The role of immune modulators in age-related macular degeneration.

We provide an overview of the expanding literature on the role of cytokines and immune mediators in pathophysiology of age-related macular degeneration (AMD). Although many immunological mediators have been linked to AMD pathophysiology, the broader mechanistic picture remains unclear with substantial variations in the levels of evidence supporting these mediators. Therefore, we reviewed the literature considering the varying levels of supporting evidence. A Medical Subject Headings (MeSH) term-based literature research was conducted in September, 2023, consisting of the MeSH terms "cytokine" and "Age-related macular degeneration" connected by the operator "AND". After screening the publications by title, abstract, and full text, a total of 146 publications were included. The proinflammatory cytokines IL-1ß (especially in basic research studies), IL-6, IL-8, IL-18, TNF-α, and MCP-1 are the most extensively characterised cytokines/chemokines, highlighting the role of local inflammasome activation and altered macrophage function in the AMD pathophysiology. Among the antiinflammatory mediators IL-4, IL-10, and TGF-ß were found to be the most extensively characterised, with IL-4 driving and IL-10 and TGF-ß suppressing disease progression. Despite the extensive literature on this topic, a profound understanding of AMD pathophysiology has not yet been achieved. Therefore, further studies are needed to identify potential therapeutic targets, followed by clinical studies.

Neutrophils in Ocular Diseases.

Neutrophils, traditionally viewed as first responders to infection or tissue damage, exhibit dynamic and diverse roles in ocular health and disease. This review elaborates on previous findings that showed how neutrophils contribute to ocular diseases. In ocular infections, neutrophils play a pivotal role in host defense by orchestrating inflammatory responses to combat pathogens. Furthermore, in optic nerve neuropathies and retinal degenerative diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR), neutrophils are implicated in neuroinflammation and tissue damage owing to their ability to undergo neutrophil extracellular trap formation (NETosis) and secretion of inflammatory molecules. Targeting neutrophil-dependent processes holds promise as a therapeutic strategy, offering potential avenues for intervention in ocular infections, cancers, and retinal degenerative diseases. Understanding the multifaceted roles of neutrophils in ocular diseases is crucial for developing targeted therapies to improve patient outcomes.

SPI1-mediated macrophage polarization aggravates age-related macular degeneration.

Objective: This study revealed a core regulator and common upstream mechanisms for the multifaceted pathological processes of age-related macular degeneration (AMD) and provided proof-of-concept for this new therapeutic target. Methods: Comprehensive gene expression analysis was performed using RNA sequencing of eye cup from old mice as well as laser-induced choroidal neovascularization (CNV) mouse model. Through integrative analysis and protein-protein interaction (PPI) analysis, common pathways and key transcription factor was identified simultaneously engaged in age-related retinal degeneration and CNV, the two typical pathological process of AMD. Subsequently, the expression changes of Spi1, the key regulator, as well as the alternation of the downstream mechanisms were validated in both models through qRT-PCR, Elisa, flow cytometry and immunofluorescence. Further, we assessed the impact of Spi1 knockdown in vitro and in vivo using gene intervention vectors carried by adeno-associated virus or lentivirus to test its potential as a therapeutic target. Results: Compared to corresponding controls, we found 1,939 and 1,319 genes differentially expressed in eye cups of old and CNV mice respectively. The integrative analysis identified a total of 275 overlapping DEGs, of which 150 genes were co-upregulated. PPI analysis verified a central transcription factor, SPI1. The significant upregulation of Spi1 expression was then validated in both models, accompanied by macrophage polarization towards the M1 phenotype. Finally, SPI1 suppression significantly inhibited M1 polarization of BMDMs and attenuated neovascularization in CNV mice. Conclusion: This study demonstrates that SPI1 exerts a pivotal role in AMD by regulation of macrophage polarization and innate immune response, offering promise as an innovative target for treating AMD.

Age-Related Macular Degeneration: A Disease of Cellular Senescence and Dysregulated Immune Homeostasis.

