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Abstract 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-1β 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-1β 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.

PurposePolymorphisms 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.DesignCollection 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.MethodsGenotyping, 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 measuresTCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion.ResultsTCC 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.ConclusionTCC 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.

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].

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.