Neuroretinal degeneration in a mouse model of systemic chronic immune activation observed by proteomics.

Blindness or vision loss due to neuroretinal and photoreceptor degeneration affects millions of individuals worldwide. In numerous neurodegenerative diseases, including age-related macular degeneration, dysregulated immune response-mediated retinal degeneration has been found to play a critical role in the disease pathogenesis. To better understand the pathogenic mechanisms underlying the retinal degeneration, we used a mouse model of systemic immune activation where we infected mice with lymphocytic choriomeningitis virus (LCMV) clone 13. Here, we evaluated the effects of LCMV infection and present a comprehensive discovery-based proteomic investigation using tandem mass tag (TMT) labeling and high-resolution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in protein regulation in the posterior part of the eye, neuroretina, and RPE/choroid were compared to those in the spleen as a secondary lymphoid organ and to the kidney as a non-lymphoid but encapsulated organ at 1, 8, and 28 weeks of infection. Using bioinformatic tools, we found several proteins responsible for maintaining normal tissue homeostasis to be differentially regulated in the neuroretina and the RPE/choroid during the degenerative process. Additionally, in the organs we observed, several important protein pathways contributing to cellular homeostasis and tissue development were perturbed and associated with LCMV-mediated inflammation, promoting disease progression. Our findings suggest that the response to a systemic chronic infection differs between the neuroretina and the RPE/choroid, and the processes induced by chronic systemic infection in the RPE/choroid are not unlike those induced in non-immune-privileged organs such as the kidney and spleen. Overall, our data provide detailed insight into several molecular mechanisms of neuroretinal degeneration and highlight various novel protein pathways that further suggest that the posterior part of the eye is not an isolated immunological entity despite the existence of neuroretinal immune privilege.

Chemokine Receptor Profile of T Cells and Progression Rate of Geographic Atrophy Secondary to Age-related Macular Degeneration.

Purpose: Geographic atrophy (GA) secondary to age-related macular degeneration is a progressive retinal degenerative disease. Systemic chemokine receptors and known risk-associated single-nucleotide polymorphisms have been associated with GA pathogenesis. Because halting progression is pivotal for patients, we investigated the association of candidate chemokine receptors and progression rate (PR) of atrophic lesions in patients with GA. Methods: This prospective observational study conducted at a single center included 85 patients with GA and 45 healthy controls. Patients were followed up after 13 months on average. Serial fundus autofluorescence images were used to determine the PR of atrophic lesions. The proportion of chemokine receptors on peripheral lymphocytes were determined by flow cytometric analysis. Results: Patients with GA had a lower proportion of CCR6 on CD8+T cells compared to healthy controls. Importantly, the proportion of CCR6 on CD4+T cells was lower in patients with fast GA progression compared to patients with slow progression of disease, suggesting that dysregulation of CCR6 could be involved in progression of GA. We also found that GA patients had a markedly higher percentage of CCR5 on CD4+ and CD8+T cells compared to healthy controls. After stratification according to ARMS2 polymorphism, we found a significantly lower level of CCR5 on CD8+T cells among patients with high-risk genotypes compared with patients with the low-risk genotype. Conclusions: Our study finds that chemokine receptors are dysregulated in patients with GA and that CCR6 might be involved in GA progression, making it a potential target for intervention.

Complement factor H Y402H polymorphism results in diminishing CD4+ T cells and increasing C-reactive protein in plasma.

