Retinal Macrophages Synthesize C3 and Activate Complement in AMD and in Models of Focal Retinal Degeneration

PURPOSE. Complement system dysregulation is strongly linked to the progression of age-related macular degeneration (AMD). Deposition of complement including C3 within the lesions in atrophic AMD is thought to contribute to lesion growth, although the contribution of local cellular sources remains unclear. We investigated the role of retinal microglia and macrophages in complement activation within atrophic lesions, in AMD and in models of focal retinal degeneration. METHODS. Human AMD donor retinas were labeled for C3 expression via in situ hybridization. Rats were subject to photo-oxidative damage, and lesion expansion was tracked over a 2- month period using optical coherence tomography (OCT). Three strategies were used to determine the contribution of local and systemic C3 in mice: total C3 genetic ablation, local C3 inhibition using intravitreally injected small interfering RNA (siRNA), and depletion of serum C3 using cobra venom factor. RESULTS. Retinal C3 was expressed by microglia/macrophages located in the outer retina in AMD eyes. In rodent photo-oxidative damage, C3-expressing microglia/macrophages and complement activation were located in regions of lesion expansion in the outer retina over 2 months. Total genetic ablation of C3 ameliorated degeneration and complement activation in retinas following damage, although systemic depletion of serum complement had no effect. In contrast, local suppression of C3 expression using siRNA inhibited complement activation and deposition, and reduced cell death. CONCLUSIONS. 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.Supported by a grant from Retina Australia, as well as the National Health and Medical Research Council (NHMRC; APP1049990). Also supported by an Australian Government Research Training Program (RTP) Scholarshi

MicroRNA-124 Dysregulation is Associated With Retinal Inflammation and Photoreceptor Death in the Degenerating Retina

PURPOSE. We sought to determine the role and retinal cellular location of microRNA-124 (miR-124) in a neuroinflammatory model of retinal degeneration. Further, we explored the anti-inflammatory relationship of miR-124 with a predicted messenger RNA (mRNA) binding partner, chemokine (C-C motif) ligand 2 (Ccl2), which is crucially involved in inflammatory cell recruitment in the damaged retina. METHODS. Human AMD donor eyes and photo-oxidative damaged (PD) mice were labeled for miR-124 expression using in situ hybridization. PDGFRa-cre RFP mice were used for Müller cell isolation from whole retinas. MIO-M1 immortalized cells and rat primary Müller cells were used for in vitro analysis of miR-124 expression and its relationship with Ccl2. Therapeutic efficacy was tested with intravitreal administration of miR-124 mimic in mice, with electroretinography used to determine retinal function. IBA1 immunohistochemistry and photoreceptor row counts were used for assessment of inflammation and cell death. RESULTS. MiR-124 expression was correlated with progressive retinal damage, inflammation, and cell death in human AMD and PD mice. In addition, miR-124 expression was inversely correlated to Ccl2 expression in mice following PD. MiR-124 was localized to both neuronal-like photoreceptors and glial (Müller) cells in the retina, with a redistribution from neurons to glia occurring as a consequence of PD. Finally, intravitreal administration of miR-124 mimics decreased retinal inflammation and photoreceptor cell death, and improved retinal function. CONCLUSIONS. 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.Supported by the National Health and Medical Research Council in Australia (APP1127705, 2017-2019), the Australian Government Research Training Program, the Gretel and Gordon Bootes Foundation (2013), and the Ophthalmic Research Institute of Australia/Eye Surgeons’ Foundation (2015)

Ablation of C3 modulates macrophage reactivity in the outer retina during photo-oxidative damage

