Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells.

Purpose: Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells. Design: Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source. Methods: Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants. Main outcome measures: TCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion. Results: TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE. Conclusion: TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology.

[Ocular side effects of modern oncological therapy : Immunological checkpoint and MEK/BRAF signal transduction inhibitors]. / Okuläre Nebenwirkungen moderner onkologischer Therapie : Immunologische Checkpoint- und MEK/BRAF-Signaltransduktionsinhibitoren.

In recent years, checkpoint inhibitors have revolutionized the treatment of previously untreatable malignant tumors, significantly improving the life expectancy as well as quality of life in many cases. Checkpoint inhibitors comprise a group of drugs with different mechanisms of action. These include immunological checkpoint inhibitors (iCPI) and intracellular signal transduction inhibitors; however, both substance classes can cause inflammatory or toxic ocular side effects. The frequency of intraocular inflammation (uveitis) is reported to be ca. 1-2%, toxic side effects were observed in up to more than 50% of the patients treated with signal transduction inhibitors. In the following article the main mechanisms of these forms of treatment are characterized. Furthermore, this article presents the currently most frequently used therapeutic agents and their typical ophthalmological side effects to increase awareness and to draw attention to these still rare but increasingly more frequent findings.

Pathogenesis of Bacterial Uveitis.

PURPOSE: To describe the pathogenesis and the general immune mechanisms of the most frequent causes of bacterial uveitis. METHODOLOGY: Narrative review. RESULTS: Both extra- and intracellular bacteria can induce uveitis, whereas intracellular bacteria are generally transported into the inner eye via cells of the innate immune system, mainly macrophages. Systemic adaptive immunity is usually induced before the bacteria are localized to the inner eye, and once T and B cells have detected the pathogens behind the blood-eye barriers they elicit an acute and/or chronic inflammatory response deteriorating visual acuity that can severely affect the non-regenerating, intraocular tissues. CONCLUSIONS: An understanding of pathogenic mechanisms, and its correlation with clinical and imaging features, can facilitate early recognition of microbial factors and institution of appropriate therapy.

Antigenic mimicry - The key to autoimmunity in immune privileged organs.

The eye and the brain are the best investigated immune privileged sites for T cell mediated autoimmune diseases, with several experimental animal models in multiple species like experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU). It was difficult to explain autoimmunity to antigens which are sequestered behind blood-organ barriers since those barriers can only be passed by already activated lymphocytes. Antigen-specific lymphocytes must therefore be activated outside of the immune privileged target organs by crossreactivity of immune receptors to similar antigens, designated as "antigenic mimicry". Crossreactivity as the basic mechanism for antigenic mimicry is generally important for the immune recognition of a huge variety of different antigens with a comparably restricted number of T cell receptors and antibodies. Here, various T cell mimotopes are discussed that can induce autoimmune diseases or immune tolerance or both. Mimotopes are antigenic epitopes with similarity to different, unrelated antigens that are not distinguished by T cell receptors. Finally, the role of the microbiome is touched briefly, it is suspected to provide many mimotopes for T cell responses that, however, are very difficult to define due to the enormous size, and individual variability of our microbiome and virome.

A new small molecule DHODH-inhibitor [KIO-100 (PP-001)] targeting activated T cells for intraocular treatment of uveitis - A phase I clinical trial.

Uveitis is a T cell-mediated, intraocular inflammatory disease and one of the main causes of blindness in industrialized countries. There is a high unmet need for new immunomodulatory, steroid-sparing therapies, since only ciclosporin A and a single TNF-α-blocker are approved for non-infectious uveitis. A new small molecule inhibitor of dihydroorotate dehydrogenase (DHODH), an enzyme pivotal for de novo synthesis of pyrimidines, has a high potency for suppressing T and B cells and has already proven highly effective for treating uveitis in experimental rat models. Systemic and intraocular application of KIO-100 (PP-001) (previously called PP-001, now KIO-100) could efficiently suppress rat uveitis in a preventive as well as therapeutic mode. Here we describe the outcome of the first clinical phase 1 trial comparing three different doses of a single intraocular injection of KIO-100 (PP-001) in patients with non-infectious posterior segment uveitis. No toxic side effects on intraocular tissues or other adverse events were observed, while intraocular inflammation decreased, and visual acuity significantly improved. Macular edema, a sight-threatening complication in uveitis, showed regression 2 weeks after intraocular KIO-100 (PP-001) injection in some patients, indicating that this novel small molecule has a high potential as a new intraocular therapy for uveitis. Clinical trial registration: [https://www.clinicaltrials.gov/ct2/show/NCT03634475], identifier [NCT03634475].