Age-related macular degeneration (AMD) is a common cause of visual loss among the elderly. Genetic variants in the gene encoding complement factor H (CFH) have been identified as an AMD susceptibility gene, however, the mechanistic link is debated. Here, we investigated the link between the CFH Y402H genotype and low-grade inflammation. We recruited 153 healthy individuals, 84 participants with dry stages of AMD, and 148 participants with neovascular AMD. All participants were subjected to detailed retinal examination, and interview regarding comorbidities and lifestyle. Blood samples were analyzed for level of C-Reactive Protein (CRP), white blood cell differential count, and stained with fluorescent antibodies to differentiate CD4+ and CD8+ T cells. CFH Y402H genotyping was performed using an allele-specific polymerase chain reaction genotyping assay. Splenocytes from young and aged wild type and Cfh null mutant C57BL/6J mice were examined for CD4+ and CD8+ T cells. Healthy individuals with the CFH Y402H at-risk polymorphism HH had higher levels of CRP and lower proportions of CD4+ T cells compared to persons with the YH or YY polymorphism (P = 0.037, Chi-square). Healthy individuals with the HH polymorphism displayed lower proportions of CD4+ T cells with ageing (P < 0.01, one-way ANOVA), whereas both young and aged Cfh null mutant mice displayed lower proportions of CD4+ T cells (P < 0.001 and P < 0.05; unpaired t test). Participants with dry AMD and the HH polymorphism had similarly lower proportions of CD4+ T cells (P = 0.024, one-way ANOVA), but no difference in CRP-levels. In the neovascular stage of AMD, there was no difference in proportion of CD4+ cells or CRP levels according to genotype. The risk-associated CFH genotype is associated with an age-related decrease in proportion of CD4+ T cells and increased levels of CRP in healthy individuals. This indicates that decreased complement regulation results in extensive changes in innate and adaptive immune compartments that precede development of AMD.

Systemic virus infection results in CD8 T cell recruitment to the retina in the absence of local virus infection.

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues.

Complement activation by RPE cells preexposed to TNFα and IFNγ.

Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.

Chemokine Profile and the Alterations in CCR5-CCL5 Axis in Geographic Atrophy Secondary to Age-Related Macular Degeneration.

Purpose: Geographic atrophy (GA) secondary to age-related macular degeneration (AMD) is a progressive disease with no treatment option. Previous studies show chemokine-mediated recruitment of immune cells in the retina, and therefore we investigated systemic levels of chemokines and chemokine receptors in patients with GA. Methods: This observational prospective study was conducted at a single center. We included 122 participants with no immune disease: 41 participants with GA and no choroidal neovascularization, 51 patients with neovascular AMD, and 30 healthy control individuals. Flow cytometric analysis was used to detect expression level of C-C chemokine receptor (CCR)1, CCR2, CCR3, CCR5, and C-X-C motif chemokine receptor (CXCR)3 on peripheral blood mononuclear cells (CD14+ monocytes, CD4+ T cells, CD8+ T cells). Plasma levels of C-C motif ligand (CCL)11, C-X-C motif chemokine (CXCL)10, and CCL5 were measured by specific immunoassays. Enlargement rate of GA lesion was measured from autofluorescence images. Results: Participants with GA have a specific chemokine profile with a higher expression of CCR5 than healthy controls in peripheral blood mononuclear cells, and a higher plasma levels of CCL-5. Further, GA was associated with higher monocytic expression of CCR2 than in neovascular AMD. We found that a high expression level of CCR5 on CD8+ T cells was associated with slower enlargement rate of atrophic lesion. Conclusions: The study showed an association between systemic chemokine profile and GA formation. Further studies are needed to fully elucidate the possible role of systemic chemokine regulation in mediating pathogenesis of GA.

Polypoidal Choroidal Vasculopathy Associate With Diminished Regulatory T Cells That Are Polarized Into a T Helper 2-Like Phenotype.

Purpose: To investigate possible roles of T helper (Th) cells, regulatory T cells (Tregs), and the recently mapped Th-like Tregs in patients with polypoidal choroidal vasculopathy (PCV). Methods: In this prospective case-control study, we obtained fresh venous blood from patients with PCV (n = 24), age-matched healthy controls (n = 32), and patients with neovascular AMD (n = 45). All participants underwent a comprehensive ocular examination including fluorescein and indocyanine green angiography for where retinal disease was suspected. Using flow cytometry, we identified Th subsets, Tregs, and Th-like Tregs. Plasma samples were stored at -80°C to investigate plasma cytokines of interest. Results: Compared to healthy controls, patients with PCV had lower percentages of Tregs (8.7% ± 2.8% vs. 7.3% ± 1.7%, P = 0.027), which were significantly more Th2-like polarized (42.6% ± 13.3% vs. 50.5% ± 13.0%, P = 0.029). These changes differed from that observed in neovascular AMD, which compared to healthy controls had fewer Th1/Th17 cells (3.6% ± 2.7% vs. 2.4% ± 2.5%, P = 0.049), comparable Treg levels, and no distinct polarization of Th-like Tregs. Because of these findings, we measured plasma IL-4 and IL-33 levels. Plasma IL-33 in patients with PCV (median 0.30 pg/mL) was twice as high compared to healthy controls (median 0.16 pg/mL; P = 0.037). Conclusions: PCV associate with diminished Tregs that are polarized more into a Th2-like phenotype. This is correlated to IL-33 levels, which we also find increased in patients with PCV. Our findings suggest a possible role for Th2-like Tregs and IL-33 in PCV.

