Retinal Inflammatory Diseases Research Articles

A Case of Concomitant Acute Zonal Occult Outer Retinopathy and Secondary Nonparaneoplastic Autoimmune Retinopathy

Introduction: Acute Zonal Occult Outer Retinopathy (AZOOR) is a rare inflammatory retinal disease with rapid outer retinal function loss, photopsias, unremarkable fundus findings, and electroretinography (ERG) abnormalities. AZOOR diagnosis can be challenging due to its overlap with other retinal conditions, such as acute idiopathic blind spot enlargement syndrome (AIBES) and autoimmune retinopathies (AIRs). Multimodal imaging, including fundus autofluorescence (FAF) and optical coherence tomography (OCT), has improved detection, revealing progressive outer retinal damage. Although the etiology of AZOOR remains uncertain, autoimmune mechanisms and viral associations have been proposed. Recent studies have identified anti-retinal antibodies, complicating differentiation from other autoimmune retinopathies.. Case Presentation: A 63-year-old male presented with photopsias, floaters, and worsening vision in his left eye. He had a prior diagnosis of autoimmune retinopathy with serum antibodies against enolase, arrestin, and heat shock protein 27 (HSP27). Despite corticosteroid therapy, his visual acuity worsened from 20/20 to 20/60. Fundus examination showed subtle changes, and multimodal imaging revealed outer retinal damage consistent with AZOOR. He was started on mycophenolate mofetil, cyclosporine, and intravenous immunoglobulin (IVIg). Over a year of follow-up, his vision improved to 20/25, and imaging showed stabilization of retinal damage. Conclusion: This case report highlights AZOOR can be associated with secondary npAIR. Multimodal imaging, electrophysiologic testing of retina and RPE, and anti-retinal antibody may be helpful for diagnosis of these patients. A combination of conventional immunomodulatory therapy and IVIg can help with controlling AZOOR and secondary npAIR.

Examining the correlation of lymphangiogenesis biomarkers with clinical condition in Age-Related Macular Degeneration (AMD)

The aim of this study is to investigate the relationship between age-related macular degeneration (AMD) and lymphangiogenesis biomarkers, namely LYVE-1, Podoplanin, VEGF-C, VEGFR-2 and VEGFR-3. This prospective and interventional study includes 30 patients with AMD which may be dry or wet type and 30 controls for whom vitrectomy and phacoemulsification was indicated due to additional pathologies (epiretinal membrane, macular hole, retinal detachment, and cataract). 0.1–0,2 ml of aqueous humor and 0.5–1 ml of vitreous sample was taken during the operations. Before the operations 1 tube serum was also taken. All the lymphangiogenesis biomarkers in the study are examined by ELISA method. LYVE-1 (p = 0.001) and Podoplanin (p = 0.004) levels in the vitreous for the patient group are found to be significantly lower than the control group. Serum (p = 0.019), vitreous (p = 0.001), aqueous (p < 0.001) levels of VEGF-C for the patient group are significantly higher than the control group. VEGF-C/VEGFR-2 (p < 0.001), VEGF-C/VEGFR-3 (p < 0.001) ratios in the vitreous for the patient group are found to be significantly higher than the control group. Especially in wet AMD patients, LYVE-1 level is significantly lower in the vitreous (p = 0.002) and aqueous (p = 0.002) than the control group. In addition, Podoplanin level is observed as significantly lower in the vitreous (p = 0.014) and serum (p = 0.002) in comparison to control group. In the wet AMD group, VEGF-C level in the vitreous (p < 0.001), aqueous (p < 0.001) and serum (p = 0.001) is higher than the control group. The result of this study indicates a valid relationship between the weakening of lymphangiogenesis and the pathophysiology of AMD, especially for the wet type. It is observed that the levels of receptors that bind VEGF-C (VEGFR-2 and VEGFR-3) do not increase at the same rate as VEGF-C to compensate for the increase in VEGF-C. The absence of an increase in VEGFR-3, which is especially necessary for lymphangiogenesis, also suggests that lymphangiogenesis is weakened or decreased in AMD. In the future interventional studies with larger series, examination of lymphangiogenic biomarkers in inflammatory retinal diseases and glaucoma may reveal unexplored details.

