Subretinal Macrophages Research Articles

Role of Adrenomedullin 2/Intermedin in the Pathogenesis of Neovascular Age-Related Macular Degeneration

Adrenomedullin 2 (AM2; also known as intermedin) is a member of the adrenomedullin (AM) peptide family. Similarly to AM, AM2 partakes in a variety of physiological activities. AM2 has been reported to exert protective effects on various organ disorders; however, its significance in the eye is unknown. We investigated the role of AM2 in ocular diseases. The receptor system of AM2 was expressed more abundantly in the choroid than in the retina. In an oxygen-induced retinopathy model, physiological and pathologic retinal angiogenesis did not differ between AM2-knockout (AM2-/-) and wild-type mice. In contrast, in laser-induced choroidal neovascularization, a model of neovascular age-related macular degeneration, AM2-/- mice had enlarged and leakier choroidal neovascularization lesions, with exacerbated subretinal fibrosis and macrophage infiltration. Contrary to this, exogenous administration of AM2 ameliorated the laser-induced choroidal neovascularization–associated pathology and suppressed gene expression associated with inflammation, fibrosis, and oxidative stress, including that of VEGF-A, VEGFR-2, CD68, CTGF, and p22-phox. The stimulation of human adult retinal pigment epithelial (ARPE) cell line 19 cells with TGF-β2 and TNF-α induced epithelial-to-mesenchymal transition (EMT), whereas AM2 expression was also elevated. The induction of EMT was suppressed when the ARPE-19 cells were pretreated with AM2. A transcriptome analysis identified 15 genes, including mesenchyme homeobox 2 (Meox2), whose expression was significantly altered in the AM2-treated group compared with that in the control group. The expression of Meox2, a transcription factor that inhibits inflammation and fibrosis, was enhanced by AM2 treatment and attenuated by endogenous AM2 knockout in the early phase after laser irradiation. The AM2 treatment of endothelial cells inhibited endothelial to mesenchymal transition and NF-κB activation; however, this effect tended to be canceled following Meox2 gene knockdown. These results indicate that AM2 suppresses the neovascular age-related macular degeneration–related pathologies partially via the upregulation of Meox2. Thus, AM2 may be a promising therapeutic target for ocular vascular diseases.

Dual angiopoietin‐2/VEGF‐A neutralisation prevents vascular leakage, cell death and subretinal macrophage infiltration in mouse models of retinal ischaemia/reperfusion injury and spontaneous CNV

AbstractPurposeExplore impact of dual angiopoietin‐2 (Ang‐2)/vascular endothelial growth factor‐A (VEGF‐A) targeting by faricimab on vessel destabilisation and retinal inflammation in 2 mouse models: retinal ischaemia/reperfusion (I/R) injury model to assess vascular permeability, and JR5558 spontaneous choroidal neovascularisation (sCNV) model to investigate inflammation, in the context of phase 2/3 clinical data on faricimab.MethodsAntibodies against Ang‐2, VEGF‐A, or both (VA2) and IgG (control) were intravitreally injected 2 days before I/R injury in the I/R model. Vascular permeability and ongoing cell death were assessed by retinal FITC‐BSA accumulation and DNA fragmentation assay, respectively, 48h after injury. JR5558 mice were treated intraperitoneally at postnatal days P45, P52 with mouse cross‐reactive tool antibodies to Ang‐2, VEGF‐A, both (VA2) and IgG. Subretinal inflammatory cell (Iba1+, CD45+, CD11b+) infiltration was evaluated by flat‐mount retinal pigment epithelium (RPE)/choroid histology at 1 (PT1), 3 (PT2), and 5 weeks (PT3) post‐treatment.ResultsPost‐I/R injury, VA2 significantly prevented retinal vascular permeability by 64%; anti–VEGF‐A by 37%; anti–Ang‐2 produced no significant change. VA2 significantly reduced ongoing cell death by 47%; VEGF‐A, Ang‐2 alone had no significant effect. In JR5558 mice, VA2, not anti–VEGF‐A or anti–Ang‐2 alone, significantly reduced Iba1+ microglia/macrophages on RPE/choroid and around lesions versus IgG at PT1 and PT2. At PT3, anti–Ang‐2, VA2, not anti–VEGF‐A, showed significant reductions in Iba1+ cells versus IgG. CD45+, CD11b+ cells on RPE/choroid and around lesions were significantly reduced only with VA2 versus IgG.ConclusionsDual Ang‐2/VEGF‐A inhibition synergistically prevented retinal vascular leakage, cell death and inflammation in retinal I/R injury and sCNV mouse models, suggesting that vascular leakage, prolonged inflammation are driven by Ang‐2, and supporting recent phase 2/3 clinical data that demonstrated sustained efficacy of faricimab versus intravitreal anti‐VEGF monotarget therapy and extended durability up to every 16 weeks.