Association of CD11b+ Monocytes and Anti-Vascular Endothelial Growth Factor Injections in Treatment of Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy.

Importance: CD11b+ immune cells have been implicated in the formation of choroidal neovascularization in experimental studies on animals and disease-association studies on humans. However, the clinical importance of such observations remains unknown. Objective: To investigate whether the proportion of CD11b+ circulating monocytes is associated with the number of anti-vascular endothelial growth factor (anti-VEGF) injections in neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). Design, Setting, and Participants: These observational cohort studies collected data from January 1, 2010, through December 31, 2013, and from January 1, 2015, through December 31, 2018. Fresh venous blood samples were acquired for flow cytometric immune studies in patients with neovascular AMD or PCV receiving treatment with aflibercept or ranibizumab as needed for 36 months. Patients (n = 81) without immune diseases were consecutively recruited from a single center in Denmark. Exposures: Proportion of CD11b+ circulating monocytes. Main Outcomes and Measures: The estimation of the number of intravitreal anti-VEGF injections given at 12, 24, and 36 months by the proportion of CD11b+ circulating monocytes and the correlation between these values. The angiogenic role of CD11b+ circulating monocytes was further evaluated by investigating the expression of the known proangiogenic receptor CCR2. Results: Eighty-one patients were included in the analysis (54% women; mean [SD] age, 76 [7] years). The proportion of CD11b+ monocytes at baseline positively estimated the future number of anti-VEGF injections at 12 (ρ = 0.77; 95% CI, 0.35-0.93; P = .004), 24 (ρ = 0.82; 95% CI, 0.44-0.95; P = .002), and 36 (ρ = 0.78; 95% CI, 0.34-0.94; P = .005) months. This association was also found retrospectively in a larger sample of patients with neovascular AMD at 12 (ρ = 0.46; 95% CI, 0.16-0.68; P = .004), 24 (ρ = 0.49; 95% CI, 0.20-0.70; P = .002), and 36 (ρ = 0.65; 95% CI, 0.41-0.80; P < .001) months and patients with PCV at 12 (ρ = 0.27; 95% CI, -0.28 to 0.68; P = .30), 24 (ρ = 0.60; 95% CI, 0.12-0.85; P = .02), and 36 (ρ = 0.70; 95% CI, 0.27-0.90; P = .005) months, suggesting that this association is not specific to AMD but rather reflects VEGF activity in neovascularization. CD11b+ monocytes highly coexpressed CCR2, an important monocytic marker of proangiogenic activity. Conclusions and Relevance: Results of this study demonstrated that the proportion of circulating CD11b+ monocytes estimated and correlated with the number of anti-VEGF injections in patients with neovascular AMD and PCV. Additional longitudinal studies are needed to determine whether these findings have clinical relevance to influence treatment algorithms or provide novel targets for medical therapy.

Systemic Levels of Interleukin-6 Correlate With Progression Rate of Geographic Atrophy Secondary to Age-Related Macular Degeneration.

Purpose: Geographic atrophy (GA) is a clinical phenotype of late age-related macular degeneration (AMD) with no current treatment available. In this study, we investigated markers of chronic inflammation in plasma of patients with GA and how these relate to progression rate. Methods: We prospectively included 42 patients with GA, 41 patients with neovascular AMD, and 27 healthy controls. We quantified levels of interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor (TNF) receptor 2, and C-reactive protein (CRP). We adapted an inflammation summary score to cluster conceptually related markers of chronic inflammation. Enlargement rate of the atrophic lesion was measured from fundus autofluorescence images performed at baseline and after 1 year. Results: Patients with GA showed an increase in proinflammatory markers of IL-6 (P = 0.009), TNF receptor 2 (P = 0.013), and CRP (P = 0.017) compared to healthy controls. We found that IL-8 levels were markedly higher in patients with GA when compared to patients with neovascular AMD (P = 0.013). The inflammation summary score was high in patients with neovascular AMD (P = 0.024), but even higher in patients with GA (<0.001), when compared to healthy controls. GA enlargement was measured in 36 patients, who completed follow-up. Plasma levels of IL-6 had a moderate but significant correlation with GA enlargement rate (R2 = 0.23, P = 0.0035). Conclusions: Markers of chronic inflammation strongly associates with presence of GA secondary to AMD. Plasma IL-6 possesses predictive ability of progression and constitutes the first known plasma biomarker of disease activity in GA. These findings shed light into a poorly understood clinical phenotype of AMD and highlights the important role of chronic inflammation in GA.

