Chemokine signalling is required for the homing of leukocytes during retinal inflammation, and is associated with pathogenesis of diseases such as age-related macular degeneration (AMD). Here, we explore the role of interleukin-1β (IL-1β) in modulating AMD-associated chemokines Ccl2, Cxcl1, and Cxcl10 during photo-oxidative retinal damage, and the effect on both the accumulation of outer-retinal macrophages, and death of photoreceptors. Inhibition of retinal IL-1β expression was performed using either siRNA or antibody neutralisation, which was intravitreally injected in SD rats prior to photo-oxidative damage. Changes in the expression and localisation of Il-1β, Ccl2, Cxcl1 and Cxcl10 genes were assessed using qPCR and in situ hybridisation, while the recruitment of retinal macrophages was detected using immunohistochemistry for IBA1. Levels of photoreceptor cell death were determined using TUNEL. Photo-oxidative damage elevated the expression of Il-1β and inflammasome-related genes, and IL-1β protein was detected in microglia infiltrating the outer retina. This was associated with increased expression of Ccl2, Cxcl1, and Cxcl10. Intravitreal IL-1β inhibitors suppressed chemokine expression following damage and reduced macrophage accumulation and photoreceptor death. Moreover, in Müller and RPE cell cultures, and in vivo, Ccl2, Cxcl1 and Cxcl10 were variously upregulated when stimulated with IL-1β, with increased macrophage accumulation detected in vivo. IL-1β is produced by retinal microglia and macrophages and promotes chemokine expression by Müller cells and RPE in retinal degeneration. Targeting IL-1β may prove efficacious in broadly suppressing chemokine-mediated inflammation in retinal dystrophies such as AMD.

The human macula is more susceptible than the peripheral retina to developing blinding conditions such as age-related macular degeneration, diabetic retinopathy. A key difference between them may be the nature of their Müller cells. We found primary cultured Müller cells from macula and peripheral retina display significant morphological and transcriptomic differences. Macular Müller cells expressed more phosphoglycerate dehydrogenase (PHGDH, a rate-limiting enzyme in serine synthesis) than peripheral Müller cells. The serine synthesis, glycolytic and mitochondrial function were more activated in macular than peripheral Müller cells. Serine biosynthesis is critical in defending against oxidative stress. Intracellular reactive oxygen species and glutathione levels were increased in primary cultured macular Müller cells which were more susceptible to oxidative stress after inhibition of PHGDH. Our findings indicate serine biosynthesis is a critical part of the macular defence against oxidative stress and suggest dysregulation of this pathway as a potential cause of macular pathology.

The human retina, which is part of the central nervous system, has exceptionally high energy demands that requires an efficient metabolism of glucose, lipids, and amino acids. Dysregulation of retinal metabolism disrupts local energy supply and redox balance, contributing to the pathogenesis of diverse retinal diseases, including age-related macular degeneration, diabetic retinopathy, inherited retinal degenerations, and Macular Telangiectasia. A better understanding of the contribution of dysregulated metabolism to retinal diseases may provide better therapeutic targets than we currently have.

Background: Activating Gα_q signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT₂R) forms a rare alternative. The role of wild-type CysLT₂R in uveal melanoma remains unknown. Methods: We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. Results: 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. Conclusions: Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT₂R an attractive therapeutic target in uveal melanoma.

: Purpose:  To establish if impression cytology combined with histochemical and immunocytochemical staining can be used to assess epithelium of the human upper lid margin.Methods:  Following an initial eye examination of 40 healthy subjects (19 soft contact lens wearers and 21 non‐contact lens wearers, aged 18–57 years), lid margin staining was assessed with lissamine green using slit lamp biomicroscopy and graded (grade 0–3). Impression cytology of the upper lid margin of both eyes was collected, fixed and stained with periodic acid Schiff (PAS) and haematoxylin for cell morphology analysis (Nelson grade) or for immunocytochemistry (keratinization‐related proteins: filaggrin, transglutaminase1 (TGase1) and cytokeratin 1/10).Results:  In 57% of all subjects, grade 0 lissamine green staining showed a thin line (the Marx line), just posterior to the meibomian gland ducts. Grade 2 or 3 lissamine green staining was observed in 17% of all subjects. There was no difference between contact lens and non‐contact lens wearers for lid margin staining or Nelson grade (p = 0.4, Fisher’s exact test). PAS/haematoxylin staining and immunocytochemistry showed transition in epithelial cell morphology, with marginal conjunctival epithelium, mucocutaneous junction and squamous epithelium, adjacent to meibomian gland ducts. This transition in epithelium was associated with differential expression of keratinization‐related proteins (filaggrin, cytokeratin 1/10 and TGase1).Conclusion:  Lid margin epithelium can be successfully sampled using impression cytology and further characterized using histochemistry and immunocytochemistry staining techniques. This approach can be applied to assess lid margin changes in conditions such as dry eye and meibomian gland dysfunction.