Haihan Jiao

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.

Spatiotemporal Cadence of Macrophage Polarisation in a Model of Light-Induced Retinal Degeneration

Background The recruitment of macrophages accompanies almost every pathogenic state of the retina, and their excessive activation in the subretinal space is thought to contribute to the progression of diseases including age-related macular degeneration. Previously, we have shown that macrophages aggregate in the outer retina following damage elicited by photo-oxidative stress, and that inhibition of their recruitment reduces photoreceptor death. Here, we look for functional insight into macrophage activity in this model through the spatiotemporal interplay of macrophage polarisation over the course of degeneration. Methods Rats were exposed to 1000 lux light damage (LD) for 24hrs, with some left to recover for 3 and 7 days post-exposure. Expression and localisation of M1- and M2- macrophage markers was investigated in light-damaged retinas using qPCR, ELISA, flow cytometry, and immunohistochemistry. Results Expression of M1- (Ccl3, Il-6, Il-12, Il-1β, TNFα) and M2- (CD206, Arg1, Igf1, Lyve1, Clec7a) related markers followed discrete profiles following light damage; up-regulation of M1 genes peaked at the early phase of cell death, while M2 genes generally exhibited more prolonged increases during the chronic phase. Moreover, Il-1β and CD206 labelled accumulations of microglia/macrophages which differed in their morphological, temporal, and spatial characteristics following light damage. Conclusions The data illustrate a dynamic shift in macrophage polarisation following light damage through a broad swathe of M1 and M2 markers. Pro-inflammatory M1 activation appears to dominate the early phase of degeneration while M2 responses appear to more heavily mark the chronic post-exposure period. While M1/M2 polarisation represents two extremes amongst a spectrum of macrophage activity, knowledge of their predominance offers insight into functional consequences of macrophage activity over the course of damage, which may inform the spatiotemporal employment of therapeutics in retinal disease.

Erratum to: Retinal metabolic events in preconditioning light stress as revealed by wide-spectrum targeted metabolomics

under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made. Erratum to: Metabolomics (2017) 13:22 DOI 10.1007/s11306-016-1156-9

Photobiomodulation with 670 nm light ameliorates Müller cell-mediated activation of microglia and macrophages in retinal degeneration

Abstract Müller cells, the supporting cells of the retina, play a key role in responding to retinal stress by releasing chemokines, including CCL2, to recruit microglia and macrophages (MG/M&PHgr;) into the damaged retina. Photobiomodulation (PBM) with 670 nm light has been shown to reduce inflammation in models of retinal degeneration. In this study, we aimed to investigate whether 670 nm light had an effect on Müller cell‐initiated inflammation under retinal photo‐oxidative damage (PD) in vivo and in vitro. Sprague‐Dawley rats were pre‐treated with 670 nm light (9J/cm2) once daily over 5 days prior to PD. The expression of inflammatory genes including CCL2 and IL‐1&bgr; was analysed in retinas. In vitro, primary Müller cells dissociated from neonatal rat retinas were co‐cultured with 661W photoreceptor cells. Co‐cultures were exposed to PD, followed by 670 nm light treatment to the Müller cells only, and Müller cell stress and inflammation were assessed. Primary MG/M&PHgr; were incubated with supernatant from the co‐cultures, and collected for analysis of inflammatory activation. To further understand the mechanism of 670 nm light, the expression of COX5a and mitochondrial membrane potential (&Dgr;&PSgr;m) were measured in Müller cells. Following PD, 670 nm light‐treated Müller cells had a reduced inflammatory activation, with lower levels of CCL2, IL‐1&bgr; and IL‐6. Supernatant from 670 nm light‐treated co‐cultures reduced activation of primary MG/M&PHgr;, and lowered the expression of pro‐inflammatory cytokines, compared to untreated PD controls. Additionally, 670 nm light‐treated Müller cells had an increased expression of COX5a and an elevated &Dgr;&PSgr;m following PD, suggesting that retrograde signaling plays a role in the effects of 670 nm light on Müller cell gene expression. Our data indicates that 670 nm light reduces Müller cell‐mediated retinal inflammation, and offers a potential cellular mechanism for 670 nm light therapy in regulating inflammation associated with retinal degenerations. HighlightsPhotobiomodulation using 670 nm lightmitigates Müller cell activation directly, through whichit reduces retinal microglia/macrophage activationmaintains mitochondrial function in Müller cells thereby supporting retinal homeostasis, andprotects photoreceptors following photo‐oxidative stressmay provide a non‐invasive and cost‐effective treatment option in retinal inflammatory conditions.

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.

Photoreceptor Survival Is Regulated by GSTO1-1 in the Degenerating Retina

Purpose Glutathione-S-transferase omega 1-1 (GSTO1-1) is a cytosolic glutathione transferase enzyme, involved in glutathionylation, toll-like receptor signaling, and calcium channel regulation. GSTO1-1 dysregulation has been implicated in oxidative stress and inflammation, and contributes to the pathogenesis of several diseases and neurological disorders; however, its role in retinal degenerations is unknown. The aim of this study was to investigate the role of GSTO1-1 in modulating oxidative stress and consequent inflammation in the normal and degenerating retina. Methods The role of GSTO1-1 in retinal degenerations was explored by using Gsto1-/- mice in a model of retinal degeneration. The expression and localization of GSTO1-1 were investigated with immunohistochemistry and Western blot. Changes in the expression of inflammatory (Ccl2, Il-1β, and C3) and oxidative stress (Nox1, Sod2, Gpx3, Hmox1, Nrf2, and Nqo1) genes were investigated via quantitative real-time polymerase chain reaction. Retinal function in Gsto1-/- mice was investigated by using electroretinography. Results GSTO1-1 was localized to the inner segment of cone photoreceptors in the retina. Gsto1-/- photo-oxidative damage (PD) mice had decreased photoreceptor cell death as well as decreased expression of inflammatory (Ccl2, Il-1β, and C3) markers and oxidative stress marker Nqo1. Further, retinal function in the Gsto1-/- PD mice was increased as compared to wild-type PD mice. Conclusions These results indicate that GSTO1-1 is required for inflammatory-mediated photoreceptor death in retinal degenerations. Targeting GSTO1-1 may be a useful strategy to reduce oxidative stress and inflammation and ameliorate photoreceptor loss, slowing the progression of retinal degenerations.

Dynamic interplay of innate and adaptive immunity during sterile retinal inflammation: Insights from the transcriptome

The pathogenesis of many retinal degenerations, such as age-related macular degeneration (AMD), is punctuated by an ill-defined network of sterile inflammatory responses. The delineation of innate and adaptive immune milieu among the broad leukocyte infiltrate, and the gene networks, which construct these responses, are poorly described in the eye. Using photo-oxidative damage in a rodent model of subretinal inflammation, we employed a novel RNA-sequencing framework to map the global gene network signature of retinal leukocytes. This revealed a previously uncharted interplay of adaptive immunity during subretinal inflammation, including prolonged enrichment of myeloid and lymphocyte migration, antigen presentation, and the alternative arm of the complement cascade involving Factor B. We demonstrate Factor B-deficient mice are protected against macrophage infiltration and subretinal inflammation. Suppressing the drivers of retinal leukocyte proliferation, or their capacity to elicit complement responses, may help preserve retinal structure and function during sterile inflammation in diseases such as AMD.