Retinal Microglia Research Articles

Ganglion cell-derived LysoPS induces retinal neovascularisation by activating the microglial GPR34-PI3K-AKT-NINJ1 axis

Retinal neovascularisation is a major cause of blindness in patients with proliferative diabetic retinopathy (PDR). It is mediated by the complex interaction between dysfunctional ganglion cells, microglia, and vascular endothelial cells. Notably, retinal microglia, the intrinsic immune cells of the retina, play a crucial role in the pathogenesis of retinopathy. In this study, we found that lysophosphatidylserines (LysoPS) released from injured ganglion cells induced microglial extracellular trap formation and retinal neovascularisation. Mechanistically, LysoPS activated the GPR34-PI3K-AKT-NINJ1 signalling axis by interacting with the GPR34 receptor on the microglia. This activation upregulated the expression of inflammatory cytokines, such as IL-6, IL-8, VEGFA, and FGF2, and facilitated retinal vascular endothelial cell angiogenesis. As a result, inhibition of the GPR34-PI3K-AKT-NINJ1 axis significantly decreased microglial extracellular trap formation and neovascularisation by suppressing LysoPS-induced microglial inflammatory responses, both in vitro and in vivo. This study reveals the crucial role of apoptotic ganglion cells in activating microglial inflammation in PDR, thereby enhancing our understanding of the pathogenesis of retinal neovascularisation.

Microglial cGAS–STING signaling underlies glaucoma pathogenesis

Characterized by progressive degeneration of retinal ganglion cells (RGCs) and vision loss, glaucoma is the primary cause of irreversible blindness, incurable and affecting over 78 million patients. However, pathogenic mechanisms leading to glaucoma-induced RGC loss are incompletely understood. Unexpectedly, we found that cGAS-STING (2'3'-cyclic GMP-AMP-stimulator of interferon genes) signaling, which surveils displaced double-stranded DNA (dsDNA) in the cytosol and initiates innate immune responses, was robustly activated during glaucoma in retinal microglia in distinct murine models. Global or microglial deletion of STING markedly relieved glaucoma symptoms and protected RGC degeneration and vision loss, while mice bearing genetic cGAS-STING supersensitivity aggravated retinal neuroinflammation and RGC loss. Mechanistically, dsDNA from tissue injury activated microglial cGAS-STING signaling, causing deleterious macroglia reactivity in retinas by cytokine-mediated microglia-macroglia interactions, progressively driving apoptotic death of RGCs. Remarkably, preclinical investigations of targeting cGAS-STING signaling by intraocular injection of TBK1i or anti-IFNAR1 antibody prevented glaucoma-induced losses of RGCs and vision. Therefore, we unravel an essential role of cGAS-STING signaling underlying glaucoma pathogenesis and suggest promising therapeutic strategies for treating this devastating disease.

MDSCs promote pathological angiogenesis in ocular neovascular disease

BackgroundOcular neovascular diseases, which contribute significantly to vision loss, lack effective preventive treatments. Recent studies have highlighted the significant involvement of immune cells in neovascular retinopathy. Myeloid-derived suppressor cells (MDSCs) promote the development of neovascularization, but it is unknown whether they participate in pathological neovascularization and whether they are expected to be a therapeutic target. MethodWe investigated the role of MDSCs in promoting pathological angiogenesis using an oxygen-induced retinopathy (OIR) model, employing flow cytometry, immunofluorescence, and smart-seq analysis. Then, we evaluated the proportion of MDSCs in patient blood samples using flow cytometry. Additionally, we assessed the effect of MDSC depletion using an anti-Gr-1 monoclonal antibody on retinal vasculopathy and alterations in retinal microglia. ResultsIn the OIR model, an elevated ratio of MDSCs was observed in both blood and retinal tissue during phase II (Neovascularization). The depletion of MDSCs resulted in reduced retinal neovascularization and vaso-obliteration, along with a decrease in microglia within the neovascularization area. Furthermore, analysis of gene transcripts associated with MDSCs indicated activation of vascular endothelial growth factor (VEGF) regulation and inflammation. Importantly, infants with ROP exhibited a higher proportion of MDSCs in their blood samples. ConclusionOur results suggested that excessive MDSCs represent an unrecognized feature of ocular neovascular diseases and be responsible for the retinal vascular inflammation and angiogenesis, providing opportunities for new therapeutic approaches to ocular neovascular disease.

Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body

BackgroundThe eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive.ResultsIn this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes.ConclusionOur study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Single-cell RNA sequencing reveals the heterogeneity and interactions of immune cells and Müller glia during zebrafish retina regeneration.

Inflammation plays a crucial role in the regeneration of fish and avian retinas. However, how inflammation regulates Müller glia (MG) reprogramming remains unclear. Here, we used single-cell RNA sequencing to investigate the cell heterogeneity and interactions of MG and immune cells in the regenerating zebrafish retina. We first showed that two types of quiescent MG (resting MG1 and MG2) reside in the uninjured retina. Following retinal injury, resting MG1 transitioned into an activated state expressing known reprogramming genes, while resting MG2 gave rise to rod progenitors. We further showed that retinal microglia can be categorized into three subtypes (microglia-1, microglia-2, and proliferative) and pseudotime analysis demonstrated dynamic changes in microglial status following retinal injury. Analysis of cell-cell interactions indicated extensive crosstalk between immune cells and MG, with many interactions shared among different immune cell types. Finally, we showed that inflammation activated Jak1-Stat3 signaling in MG, promoting their transition from a resting to an activated state. Our study reveals the cell heterogeneity and crosstalk of immune cells and MG in zebrafish retinal repair, and may provide valuable insights into future mammalian retina regeneration.

CX3CL1-CX3CR1 axis protects retinal ganglion cells by inhibiting microglia activation in a distal optic nerve trauma model

BackgroundThe chemokine CX3CL1 has been reported to play an important role in optic nerve protection, but the underlying mechanism is still unclear. CX3CR1, the only receptor of CX3CL1, is specifically expressed on retinal microglia, whose activation plays a role in the pathological process of optic nerve injury. This study aimed to evaluate whether CX3CL1 exerts optic neuroprotection by affecting the activation of microglia by combining with CX3CR1.MethodsA mouse model of distal optic nerve trauma (ONT) was used to evaluate the effects of the CX3CL1-CX3CR1 axis on the activation of microglia and survival or axonal regeneration of retinal ganglion cells (RGCs). The activation of microglia, loss of RGCs, and damage to visual function were detected weekly till 4 weeks after modeling. CX3CL1 was injected intravitreally immediately or delayed after injury and the status of microglia and RGCs were examined.ResultsIncreases in microglia activation and optic nerve damage were accompanied by a reduced production of the CX3CL1-CX3CR1 axis after the distal ONT modeling. Both immediate and delayed intravitreal injection of CX3CL1 inhibited microglia activation, promoted survival of RGCs, and improved axonal regenerative capacity. Injection with CX3CL1 was no longer effective after 48 h post ONT. The CX3CL1-CX3CR1 axis promotes survival and axonal regeneration, as indicated by GAP43 protein and gene expression, of RGCs by inhibiting the microglial activation after ONT.ConclusionsThe CX3CL1-CX3CR1 axis could promote survival and axonal regeneration of RGCs by inhibiting the microglial activation after optic nerve injury. The CX3CL1-CX3CR1 axis may become a potential target for the treatment of optic nerve injury. Forty-eight hours is the longest time window for effective treatment after injury. The study is expected to provide new ideas for the development of targeted drugs for the repair of optic nerve.

Effects of Fucoidans on Activated Retinal Microglia.