Age-related macular degeneration (AMD) is a degenerative ocular disease primarily affecting central vision in the elderly. Its pathogenesis is complex, involving cellular senescence and immune homeostasis dysregulation. This review investigates the interaction between these two critical biological processes in AMD pathogenesis and their impact on disease progression. Initially, cellular senescence is analyzed, with particular emphasis on retinal damage induced by senescent retinal pigment epithelial cells. Subsequently, the occurrence of immune homeostasis dysregulation within the retina and its mechanistic role in AMD areis explored. Furthermore, the paper also discusses in detail the interplay between cellular senescence and immune responses, forming a vicious cycle that exacerbates retinal damage and may influence treatment outcomes. In summary, a deeper understanding of the interrelation between cellular senescence and immune dysregulation is vital for the developing innovative therapeutic strategies for AMD.

Complement regulation in the eye: implications for age-related macular degeneration.

Careful regulation of the complement system is critical for enabling complement proteins to titrate immune defense while also preventing collateral tissue damage from poorly controlled inflammation. In the eye, this balance between complement activity and inhibition is crucial, as a low level of basal complement activity is necessary to support ocular immune privilege, a prerequisite for maintaining vision. Dysregulated complement activation contributes to parainflammation, a low level of inflammation triggered by cellular damage that functions to reestablish homeostasis, or outright inflammation that disrupts the visual axis. Complement dysregulation has been implicated in many ocular diseases, including glaucoma, diabetic retinopathy, and age-related macular degeneration (AMD). In the last two decades, complement activity has been the focus of intense investigation in AMD pathogenesis, leading to the development of novel therapeutics for the treatment of atrophic AMD. This Review outlines recent advances and challenges, highlighting therapeutic approaches that have advanced to clinical trials, as well as providing a general overview of the complement system in the posterior segment of the eye and selected ocular diseases.

Past history of obesity triggers persistent epigenetic changes in innate immunity and exacerbates neuroinflammation.

Age-related macular degeneration is a prevalent neuroinflammatory condition and a major cause of blindness driven by genetic and environmental factors such as obesity. In diseases of aging, modifiable factors can be compounded over the life span. We report that diet-induced obesity earlier in life triggers persistent reprogramming of the innate immune system, lasting long after normalization of metabolic abnormalities. Stearic acid, acting through Toll-like receptor 4 (TLR4), is sufficient to remodel chromatin landscapes and selectively enhance accessibility at binding sites for activator protein-1 (AP-1). Myeloid cells show less oxidative phosphorylation and shift to glycolysis, ultimately leading to proinflammatory cytokine transcription, aggravation of pathological retinal angiogenesis, and neuronal degeneration associated with loss of visual function. Thus, a past history of obesity reprograms mononuclear phagocytes and predisposes to neuroinflammation.

Obesity confers macrophage memory.

Epigenetic programming of myeloid cells in obesity contributes to macular degeneration.

Inflammatory Factors of Macular Atrophy in Eyes With Neovascular Age-Related Macular Degeneration Treated With Aflibercept.