Roles of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor 2 in Endotoxin-Induced Acute Retinal Inflammation

ABSTRACT Purpose To investigate the roles of sphingosine kinases (SphKs) and sphingosine-1-phosphate receptors (S1PRs) in endotoxin-induced uveitis (EIU) mice. Methods EIU model was induced using an intraperitoneal injection of lipopolysaccharide (LPS). The expression of SphKs and S1PRs in the retina was assessed using quantitative polymerase chain reaction (qPCR) and immunofluorescence. The effects of S1PR antagonists on the expression of inflammatory cytokines in the retina were evaluated using qPCR and western blotting. Effects of leukocyte infiltration of the retinal vessels were evaluated to determine the effects of the S1PR2 antagonist and genetic deletion of S1PR2 on retinal inflammation. Results Retinal SphK1 expression was significantly upregulated in EIU. SphK1 was expressed in the GCL, IPL, and OPL and S1PR2 was expressed in the GCL, INL, and OPL. Positive cells in IPL and OPL of EIU retina were identified as endothelial cells. S1PR2 antagonist and genetic deletion of S1PR2 significantly suppressed the expression of IL-1α, IL-6, TNF-α, and ICAM-1, whereas S1PR1/3 antagonist did not. Use of S1PR2 antagonist and S1PR2 knockout in mice significantly ameliorated leukocyte adhesion induced by LPS. Conclusion SphK1/S1P/S1PR2 signaling was upregulated in EIU and S1PR2 inhibition suppressed inflammatory response. Targeting this signaling pathway has potential for treating retinal inflammatory diseases.

Co-cultivation of primary porcine RPE cells and neuroretina induces inflammation: a potential inflammatory AMD-model

One common aspect in the pathology of many retinal diseases like age-related macular degeneration (AMD) is the death of retinal pigment epithelium (RPE) cells. RPE cells are essential for photoreceptor survival as they recycle and remove compounds of the visual cycle and secrete protective cytokines. Studying RPE cells is crucial to improve our understanding of retinal pathologies, yet only a few retinal ex vivo models include them or do so only indirectly. Besides the positive effects in indirect co-cultivation models, also a slight inflammation was observed. In this study we developed an ex vivo model consisting of a primary porcine RPE monolayer directly co-cultured with porcine retinal organ cultures, to investigate and simulate inflammatory retinal diseases, such as (dry) AMD. The direct co-cultivation resulted in immune reactivity (enhanced expression of pro-inflammatory cytokines e.g., IL-1β, IL-6,IL-8) and cell death. These effects were evaluated for the retinal explant as well as for the RPE-monolayer to further understand the complex interactions between these two compartments. Taken together, this ex vivo model can be used to study inflammatory retinal diseases like AMD as well as the rejection observed after RPE-transplantation.

Lithium Chloride Exerts Anti-Inflammatory and Neuroprotective Effects by Inhibiting Microglial Activation in LPS-Induced Retinal Injury.

To explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury. In vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography. In vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage. This study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.

Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure

BackgroundClustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation.MethodsC57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre−posTlr4loxP/loxP) and TekCre−negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function.ResultsActivation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre−negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge.ConclusionsThe findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia–endothelial cell interaction in inflammatory retinal disease.

Anti-Inflammatory Effects of GLP-1R Activation in the Retina.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone, mainly produced by enteroendocrine L cells, which participates in the regulation of glucose homeostasis, and in reduction in body weight by promoting satiety. Actions of GLP-1 are mediated by activation of its receptor GLP-1R, which is widely expressed in several tissues including the retina. The effects of GLP-1R activation are useful in the management of type 2 diabetes mellitus (T2DM). In addition, the activation of GLP-1R has anti-inflammatory effects in several organs, suggesting that it may be also useful in the treatment of inflammatory diseases. Inflammation is a common element in the pathogenesis of several ocular diseases, and the protective effects of treatment with GLP-1 emerged also in retinal diseases. In this review we highlight the anti-inflammatory effects of GLP-1R activation in the retina. Firstly, we summarized the pathogenic role of inflammation in ocular diseases. Then, we described the pleiotropic effects of GLP-1R activation on the cellular components of the retina which are mainly involved in the pathogenesis of inflammatory retinal diseases: the retinal ganglion cells, retinal pigment epithelial cells and endothelial cells.