Sulfated marine polysaccharides, so-called fucoidans, have been shown to exhibit anti-inflammatory and immunomodulatory activities in retinal pigment epithelium (RPE). In this study, we tested the effects of different fucoidans (and of fucoidan-treated RPE cells) on retinal microglia to investigate whether its anti-inflammatory effect can be extrapolated to the innate immune cells of the retina. In addition, we tested whether fucoidan treatment influenced the anti-inflammatory effect of RPE cells on retinal microglia. Three fucoidans were tested (FVs from Fucus vesiculosus, Fuc1 and FucBB04 from Laminaria hyperborea) as well as the supernatant of primary porcine RPE treated with fucoidans for their effects on inflammatory activated (using lipopolysaccharide, LPS) microglia cell line SIM-A9 and primary porcine retinal microglia. Cell viability was detected with a tetrazolium assay (MTT), and morphology by Coomassie staining. Secretion of tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and interleukin 8 (IL8) was detected with ELISA, gene expression (NOS2 (Nitric oxide synthase 2), and CXCL8 (IL8)) with qPCR. Phagocytosis was detected with a fluorescence assay. FucBB04 and FVs slightly reduced the viability of SIM-A9 and primary microglia, respectively. Treatment with RPE supernatants increased the viability of LPS-treated primary microglia. FVs and FucBB04 reduced the size of LPS-activated primary microglia, indicating an anti-inflammatory phenotype. RPE supernatant reduced the size of LPS-activated SIM-A9 cells. Proinflammatory cytokine secretion and gene expression in SIM-A9, as well as primary microglia, were not significantly affected by fucoidans, but RPE supernatants reduced the secretion of LPS-induced proinflammatory cytokine secretion in SIM-A9 and primary microglia. The phagocytosis ability of primary microglia was reduced by FucBB04. In conclusion, fucoidans exhibited only modest effects on inflammatorily activated microglia by maintaining their cell size under stimulation, while the anti-inflammatory effect of RPE cells on microglia irrespective of fucoidan treatment could be confirmed, stressing the role of RPE in regulating innate immunity in the retina.

Paeoniflorin alleviates TGF-β2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-β2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-β2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-β2 (5ng/mL). PAE (300μM) was added to serum-starved confluent cultures of HTM cells for 2h, followed by incubation with TGF-β2 for 22h. SB-431542, a TGF-β receptor inhibitor (10μM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-β2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. PAE attenuated TGF-β2-induced oxidative stress and suppressed TGF-β2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-β2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-β2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. PAE alleviates TGF-β2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

YY1 Lactylation Aggravates Autoimmune Uveitis by Enhancing Microglial Functions via Inflammatory Genes.

Activated microglia in the retina are essential for the development of autoimmune uveitis. Yin-Yang 1 (YY1) is an important transcription factor that participates in multiple inflammatory and immune-mediated diseases. Here, an increased YY1 lactylation in retinal microglia within in the experimental autoimmune uveitis (EAU) group is observed. YY1 lactylation contributed to boosting microglial activation and promoting their proliferation and migration abilities. Inhibition of lactylation suppressed microglial activation and attenuated inflammation in EAU. Mechanistically, cleavage under targets & tagmentation（CUT&Tag）analysis revealed that YY1 lactylation promoted microglial activation by regulating the transcription of a set of inflammatory genes, including STAT3, CCL5, IRF1, IDO1, and SEMA4D. In addition, p300 is identified as the writer of YY1 lactylation. Inhibition of p300 decreased YY1 lactylation and suppressed microglial inflammation in vivo and in vitro. Collectively, the results showed that YY1 lactylation promoted microglial dysfunction in autoimmune uveitis by upregulating inflammatory cytokine secretion and boosting cell migration and proliferation. Therapeutic effects can be achieved by targeting the lactate/p300/YY1 lactylation/inflammatory genes axis.

Neuroprotective and Anti-Inflammatory Activities of Hybrid Small-Molecule SA-10 in Ischemia/Reperfusion-Induced Retinal Neuronal Injury Models.