In vivo analysis of onset and progression of retinal degeneration in the Nr2e3rd7/rd7 mouse model of enhanced S-cone sensitivity syndrome

The photoreceptor-specific nuclear receptor Nr2e3 is not expressed in Nr2e3rd7/rd7 mice, a mouse model of the recessively inherited retinal degeneration enhanced S-cone sensitivity syndrome (ESCS). We characterized in detail C57BL/6J Nr2e3rd7/rd7 mice in vivo by fundus photography, optical coherence tomography and fluorescein angiography and, post mortem, by histology and immunohistochemistry. White retinal spots and so-called ‘rosettes’ first appear at postnatal day (P) 12 in the dorsal retina and reach maximal expansion at P21. The highest density in ‘rosettes’ is observed within a region located between 100 and 350 µM from the optic nerve head. ‘Rosettes’ disappear between 9 to 12 months. Non-apoptotic cell death markers are detected during the slow photoreceptor degeneration, at a rate of an approximately 3% reduction of outer nuclear layer thickness per month, as observed from 7 to 31 months of age. In vivo analysis of Nr2e3rd7/rd7 Cx3cr1gfp/+ retinas identified microglial cells within ‘rosettes’ from P21 on. Subretinal macrophages were observed in vivo and by confocal microscopy earliest in 12-months-old Nr2e3rd7/rd7 retinas. At P21, S-opsin expression and the number of S-opsin expressing dorsal cones was increased. The dorso-ventral M-cone gradient was present in Nr2e3rd7/rd7 retinas, but M-opsin expression and M-opsin expressing cones were decreased. Retinal vasculature was normal.

Effects of age on retinal macrophage responses to acute elevation of intraocular pressure

There is accumulating evidence that aging shifts the central nervous system milieu towards a proinflammatory state, with increased reactivity of microglia in the aging eye and brain having been implicated in the development of age-related neurodegenerative conditions. Indeed, alterations to microglial morphology and function have been recognized as a part of normal aging. Here, we sought to assess the effects of age on the retinal microglial and macrophage response to acute intraocular pressure (IOP) elevation. Further, we performed experiments whereby bone marrow from young or middle-aged mice was used to reconstitute the bone marrow of whole-body irradiated 12 month old mice. Bone marrow chimeric mice then underwent cannulation and IOP elevation 8 weeks after whole-body irradiation and bone marrow transplantation in order to determine whether the age of bone marrow alters the macrophage response to retinal injury. Our data show retinal macrophage reactivity and microglial morphological changes were enhanced in older mice when compared to younger mice in response to injury. When IOP elevation was performed after whole-body irradiation and bone marrow rescue, we noted subretinal macrophage accumulation and glial reactivity was reduced compared to non-irradiated mice that had also undergone IOP elevation. This effect was evident in both groups of chimeric mice that had received either young or middle-aged bone marrow, suggesting irradiation itself may alter the macrophage and glial response to injury rather than the age of bone marrow.

Fundus autofluorescence, spectral-domain optical coherence tomography, and histology correlations in a Stargardt disease mouse model.

