Adaptive Müller cell responses to microglial activation mediate neuroprotection and coordinate inflammation in the retina

<p>Abstract</p> <p>Purpose</p> <p>Microglia and Müller cells are prominent participants in retinal responses to injury and disease that shape eventual tissue adaptation or damage. This investigation examined how microglia and Müller cells interact with each other following initial microglial activation.</p> <p>Methods</p> <p>Mouse Müller cells were cultured alone, or co-cultured with activated or unactivated retinal microglia, and their morphological, molecular, and functional responses were evaluated. Müller cell-feedback signaling to microglia was studied using Müller cell-conditioned media. Corroborative <it>in vivo </it>analyses of retinal microglia-Müller cell interactions in the mouse retina were also performed.</p> <p>Results</p> <p>Our results demonstrate that Müller cells exposed to activated microglia, relative to those cultured alone or with unactivated microglia, exhibit marked alterations in cell morphology and gene expression that differed from those seen in chronic gliosis. These Müller cells demonstrated <it>in vitro </it>(1) an upregulation of growth factors such as GDNF and LIF, and provide neuroprotection to photoreceptor cells, (2) increased pro-inflammatory factor production, which in turn increased microglial activation in a positive feedback loop, and (3) upregulated chemokine and adhesion protein expression, which allowed Müller cells to attract and adhere to microglia. <it>In vivo </it>activation of microglia by intravitreal injection of lipopolysaccharide (LPS) also induced increased Müller cell-microglia adhesion, indicating that activated microglia may translocate intraretinally in a radial direction using Müller cell processes as an adhesive scaffold.</p> <p>Conclusion</p> <p>Our findings demonstrate that activated microglia are able to influence Müller cells directly, and initiate a program of bidirectional microglia-Müller cell signaling that can mediate adaptive responses within the retina following injury. In the acute aftermath following initial microglia activation, Müller cell responses may serve to augment initial inflammatory responses across retinal lamina and to guide the intraretinal mobilization of migratory microglia using chemotactic cues and adhesive cell contacts. Understanding adaptive microglia-Müller cell interactions in injury responses can help discover therapeutic cellular targets for intervention in retinal disease.</p

7-Ketocholesterol increases retinal microglial migration, activation, and angiogenicity: a potential pathogenic mechanism underlying age-related macular degeneration.

Age-related macular degeneration (AMD) has been associated with both accumulation of lipid and lipid oxidative products, as well as increased neuroinflammatory changes and microglial activation in the outer retina. However, the relationships between these factors are incompletely understood. 7-Ketocholesterol (7KCh) is a cholesterol oxidation product localized to the outer retina with prominent pro-inflammatory effects. To explore the potential relationship between 7KCh and microglial activation, we localized 7KCh and microglia to the outer retina of aged mice and investigated 7KCh effects on retinal microglia in both in vitro and in vivo systems. We found that retinal microglia demonstrated a prominent chemotropism to 7KCh and readily internalized 7KCh. Sublethal concentrations of 7KCh resulted in microglial activation and polarization to a pro-inflammatory M1 state via NLRP3 inflammasome activation. Microglia exposed to 7KCh reduced expression of neurotrophic growth factors but increased expression of angiogenic factors, transitioning to a more neurotoxic and pro-angiogenic phenotype. Finally, subretinal transplantation of 7KCh-exposed microglia promoted choroidal neovascularization (CNV) relative to control microglia in a Matrigel-CNV model. The interaction of retinal microglia with 7KCh in the aged retina may thus underlie how outer retinal lipid accumulation in intermediate AMD results in neuroinflammation that ultimately drives progression towards advanced AMD

Autoimmune Retinopathy in Systemic Lupus Erythematosus: Histopathologic Features

The ocular pathology of autoimmune retinopathy is demonstrated in a 62-year-old female patient with systemic lupus erythematosus (SLE) who presented with typical clinical autoimmune retinopathy. Macroscopically, there were multiple depigmented lesions in the peripheral retina and choroid and scattered pigmentary bone-spickling at the equator and periphery. Microscopically, there were generalized loss of photoreceptors and thinning of the outer plexiform layer. Many peripheral retinal vessels were sclerotic and occluded, some surrounded by pigment granules and RPE cells. Cobblestone degeneration was prominent in the periphery. Macrophages were seen in the retina, particularly in areas of photoreceptor degeneration. Rare, scattered T- lymphocytes were present in the retina and choroid, while B-cells were notably absent. The optic nerve showed loss of axons and thickened septae. Serum autoantibodies against normal retinal nuclei were detected. These pathological changes represent both known SLE-associated ocular complications as well as possible features of autoimmune retinopathy secondary to SLE

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

Autoimmune Retinopathy in Systemic Lupus Erythematosus: Histopathologic Features

The ocular pathology of autoimmune retinopathy is demonstrated in a 62-year-old female patient with systemic lupus erythematosus (SLE) who presented with typical clinical autoimmune retinopathy. Macroscopically, there were multiple depigmented lesions in the peripheral retina and choroid and scattered pigmentary bone-spickling at the equator and periphery. Microscopically, there were generalized loss of photoreceptors and thinning of the outer plexiform layer. Many peripheral retinal vessels were sclerotic and occluded, some surrounded by pigment granules and RPE cells. Cobblestone degeneration was prominent in the periphery. Macrophages were seen in the retina, particularly in areas of photoreceptor degeneration. Rare, scattered T- lymphocytes were present in the retina and choroid, while B-cells were notably absent. The optic nerve showed loss of axons and thickened septae. Serum autoantibodies against normal retinal nuclei were detected. These pathological changes represent both known SLE-associated ocular complications as well as possible features of autoimmune retinopathy secondary to SLE

Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3β phosphorylation

Platelet-derived growth factor CC (PDGF-CC) is the third member of the PDGF family discovered after more than two decades of studies on the original members of the family, PDGF-AA and PDGF-BB. The biological function of PDGF-CC remains largely to be explored. We report a novel finding that PDGF-CC is a potent neuroprotective factor that acts by modulating glycogen synthase kinase 3β (GSK3β) activity. In several different animal models of neuronal injury, such as axotomy-induced neuronal death, neurotoxin-induced neuronal injury, 6-hydroxydopamine–induced Parkinson’s dopaminergic neuronal death, and ischemia-induced stroke, PDGF-CC protein or gene delivery protected different types of neurons from apoptosis in both the retina and brain. On the other hand, loss-of-function assays using PDGF-C null mice, neutralizing antibody, or short hairpin RNA showed that PDGF-CC deficiency/inhibition exacerbated neuronal death in different neuronal tissues in vivo. Mechanistically, we revealed that the neuroprotective effect of PDGF-CC was achieved by regulating GSK3β phosphorylation and expression. Our data demonstrate that PDGF-CC is critically required for neuronal survival and may potentially be used to treat neurodegenerative diseases. Inhibition of the PDGF-CC–PDGF receptor pathway for different clinical purposes should be conducted with caution to preserve normal neuronal functions

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

Silencing of the CHM Gene Alters Phagocytic and Secretory Pathways in the Retinal Pigment Epithelium

The pathogenesis of choroideremia (CHM), an X-linked retinopathy, remains poorly defined. Silencing of the CHM gene in the retinal pigment epithelium in vitro alters phagocytic and secretory pathways and may indicate how the disorder leads to retinal degeneration