ionotropic Glutamatergic and GABAergic Neurotransmission

PURPOSE: Microglia represent the primary resident immune cells in the CNS, and have been implicated in the pathology of neurodegenerative diseases. Under basal or "resting" conditions, microglia possess ramified morphologies and exhibit dynamic surveying movements in their processes. Despite the prominence of this phenomenon, the function and regulation of microglial morphology and dynamic behavior are incompletely understood. We investigate here whether and how neurotransmission regulates "resting" microglial morphology and behavior. METHODS: We employed an ex vivo mouse retinal explant system in which endogenous neurotransmission and dynamic microglial behavior are present. We utilized live-cell time-lapse confocal imaging to study the morphology and behavior of GFP-labeled retinal microglia in response to neurotransmitter agonists and antagonists. Patch clamp electrophysiology and immunohistochemical localization of glutamate receptors were also used to investigate direct-versus-indirect effects of neurotransmission by microglia. RESULTS: Retinal microglial morphology and dynamic behavior were not cell-autonomously regulated but are instead modulated by endogenous neurotransmission. Morphological parameters and process motility were differentially regulated by different modes of neurotransmission and were increased by ionotropic glutamatergic neurotransmission and decreased by ionotropic GABAergic neurotransmission. These neurotransmitter influences on retinal microglia were however unlikely to be directly mediated; local applications of neurotransmitters were unable to elicit electrical responses on microglia patch-clamp recordings and ionotropic glutamatergic receptors were not located on microglial cell bodies or processes by immunofluorescent labeling. Instead, these influences were mediated indirectly via extracellular ATP, released in response to glutamatergic neurotransmission through probenecid-sensitive pannexin hemichannels. CONCLUSIONS: Our results demonstrate that neurotransmission plays an endogenous role in regulating the morphology and behavior of "resting" microglia in the retina. These findings illustrate a mode of constitutive signaling between the neural and immune compartments of the CNS through which immune cells may be regulated in concert with levels of neural activity

DNA primers used for RT-PCR.

<p>DNA primers used for RT-PCR.</p

<i>In vivo</i> subretinal transplantation of retinal microglia promotes choroidal neovascularization, RPE disorganization, and microglia recruitment.

<p>The tissue effects of juxtaposing microglia with RPE cells were assessed in an <i>in vivo</i> mouse model by the injection of cultured microglia with factor-reduced Matrigel into the subretinal space of adult, wild-type mice. Injected microglia, derived from CX3CR1^+/GFP mice, were marked by GFP expression. Contralateral eyes in experimental animals served as controls (injected with factor-reduced Matrigel only without microglia). Four days after transplantation, mice were euthanized and perfused with DiI to mark vascular structures. (A) Representative RPE flatmount from a control eye. Arrowhead marks the injection entry site, and ellipse outlines the area of the subretinal bleb formed. Little or no associated CNV (marked in red by DiI perfusion) formation were observed in control eyes. (B) Representative RPE flatmount from an eye injected with a subretinal injection of Matrigel containing microglia (green). DiI perfusion reveals presence of a feeding vessel (arrow) growing from the injection entry site (arrowhead) to form a branching CNV complex that is centered at the site of microglia cell accumulation. Scale bar (A, B) = 300 µm (C) Magnification of CNV complex (inset in B) showing location of the CNV complex between the RPE and the overlying microglia in the subretinal space (side panels). (D) Quantification of the area of CNV membranes formed in control eyes versus eyes with subretinal microglia showed a significant increased in CNV formation (n = 10 animals, p<0.003, paired t-test). (E–F) Phalloidin staining of F-actin in RPE flat-mounts in the area of subretinal injection. The RPE layer in Matrigel controls demonstrated a predominantly regular array of uniformly hexagonal cells with normal cell-cell relations (E), while that in the area of subretinal microglia transplantation showed pleomorphic and irregularly-spaced cells in which the location of F-actin appeared disorganized (F). (G–H) Iba-1 staining of microglia in the outer retina (retinal flat-mounts, mounted photoreceptor-side up) in control (G) and microglia-transplanted eyes (H). The recruitment of endogenous microglia (labeled in red only) to the outer retina was increased in the region of transplanted GFP+ microglia (green). (I) Migration of endogenous microglia (red) was observed from the inner retina to the outer nuclear layer (ONL) and subretinal space (SRS) (arrowheads) in the area of subretinal microglia transplantation (green). Scale bars (E–I) = 50 µm.</p

Conditioned supernatants from RPE cells co-cultured with retinal microglia (RMG) increase angiogenesis in <i>in vitro</i> models.

<p>(A) Primary endothelial cells show higher rates of proliferation when exposed to supernatants of RPE cells co-cultured with activated RMG (black bar) compared to supernatants from unexposed RPE cells (white bar) (*p = 0.007, unpaired t-test, n = 7 replicates). Proliferation of endothelial cells exposed to supernatants of RPE cells co-cultured with unactivated RMG (gray bar) was slightly but not significantly elevated. (B) Primary endothelial cell migration, as evaluated by a scratch wound assay, was increased in the presence of culture supernatants from RPE cells co-cultured with either unactivated (gray bar) or activated RMG (black bar), relative to that from unexposed RPE (white bar). The assay was scored according to the ability of endothelial cells to migrate into a scratch wound of uniform width marked on a confluent layer of endothelial cells (lower panels, cells stained with CD31 (red) and DAPI (blue), n = 9 replicates) (C) Sprouting angiogenesis, as evaluated by an aortic ring assay, was increased in the presence of culture supernatant from RPE cells co-cultured with activated RMG (black bar), compared to that from unexposed RPE (white bar). Measurements were slightly but non-signficantly increased with supernatants from RPE cells co-cultured with unactivated RMG (gray bar). Endothelial cell nuclei from vessels sprouting from sections of vascular aortic rings were scored (n> = 6 replicates).</p

Primary RPE cells increase expression and secretion of pro-angiogenic factors following co-culture with retinal microglia (RMG).

<p>(A) Quantitative RT-PCR comparing mRNA levels of metalloproteinases (MMP1, MMP2, MMP9), and growth factors, VEGF and PEDF, in RPE cells co-cultured with unactivated (gray bars) and activated RMG (black bars) relative to control (unexposed RPE cells, white bars). (B) Protein analyses of RPE cell lysates showing that levels of MMP2, MMP9, and VEGF, as analyzed by ELISA, were increased following RMG co-culture (left). MMP1 levels, as analyzed by Western blotting, were also significantly elevated (right). (C) Protein analyses of RPE supernatants showing similar significant increases by ELISA analyses (MMP9, VEGF, left) and by Western blotting (MMP1, right). Levels of PEDF, an anti-angiogenic factor, were unchanged by co-culture in both RPE cell lysates and supernatants. Significant differences (p<0.05) are indicated with *.</p