Stargardt disease (STGD1), known as inherited retinal dystrophy, is caused by ABCA4 mutations. The pigmented Abca4-/- mouse strain only reflects the early stage of STGD1 since it is devoid of retinal degeneration. This blue light-illuminated pigmented Abca4-/- mouse model presented retinal pigment epithelium (RPE) and photoreceptor degeneration which was similar to the advanced STGD1 phenotype. In contrast, wild-type mice showed no RPE degeneration after blue light illumination. In Abca4-/- mice, the acute blue light diminished the mean autofluorescence (AF) intensity in both fundus short-wavelength autofluorescence (SW-AF) and near-infrared autofluorescence (NIR-AF) modalities correlating with reduced levels of bisretinoid-fluorophores. Blue light-induced RPE cellular damage preceded the photoreceptors loss. In late-stage STGD1-like patient and blue light-illuminated Abca4-/- mice, lipofuscin and melanolipofuscin granules were found to contribute to NIR-AF, indicated by the colocalization of lipofuscin-AF and NIR-AF under the fluorescence microscope. In this mouse model, the correlation between in vivo and ex vivo assessments revealed histological characteristics of fundus AF abnormalities. The flecks which are hyper AF in both SW-AF and NIR-AF corresponded to the subretinal macrophages fully packed with pigment granules (lipofuscin, melanin, and melanolipofuscin). This mouse model, which has the phenotype of advanced STGD1, is important to understand the histopathology of Stargardt disease.

IL-1\u03b2 induces rod degeneration through the disruption of retinal glutamate homeostasis

BackgroundAge-related macular degeneration is characterized by the accumulation of subretinal macrophages and the degeneration of cones, but mainly of rods. We have previously shown that Mononuclear Phagocytes-derived IL-1β induces rod photoreceptor cell death during experimental subretinal inflammation and in retinal explants exposed to IL-1β but the mechanism is unknown.MethodsRetinal explants were culture in the presence of human monocytes or IL-1β and photoreceptor cell survival was analyzed by TUNEL labeling. Glutamate concentration and transcription levels of gene involved in the homeostasis of glutamate were analyzed in cell fractions of explant cultured or not in the presence of IL-1β. Glutamate receptor antagonists were evaluated for their ability to reduce photoreceptor cell death in the presence of IL1-β or monocytes.ResultsWe here show that IL-1β does not induce death in isolated photoreceptors, suggesting an indirect effect. We demonstrate that IL-1β leads to glutamate-induced rod photoreceptor cell death as it increases the extracellular glutamate concentrations in the retina through the inhibition of its conversion to glutamine in Müller cells, increased release from Müller cells, and diminished reuptake. The inhibition of non-NMDA receptors completely and efficiently prevented rod apoptosis in retinal explants cultured in the presence of IL-1β or, more importantly, in vivo, in a model of subretinal inflammation.ConclusionsOur study emphasizes the importance of inflammation in the deregulation of glutamate homeostasis and provides a comprehensive mechanism of action for IL-1β-induced rod degeneration.

Identification of aUnique Subretinal Microglia Type in Retinal Degeneration Using Single Cell RNA-Seq.

As the resident macrophages of central nervous system, microglia reside in the plexiform and nerve fiber layers of the retina. In degenerative diseases, monocyte-derived macrophages can be recruited to the retina, and histopathology shows abnormal accumulation of macrophages subretinally. However, due to lack of known markers, recruited cells and resident microglia are phenotypically indistinguishable, leaving a major knowledge gap about their potentially independent roles. Here, we used single cell RNA-seq and analyzed over 10,000 immune cells of mouse retinas from normal control and light damage-induced retinal degeneration. We observed ten major macrophage clusters. Moreover, combining trajectory analysis and in situ validation allowed us to pinpoint that subretinal phagocytes are microglia-derived and express high levels of Gal3, Cd68, and Lpl but not P2ry12. Hence, we have identified novel subretinal macrophage markers indicative of their origin and phenotype, which may be useful in other degeneration models and human specimens.