To induce I/R injury and inflammation, we subjected R28 and primary microglial cells to oxygen glucose deprivation (OGD) for 6 h in vitro or treated these cells with a cocktail of TNF-α, IL-1β and IFN-γ for 1 h, followed by the addition of SA-10 (10 µM). Inhibition of microglial activation, ROS scavenging, cytoprotective and anti-inflammatory activities were measured. In vivo I/R-injured mouse retinas were treated with either PBS or SA-10 (2%) intravitreally, and pattern electroretinogram (ERG), spectral-domain optical coherence tomography, flash ERG and retinal immunocytochemistry were performed. SA-10 significantly inhibited microglial activation and inflammation in vitro. Compared to the control, the compound SA-10 significantly attenuated cell death in both microglia (43% vs. 13%) and R28 cells (52% vs. 17%), decreased ROS (38% vs. 68%) production in retinal microglia cells, preserved neural retinal function and increased SOD1 in mouse eyes. SA-10 is protective to retinal neurons by decreasing oxidative stress and inflammatory cytokines.

The fluorescent ligand bTVBT2 reveals increased p-tau uptake by retinal microglia in Alzheimer's disease patients and AppNL-F/NL-F mice.

Amyloid beta (Aβ) deposits and hyperphosphorylated tau (p-tau) accumulation have been identified in the retina of Alzheimer's disease (AD) patients and transgenic AD mice. Previous studies have shown that retinal microglia engulf Aβ, but this property decreases in AD patients. Whether retinal microglia also take up p-tau and if this event is affected in AD is yet not described. In the current study, we use the p-tau-specific thiophene-based ligand bTVBT2 to investigate the relationship between disease progression and p-tau uptake by microglia in the retina of AD patients and AppNL-F/NL-F knock-in mice, an AD mouse model known to demonstrate extracellular Aβ plaques and dystrophic neurites in the brain from 6months of age. Evaluation of bTVBT2 specificity and its presence within microglia was assessed by immunofluorescent staining of hippocampal sections and flat-mount retina samples from non-demented controls, AD patients, 3-, 9-, and 12-month-old AppNL-F/NL-F knock-in mice and 12- and 18-month-old wild type (WT) mice. We used ImageJ to analyze the amount of bTVBT2 inside Iba1-positive microglia. Co-localization between the ligand and p-tau variant Ser396/Ser404 (PHF-1), Aβ, phosphorylated TAR DNA binding protein 43 (pTDP-43), and islet amyloid polypeptide (IAPP) in the brain and retina was analyzed using confocal imaging. Confocal imaging analysis showed that bTVBT2 binds to PHF-1- and AT8-positive aggregates inside retinal microglia, and not to Aβ, pTDP-43, or IAPP. The density of bTVBT2-positive microglia was higher in cases with a high Aβ load compared to those with a low Aβ load. This density correlated with the neurofibrillary tangle load in the brain, but not with retinal levels of high molecular weight (aggregated) Aβ40 or Aβ42. Analysis of AppNL-F/NL-F knock-in mouse retina further showed that 50% of microglia in 3-month-old AppNL-F/NL-F knock-in mice contained bTVBT2. The percentage significantly increased in 9- and 12-month-old mice. Our study suggests that the microglial capability to uptake p-tau in the retina persists and intensifies with AD progression. These results also highlight bTVBT2 as a ligand of interest in future monitoring of retinal AD pathology.

Non-invasive in vivo imaging of brain and retinal microglia in neurodegenerative diseases.

