MicroRNA as a diagnostic and therapeutic tool in mononuclear phagocyte mediated retinal inflammation and degeneration

Abstract

Age-related macular degeneration (AMD) is the leading cause of blindness in the industrialised world. Current estimates predict the prevalence of AMD to reach 280 million by 2040, and with no treatments currently available for the more prevalent atrophic form, this number is expected to rise. Underlying disease pathology is increased oxidative stress and non-resolving inflammation attributed to the sustained activation of microglia and macrophages (mononuclear phagocytes (MP)), ultimately leading to photoreceptor cell death and vision loss. Due to the complex etiology of AMD, diagnostic and therapeutic strategies targeting the expression and regulation of broad disease-associated pathways are gaining traction. Of particular interest are microRNA (miRNA), small endogenous gene repressors that play a central role in regulating biological processes, including inflammation and the associated activation of MP. However, the role of miRNA in the retina and their diagnostic and therapeutic potential is currently unclear. Therefore, this thesis explores the use of miRNA as a diagnostic and therapeutic tool, with a specific focus on the role and regulation of miRNA in MP-mediated inflammation. Firstly, I will explore the use of miRNA as biomarker for retinal degenerations (Chapter 3), secondly I will look at modulating retinal inflammation using miR-155, a known pro-inflammatory miRNA (Chapter 4) and lastly I will present a new protocol for culturing adult primary retinal MP for use as a screening tool in studying retinal inflammation (Chapter 5). The relatively asymptomatic early stages of AMD disease progression often delays clinical diagnosis until severe pathology ensues. Therefore, to develop a diagnostic tool for early AMD detection, I present findings in Chapter 3 characterising a novel panel of circulating miRNA over progressive degeneration, Results from this work showed that miRNA modulation was detected from early-stage degeneration, and that miRNA at all stages was associated with the inflammatory response. These results suggest that circulating miRNA may prove effective as a diagnostic panel for AMD including at early stages. As inflammation is evidenced to play a significant role in retinal degenerations, the pro-inflammatory miRNA, miR-155, was targeted in the retina to alleviate MP mediated inflammation (Chapter 4). It was demonstrated that miR-155 was expressed by both macroglia and microglia in the retina, with inhibition of miR-155 found to alter the characteristic chronic inflammatory response during degeneration, leading to functional protection and photoreceptor survivability. These results combined suggest a pro-inflammatory MP-polarising role for miR-155, with inhibition inducing a homeostatic state and lessening the progression of degeneration. MP modulation may therefore represent an effective therapeutic strategy for the treatment of AMD. Finally, as a platform to investigate the development of MP targeted therapeutics, in Chapter 5 I describe the methodology and characterisation of a novel culture system of activated retinal MP. MP from murine retinas were cultured in medium supplemented with GM-CSF and M-CSF. The molecular identity of these cultures was determined by comparing RNA sequencing to publicly available single cell sequencing data from degenerating rodent retinas, revealing that our primary cultures were most analogous to a population of degeneration-specific subretinal activated microglia. This culture paradigm therefore represents an ideal in vitro model for the development of anti-inflammatory therapeutics, in particular for diseases such as AMD in which MP activation is a key pathogenic feature. Together these findings identify a role for miRNA in both systemic signalling and propagation of the immune response during retinal degeneration. Ultimately, this thesis extrapolates current understandings surrounding the use of miRNA as both a diagnostic tool and therapeutic target for AMD.