Subretinal macrophages produce classical complement activator C1q leading to the progression of focal retinal degeneration

BackgroundThe role of the alternative complement pathway and its mediation by retinal microglia and macrophages, is well-established in the pathogenesis of Age-Related Macular Degeneration (AMD). However, the contribution of the classical complement pathway towards the progression of retinal degenerations is not fully understood, including the role of complement component 1q (C1q) as a critical activator molecule of the classical pathway. Here, we investigated the contribution of C1q to progressive photoreceptor loss and neuroinflammation in retinal degenerations.MethodsWild-type (WT), C1qa knockout (C1qa−/−) and mice treated with a C1q inhibitor (ANX-M1; Annexon Biosciences), were exposed to photo-oxidative damage (PD) and were observed for progressive lesion development. Retinal function was assessed by electroretinography, followed by histological analyses to assess photoreceptor degeneration. Retinal inflammation was investigated through complement activation, macrophage recruitment and inflammasome expression using western blotting, qPCR and immunofluorescence. C1q was localised in human AMD donor retinas using immunohistochemistry.ResultsPD mice had increased levels of C1qa which correlated with increasing photoreceptor cell death and macrophage recruitment. C1qa−/− mice did not show any differences in photoreceptor loss or inflammation at 7 days compared to WT, however at 14 days after the onset of damage, C1qa−/− retinas displayed less photoreceptor cell death, reduced microglia/macrophage recruitment to the photoreceptor lesion, and higher visual function. C1qa−/− mice displayed reduced inflammasome and IL-1β expression in microglia and macrophages in the degenerating retina. Retinal neutralisation of C1q, using an intravitreally-delivered anti-C1q antibody, reduced the progression of retinal degeneration following PD, while systemic delivery had no effect. Finally, retinal C1q was found to be expressed by subretinal microglia/macrophages located in the outer retina of early AMD donor eyes, and in mouse PD retinas.ConclusionsOur data implicate subretinal macrophages, C1q and the classical pathway in progressive retinal degeneration. We demonstrate a role of local C1q produced by microglia/macrophages as an instigator of inflammasome activation and inflammation. Crucially, we have shown that retinal C1q neutralisation during disease progression may slow retinal atrophy, providing a novel strategy for the treatment of complement-mediated retinal degenerations including AMD.

The expression of C1 inhibitor (C1INH) in macrophages is upregulated by retinal pigment epithelial cells - implication in subretinal immune privilege in the aging eye.

Age-related para-inflammation in the retina-choroidal interface is featured by low-levels of complement activation and subretinal macrophage accumulation. This study aimed to understand how complement expression in macrophages is regulated by retinal pigment epithelium (RPE). Bone marrow-derived macrophages (BMDMs) and RPE cells were cultured from 8-10 weeks old C57BL/6J mice. The BMDMs were co-cultured with normal RPE, or oxidized photoreceptor outer segment (oxPOS) or TNF-α pre-treated RPE, or apoptotic RPE, or RPE-choroid eyecups. Macrophages were then isolated and processed for real-time RT-PCR. The expression of complement inhibitor C1INH in BMDMs was significantly upregulated by RPE and RPE-choroid eyecups. The eyecups also upregulated CFH, CD59a, and Crry in BMDMs. oxPOS pre-treated RPE upregulated C1qb but down-regulated C3 expression in BMDMs. TNF-α pre-treated RPE enhanced C1INH and CFB expression. When BMDMs were treated with apoptotic RPE, the expression of C1qb, CFH, and CD59a was reduced, whereas the expression of C3, CFB and C1INH was increased. Our results suggest that RPE can modulate macrophages complement expression at the retina-choroidal interface even under aging or oxidative conditions. However, during inflammation, they may promote the alternative pathway of complement activation through down-regulating CFH and CD59a and upregulating CFB and C3.