Microglia play crucial roles in immune responses and contribute to fundamental biological processes within the central nervous system (CNS). In neurodegenerative diseases, microglia undergo functional changes and can have both protective and pathogenic roles. Microglia in the retina, as an extension of the CNS, have also been shown to be affected in many neurological diseases. While our understanding of how microglia contribute to pathological conditions is incomplete, non-invasive in vivo imaging of brain and retinal microglia in living subjects could provide valuable insights into their role in the neurodegenerative diseases and open new avenues for diagnostic biomarkers. This mini-review provides an overview of the current brain and retinal imaging tools for studying microglia in vivo. We focus on microglia targets, the advantages and limitations of in vivo microglia imaging approaches, and applications for evaluating the pathogenesis of neurological conditions, such as Alzheimer's disease and multiple sclerosis.

Retinal microglia and ganglion cells in a cuprizone (CPZ) induced mouse model of multiple sclerosis (MS)

Aims/Purpose: Up to 90% of MS patients have ocular manifestations. Activated microglia (mg) are associated with age and diseases like MS. Mg change their activation states (ramified, hyper‐ramified, activated, amoeboid and rod) in response to stimuli, to exert either neuro‐protective or ‐toxic effects, leading to the rescue or loss of neurons. Here, we characterize retinal ganglion cell (RGC) densities and retinal mg morphotype proportions in eyes of a CPZ induced MS model and age‐matched controls.Methods: Male C57 mice were fed CPZ (0.2%w/w) mixed chow for 4 months (m) until sacrificed at 6 m (6mCPZ) or 28 m (28mCPZ) old. Age‐matched controls (6mCtrl; 28mCtrl) were fed normal chow. Enucleated eyes were fixed in 4% PFA. Whole retinas were dissected, stained (RBPMS+RGCs; iba‐1+mg), mounted and imaged. A published retinal mg morphotyping algorithm and an automated RBPMS counting script were used. Two‐way ANOVA with paired analysis (PA) was used (mg n: 6mCtrl = 15; 6mCPZ = 7; 28mCtrl = 7; 28mCPZ = 10; and RGC n: 6mCtrl = 4; 6mCPZ = 3; 28mCtrl = 3; 28mCPZ = 4).Results: There was a main effect of age for activated (p = 0.0282) and rod (p < 0.0001), and disease state for rod (p = 0.04) mg. PA showed higher activated mg in 28mCtrl versus 6mCtrl (p = 0.0485), and lower rod mg in 28mCtrl versus 6mCtrl (p = 0.0374) and in 28mCPZ versus 6mCPZ (p = 0.0007). There was a main effect of age (p = 0.0001) and disease state (p = 0.04) on RBPMS density. PA showed lower RGC density in 28mCtrl versus 6mCtrl (p = 0.0025) and in 28mCPZ versus 6mCPZ (p = 0.025).Conclusions: Our results show that there is age‐related loss of rod mg and RGCs in healthy and CPZ groups, supporting reports that rod mg are involved in protecting less damaged neurons. Age‐related increased activated mg may cause RGC loss as transcription factors like TNFα, bind to RGC receptors, inducing apoptosis. Further transcriptomic analysis may clarify functional interpretations.

Microbial infection and associated inflammasome in the retina of Alzheimer’s disease patients