Retinal Proteomic Analysis in a Mouse Model of Endotoxin-Induced Uveitis Using Data-Independent Acquisition-Based Mass Spectrometry

Uveitis is a group of sight-threatening ocular inflammatory diseases, potentially leading to permanent vision loss in patients. However, it remains largely unknown how uveitis causes retinal malfunction and vision loss. Endotoxin-induced uveitis (EIU) in rodents is a good animal model to study uveitis and associated acute retinal inflammation. To understand the pathogenic mechanism of uveitis and screen potential targets for treatment, we analyzed the retinal proteomic profile of the EIU mouse model using a data-independent acquisition-based mass spectrometry (SWATH-MS). After systemic LPS administration, we observed activation of microglial cells accompanied with the elevation of pro-inflammatory mediators and visual function declines. In total, we observed 79 upregulated and 90 downregulated differentially expressed proteins (DEPs). Among the DEPs, we found that histone family members (histone H1, H2A, H2B) and blood proteins including haptoglobin (HP), hemopexin (HPX), and fibrinogen gamma chain (FGG) were dramatically increased in EIU groups relative to those in control groups. We identified phototransduction and synaptic vesicle cycle as the top two significant KEGG pathways. Moreover, canonical pathway analysis on DEPs using Ingenuity Pathway Analysis revealed top three most significant enriched pathways related to acute phase response signaling, synaptogenesis signaling, and eif2 signaling. We further confirmed upregulation of several DEPs associated with the acute phase response signaling including HP, HPX, and FGG in LPS-treated retinas by qPCR and Western blot. In summary, this study serves as the first report to detect retinal proteome changes in the EIU model. The study provides several potential candidates for exploring the mechanism and novel therapeutic targets for uveitis and other retinal inflammatory diseases.

Regulations of Retinal Inflammation: Focusing on Müller Glia.

Retinal inflammation underlies multiple prevalent retinal diseases. While microglia are one of the most studied cell types regarding retinal inflammation, growing evidence shows that Müller glia play critical roles in the regulation of retinal inflammation. Müller glia express various receptors for cytokines and release cytokines to regulate inflammation. Müller glia are part of the blood-retinal barrier and interact with microglia in the inflammatory responses. The unique metabolic features of Müller glia in the retina makes them vital for retinal homeostasis maintenance, regulating retinal inflammation by lipid metabolism, purine metabolism, iron metabolism, trophic factors, and antioxidants. miRNAs in Müller glia regulate inflammatory responses via different mechanisms and potentially regulate retinal regeneration. Novel therapies are explored targeting Müller glia for inflammatory retinal diseases treatment. Here we review new findings regarding the roles of Müller glia in retinal inflammation and discuss the related novel therapies for retinal diseases.

The topical ocular delivery of rapamycin to posterior eye tissues and the suppression of retinal inflammatory disease

Treatment of posterior eye diseases with intravitreal injections of drugs, while effective, is invasive and associated with side effects such as retinal detachment and endophthalmitis. In this work, we have formulated a model compound, rapamycin (RAP), in nanoparticle-based eye drops and evaluated the delivery of RAP to the posterior eye tissues in a healthy rabbit. We have also studied the formulation in experimental autoimmune uveitis (EAU) mouse model with retinal inflammation. Aqueous RAP eye drops were prepared using N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (Molecular Envelope Technology – MET) containing 0.23 ± 0.001% w/v RAP with viscosity, osmolarity, and pH within the ocular comfort range, and the formulation (MET-RAP) was stable in terms of drug content at both refrigeration and room temperature for one month. The MET-RAP eye drops delivered RAP to the choroid-retina with a Cmax of 145 ± 49 ng/g (tmax = 1 h). The topical application of the MET-RAP eye drops to the EAU mouse model resulted in significant disease suppression compared to controls, with activity similar to dexamethasone eye drops. The MET-RAP eye drops also resulted in a reduction of RORγt and an increase in both Foxp3 expression and IL-10 secretion, indicating a mechanism involving the inhibition of Th17 cells and the up-regulation of T-reg cells. The MET-RAP formulation delivers RAP to the posterior eye segments, and the formulation is active in EAU.