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration.

BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1ß and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1ß to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Immunogenicity of Novel AAV Capsids for Retinal Gene Therapy.

OBJECTIVES: AAV vectors are widely used in gene therapy, but the prevalence of neutralizing antibodies raised against AAV serotypes in the course of a natural infection, as well as innate and adaptive immune responses induced upon vector administration, is still considered an important limitation. In ocular gene therapy, vectors applied subretinally bear the risk of retinal detachment or vascular leakage. Therefore, new AAV vectors that are suitable for intravitreal administration for photoreceptor transduction were developed. METHODS: Here, we compared human immune responses from donors with suspected previous AAV2 infections to the new vectors AAV2.GL and AAV2.NN-two capsid peptide display variants with an enhanced tropism for photoreceptors-with the parental serotype AAV2 (AAV2 WT). We investigated total and neutralizing antibodies, adaptive and innate cellular immunogenicity determined by immunofluorescence staining and flow cytometry, and cytokine secretion analyzed with multiplex beads. RESULTS: While we did not observe obvious differences in overall antibody binding, variants-particularly AAV2.GL-were less sensitive to neutralizing antibodies than the AAV2 WT. The novel variants did not differ from AAV2 WT in cellular immune responses and cytokine production in vitro. CONCLUSION: Due to their enhanced retinal tropism, which allows for dose reduction, the new vector variants are likely to be less immunogenic for gene therapy than the parental AAV2 vector.

Uveitis in Tumor Patients Treated with Immunological Checkpoint- and Signal Transduction Pathway-Inhibitors.

PURPOSE: New tumor therapies like immune checkpoint inhibitors and small molecule inhibitors of MEK and BRAF have increased the patient's survival rate but can be burdened with severe side-effects including uveitis. Here, we show the spectrum, treatment, and outcome of uveitis types induced by tumor treatment. METHODS: In this retrospective study, we have included 54 patients from different centers who were developing uveitis under tumor therapy. A 16-item questionnaire was analyzed for type, treatment, and outcome of uveitis and type of tumor treatment, which we have correlated here. RESULTS: Irrespective of the tumor treatment, most patients developed anterior uveitis. All patients received corticosteroids and some additional immunosuppressive treatments. Cessation of tumor therapy was necessary only in a minority of cases. CONCLUSIONS: Ocular autoimmunity should be differentiated from toxic effects of cancer treatment and timely recognized since it can be generally well controlled by anti-inflammatory treatment, preserving the patient's vision without cessation of the tumor treatment.

Tumors, tumor therapies, autoimmunity and the eye.

The eye as an immune privileged organ is mostly spared from (auto)immune attacks. Intraocular inflammation like autoimmune uveitis is a rare event. Nevertheless, tumor-related destructive autoimmune responses can affect the eye, as observed in the case of cancer- associated retinopathy (CAR), an autoantibody-mediated destruction of retinal cells induced by the ectopic expression of ocular antigens by peripheral tumors. The new tumor therapies targeting immune checkpoints to enhance anti-tumor responses can also induce autoimmune responses and result in autoimmune diseases even in immune privileged organs like the eyes. Even MEK/BRAF-inhibitor therapies using small molecules to block tumor-specific signal transduction molecules have turned out to not just inhibit tumor growth and survival and render tumors more susceptible for immune recognition, but to have additional toxic effects on non-dividing retinal cells, destroying and making them potential targets of autoimmunity.

Posterior subcapsular cataracts are a late effect after acute exposure to 0.5 Gy ionizing radiation in mice.