AbstractBackgroundRecent advances in Alzheimer’s disease (AD) research revealed that AD pathology is not confined to the brain but also occur in the neurosensory retina. Data have further suggested parallels between retinal and brain AD biomarkers. While the exact etiology of AD pathogenesis is undefined, an emerging theory has linked AD with microbial infection as a plausible cause for AD progression. The presence of several microbes including Chlamydia pneumoniae (Cp), Borrelia burgdorferi, Porphyromonas gingivalis, and Toxoplasma gondii have been reported in the post‐mortem brain of AD patients and murine models. However, the existence and role of microbial infection in AD retina has never been studied.MethodWe employed a range of histological, biochemical, and genetic techniques to detect bacterial infection and other associated biomarkers in postmortem retinas of patients with mild cognitive impairment (MCI) or AD dementia, compared to retinas from age‐matched individuals who displayed normal cognition (NC). We assessed the presence and burden of Cp and correlated with gliosis, inflammasome, and apoptosis markers.ResultWe discovered the presence of Cp inclusions in postmortem retinas of patients with MCI and AD as compared to age‐ and sex‐matched NC individuals. Our experimental evidence revealed Cp‐infected retinal neuronal cells in AD patients, with no increase in retinal Cp infection in MCI versus CN and a significant 4‐fold increase in of Cp immunoreactivity in retinas of AD patients versus MCI and CN subjects. These data possibly indicate that Cp infection mainly occur during the dementia phase of AD and is less observable at the early functional impairment stages of AD. Retinal microglia were found to aggregate around and engulf Cp‐infected cells. Alongside Cp infection, retinal tissues from MCI and AD patients exhibited increased NLRP3 inflammasome and gliosis. Our data suggest Cp‐triggered inflammasome activation, that can lead to neuronal degeneration. We further found a relationship between the severity of retinal Cp infection and brain pathology; retinal Cp burden was associated with brain neurofibrillary tangles, neuropil threads, Braak stage.ConclusionCp‐triggered chronic inflammation could be a detrimental process leading to retinal degeneration in AD, suggesting potential new therapeutic avenues involving early antibiotic treatment or inflammasome attenuation.

COG1410 regulates microglial states and protects retinal ganglion cells in retinal ischemia-reperfusion injury

Progressive loss of retinal ganglion cells (RGCs) caused by retinal ischemia-reperfusion (IR) injury can lead to irreversible vision impairment, with neuroinflammatory responses playing an important role in this process. COG1410, a mimetic peptide of apolipoprotein E, has demonstrated protective potential in the central nervous system, but its effects on retinal IR injury remain unexplored. In this study, we established a mouse model of retinal IR injury to investigate the effects of COG1410 on retinal microglia and RGCs. We observed CD16/32-marked and CD206-marked microglia and RGCs using immunofluorescence staining, detected the expression of inflammatory factors by PCR, and evaluated retinal apoptosis with TUNEL staining. We further investigated the potential mechanism by detecting the expression of key proteins via Western blot. The results reveal that COG1410 decreased the number of CD16/32-marked microglia and increased the number of CD206-marked microglia, alleviated the expression of IL-1β and TNF-α, and reduced the loss of RGCs by inhibiting the mitochondrial-related apoptotic pathway. COG1410 was found to increase the expression of ERK1/2 and Nr4a1 but decrease the expression of NF-κB. The expression of TREM2 showed an increasing trend after COG1410 administration, but it was not statistically significant. In conclusion, COG1410 regulates microglial states and protects RGCs in retinal IR injury, showing promising potential for the treatment of eye diseases.

Glutathione trisulfide prevents lipopolysaccharide-induced retinal inflammation via inhibition of proinflammatory cytokine production in glial cells

We aimed to investigate the impact of glutathione trisulfide (GSSSG) on lipopolysaccharide (LPS)-induced inflammation in retinal glia. Inflammatory responses in mouse-derived glial cells and Wistar rat retinas were stimulated with administration of LPS. Cell survival and proinflammatory cytokine production were examined using the Calcein-AM assay, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Retinal microglia were visualized with immunohistochemistry for Iba1. Administration of LPS (10 µg/mL) or GSSSG (less than 100 µM) did not affect survival of cultured primary Müller cells and established microglial cells (BV-2). RT-qPCR and ELISA indicated that GSSSG inhibited LPS-induced gene upregulation and protein secretion of proinflammatory cytokines in these glial cells and rat retinas. GSSSG inhibited LPS-induced activation of TGF-β-activated kinase 1 (TAK1), which is an upstream kinase of NF-κB, in BV-2 cells. Finally, in vivo experiments indicated that intravitreal administration of GSSSG but not its relative glutathione disulfide (GSSG) inhibited LPS (500 ng)-induced accumulation of Iba1-immunopositive microglia in rat retinas. Taken together, GSSSG has the potential to prevent pathogenesis of inflammation-associated ocular diseases by inhibiting proinflammatory cytokine expression in retinal glial cells.