Purpose Dysregulation of the complement cascade contributes to a variety of retinal dystrophies, including age-related macular degeneration (AMD). The central component of complement, C3, is expressed in abundance by macrophages in the outer retina, and its ablation suppresses photoreceptor death in experimental photo-oxidative damage. Whether this also influences macrophage reactivity in this model system, however, is unknown. We investigate the effect of C3 ablation on macrophage activity and phagocytosis by outer retinal macrophages during photo-oxidative damage. Methods Age-matched C3 knockout (KO) mice and wild-type (WT) C57/Bl6 mice were subjected to photo-oxidative damage. Measurements of the outer nuclear layer (ONL) thickness and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were used to assess pathology and photoreceptor apoptosis, respectively. Macrophage abundance and phagocytosis were assessed with immunolabeling for pan-macrophage and phagocytic markers, in conjunction with TUNEL staining in cohorts of C3 KO and WT mice. Results The C3 KO mice exhibited protection against photoreceptor cell death following photo-oxidative damage, which was associated with a reduction in immunoreactivity for the stress-related factor GFAP. In conjunction, there was a reduction in IBA1-positive macrophages in the outer retina compared to the WT mice and a decrease in the number of CD68-positive cells in the outer nuclear layer and the subretinal space. In addition, the engulfment of TUNEL-positive and -negative photoreceptors by macrophages was significantly lower in the C3 KO mice cohort following photo-oxidative damage compared to the WT cohort. Conclusions The results show that the absence of C3 mitigates the phagocytosis of photoreceptors by macrophages in the outer retina, and the net impact of C3 depletion is neuroprotective in the context of photo-oxidative damage. These data improve our understanding of the impact of C3 inhibition in subretinal inflammation and inform the development of treatments for targeting complement activation in diseases such as AMD.This study was supported by project grants from the National Health and Medical Research Council (APP1165599; APP1127705), Australian National University Translational Fellowship and Australian Government Research Training Program Scholarship

Retinal monocyte-derived complement, not systemically derived complement contributes to the early onset of focal retinal degeneration

Purpose : The complement cascade is associated with pathogenesis of retinal dystrophies including age-related macular degeneration (AMD) although the cellular events that initiate the cascade remains unclear. In this study, we aimed to determine the functional significance of C3 derived from local/retinal sources and systemically-derived C3, and compared the findings with C3 localized in human donor eyes Methods : Photo-oxidative damage was used for a focal lesion in rat and mouse retinas, according to published protocols (Rutar et al., 2011;Natoli et al., 2016). The effect of C3 inhibition on the focal retinal degeneration was studied using C3 mice, and intravitreal C3-specific siRNA in wildtype mice to locally deplete C3, and intraperitoneally administered cobra venom factor CVF to deplete C3 systemically. Animals were assessed for using electroretinogram(ERG), immunohistochemistry, in situ hybridisation, and qRT-PCR. Human retinas were assessed using immunohistochemistry and in situ hybridization for C3 -/- This site uses cookies. By continuing to use our website, you are agreeing to our privacy policy. | Accept Retinal monocyte-derived complement, not systemically derived comp… early onset of focal retinal degeneration | IOVS | ARVO Journals 19/6/18, 1(29 pm http://iovs.arvojournals.org/article.aspx?articleid=2639479 Page 2 of 2 Results : Human retinas from AMD patients with atrophic lesions showed C3 mRNA expressed by retinal monocytes within lesions and at the lesions edge, in the subretinal space, optic nerve head and inner retina. In focal lesions of photo-oxidative damaged rodent retina, complement gene expression increased significantly (P=0.0244), with strong immunoreactivity to C3d in retinal lesions in cells that co-localized the monocytes. C3 retinas had significantly reduced photoreceptor cell death post damage (P=0.0014), a better preserved photoreceptor layer and improved retinal function compared to wildtypes (P=0.005). Local C3 inhibition using siRNA showed significantly reduced C3 gene expression in retinas post damage (P=0.034), accompanied by reduced C3 deposits in the outer retina, thicker photoreceptor layer and higher ERG responses compared to negative-siRNA controls (P=0.036). Systemic complement depletion by CVF had no effect on complement gene expression and did not mitigate the effects of photooxidative damage on retinal morphology or function (P=0.43) Conclusions : Local C3 deposition by retinal monocytes, not systemic complement contributes to the progression of retinal degeneration. This study emphasises that targeting local complement specifically on retinal monocytes population could be a potential approach to slow down the progression of retinal dystrophy