AB006. The co-receptor CD36 as a target in regulation of subretinal inflammation

: Subretinal inflammation plays a critical role in retinal degenerative diseases. Although activated macrophages have been shown to play a key role in the progression of retinopathies and specifically in age-related macular degeneration, little is known about the mechanisms involved in the loss of photoreceptors leading to vision impairment. In our study on retinal damages induced by photo-oxidative stress, we have observed that CD36-deficient mice featured less subretinal macrophage accumulation with attenuated photoreceptor degeneration compared to wild-type (WT) mice. Treatment with CD36-selective azapeptide ligand (labelled MPE-001) as modulator of the inflammatory environment of the retina reduced subretinal macrophage/activated microglia accumulation with preservation of photoreceptor layers and function assessed by ERG in WT, in a CD36-dependent manner. The azapeptide modulated the transcriptome of subretinal macrophage/activated microglia by reducing pro-inflammatory markers. In isolated macrophages, the CD36-selective azapeptide induced dissociation of the CD36-TLR2/6 heterodimer complex (using FRET) altering the TLR2 signaling pathway, thus decreasing NF-KB activation and inflammasome activity. The azapeptide also incurred cytoprotection against photoreceptor apoptosis elicited by activated macrophages. These findings suggest that the azapeptide as ligand of co-receptor CD36 decreases the inflammatory response by modulating CD36-TLR2/6 complex signaling pathway in macrophages, and suggests its potential application in the treatment of retinal degenerative diseases.

PHOTORECEPTOR INNER SEGMENT MORPHOLOGY IN BEST VITELLIFORM MACULAR DYSTROPHY.

To characterize outer retina structure in best vitelliform macular dystrophy (BVMD) and to determine the effect of macular lesions on overlying and adjacent photoreceptors. Five individuals with BVMD were followed prospectively with spectral domain optical coherence tomography and confocal and nonconfocal split-detector adaptive optics scanning light ophthalmoscopy (AOSLO). The AOSLO cone photoreceptor mosaic images were obtained within and around retinal lesions. Cone density was measured inside and outside lesions. In 2 subjects, densities were compared with published measurements acquired ∼2.5 years before. One subject was imaged 3 times over a 5-month period. The AOSLO imaging demonstrated that photoreceptor morphology within BVMD retinal lesions was highly variable depending on the disease stage, with photoreceptor structure present even in advanced disease. The AOSLO imaging was repeatable even in severe disease over short-time and long-time intervals. Photoreceptor density was normal in retinal areas immediately adjacent to lesions and stable over ∼2.5 years. Mobile disk-like structures possibly representing subretinal macrophages were also observed. Combined confocal and nonconfocal split-detector AOSLO imaging reveals substantial variability within clinical lesions in all stages of BVMD. Longitudinal cellular photoreceptor imaging could prove a powerful tool for understanding disease progression and monitoring emerging therapeutic treatment response in inherited degenerations such as BVMD.

Loss of Zebrafish Mfrp Causes Nanophthalmia, Hyperopia, and Accumulation of Subretinal Macrophages

PurposeMutations in membrane frizzled-related protein (MFRP) are associated with nanophthalmia, hyperopia, foveoschisis, irregular patches of RPE atrophy, and optic disc drusen in humans. Mouse mfrp mutants show retinal degeneration but no change in eye size or refractive state. The goal of this work was to generate zebrafish mutants to investigate the loss of Mfrp on eye size and refractive state, and to characterize other phenotypes observed.MethodsClustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methods were used to generate multiple frameshift mutations in zebrafish mfrp causing premature translational stops in Mfrp. Spectral-domain optical coherence tomography (SD-OCT) was used to measure eye metrics and refractive state, and immunohistochemistry was used to study adult eyes. Gene expression levels were measured using quantitative PCR.ResultsZebrafish Mfrp was shown to localize to apical and basal regions of RPE cells, as well as the ciliary marginal zone. Loss of Mfrp in mutant zebrafish was verified histologically. Zebrafish eyes that were mfrp mutant showed reduced axial length causing hyperopia, RPE folding, and macrophages were observed subretinally. Visual acuity was reduced in mfrp mutant animals.ConclusionsMutation of zebrafish mfrp results in hyperopia with subretinal macrophage infiltration, phenocopying aspects of human and mouse Mfrp deficiency. These mutant zebrafish will be useful in studying the onset and progression of Mfrp-related nanophthalmia, the cues that initiate the recruitment of macrophages, and the mechanisms of Mfrp function.

Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF-α.

SummaryOrthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14+ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human‐induced pluripotent stem cell‐derived RPE cells, we show that activated Mos markedly inhibit RPE OTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF‐α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF‐α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age‐related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.

Aged complement factor H knockout mice kept in a clean barriered environment have reduced retinal pathology

Age-related macular degeneration (AMD) is the largest cause of visual loss in those over 60 years in the West and is a condition increasing in prevalence. Many diseases result from genetic/environmental interactions and 50% of AMD cases have an association with polymorphisms of the complement system including complement factor H. Here we explore interactions between genetic predisposition and environmental conditions in triggering retinal pathology in two groups of aged complement factor H knock out (Cfh−/−) mice. Mice were maintained over 9 months in either a conventional open environment or a barriered pathogen free environment. Open environment Cfh−/− mice had significant increases in subretinal macrophage numbers, inflammatory and stress responses and reduced photoreceptor numbers over mice kept in a pathogen free environment. Hence, environmental factors can drive retinal disease in these mice when linked to complement deficits impairing immune function. Both groups of mice had similar levels of retinal amyloid beta accumulation. Consequently there is no direct link between this and inflammation in Cfh−/− mice.

A model of progressive photo-oxidative degeneration and inflammation in the pigmented C57BL/6J mouse retina

Light–induced degeneration in rodent retinas is an established model for of retinal degeneration, including the roles of oxidative stress and neuroinflammatory activity. In these models, photoreceptor death is elicited via photo-oxidative stress, and is exacerbated by recruitment of subretinal macrophages and activation of immune pathways including complement propagation. Existing light damage models have relied heavily on albino rodents, and mostly using acute light stimuli. These albino models have proven valuable in uncovering the pathogenic mechanisms of such pathways in the context of retinal disease. However, their inherent albinism hinders comparability to normal retinal physiology, and also makes gene technology analysis time-consuming due to the predominance of the pigmented mouse strains in these applications. In this study, we characterise a new light damage model utilising C57BL/6J mice over a 7 day period of chronic light exposure. We use high-efficiency LED technology to deliver a sustained intensity of 100 k lux with negligible modulation of ambient temperature. We show that in the C57BL/6J mouse, chronic light exposure elicits the cardinal features of light damage including photoreceptor degeneration, atrophy of the choriocapillaris, decreased retinal function and increases in oxidative stress markers 4-HNE and 8-OHG, which emerge progressively over the 7 day period of exposure. These changes are accompanied by robust recruitment of IBA1+ and F4/80 + microglia/macrophages to the ONL and subretinal space, followed the strong up-regulation of monocyte-chemoattractants Ccl2, Ccl3, and Ccl12, as well as increases in expression of complement component C3. These findings are in agreement with prior damage models conducted in albino rodents such as Balb/c mice, and support the use of this new model in further investigating the causative features of oxidative stress and inflammation in retinal disease.

The broad-spectrum chemokine inhibitor NR58-3.14.3 modulates macrophage-mediated inflammation in the diseased retina.