Integrating human iPSC-derived macrophage progenitors into retinal organoids to generate a mature retinal microglial niche.

In the retina, microglia are resident immune cells that are essential for development and function. Retinal microglia play a central role in mediating pathological degeneration in diseases such as glaucoma, retinitis pigmentosa, age-related neurodegeneration, ischemic retinopathy, and diabetic retinopathy. Current models of mature human retinal organoids (ROs) derived from iPS cell (hiPSC) do not contain resident microglia integrated into retinal layers. Increasing cellular diversity in ROs by including resident microglia would more accurately represent the native retina and better model diseases in which microglia play a key role. In this study, we develop a new 3D in vitro tissue model of microglia-containing retinal organoids by co-culturing ROs and hiPSC-derived macrophage precursor cells (MPCs). We optimized the parameters for successful integration of MPCs into retinal organoids. We show that while in the ROs, MPCs migrate to the equivalent of the outer plexiform layer where retinal microglia cells reside in healthy retinal tissue. While there, they develop a mature morphology characterized by small cell bodies and long branching processes which is only observed in vivo. During this maturation process these MPCs cycle through an activated phase followed by a stable mature microglial phase as seen by the down regulation of pro-inflammatory cytokines and upregulation of anti-inflammatory cytokines. Finally, we characterized mature ROs with integrated MPCs using RNAseq showing an enrichment of cell-type specific microglia markers. We propose that this co-culture system may be useful for understanding the pathogenesis of retinal diseases involving retinal microglia and for drug discovery directly in human tissue.

Neuroprotection of Retinal Ganglion Cells Suppresses Microglia Activation in a Mouse Model of Glaucoma.

Microglial activation has been implicated in many neurodegenerative eye diseases, but the interrelationship between cell loss and microglia activation remains unclear. In glaucoma, there is no consensus yet whether microglial activation precedes or is a consequence of retinal ganglion cell (RGC) degeneration. We therefore investigated the temporal and spatial appearance of activated microglia in retina and their correspondence to RGC degeneration in glaucoma. We used an established microbead occlusion model of glaucoma in mouse whereby intraocular pressure (IOP) was elevated. Specific antibodies were used to immunolabel microglia in resting and activated states. To block retinal gap junction (GJ) communication, which has been shown previously to provide significant neuroprotection of RGCs, the GJ blocker meclofenamic acid was administered or connexin36 (Cx36) GJ subunits were ablated genetically. We then studied microglial activation at different time points after microbead injection in control and neuroprotected retinas. Histochemical analysis of flatmount retinas revealed major changes in microglia morphology, density, and immunoreactivity in microbead-injected eyes. An early stage of microglial activation followed IOP elevation, as indicated by changes in morphology and cell density, but preceded RGC death. In contrast, the later stage of microglia activation, associated with upregulation of major histocompatibility complex class II expression, corresponded temporally to the initial loss of RGCs. However, we found that protection of RGCs afforded by GJ blockade or genetic ablation largely suppressed microglial changes at all stages of activation in glaucomatous retinas. Together, our data strongly suggest that microglia activation in glaucoma is a consequence, rather than a cause, of initial RGC degeneration and death.