A modified high-yield method for primary culture of rat retinal microglial cells

Microglial cells are the main immune cells of the retina. The primary culture of the retinal microglia is critically important in investigating the cells' properties and behaviors in neurodegenerative and inflammatory retinal disease. Here, we described a modified protocol of a microglial cell culture from the neonatal rat retina. In our culture protocol, the retina was isolated from the neonatal rat eye from postnatal day 1 to day 3 and trypsinized into a single-cell suspension. The cells were seeded into a T75 flask, which was pre-coated with poly-D-lysine (PDL) and cultured with dulbecco's modified eagle medium-F12 (DMEM/F12) that contained 10% fetal bovine serum (FBS) with different concentrations. Small bright rounded cells were observed on the top of mixed glial cells on the seventh day, and attained the maximum cell number on the 14th day. Then, the isolation was performed by a shaking method and isolated cells were identified with microglia markers ionized calcium-binding adaptor molecule 1 (IBA1), transmembrane protein 119 (TMEM119), cluster of differentiation 11b (CD11b), as well as astrocyte marker glial fibrillary acidic protein (GFAP) by immunofluorescence staining. Additionally, the initial plating ratio of the mixed glial cell, culture period of isolation, procedures of the isolation, as well as the purification procedure, were optimized for our primary microglial cell culture. The morphological changes and phagocytic function were performed after lipopolysaccharide (LPS) stimulation. Moreover, the release of pro-inflammatory cytokines at different time points of LPS activation were measured. In the present study, we found that the concentration of one retina/T75 flask could harvest the largest number of microglial cells. Besides, we continuously cultured the mixed glial cells as long as one month and isolated the mixed glial cells as much as three times. In our study, we used an isolation-shaking rate of 200 rpm for 2h, which guaranteed the steady rate and resulted in high purification of the primary retinal-microglial cells, with no need of an additional purification procedure. In conclusion, we provided a high-producing protocol for the primary culture of purified rat retinal-microglial cells.

CD4+ T-Cell Plasticity in Non-Infectious Retinal Inflammatory Disease.

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4+ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4+FoxP3+CD25+ regulatory T cells (Tregs) were known to suppress function of effector CD4+ T cells and contribute to resolution of disease. It has been recently reported that some CD4+ T-cell subsets demonstrate shared phenotypes with another CD4+ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4+ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

The Chx10-Traf3 Knockout Mouse as a Viable Model to Study Neuronal Immune Regulation.

Uncontrolled inflammation is associated with neurodegenerative conditions in central nervous system tissues, including the retina and brain. We previously found that the neural retina (NR) plays an important role in retinal immunity. Tumor necrosis factor Receptor-Associated Factor 3 (TRAF3) is a known immune regulator expressed in the retina; however, whether TRAF3 regulates retinal immunity is unknown. We have generated the first conditional NR-Traf3 knockout mouse model (Chx10-Cre/Traf3f/f) to enable studies of neuronal TRAF3 function. Here, we evaluated NR-Traf3 depletion effects on whole retinal TRAF3 protein expression, visual acuity, and retinal structure and function. Additionally, to determine if NR-Traf3 plays a role in retinal immune regulation, we used flow cytometry to assess immune cell infiltration following acute local lipopolysaccharide (LPS) administration. Our results show that TRAF3 protein is highly expressed in the NR and establish that NR-Traf3 depletion does not affect basal retinal structure or function. Importantly, NR-Traf3 promoted LPS-stimulated retinal immune infiltration. Thus, our findings propose NR-Traf3 as a positive regulator of retinal immunity. Further, the NR-Traf3 mouse provides a tool for investigations of neuronal TRAF3 as a novel potential target for therapeutic interventions aimed at suppressing retinal inflammatory disease and may also inform treatment approaches for inflammatory neurodegenerative brain conditions.

The Major Role of Anti-Vascular Endothelial Growth Factor for Management of Macular Edema Secondary to Retinal Vein Occlusion

The goal of this study is to evaluate the efficacy, safety, and injection frequency of vascular endothelial growth factor (VEGF) antagonists in the treatment of macular edema secondary to retinal vein occlusion (RVO) in clinical practice.Macular edema is a major complication of several vascular and inflammatory retinal diseases Multiple mechanisms are implicated in its development and lead to visual impairment that could be reversible (the acute stages) or not reversible (long-standing ME). This study was conducted on 30 patients with retinal vein occlusion .The study population were also randomly assigned into two groups regarding the management regimens: 15 subjects as central RVO ,15 subjects as branch RVO. Recently, antiangiogenic therapy with anti-vascular endothelialyy growth factor (anti-VEGF) has been used successfully to treat MO resulting from a variety of causes. As elevated intraocular levels of VEGF have been demonstrated in patients with retinal vein occlusions there is a strong basis for the hypothesis that anti-VEGF agents may be beneficial in the treatment of vascular leakage and MO.