Chemokine Expression in Murine RPE/Choroid in Response to Systemic Viral Infection and Elevated Levels of Circulating Interferon-γ.

Purpose: To examine how circulating immune mediators in vivo may affect gene and protein expression at the RPE/choroid interface. Methods: Young mice were systemically infected with lymphocytic choriomeningitis virus (LCMV) or continuously infused with IFN-γ. RPE/choroid was isolated and analyzed with whole-transcriptome gene expression microarrays. Selected gene expression findings were validated at the protein level. Results: Both the systemic immune activation from virus infection and the sterile systemically increased level of IFN-γ resulted in increased expression of chemokine ligands, chemokine receptors, and early complement components in isolates of RPE/choroid. These findings were largely absent from LCMV-infected mice deficient in either the interferon α/ß receptor or IFN-γ. Conclusions: Together, these findings demonstrate that acute systemic immune activation results in a local response at the RPE/choroid interface that may include chemokine-dependent recruitment of inflammatory cells and engagement of the complement system. This may represent a link between the systemic low-grade inflammation and the retinal pathology observed in several multifactorial entities such as aging, AMD, and diabetes.

Plasma markers of chronic low-grade inflammation in polypoidal choroidal vasculopathy and neovascular age-related macular degeneration.

PURPOSE: Ageing is the strongest predictor of neovascular age-related macular degeneration (AMD), where neuroinflammation is known to play a major role. Less is known about polypoidal choroidal vasculopathy (PCV), which is an important differential diagnosis to neovascular AMD. Here, we report plasma markers of inflammation with age (inflammaging) in patients with PCV, patients with neovascular AMD and a healthy age-matched control group. METHODS: We isolated plasma from fresh venous blood obtained from participants (n = 90) with either PCV, neovascular AMD, or healthy maculae. Interleukin(IL)-1ß, IL-6, IL-8, IL-10 and tumour necrosis factor receptor 2 (TNF-R2) were measured using U-PLEX Human Assays. Routine plasma C-reactive protein (CRP) was measured using Dimension Vista 1500. RESULTS: Patients with PCV had plasma levels of IL-1ß, IL-6, IL-8, IL-10 and TNF-R2 similar to that in healthy controls. Patients with neovascular AMD had significantly higher plasma IL-1ß, IL-6 and IL-10 than healthy controls, whereas no significant differences were observed for plasma IL-8 and TNF-R2. Differences between plasma IL-1ß, IL-6 and IL-10 possessed a positive but weak ability in discriminating neovascular AMD from PCV. Both patients with PCV and patients with neovascular AMD had significantly higher levels of routine plasma CRP. CONCLUSION: Patients with PCV differ from patients with neovascular AMD in terms of plasma inflammaging profile. Apart from increased CRP, no signs of inflammaging were observed in patients with PCV. In patients with neovascular AMD, we find a specific angiogenesis-twisted inflammaging profile.

Imbalances in tissue inhibitors of metalloproteinases differentiate choroidal neovascularization from geographic atrophy.

PURPOSE: Tissue inhibitor of metalloproteinase (TIMP) is known to play a role in age-related macular degeneration (AMD). We wished to investigate alterations in different late stages of AMD: neovascular AMD and geographic atrophy (GA). METHODS: This was a prospective case-control study. A total of 125 participants were included consecutively during a period of 18 months. We included 46 patients with neovascular AMD, 46 patients with GA without any sign of choroidal neovascularization in either eye, and 33 healthy aged controls. Patients with immune-affecting disorders were not included. Commercial immunoassay kits were used to quantify levels of TIMP-1, TIMP-3, MMP-2 and MMP-9 in blood plasma. RESULTS: We found that patients with neovascular AMD had lower plasma concentration of TIMP-3 (p = 0.028) than healthy controls. Patients with GA had higher plasma levels of TIMP-1 (p < 0.001) and MMP-9 (p = 0.022) compared to healthy controls. Also, we found that TIMP-1 levels in patients with GA increased with age (Spearman's rho = 0.04, p = 0.006). CONCLUSION: Matrix metalloproteinases (MMPs) and TIMPs, which are known to be involved in age-related changes in Bruch's membrane, are significantly altered systemically, suggesting the presence of an imbalance in the homeostasis of the extracellular matrix. These imbalances may explain differences in the clinical manifestation of late AMD.