The BET PROTAC inhibitor dBET6 protects against retinal degeneration and inhibits the cGAS-STING in response to light damage

BackgroundChronic inflammation significantly contributes to photoreceptor death in blinding retinal diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Bromodomain and extraterminal domain (BET) proteins are epigenetic readers that act as key proinflammatory factors. We recently found the first-generation BET inhibitor JQ1 alleviated sodium iodate-induced retinal degeneration by suppressing cGAS-STING innate immunity. Here, we investigated the effects and mechanism of dBET6, a proteolysis‑targeting chimera (PROTAC) small molecule that selectively degrades BET by the ubiquitin‒proteasome system, in light-induced retinal degeneration.MethodsMice were exposed to bright light to induce retinal degeneration, and the activation of cGAS-STING was determined by RNA-sequencing and molecular biology. Retinal function, morphology, photoreceptor viability and retinal inflammation were examined in the presence and absence of dBET6 treatment.ResultsIntraperitoneal injection of dBET6 led to the rapid degradation of BET protein in the retina without detectable toxicity. dBET6 improved retinal responsiveness and visual acuity after light damage (LD). dBET6 also repressed LD-induced retinal macrophages/microglia activation, Müller cell gliosis, photoreceptor death and retinal degeneration. Analysis of single-cell RNA-sequencing results revealed cGAS-STING components were expressed in retinal microglia. LD led to dramatic activation of the cGAS-STING pathway, whereas dBET6 suppressed LD-induced STING expression in reactive macrophages/microglia and the related inflammatory response.ConclusionsThis study indicates targeted degradation of BET by dBET6 exerts neuroprotective effects by inhibiting cGAS-STING in reactive retinal macrophages/microglia, and is expected to become a new strategy for treatment of retinal degeneration.

Interleukin-17A modulates retinal inflammation by regulating microglial activation via the p38 MAPK pathway in experimental glaucoma neuropathy.

As a prototypical member of the IL-17 family, interleukin-17A (IL-17A) has received increasing attentions for its potent proinflammatory role as well as potential to be a key therapeutic target in human autoimmune inflammatory diseases; however, its roles in other pathological scenarios like neuroinflammations are not fully elucidated yet but appear essentially correlating and promising. Glaucoma is the leading cause of irreversible blindness with complicated pathogenesis still to be understood, where neuroinflammation was reported to be critically involved in its both initiation and progression. Whether IL-17A takes part in the pathogenesis of glaucoma through interfering neuroinflammation due to its potent proinflammatory effect is still unknown. In the present study, we investigated the role of IL-17A in the pathological process of glaucoma neuropathy as well as its relationship with the predominant immune inflammation mediator microglia in retina, trying to elucidate the underlying mechanisms from the view of inflammation modulation. In our study, RNA sequencing was performed for the retinas of chronic ocular hypertension (COH) and control mice. Western blot, RT-PCR, immunofluorescence, and ELISA were used to evaluate the microglial activation and proinflammatory cytokines release at conditioned levels of IL-17A, along with assessment of optic nerve integrity including retinal ganglion cells (RGCs) counting, axonal neurofilament quantification, and flash visual-evoked potential (F-VEP) examination. And the possibly involved signaling pathways were screened out to go through further validation in scenarios with conditioned IL-17A. Subsequently, IL-17A was found to be significantly upregulated in COH retina. Furthermore, suppression of IL-17A effectively diminished the loss of RGCs, improved axonal quality, and F-VEP performance in COH mice. Mechanistically, IL-17A promoted microglial activation and proinflammatory cytokines release along with enhanced phenotypic conversion of activated microglia to M2-type in early stage and to M1-type in late stage in glaucomatous retinas. Microglia elimination decreased the proinflammatory factors secretion, enhanced the RGCs survival and axonal quality mediated by IL-17A. Furthermore, IL-17A-induced the overactivation of microglia in glaucomatous condition was alleviated after blocking the p38 MAPK pathway. Taken together, IL-17A is involved in the regulation of retinal immune response and RGCs cell death in experimental glaucoma by essentially promoting retinal microglial activation via p38 MAPK signaling pathway. IL-17A dynamically regulates the phenotypic conversion of retinal microglia in experimental glaucoma partly depending on the duration of elevated intraocular pressure. Suppression of IL-17A contributes to alleviate glaucoma neuropathy and exhibits promising potential as an innovative target for therapeutic strategy in glaucoma.