PURPOSE: The long-term effect of low and moderate doses of ionizing radiation on the lens is still a matter of debate and needs to be evaluated in more detail. MATERIAL AND METHODS: We conducted a detailed histological analysis of eyes from B6C3F1 mice cohorts after acute gamma irradiation (60Co source; 0.063 Gy/min) at young adult age of 10 weeks with doses of 0.063, 0.125, and 0.5 Gy. Sham irradiated (0 Gy) mice were used as controls. To test for genetic susceptibility heterozygous Ercc2 mutant mice were used and compared to wild-type mice of the same strain background. Mice of both sexes were included in all cohorts. Eyes were collected 4 h, 12, 18 and 24 months after irradiation. For a better understanding of the underlying mechanisms, metabolomics analyses were performed in lenses and plasma samples of the same mouse cohorts at 4 and 12 h as well as 12, 18 and 24 months after irradiation. For this purpose, a targeted analysis was chosen. RESULTS: This analysis revealed histological changes particularly in the posterior part of the lens that rarely can be observed by using Scheimpflug imaging, as we reported previously. We detected a significant increase of posterior subcapsular cataracts (PSCs) 18 and 24 months after irradiation with 0.5 Gy (odds ratio 9.3; 95% confidence interval 2.1-41.3) independent of sex and genotype. Doses below 0.5 Gy (i.e. 0.063 and 0.125 Gy) did not significantly increase the frequency of PSCs at any time point. In lenses, we observed a clear effect of sex and aging but not of irradiation or genotype. While metabolomics analyses of plasma from the same mice showed only a sex effect. CONCLUSIONS: This article demonstrates a significant radiation-induced increase in the incidence of PSCs, which could not be identified using Scheimpflug imaging as the only diagnostic tool.

Molecular Mimicry and Uveitis.

Molecular or antigenic mimicry is a term for the similarity of different antigens, which can be confused by the immune system. Antigen recognition by antibodies and T cell receptors is specific, but not restricted to a single antigen. Both types of receptors specifically recognize antigens and are expressed with a very high but still restricted variability compared to the number of different antigens they potentially could bind. T cell receptors only can bind to antigen peptides presented on certain self-MHC-molecules by screening only some amino acid side chains on both the presented peptides and the MHC molecule. The other amino acids of the peptide are not directly perceived by the T cell, offering the opportunity for a single T cell to recognize a variety of different antigens with the same receptor, which significantly increases the immune repertoire. The immune system is usually tolerant to autoantigens, especially to those of immune privileged sites, like the eye. Therefore, autoimmune diseases targeting these organs were hard to explain, unless a T cell is activated by an environmental peptide (e.g. pathogen) that is similar, but not necessarily identical with an autoantigen. Here we describe antigenic mimicry of retinal autoantigens with a variety of non-ocular antigens resulting in the induction of intraocular inflammation. T cells that are activated by mimotopes outside of the eye can pass the blood-retina barrier and enter ocular tissues. When reactivated in the eye by crossreaction with autoantigens they induce uveitis by recruiting inflammatory cells.

Is glaucoma an autoimmune disease?

There is increasing evidence from animal and human studies that glaucoma is an autoimmune disease. Evidence for this hypothesis includes the fact that antibodies as well as T-cell responses to heat-shock proteins (HSPs) are detectable in some patients with glaucoma and in an animal model of the disease. As in the human disease, experimental animal models of glaucoma have been found to demonstrate neurodegenerative changes in the optic nerve associated with immunoglobulin and T-cell infiltration. Although there is still insufficient evidence in humans to classify all cases of glaucoma as autoimmune diseases, the implications of this hypothesis have major impact on the diagnosis and treatment of glaucoma.

Resolution of uveitis.

Autoimmune uveitis is a sight-threatening, rare disease, potentially leading to blindness. Uveitis is a synonym for intraocular inflammation, presenting as various clinical phenotypes with different underlying immune responses in patients, whereas different animal models usually represent one certain clinical and immunological type of uveitis due to genetic uniformity and the method of disease induction. T cells recognizing intraocular antigens initiate the disease, recruiting inflammatory cells (granulocytes, monocytes/macrophages) to the eyes, which cause the damage of the tissue. The treatment of uveitis so far aims at downregulation of inflammation to protect the ocular tissues from damage, and at immunosuppression to stop fueling T cell reactivity. Uveitis is usually prevented by specific mechanisms of the ocular immune privilege and the blood-eye-barriers, but once the disease is induced, mechanisms of the immune privilege as well as a variety of novel regulatory features including new Treg cell populations and suppressive cytokines are induced to downregulate the ocular inflammation and T cell responses and to avoid relapses and chronicity. Here we describe mechanisms of regulation observed in experimental animal models as well as detected in studies with peripheral lymphocytes from patients.