BackgroundThe activity of macrophages is implicated in the progression of retinal pathologies such as atrophic age-related macular degeneration (AMD), where they accumulate among the photoreceptor layer and subretinal space. This process is aided by the local expression of chemokines, which furnish these cells with directional cues that augment their migration to areas of retinal injury. While these qualities make chemokines a potential therapeutic target in curtailing damaging retinal inflammation, their wide variety and signalling redundancy pose challenges in broadly modulating their activity. Here, we examine the efficacy of the broad-spectrum chemokine inhibitor NR58-3.14.3—a suppressor of Ccl- and Cxcl- chemokine pathways—in suppressing macrophage activity and photoreceptor death, using a light-induced model of outer retinal atrophy and inflammation.MethodsPhoto-oxidative damage was induced in SD rats via exposure to 1000 lux of light for 24 h, after which animals were euthanized at 0- or 7-day post-exposure time points. Prior to damage, NR58-3.14.3 was injected intravitreally. Retinas were harvested and evaluated for the effect of NR58-3.14.3 on subretinal macrophage accumulation and cytokine expression profile, as well as photoreceptor degeneration.ResultsWe report that intravitreal administration of NR58-3.14.3 reduces the accumulation of macrophages in the outer retina following exposure to light damage, at both 0- and 7-day post-exposure time points. Injection of NR58-3.14.3 also reduced the up-regulation of inflammatory markers including of Il6, Ccl3, and Ccl4 in infiltrating macrophages, which are promoters of their pathogenic activity in the retina. Finally, NR58-3.14.3-injected retinas displayed markedly reduced photoreceptor death following light damage, at both 0 and 7 days post-exposure.ConclusionsOur findings indicate that NR58-3.14.3 is effective in inhibiting subretinal macrophage accumulation in light-induced retinal degeneration and illustrate the potential of broad-spectrum chemokine inhibitors as novel therapeutic agents in thwarting retinal inflammation. Although broad-spectrum chemokine inhibitors may not be appropriate for all retinal inflammatory conditions, our results suggest that they may be beneficial for retinal dystrophies in which chemokine expression and subretinal macrophage accumulation are implicated, such as advanced AMD.

Cd59a deficiency in mice leads to preferential innate immune activation in the retinal pigment epithelium–choroid with age

Dysregulation of the complement system has been implicated in the pathogenesis of age-related macular degeneration. To investigate consequences of altered complement regulation in the eye with age, we examined Cd59a complement regulator deficient (Cd59a−/−) mice between 4 and 15 months. In vivo imaging revealed an increased age-related accumulation of autofluorescent spots in Cd59a−/− mice, a feature that reflects accumulation of subretinal macrophages and/or microglia. Despite this activation of myeloid cells in the eye, Cd59a−/− mice showed normal retinal histology and function as well as normal choroidal microvasculature. With age, they revealed increased expression of activators of the alternative complement pathway (C3, Cfb, Cfd), in particular in the retinal pigment epithelium (RPE)-choroid but less in the retina. This molecular response was not altered by moderately-enhanced light exposure. Cd59a deficiency therefore leads to a preferential age-related dysregulation of the complement system in the RPE-choroid, that alone or in combination with light as a trigger, is not sufficient to cause choroidal vascular changes or retinal degeneration and dysfunction. This data emphasizes the particular vulnerability of the RPE-choroidal complex to dysregulation of the alternative complement pathway during aging.

Effect of Anterior Chamber Cannulation and Acute IOP Elevation on Retinal Macrophages in the Adult Mouse

To assess the retinal macrophage response to cannulation of the anterior chamber (AC) and acute elevation of intraocular pressure (IOP) in adult mice. Eyes from 12-month-old C57BL/6J WT mice were subject to IOP increase (50 mm Hg for 30 minutes) by direct cannulation of the AC. Fellow eyes were either cannulated without pressure increase or left untreated. Electroretinography was carried out prior to IOP elevation and 1 week later. Immunofluorescence staining was performed on frozen sections and retinal wholemounts 1 week after sham and IOP elevation. Eyes were assessed by epifluorescence and confocal microscopy and the density of vitreal hyalocytes and subretinal macrophages was calculated. The density of hyalocytes and subretinal macrophages was significantly increased 1 week after IOP elevation and AC cannulation compared with naïve eyes. CD68 and MHC Class II expression was upregulated in both cannulated eyes and eyes with elevated IOP. Electroretinographic signals derived from retinal ganglion cells were significantly reduced in response to acute IOP elevation, but not in response to cannulation alone. Cannulation of the AC causes an increase in hyalocyte density, microglial activation, and accumulation of macrophages in the subretinal space. These macrophage changes are similar to those observed in eyes subject to IOP elevation. Additional IOP elevation led to significant Müller cell activation, which was not evident after cannulation alone. These data highlight the importance of using appropriate controls in models of acute retinal injury.