Background: Neovascular age-related macular degeneration (nAMD) is a leading cause of blindness in older people. Low-grade inflammation is well-known as one of the pathogenic mechanisms in nAMD. Anti-vascular endothelial growth factor (VEGF) therapy is the first-line treatment for nAMD, although macula atrophy (MA) developed under anti-VEGF therapy causes irreversible visual function impairment and is recognized as a serious disorder. Here, we show specific expression patterns of aqueous humor (AH) cytokines in nAMD eyes developing MA under intravitreal injection of aflibercept (IVA) as an anti-VEGF antibody and present predictive cytokines as biomarkers for the incidence of MA in nAMD eyes under IVA treatment. Methods: Twenty-eight nAMD patients received three consecutive monthly IVA, followed by a pro re nata regimen for 2 years. AH specimens were collected before first IVA (pre-IVA) and before third IVA (post-IVA). AH cytokine levels, visual acuity (VA), and central retinal thickness (CRT) were measured. Results: Two-year incidence of MA was 21.4%. In nAMD eyes developing MA [MA (+) group], pre-IVA levels of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1ß, VEGF and post-IVA level of MCP-1 were higher than those in nAMD eyes without MA [MA (-) group]. In hierarchical cluster analysis, pre-IVA MCP-1 and VEGF were grouped into the same subcluster, as were post-IVA MCP-1 and CRT. In principal component analysis, principal component loading (PCL) of pre-IVA interferon-γ-inducible protein 10 (IP-10) was 0.61, but PCL of post-IVA IP-10 decreased to -0.09. In receiver operating characteristic analysis and Kaplan-Meier curves, pre-IVA MCP-1, MIP-1ß, and VEGF and post-IVA interleukin-6, MCP-1, and MIP-1ß were detected as predictive factors for MA incidence. In 2-year clinical course, changes of VA in groups with high levels of pre-IVA MIP-1ß (over 39.9 pg/ml) and VEGF (over 150.4 pg/ml) were comparable to those in MA (+) group. Conclusion: Substantial loss of IP-10 effects and persistent inflammation contribute to incidence of MA, and screening of AH cytokine levels could be a useful method to predict MA incidence in nAMD eyes under anti-VEGF therapy.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

A2E-induced inflammation and angiogenesis in RPE cells in vitro are modulated by PPAR-α, -ß/δ, -γ, and RXR antagonists and by norbixin.

N-retinylidene-N-retinylethanolamine (A2E) plays a central role in age-related macular degeneration (AMD) by inducing angiogenesis and inflammation. A2E effects are mediated at least partly via the retinoic acid receptor (RAR)-α. Here we show that A2E binds and transactivates also peroxisome proliferator-activated receptors (PPAR) and retinoid X receptors (RXR). 9'-cis-norbixin, a di-apocarotenoid is also a ligand of these nuclear receptors (NR). Norbixin inhibits PPAR and RXR transactivation induced by A2E. Moreover, norbixin reduces protein kinase B (AKT) phosphorylation, NF-κB and AP-1 transactivation and mRNA expression of the inflammatory interleukins (IL) -6 and -8 and of vascular endothelial growth factor (VEGF) enhanced by A2E. By contrast, norbixin increases matrix metalloproteinase 9 (MMP9) and C-C motif chemokine ligand 2 (CCL2) mRNA expression in response to A2E. Selective PPAR-α, -ß/δ and -γ antagonists inhibit the expression of IL-6 and IL-8 while only the antagonist of PPAR-γ inhibits the transactivation of NF-κB following A2E exposure. In addition, a cocktail of all three PPARs antagonists and also HX531, an antagonist of RXR reproduce norbixin effects on inflammation. Altogether, A2E's deleterious biological effects could be inhibited through PPAR and RXR regulation. Moreover, the modulation of these NR by norbixin may open new avenues for the treatment of AMD.

CFH Loss in Human RPE Cells Leads to Inflammation and Complement System Dysregulation via the NF-κB Pathway.

Age-related macular degeneration (AMD), the leading cause of vision loss in the elderly, is a degenerative disease of the macula, where retinal pigment epithelium (RPE) cells are damaged in the early stages of the disease, and chronic inflammatory processes may be involved. Besides aging and lifestyle factors as drivers of AMD, a strong genetic association to AMD is found in genes of the complement system, with a single polymorphism in the complement factor H gene (CFH), accounting for the majority of AMD risk. However, the exact mechanism of CFH dysregulation confers such a great risk for AMD and its role in RPE cell homeostasis is unclear. To explore the role of endogenous CFH locally in RPE cells, we silenced CFH in human hTERT-RPE1 cells. We demonstrate that endogenously expressed CFH in RPE cells modulates inflammatory cytokine production and complement regulation, independent of external complement sources, or stressors. We show that loss of the factor H protein (FH) results in increased levels of inflammatory mediators (e.g., IL-6, IL-8, GM-CSF) and altered levels of complement proteins (e.g., C3, CFB upregulation, and C5 downregulation) that are known to play a role in AMD. Moreover, our results identify the NF-κB pathway as the major pathway involved in regulating these inflammatory and complement factors. Our findings suggest that in RPE cells, FH and the NF-κB pathway work in synergy to maintain inflammatory and complement balance, and in case either one of them is dysregulated, the RPE microenvironment changes towards a proinflammatory AMD-like phenotype.