The Role of IFN-alpha in Experimental and Clinical Uveitis.

PURPOSE: IFN-α is the only treatment capable of inducing long-term remission in some patients with ocular Behçet's disease. In this review, we focus on immune mechanisms of IFN-α in animal models and patients and compare the outcome of different clinical studies. METHODS: Review of literature using PubMed and Google and original data from rat models with monophasic/chronic or relapsing experimental autoimmune uveitis treated with IFN-α. RESULTS: The role of IFN-α and its effect on various cell types were investigated, in some cases with contradictory results. Some patients respond very well to IFN-α treatment, while others are non-responders, which was reflected in the uveitis rat models: relapsing uveitis was ameliorated, the monophasic/chronic disease even aggravated. CONCLUSIONS: Despite intensive investigations in patients and animal models, the immune mechanisms explaining the therapeutic effect of IFN-α in ocular Behçet's disease are not yet fully understood and need further investigation.

Are rats more human than mice?

In contrast to rats, mouse models are nowadays generally used for the investigation of immune responses and immune-mediated diseases, there are many different strains and mouse-specific tools available, and it is easy to generate transgenic and constitutive or inducible knockout mice for any gene. Many immune markers and mechanisms have been detected in mice and have been introduced as gold standard in immunology, however, some turned out to be not unconditionally transferable to the human immune system. Rats have been used more frequently in former days but are mostly outstripped by mice due to the fact that fewer strains are available, they need more space than mice, are more expensive to maintain and breed, and it is extremely difficult to generate transgenic or ko-rats. Consequently, the choice of rat-specific diagnostic tools like antibodies is quite poor and most researchers have switched to mouse models for the investigation of immune mechanisms, while rats are still widely used for toxicology by the pharmaceutical industry. However, it should be taken into consideration that there are some immunological similarities between rats and humans that are not presented in mice. Some of them like MHC class II and Foxp3 expression by activated effector T cells we have detected during our research on the immune response of rat models of experimental autoimmune uveitis.

The immunopathogenesis of chronic and relapsing autoimmune uveitis - Lessons from experimental rat models.

Autoimmune diseases usually follow a relapsing-remitting or a chronic progressive course. To understand the underlying immunopathogenesis we investigated experimental Lewis rat models displaying both disease types, which were only dependent on the autoantigen peptide used for immunization. Retinal S-Antigen-peptide PDSAg induces chronic, monophasic disease, whilst interphotoreceptor retinoid-binding protein (IRBP)-peptide R14 causes a spontaneously relapsing-remitting course. R14-mediated uveitis can be re-induced by immunization; PDSAg-induced disease is even preventable by prior CFA-injection. T cells with different antigen specificities preferentially infiltrate the eyes from different sites, e.g. choroid or retinal vessels, they remain in the retina after resolution of inflammation for many weeks. The major inflammatory cell populations in the eyes during rat uveitis are CD4+ or CD8+ monocytes/macrophages. Chemokine mutants only suppress PDSAg-mediated EAU, while IFN-α-treatment ameliorated R14-, but worsened PDSAg-induced disease. Comparison of T cells revealed upregulated expression of 26 genes related to various signal transduction pathways upstream and downstream of IFN-γ only in T cells causing relapsing EAU. Intraocular injection of IFN-γ induces synchronized relapses in R14-mediated uveitis, while VEGF-expression of PDSAg-specific T cells causing chronic disease induced chorioretinal neovascularization that is suppressed by anti-CD146 antibody. Intraocular T cells from rat eyes during EAU express IL-17, IFN-γ or IL-10, with dynamic changes of the cell populations during the disease course, differing in both disease types. Immunization of animals with a mixture of both antigens suppressed relapses, indicating a dominance of the monophasic disease. Understanding the exact pathogenesis of both disease courses is key to developing novel therapies for autoimmune diseases.

Intraocular DHODH-inhibitor PP-001 suppresses relapsing experimental uveitis and cytokine production of human lymphocytes, but not of RPE cells.