Ccl2, Cx3cr1 and Ccl2/Cx3cr1 chemokine deficiencies are not sufficient to cause age-related retinal degeneration

Monocytes, macrophages, dendritic cells and microglia play critical roles in the local immune response to acute and chronic tissue injury and have been implicated in the pathogenesis of age-related macular degeneration. Defects in Ccl2-Ccr2 and Cx3cl1-Cx3cr1 chemokine signalling cause enhanced accumulation of bloated subretinal microglia/macrophages in senescent mice and this phenomenon is reported to result in the acceleration of age-related retinal degeneration. The purpose of this study was to determine whether defects in CCL2-CCR2 and CX3CL1-CX3CR1 signalling pathways, alone or in combination, cause age-dependent retinal degeneration. We tested whether three chemokine knockout mouse lines, Ccl2−/−, Cx3cr1−/− and Ccl2−/−/Cx3cr1−/−, in comparison to age-matched C57Bl/6 control mice show differences in subretinal macrophage accumulation and loss of adjacent photoreceptor cells at 12–14 months of age. All mouse lines are derived from common parental strains and do not carry the homozygous rd8 mutation in the Crb1 gene that has been a major confounding factor in previous reports. We quantified subretinal macrophages by counting autofluorescent lesions in fundus images obtained by scanning laser ophthalmoscopy (AF-SLO) and by immunohistochemistry for Iba1 positive cells. The accumulation of subretinal macrophages was enhanced in Ccl2−/−, but not in Cx3cr1−/− or Ccl2−/−/Cx3cr1−/− mice. We identified no evidence of retinal degeneration in any of these mouse lines by TUNEL staining or semithin histology. In conclusion, CCL2-CCR2 and/or CX3CL1-CX3CR1 signalling defects may differentially affect the trafficking of microglia and macrophages in the retina during ageing, but do not appear to cause age-related retinal degeneration in mice.

Macrophages and the ageing and diseased retina

AbstractPurpose Recruitment and activation of microglia and macrophages are controlled by local cytokine and chemokine signals that also determine neurotoxic or protective responses of these cells. I will review our current understanding of how the behaviour of macrophages and microglia in the retina is controlled by Ccl2‐Ccr2 and Cx3cl1‐Cx3cr1 chemokine signalling during ageing, in retinal degenerations and choroidal neovascularisation (CNV).Methods We investigated recruitment and activation of myeloid cells in wildtype and chemokine single‐(Ccl2, Cx3cr1) and double‐knockout (Ccl2/Cx3cr1) mice in vivo (by AF‐SLO, OCT, fluorescein angiography) and ex vivo (by FACS, immunohistochemistry, morphometric semithin histology and 3D serial block‐face scanning electron microscopy). We studied these mice during ageing, in combination with a monogenetic inherited retinal degeneration (RD8) and in the laser‐induced CNV model.Results Ageing is associated with alterations in myeloid cell populations in the retina and the choroid that are specifically altered by chemokine signalling dysfunctions. Ccl2 deficiency leads to an age‐related accumulation of subretinal macrophages and an attenuated CNV response with reduced infiltration of neutrophils, but no significant effect on degenerative processes in inherited (RD8) or age‐related retinal degenerations. In contrast, and despite an apparently similar accumulation of subretinal macrophages, Cx3cr1 deficiency leads to a more pronounced degenerative phenotype caused by the RD8 mutation, which was similar in Ccl2/Cx3cr1 knockout mice.Conclusion Myeloid cell behaviour, controlled by Ccl2‐Ccr2 and Cx3cl1‐Cx3cr1 chemokine signalling, differentially modulate the manifestation of inherited and age‐related retinal degenerations and contribute to the increase of CNV lesions with age.