Extended HLA-G haplotypes in patients with age-related macular degeneration.

The study aims to determine if genetic polymorphisms in the human leukocyte antigen (HLA)-G gene are associated with age-related macular degeneration (AMD). HLA-G is important for immunological tolerance, and it is also known to have angiogenic effects. Polymorphisms in the 5'-upstream regulatory region (URR) and 3'-untranslated region (UTR) of HLA-G have been associated with a number of diseases, especially with respect to a 14 bp insertion/deletion (ins/del) polymorphism in the 3'UTR. Full gene sequencing was performed on a cohort of 146 AMD patients and 63 healthy controls aged 60 years or older and HLA-G haplotypes were determined. Analyses were performed on a publicly available gene expression dataset from the NCBI GEO database (accession number GSE29801) from which expression data for HLA-G, -C and -A were extracted. Analysis of the GEO dataset showed that both HLA-G and -C was expressed in the back of the eye and that expression was upregulated in the macular area of AMD. No differences were observed between patients and controls when analysing the distribution of haplotypes in the HLA-G promoter, coding region, 3'UTR or the 14 bp ins/del polymorphism of the 3'UTR. The increased expression of HLA-G in the macula of AMD patients indicates a role of HLA-G in the micro environment as part of the AMD pathogenesis. This is supported by the expression of HLA-C, which has previously been shown to play a role in AMD. The HLA-G haplotype distribution did not display any differences between AMD patients and controls. This article is protected by copyright. All rights reserved.

T-cell differentiation and CD56+ levels in polypoidal choroidal vasculopathy and neovascular age-related macular degeneration.

Polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (AMD) are prevalent age-related diseases characterized by exudative changes in the macula. Although they share anatomical and clinical similarities, they are also distinctly characterized by their own features, e.g. vascular abnormalities in PCV and drusen-mediated progression in neovascular AMD. PCV remains etiologically uncharacterized, and ongoing discussion is whether PCV and neovascular AMD share the same etiology or constitute two substantially different diseases. In this study, we investigated T-cell differentiation and aging profile in human patients with PCV, patients with neovascular AMD, and age-matched healthy control individuals. Fresh venous blood was prepared for flow cytometry to investigate CD4+ and CD8+ T-cell differentiation (naïve, central memory, effector memory, effector memory CD45ra+), loss of differentiation markers CD27 and CD28, and expression of aging marker CD56. Patients with PCV were similar to the healthy controls in all aspects. In patients with neovascular AMD we found significantly accelerated T-cell differentiation (more CD28-CD27- cells) and aging (more CD56+ cells) in the CD8+ T-cell compartment. These findings suggest that PCV and neovascular AMD are etiologically different in terms of T cell immunity, and that neovascular AMD is associated with T-cell immunosenescence.

CD11b and CD200 on Circulating Monocytes Differentiate Two Angiographic Subtypes of Polypoidal Choroidal Vasculopathy.