Mechanism of Nucleic Acid Sensing in Retinal Pigment Epithelium (RPE): RIG-I Mediates Type I Interferon Response in Human RPE.

Age-related macular degeneration (AMD), a degenerative disease of the outer retina, is the leading cause of blindness among the elderly. A hallmark of geographic atrophy (GA), an advanced type of nonneovascular AMD (dry AMD), is photoreceptor and retinal pigment epithelium (RPE) cell death. Currently, there are no FDA-approved therapies for GA due to a lack of understanding of the disease-causing mechanisms. Increasing evidence suggests that chronic inflammation plays a predominant role in the pathogenesis of dry AMD. Dead or stressed cells release danger signals and inflammatory factors, which causes further damage to neighboring cells. It has been reported that type I interferon (IFN) response is activated in RPE cells in patients with AMD. However, how RPE cells sense stress to initiate IFN response and cause further damage to the retina are still unknown. Although it has been reported that RPE can respond to extracellularly added dsRNA, it is unknown whether and how RPE detects and senses internally generated or internalized nucleic acids. Here, we elucidated the molecular mechanism by which RPE cells sense intracellular nucleic acids. Our data demonstrate that RPE cells can respond to intracellular RNA and induce type I IFN responses via the RIG-I (DExD/H-box helicase 58, DDX58) RNA helicase. In contrast, we showed that RPE cells were unable to directly sense and respond to DNA through the cGAS-STING pathway. We demonstrated that this was due to the absence of the cyclic GMP-AMP synthase (cGAS) DNA sensor in these cells. The activation of IFN response via RIG-I induced expression of cell death effectors and caused barrier function loss in RPE cells. These data suggested that RPE-intrinsic pathways of nucleic acid sensing are biased toward RNA sensing.

IL-9 and IL-10 Single-Nucleotide Variants and Serum Levels in Age-Related Macular Degeneration in the Caucasian Population.

Considering the immunological impairment in age-related macular degeneration (AMD), we aimed to determine the associations of IL-9 rs1859430, rs2069870, rs11741137, rs2069885, and rs2069884 and IL-10 rs1800871, rs1800872, and rs1800896 polymorphisms and their haplotypes, as well as the serum levels of IL-9 and IL-10 with AMD. 1209 participants were enrolled in our study. SNPs were genotyped using TaqMan SNP genotyping assays by real-time PCR method. IL-9 and IL-10 serum levels were evaluated using ELISA kits. Our study results have shown that haplotypes A-G-C-G-G and G-A-T-A-T of IL-9 SNPs are associated with the decreased odds of early AMD occurrence (p = 0.035 and p = 0.015, respectively). A set of rare haplotypes was associated with the decreased odds of exudative AMD occurrence (p = 0.033). Also, IL-10 serum levels were lower in exudative AMD than in controls (p = 0.049), patients with early AMD (p = 0.017), and atrophic AMD (p = 0.008). Furthermore, exudative AMD patients with IL-10 rs1800896 CT and TT genotypes had lower IL-10 serum concentrations than those with wild-type (CC) genotype (p = 0.048). In conclusion, our study suggests that IL-10 serum levels can be associated with a minor allele at IL-10 rs1800896 and exudative AMD. The haplotypes of IL-9 SNPs were also associated with the decreased odds of early and exudative AMD.

A Family Affair: Addressing the Challenges of Factor H and the Related Proteins.

