BackgroundChemokine 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.MethodsInhibition 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.ResultsPhoto-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.ConclusionsIL-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.

These findings implicate C3, produced locally by retinal microglia/macrophages, as contributing causally to retinal degeneration. Consequently, this suggests that C3-targeted gene therapy may prove valuable in slowing the progression of AMD.

This study has provided an understanding of the mechanism behind miR-124 in the degenerating retina and demonstrates the usefulness of miR-124 mimics for the modulation of retinal degenerations.

Photoreceptor cell death and inflammation are known to occur progressively in retinal degenerative diseases such as age-related macular degeneration (AMD). However, the molecular mechanisms underlying these biological processes are largely unknown. Extracellular vesicles (EV) are essential mediators of cell-to-cell communication with emerging roles in the modulation of immune responses. EVs, including exosomes, encapsulate and transfer microRNA (miRNA) to recipient cells and in this way can modulate the environment of recipient cells. Dysregulation of EVs however is correlated to a loss of cellular homeostasis and increased inflammation. In this work we investigated the role of isolated retinal small-medium sized EV (s-mEV) which includes exosomes in both the healthy and degenerating retina. Isolated s-mEV from normal retinas were characterized using dynamic light scattering, transmission electron microscopy and western blotting, and quantified across 5 days of photo-oxidative damage-induced degeneration using nanotracking analysis. Small RNAseq was used to characterize the miRNA cargo of retinal s-mEV isolated from healthy and damaged retinas. Finally, the effect of exosome inhibition on cell-to-cell miRNA transfer and immune modulation was conducted using systemic daily administration of exosome inhibitor GW4869 and in situ hybridization of s-mEV-abundant miRNA, miR-124-3p. Electroretinography and immunohistochemistry was performed to assess functional and morphological changes to the retina as a result of GW4869-induced exosome depletion. Results demonstrated an inverse correlation between s-mEV concentration and photoreceptor survivability, with a decrease in s-mEV numbers following degeneration. Small RNAseq revealed that s-mEVs contained uniquely enriched miRNAs in comparison to in whole retinal tissue, however, there was no differential change in the s-mEV miRNAnome following photo-oxidative damage. Exosome inhibition via the use of GW4869 was also found to exacerbate retinal degeneration, with reduced retinal function and increased levels of inflammation and cell death demonstrated following photo-oxidative damage in exosome-inhibited mice. Further, GW4869-treated mice displayed impaired translocation of photoreceptor-derived miR-124-3p to the inner retina during damage. Taken together, we propose that retinal s-mEV and their miRNA cargo play an essential role in maintaining retinal homeostasis through immune-modulation, and have the potential to be used in targeted gene therapy for retinal degenerative diseases.

Activation of the inflammasome is involved in the progression of retinal degenerative diseases, in particular, in the pathogenesis of Age-Related Macular Degeneration (AMD), with NLRP3 activation the focus of many investigations. In this study, we used genetic and pharmacological approaches to explore the role of the inflammasome in a mouse model of retinal degeneration. We identify that Casp1/11 −/− mice have better-preserved retinal function, reduced inflammation and increased photoreceptor survivability. While Nlrp3 −/− mice display some level of preservation of retinal function compared to controls, pharmacological inhibition of NLRP3 did not protect against photoreceptor cell death. Further, Aim2 −/− , Nlrc4 −/− , Asc −/− , and Casp11 −/− mice show no substantial retinal protection. We propose that CASP-1-associated photoreceptor cell death occurs largely independently of NLRP3 and other established inflammasome sensor proteins, or that inhibition of a single sensor is not sufficient to repress the inflammatory cascade. Therapeutic targeting of CASP-1 may offer a more promising avenue to delay the progression of retinal degenerations. Age-Related Macular Degeneration (AMD) is a chronic inflammatory disease that is characterised by central vision loss due to retinal pigmented epithelium (RPE) and photoreceptor cell death in the macular region of the retina 1,2. While environmental, lifestyle and genetic risk factors are well established 2-4 , it is increasingly clear that central to disease progression is the accumulation of oxidative stress 5 and inflammation 6. A central component of inflammation is the inflammasome, multi-protein oligomers which form part of the innate immune system, acting in the first line of defence, sensing pathogen-derived or danger signals from pathogen invasion, or cellular stress signals including extracellular ATP or host dsDNA 1,7-9. Inflammasomes are composed of a pattern recognition receptor (PRR) sensor protein, including NACHT, LRR and PYD domains-containing protein 1 (NLRP1), NLRP3, NAIP-NLRC4, Absent in Melanoma 2 (AIM2), and Pyrin. Following activation, these sensor proteins form a complex with the adaptor protein Apoptosis-associated speck-like protein containing a CARD (ASC, or PYCARD) and protease enzyme Caspase-1 (CASP-1) 10,11. Activated CASP-1 in turn, cleaves and activates pro-inflammatory cytokines including interleukin-1β (IL-1β), large amounts of which are known to cause microglial activation and macrophage recruitment from the periphery, and ultimately photoreceptor cell death, characteristic features of AMD pathogenesis 3,8,12-16. Inhibition of IL-1β has been shown to reduce inflammation and further cell death 14 , highlighting the key role of the inflammasome in the progression of retinal degenerative diseases such as AMD.