Purpose: To investigate surface expression of CD11b and CD200 on circulating monocytes in patients with polypoidal choroidal vasculopathy (PCV). Methods: This was a prospective case-control study of patients with PCV (n = 27), age-matched healthy controls (n = 27), and patients with neovascular AMD (n = 49). All participants underwent a comprehensive ocular examination. Fluorescein and indocyanine green angiography were performed in patients suspected of neovascular AMD or PCV. Polypoidal choroidal vasculopathy was angiographically categorized into those with a strong presence of a branching vascular network (BVN) (type 1) or with a faint/no clear presence of a BVN (type 2). Fresh venous blood was stained with fluorescent antibodies for flow cytometric analyses. We compared the percentages of CD11b+, CD200+, and CD11b+CD200+ monocytes between groups of diagnosis and between different angiographic subtypes of PCV. Results: Overall, CD11b+ monocytes were both increased in patients with PCV and neovascular AMD. CD200+ and CD11b+CD200+ monocytes were increased in patients with neovascular AMD. An age-related increase in CD11b+CD200+ monocytes was absent in patients with PCV and neovascular AMD. Patients with PCV type 1 had significantly higher CD11b+, CD200+, and CD11b+CD200+ monocytes, whereas patients with PCV type 2 had levels similar to that in healthy controls. Conclusions: We found that PCV is immunologically heterogeneous with significant differences between angiographic subtypes. Increased CD11b+ and CD200+ monocytes in those with a strong presence of BVN indicate that BVN development may be associated with retinal injury and a VEGF-mediated process that is either reflected or propelled by systemic changes in monocytes.

Expression and differential regulation of HLA-G isoforms in the retinal pigment epithelial cell line, ARPE-19.

The purpose of this study was to examine if HLA-G is expressed in the retinal pigment epithelium (RPE) cells of the eye. The RPE comprises the outer most layer of the retina and as such defines the interface to the blood and contributes to the immune privilege in the posterior part of the eye. One way the RPE might be regulating the immune system could be by expressing the non-classical human leukocyte antigen (HLA) molecule, HLA-G. We therefore sought to define if the RPE cell line, ARPE-19, expressed HLA-G and analyse the regulation as a response to pro-inflammatory cytokines. This was done by digital droplet PCR, measuring the gene expression of HLA-G in total RNA. The protein expression was analysed by immunohistochemistry and by immunofluorescence followed by confocal microscopy and the expression of the HLA-G isoforms was explored by fragment analysis. In the current study, we show that HLA-G is expressed by ARPE-19 cells and is upregulated as a response to pro-inflammatory cytokines. Moreover, we are the first to describe a differential regulation of the HLA-G isoforms as a direct response to stimulation. These results might indicate that HLA-G can be part of the immune privilege of the posterior part of the eye, but further experiments on primary RPE cells are needed.

Systemic and Ocular Long Pentraxin 3 in Patients with Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) has been associated with both systemic and ocular alterations of the immune system. In particular dysfunction of complement factor H (CFH), a soluble regulator of the alternative pathway of the complement system, has been implicated in AMD pathogenesis. One of the ligands for CFH is long pentraxin 3 (PTX3), which is produced locally in the retinal pigment epithelium (RPE). To test the hypothesis that PTX3 is relevant to retinal immunohomeostasis and may be associated with AMD pathogenesis, we measured plasma PTX3 protein concentration and analyzed the RPE/choroid PTX3 gene expression in patients with AMD. To measure the ability of RPE cells to secrete PTX3 in vitro, polarized ARPE-19 cells were treated with activated T cells or cytokines (interferon (IFN)-gamma and/or tumor necrosis factor (TNF)-alpha) from the basolateral side; then PTX3 protein concentration in supernatants and PTX3 gene expression in tissue lysates were quantified. Plasma levels of PTX3 were generally low and did not significantly differ between patients and controls (P=0.307). No statistically significant difference was observed between dry and exudative AMD nor was there any correlation with hsCRP or CFH genotype. The gene expression of PTX3 increased in RPE/choroid with age (P=0.0098 macular; P=0.003 extramacular), but did not differ between aged controls and AMD patients. In vitro, ARPE-19 cells increased expression of the PTX3 gene as well PTX3 apical secretions after stimulation with TNF-alpha or activated T cells (P<0.01). These findings indicate that PTX3 expressed in the eye cannot be detected systemically and systemic PTX3 may have little or no impact on disease progression, but our findings do not exclude that locally produced PTX3 produced in the posterior segment of the eye may be part of the AMD immunopathogenesis.

Early and exudative age-related macular degeneration is associated with increased plasma levels of soluble TNF receptor II.