Inflammation is a common denominator of diseases. The complement system, an intrinsic part of the innate immune system, is a key driver of inflammation in numerous disorders. Recently, a family of proteins has been suggested to be of vital importance in conditions characterized by complement dysregulation: the human Factor H (FH) family. This group of proteins consists of FH, Factor H-like protein 1 and five Factor H-related proteins. The FH family has been linked to infectious, vascular, eye, kidney and autoimmune diseases. In contrast to FH, the functions of the other highly homologous proteins are largely unknown and, hence, their role in the different disease-specific pathogenic mechanisms remains elusive. In this perspective review, we address the major challenges ahead in this emerging area, including 1) the controversies about the functional roles of the FH protein family, 2) the discrepancies in quantification of the FH protein family, 3) the unmet needs for validated tools and 4) limitations of animal models. Next, we also discuss the opportunities that exist for the immunology community. A strong multidisciplinary approach is required to solve these obstacles and is only possible through interdisciplinary collaboration between biologists, chemists, geneticists and physicians. We position this review in light of our own perspective, as principal investigators of the SciFiMed Consortium, a consortium aiming to create a comprehensive analytical system for the quantitative and functional assessment of the entire FH protein family.

The Benefits of Complement Measurements for the Clinical Practice.

The complement cascade is an evolutionary ancient innate immune defense system, playing a major role in the defense against infections. Its function in maintaining host homeostasis on activated cells has been emphasized by the crucial role of its overactivation in ever growing number of diseases, such as atypical hemolytic uremic syndrome (aHUS), autoimmune diseases as systemic lupus erythematosus (SLE), C3 glomerulopathies (C3GN), age-related macular degeneration (AMD), graft rejection, Alzheimer disease, and cancer, to name just a few. The last decade of research on complement has extended its implication in many pathological processes, offering new insights to potential therapeutic targets and asserting the necessity of reliable, sensitive, specific, accurate, and reproducible biomarkers to decipher complement role in pathology. We need to evaluate accurately which pathway or role should be targeted pharmacologically, and optimize treatment efficacy versus toxicity. This chapter is an introduction to the role of complement in human diseases and the use of complement-related biomarkers in the clinical practice. It is a part of a book intending to give reliable and standardized methods to evaluate complement according to nowadays needs and knowledge.

New insights into oxidative stress and immune mechanisms involved in age-related macular degeneration tackled by novel therapies.

The prevalence of age-related macular degeneration (AMD) has increased in the last years. Although anti-VEGF agents have improved the prognosis of exudative AMD, dry AMD has still devastating effects on elderly people vision. Oxidative stress and inflammation are mechanisms involved in AMD pathogenesis and its progression. Molecular pathways involving epidermal growth factor receptor (EGFR), bone morphogenetic protein (BMP4) and the nuclear erythroid related factor 2 (Nrf2) are behind oxidative stress in AMD due to their participation in antioxidant cellular pathways. As a consequence of the disbalance produced in the antioxidant mechanisms, there is an activation of innate and adaptative immune response with cell recruitment, changes in complement factors expression, and modification of cellular milieu. Different therapies are being studied to treat dry AMD based on the possible effects on antioxidant molecular pathways or their action on the immune response. There is a wide range of treatments presented in this review, from natural antioxidant compounds to cell and gene therapy, based on their mechanisms. Finally, we hypothesize that alpha-1-antitrypsin (AAT), an anti-inflammatory and immunomodulatory molecule that can also modulate antioxidant cellular defenses, could be a good candidate for testing in AMD. This article is part of the special ssue on 'The Quest for Disease-Modifying Therapies for Neurodegenerative Disorders'.

The complement system in age-related macular degeneration.

Age-related macular degeneration (AMD) is a chronic and progressive degenerative disease of the retina, which culminates in blindness and affects mainly the elderly population. AMD pathogenesis and pathophysiology are incredibly complex due to the structural and cellular complexity of the retina, and the variety of risk factors and molecular mechanisms that contribute to disease onset and progression. AMD is driven by a combination of genetic predisposition, natural ageing changes and lifestyle factors, such as smoking or nutritional intake. The mechanism by which these risk factors interact and converge towards AMD are not fully understood and therefore drug discovery is challenging, where no therapeutic attempt has been fully effective thus far. Genetic and molecular studies have identified the complement system as an important player in AMD. Indeed, many of the genetic risk variants cluster in genes of the alternative pathway of the complement system and complement activation products are elevated in AMD patients. Nevertheless, attempts in treating AMD via complement regulators have not yet been successful, suggesting a level of complexity that could not be predicted only from a genetic point of view. In this review, we will explore the role of complement system in AMD development and in the main molecular and cellular features of AMD, including complement activation itself, inflammation, ECM stability, energy metabolism and oxidative stress.