Dynamics of Cyclooxygenase-1 Positive Microglia/Macrophage in the Retina of Pathological Model Mice as a Biomarker of the Retinal Inflammatory Diseases.

In an intraocular inflammatory state, microglia residing in the retina become active and migrate inside the retina. In this study, we investigated whether cyclooxygenase-1 (COX-1) expressed by retinal microglia/macrophage can be a biomarker for the diagnosis of retinal diseases. COX-1 was immunopositive in microglia/macrophage and neutrophils, while COX-2 was immunopositive in astrocytes and neurons in the inner layer of normal retina. The number of COX-1 positive cells per section of the retinal tissue was 14 ± 2.8 (mean ± standard deviation) in normal mice, which showed significant increase in the lipopolysaccharide (LPS)-administrated model (62 ± 5.0, p = 8.7 × 10−9). In addition to microglia, we found neutrophils that were positive for COX-1. In the early stage of inflammation in the experimental autoimmune uveoretinitis (EAU), COX-1 positive cells, infiltrating from the ciliary body into the retinal outer nuclear layer, were observed. The number of infiltrating COX-1 positive cells correlated with the severity of EAU. Taken together, the increased number of COX-1 positive microglia/macrophage with morphological changes were observed in the retinas of retinal inflammatory disease models. This suggests that COX-1 can be a marker of disease-related activities of microglia/macrophage, which should be useful for the diagnosis of retinal diseases.

Immune-Mediated Retinal Vasculitis in Posterior Uveitis and Experimental Models: The Leukotriene (LT)B4-VEGF Axis.

Retinal vascular diseases have distinct, complex and multifactorial pathogeneses yet share several key pathophysiological aspects including inflammation, vascular permeability and neovascularisation. In non-infectious posterior uveitis (NIU), retinal vasculitis involves vessel leakage leading to retinal enlargement, exudation, and macular oedema. Neovascularisation is not a common feature in NIU, however, detection of the major angiogenic factor—vascular endothelial growth factor A (VEGF-A)—in intraocular fluids in animal models of uveitis may be an indication for a role for this cytokine in a highly inflammatory condition. Suppression of VEGF-A by directly targeting the leukotriene B4 (LTB4) receptor (BLT1) pathway indicates a connection between leukotrienes (LTs), which have prominent roles in initiating and propagating inflammatory responses, and VEGF-A in retinal inflammatory diseases. Further research is needed to understand how LTs interact with intraocular cytokines in retinal inflammatory diseases to guide the development of novel therapeutic approaches targeting both inflammatory mediator pathways.

Molecular Surgery: Proteomics of a Rare Genetic Disease Gives Insight into Common Causes of Blindness.

SummaryRare diseases are an emerging global health priority. Although individually rare, the prevalence of rare “orphan” diseases is high, affecting approximately 300 million people worldwide. Treatments for these conditions are often inadequate, leaving the disease to progress unabated. Here, we review the clinical features and pathophysiology of neovascular inflammatory vitreoretinopathy (NIV), a rare inflammatory retinal disease caused by mutations in the CAPN5 gene. Although the prevalence of NIV is low (1 in 1,000,000 people), the disease mimics more common causes of blindness (e.g. uveitis, retinitis pigmentosa, proliferative diabetic retinopathy, and proliferative vitreoretinopathy) at distinct clinical stages. There is no cure for NIV to date. We highlight how personalized proteomics helped identify potential stage-specific biomarkers and drug targets in liquid vitreous biopsies. The NIV vitreous proteome revealed enrichment of molecular pathways associated with common retinal pathologies and implicated superior targets for therapeutic drug repositioning. In addition, we review our pipeline for collecting, storing, and analyzing ophthalmic surgical samples. This approach can be adapted to treat a variety of rare genetic diseases.

Innate immune response in retinal homeostasis and inflammatory disorders.

Innate immune cells such as neutrophils, monocyte-macrophages and microglial cells are pivotal for the health and disease of the retina. For the maintenance of retinal homeostasis, these cells and immunosuppressive molecules in the eye actively regulate the induction and the expression of inflammation in order to prevent excessive activation and subsequent tissue damage. In the disease context, these regulatory mechanisms are modulated genetically and/or by environmental stimuli such as damage-associated molecular patterns (DAMPs), and a chronic innate immune response regulates or contributes to the formation of diverse retinal disorders such as uveitis, retinitis pigmentosa, retinal vascular diseases and retinal fibrosis. Here we summarize the recent knowledge regarding the innate immune response in both ocular immune regulation and inflammatory retinal diseases, and we describe the potential of the innate immune response as a biomarker and therapeutic target.