BACKGROUND: Uveitis is a potentially blinding inflammatory disease of the inner eye with a high unmet need for new therapeutic interventions. Here, we wanted to investigate the suppressive effect of the intraocular application of the small molecule dihydroorotate dehydrogenase (DHODH)-inhibitor PP-001 on experimental relapsing rat uveitis and furthermore determine its effect on proliferation and cytokine secretion of human peripheral blood lymphocytes (PBL) and human retinal pigment epithelial (RPE) cells in vitro. METHODS: Spontaneously relapsing uveitis was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) peptide R14. PP-001 was injected intravitreally after resolution of the primary disease to investigate further relapses. Proliferation and metabolic activity of phytohemagglutinin (PHA)-stimulated human peripheral lymphocytes with and without PP-001 and cytokine secretion were determined by XTT assay and bioplex bead assay. The RPE cell line ARPE-19 as well as primary human RPE cells treated with PP-001 or anti-vascular endothelial growth factor (VEGF) antibody bevacizumab were also investigated for metabolic activity and cytokine/chemokine secretion. RESULTS: Injection of PP-001 into rat eyes reduced the number of relapses by 70%, from 20 relapses (57% of the rats affected) in the control group to 6 relapses (33% of the rats) in the treatment group. In human PBL cultures, PP-001 reduced the proliferation in a dose-dependent manner. The secretion of several cytokines such as IL-17, IFN-γ, and VEGF was suppressed by PP-001, as previously observed with rat T cells in the experimental autoimmune uveitis (EAU) model. In contrast, human RPE cells were not affected by PP-001, while the anti-VEGF antibody bevacizumab severely impaired the secretion of various cytokines including VEGF. CONCLUSIONS: For the first time, intravitreal injection of PP-001 demonstrated an effective, but transient reduction of relapses in the rat EAU model. In vitro PP-001 suppressed proliferation and cytokine/chemokine secretion of human lymphocytes, while neither human RPE cell line ARPE-19 nor primary RPE cells were affected.

Anaphylatoxins Activate Ca2+, Akt/PI3-Kinase, and FOXO1/FoxP3 in the Retinal Pigment Epithelium.

PURPOSE: The retinal pigment epithelium (RPE) is a main target for complement activation in age-related macular degeneration (AMD). The anaphylatoxins C3a and C5a have been thought to mostly play a role as chemoattractants for macrophages and immune cells; here, we explore whether they trigger RPE alterations. Specifically, we investigated the RPE as a potential immunoregulatory gate, allowing for active changes in the RPE microenvironment in response to complement. DESIGN: In vitro and in vivo analysis of signaling pathways. METHODS: Individual activities of and interaction between the two anaphylatoxin receptors were tested in cultured RPE cells by fluorescence microscopy, western blot, and immunohistochemistry. MAIN OUTCOME MEASURES: Intracellular free calcium, protein phosphorylation, immunostaining of tissues/cells, and multiplex secretion assay. RESULTS: Similar to immune cells, anaphylatoxin exposure resulted in increases in free cytosolic Ca2+, PI3-kinase/Akt activation, FoxP3 and FOXO1 phosphorylation, and cytokine/chemokine secretion. Differential responses were elicited depending on whether C3a and C5a were co-administered or applied consecutively, and response amplitudes in co-administration experiments ranged from additive to driven by C5a (C3a + C5a = C5a) or being smaller than those elicited by C3a alone (C3a + C5a < C3a). CONCLUSION: We suggest that this combination of integrative signaling between C3aR and C5aR helps the RPE to precisely adopt its immune regulatory function. These data further contribute to our understanding of AMD pathophysiology.

Inflammatory eye disease: Pre-treatment assessment of patients prior to commencing immunosuppressive and biologic therapy: Recommendations from an expert committee.

AIM: To outline recommendations from an expert committee on the assessment and investigation of patients with severe inflammatory eye disease commencing immunosuppressive and/or biologic therapy. METHOD: The approach to assessment is based on the clinical experience of an expert committee and a review of the literature with regard to corticosteroids, immunosuppressive drug and biologic therapy and other adjunct therapy in the management of patients with severe sight-threatening inflammatory eye disease. CONCLUSION: We recommend a careful assessment and consultative approach by ophthalmologists or physicians experienced in the use of immunosuppressive agents for all patients commencing immunosuppressive and/or biologic therapy for sight threatening inflammatory eye disease with the aim of preventing infection, cardiovascular, metabolic and bone disease and reducing iatrogenic side effects.