Functional Analysis of Variants in Complement Factor I Identified in Age-Related Macular Degeneration and Atypical Hemolytic Uremic Syndrome.

Complement factor I (FI) is a central inhibitor of the complement system, and impaired FI function increases complement activation, contributing to diseases such as age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (aHUS). Genetic variation in complement factor I (CFI) has been identified in both AMD and aHUS, with more than half of these variants leading to reduced FI secretion levels. For many of the variants with normal FI secretion, however, functional implications are not yet known. Here we studied 11 rare missense variants, with FI secretion levels comparable to wildtype, but a predicted damaging effects based on the Combined Annotation Dependent Depletion (CADD) score. Three variants (p.Pro50Ala, p.Arg339Gln, and p.Ser570Thr) were analyzed in plasma and serum samples of carriers affected by AMD. All 11 variants (nine for the first time in this study) were recombinantly expressed and the ability to degrade C3b was studied with the C3b degradation assay. The amount of degradation was determined by measuring the degradation product iC3b with ELISA. Eight of 11 (73%) mutant proteins (p.Pro50Ala, p.Arg339Gln, p.Ile340Thr, p.Gly342Glu, p.Gly349Arg, p.Arg474Gln, p.Gly487Cys, and p.Gly512Ser) showed significantly impaired C3b degradation, and were therefore classified as likely pathogenic. Our data indicate that genetic variants in CFI with a CADD score >20 are likely to affect FI function, and that monitoring iC3b in a degradation assay is a useful tool to establish the pathogenicity of CFI variants in functional studies.

Distinct effects of complement and of NLRP3- and non-NLRP3 inflammasomes for choroidal neovascularization.

NLRP3 inflammasome activation and complement-mediated inflammation have been implicated in promoting choroidal neovascularization (CNV) in age-related macular degeneration (AMD), but central questions regarding their contributions to AMD pathogenesis remain unanswered. Key open questions are (1) whether NLRP3 inflammasome activation mainly in retinal pigment epithelium (RPE) or rather in non-RPE cells promotes CNV, (2) whether inflammasome activation in CNV occurs via NLRP3 or also through NLRP3-independent mechanisms, and (3) whether complement activation induces inflammasome activation in CNV. Here we show in a neovascular AMD mouse model that NLRP3 inflammasome activation in non-RPE cells but not in RPE cells promotes CNV. We demonstrate that both NLRP3-dependent and NLRP3-independent inflammasome activation mechanisms induce CNV. Finally, we find that complement and inflammasomes promote CNV through independent mechanisms. Our findings uncover an unexpected role of non-NLRP3 inflammasomes for CNV and suggest that combination therapies targeting inflammasomes and complement may offer synergistic benefits to inhibit CNV.

Function and Dysfunction of Complement Factor H During Formation of Lipid-Rich Deposits.

Complement-mediated inflammation or dysregulation in lipid metabolism are associated with the pathogenesis of several diseases. These include age-related macular degeneration (AMD), C3 glomerulonephritis (C3GN), dense deposit disease (DDD), atherosclerosis, and Alzheimer's disease (AD). In all these diseases, formation of characteristic lipid-rich deposits is evident. Here, we will discuss molecular mechanisms whereby dysfunction of complement, and especially of its key regulator factor H, could be involved in lipid accumulation and related inflammation. The genetic associations to factor H polymorphisms, the role of factor H in the resolution of inflammation in lipid-rich deposits, modification of macrophage functions, and complement-mediated clearance of apoptotic and damaged cells indicate that the function of factor H is crucial in limiting inflammation in these diseases.