Emerging therapies in the management of macular edema: a review.

Macular edema (ME) is a major complication of several vascular and inflammatory retinal diseases. Multiple mechanisms are implicated in its development and lead to visual impairment that could be reversible (the acute stages) or not reversible (long-standing ME). For this reason, an effective approach to the treatment of ME is of paramount importance in order to prevent irreversible damage of visual function. In this review, we discuss the management of ME and, in particular, current data of studies and clinical trials about drugs that have already been evaluated or are under investigation in the management of ME. Although several diseases could lead to the development of ME, we focus on the three main causes: diabetic retinopathy (DR), retinal vein occlusion (RVO), and uveitis. The introduction into clinical practice of anti-vascular endothelial growth factor injections (ranibizumab and aflibercept) and dexamethasone implants has revolutionized the treatment of ME secondary to DR and RVO. However, new drugs are needed in the treatment of resistant forms of ME secondary to DR and RVO. A fluocinolone acetonide implant has been approved by the US Food and Drug Administration for the treatment of diabetic ME but not for RVO. Furthermore, brolucizumab and abicipar pegol have been shown to be effective in preliminary studies and have the chance to be approved soon for diabetic ME treatment. In ME secondary to uveitis, a crucial role is played by corticosteroids and non-biologic immunomodulatory drugs. However, several new biologic agents are under investigation in different clinical trials and could be important new therapeutic options in cases with a low response to first-line therapy. However, only a few of these drugs will enter the market after proving their safety and efficacy. Only after that will we be able to offer a new therapeutic option to patients affected by uveitic ME.

Lipopolysaccharide-Induced Autophagy Mediates Retinal Pigment Epithelium Cells Survival. Modulation by the Phospholipase D Pathway.

Inflammation and oxidative stress are common factors involved in the pathogenesis of retinal diseases, such as aged-related macular degeneration (AMD) and diabetic retinopathy (DR). Autophagy is a catabolic process essential to cell survival in response to stress. This process is highly active in retinal pigment epithelium (RPE) cells. Our previous findings demonstrated that lipopolysaccharide (LPS) induces an inflammatory response of RPE cells that implies classical phospholipases D (PLD1 and 2) activation, cyclooxygenase-2 (COX-2) expression, prostaglandin E2 (PGE2) production and reduced cell viability. In this work, we studied the autophagic process and its modulation by the PLD pathway in D407 and ARPE-19 RPE cells exposed to LPS. LPS (10 μg/ml or 25 μg/ml) exposure for 24 h increased light chain 3B-II (LC3B-II) content (an autophagy marker) and LC3B-positive punctate structures in both RPE cell lines studied. Next, the drug bafilomycin A1 (BAF, 50 nM) was used to block the autophagic flux. In cells pre-incubated with BAF, LC3B-II and sequestosome 1 (SQSTM1/p62) levels and autophagosome-like structures were increased by LPS, demonstrating that the inflammatory injury increases the autophagic process in RPE cells. To study the role of the PLD pathway, cells were pre-incubated for 1 h with selective PLD1 (VU0359595) or PLD2 (VU0285655-1) inhibitors prior to LPS addition. Under control condition, LC3B-positive punctate structures were increased in cells pre-incubated with PLD2 inhibitor while with PLD1 inhibitor were increased in cells exposed to LPS. MTT reduction assays showed that early autophagy inhibitors, 3-methyladenin (3-MA) or LY294002, enhanced the loss in cell viability induced by LPS exposure for 48 h. On the contrary, the inhibition of PLD1 and PLD2 prevented the loss in cell viability induced by LPS. In conclusion, our results show that even though LPS treatment promotes an inflammatory response in RPE cells, it also triggers the activation of the autophagic process which in turn may serve as a protective mechanism for the cells. In addition, we demonstrate that the PLD pathway modulates the autophagic process in RPE cells. Our findings contribute to the knowledge of the molecular basis of retinal inflammatory and degenerative diseases and open new avenues for potential therapeutic exploration.