PURPOSE: We have recently identified homeostatic alterations in the circulating T cells of patients with age-related macular degeneration (AMD). In cultures of retinal pigment epithelial cells, we have demonstrated that T-cell-derived cytokines induced the upregulation of complement, chemokines and other proteins implicated in AMD pathogenesis. The purpose of this study was to test whether increased plasma levels of cytokines were present in patients with AMD. METHODS: We conducted a case-control study. Age-related macular degeneration status was assessed using standardized multimodal imaging techniques. Plasma was isolated from freshly drawn peripheral venous blood samples and analysed for interleukin (IL)15, IL18, interferon (IFN)γ, soluble tumour necrosis factor (TNF) receptor II (sTNFRII) and complement factor H (CFH) Y402H genotype. RESULTS: We included 136 individuals with early or late forms of AMD and 74 controls. Significantly increased levels of sTNFRII were observed in patients with early or exudative AMD (p < 0.01). After adjusting for CFH Y402H genotype, age, sex and smoking history, the level of sTNFRII remained a significant predictor for prevalence of AMD with odds ratios at 3.0 in the middle and 3.6 in the highest tertiles. Levels of IL15, IL18 and IFNγ were low and not associated with AMD. CONCLUSIONS: Increased plasma level of sTNFRII is found to be associated with AMD. The data supports the observations of low-grade, systemic inflammatory alterations in patients with AMD. However, it remains to be determined whether increased levels of TNFα can be found, which directly reflects an increased activity of macrophages and T cells.

CX3CL1/CX3CR1 and CCL2/CCR2 chemokine/chemokine receptor complex in patients with AMD.

PURPOSE: The chemokine receptors CX3CR1 and CCR2 have been implicated in the development of age-related macular degeneration (AMD). The evidence is mainly derived from experimental cell studies and murine models of AMD. The purpose of this study was to investigate the association between expression of CX3CR1 and CCR2 on different leukocyte subsets and AMD. Furthermore we measured the plasma levels of ligands CX3CL1 and CCL2. METHODS: Patients attending our department were asked to participate in the study. The diagnosis of AMD was based on clinical examination and multimodal imaging techniques. Chemokine plasma level and chemokine receptor expression were measured by flow-cytometry. RESULTS: A total of 150 participants were included. We found a significantly lower expression of CX3CR1 on CD8+ T cells in the neovascular AMD group compared to the control group (p = 0.04). We found a significant positive correlation between CCR2 and CX3CR1 expression on CD8+ cells (r = 0.727, p = 0.0001). We found no difference in plasma levels of CX3CL1 and CCL2 among the groups. CONCLUSIONS: Our results show a down regulation of CX3CR1 on CD8+ cells; this correlated to a low expression of CCR2 on CD8+ cells. Further studies are needed to elucidate the possible role of this cell type in AMD development.

Calcium-independent phospholipase A2, group VIA, is critical for RPE cell survival.

PURPOSE: To investigate the significance of calcium-independent phospholipase A2, group VIA (iPLA2-VIA), in RPE cell survival following responses to sodium iodate (SI) in cell cultures. METHODS: The human retinal pigment epithelium (RPE) cell line (ARPE-19) cells and primary mouse-RPE cultures were treated with SI to induce cell death. Cells were transfected with an iPLA2-VIA promoter-luciferase construct to evaluate the regulation of iPLA-VIA after exposure to SI. PCR analysis, western blot analysis, and activity assays were performed to evaluate the mRNA level, protein level, and activity levels of iPLA2-VIA after SI exposure. Inhibitors of iPLA2-VIA were used to explore a potential protective role in cells exposed to SI. Primary RPE cell cultures were grown from iPLA2-VIA knockout mice and wild-type mice. The cultures were exposed to SI to investigate a possible increased protection against SI in iPLA2-VIA knockout mice compared to wild-type mice. RESULTS: The study revealed upregulation of iPLA2-VIA expression (promoter activity, iPLA2-VIA mRNA, iPLA2-VIA protein, and iPLA2-VIA protein activity) in ARPE-19 cells exposed to SI. SI-induced cell death was shown to be inhibited by iPLA2-VIA-specific inhibitors in ARPE-19 cell cultures. RPE cultures from iPLA2-VIA knockout mice were less vulnerable to SI-induced cell death compared to RPE cultures from wild-type mice. CONCLUSIONS: SI -induced RPE cell death involves iPLA2-VIA upregulation and activation, and amelioration of SI-induced RPE cell death can be facilitated by inhibitors of iPLA2-VIA. Thus, we suggest iPLA2-VIA as a possible pharmaceutical target to treat RPE-related retinal diseases.