Biochemical and Biophysical Research Communications
Activated microglia trigger inflammasome activation and lysosomal destabilization in human RPE cells
Introduction
Age-related macular degeneration (AMD) is the leading cause of legal blindness world wide [1]. The disease primarily affects the retinal pigment epithelium (RPE), Bruch's membrane, photoreceptors and the choroid. A hallmark of early AMD is the accumulation of retinal lipofuscin and the appearance of drusen [2]. In late stage disease, wet or neovascular AMD can be distinguished from dry AMD with geographic atrophy, the latter constituting 80% of all cases [3]. In wet AMD, blood vessels grow from the subretinal space into the retina. Especially the macula lutea and fovea centralis are affected. Production of vascular endothelial growth factor (VEGF) seems to promote this process [4]. Hence, anti-VEGF medication is currently the gold standard in the therapy of wet AMD. However, sustained VEGF inhibition can lead to retinal and systemic adverse effects. Therefore, there is an urgent need to search for new therapy options for both forms of AMD [5].
Microglia are tissue-resident immune cells of the central nervous system (CNS) including the retina, that are constantly surveying their microenvironment with their long protrusions [6]. Microglia are different from other macrophage populations concerning their specific locations and morphology [7] and they regulate immune homeostasis in the healthy and degenerating CNS [8]. Microglia detect damage-associated molecular patterns (DAMPs) from dying neurons and this response is a common hallmark of degenerative diseases of the brain including Multiple Sclerosis [9], Parkinson's disease [10], and Alzheimer's disease [11]. Chronic microglia/macrophage reactivity and dysregulated complement factors also negatively influence disease progression in the retina [12], [13], [14], [15].
The RPE constitutes a single-cell layer of post-mitotic cells that separates the choroid and the neuroretina. The RPE has pleiotropic functions including transport of nutrients, ions and water, absorption of light, protection against photo-oxidation, sustaining the visual cycle, and phagocytosis of shed photoreceptor outer segments [16]. In addition, the RPE is part of the innate immune response mainly controlled by the NLRP3 inflammasome [17], [18]. It is composed of intracellular protein complexes that consist of NLRP3 (NACHT, LRR and PYD domains-containing protein 3), ASC (Apoptosis-associated speck-like protein containing CARD), and caspase-1 (cysteine-aspartic protease-1) [19]. The RPE inflammasome can be activated by various stimuli and is consequently found in human AMD and model systems [20], [21], [22], [23]. Inflammasome activation needs two distinct signals, whereby the first microbial or pro-inflammatory signal results in NLRP3 priming, while the second signal such as the destabilization of lysosomal membranes leads to the formation of the NLRP3 inflammasome complex [24].
In this study, we investigated whether conditioned media from activated human microglia can act as priming step together with the lysosomal destabilization agent Leu-Leu-OMe (LLOMe) to trigger NLRP3 inflammasome activation in human ARPE-19 cells.
Section snippets
Cell culture
Human retinal pigment epithelial cells (ARPE-19) were cultured in DMEM/F-12 containing 10% fetal calf serum (FCS) and 1% Pen/Strep. Human iPS-derived microglia cells (iPSdM) were kindly provided by Prof. Harald Neumann, University of Bonn [25]. iPSdM were grown in DMEM/F-12 with 1% N2-Supplement, 1% Pen/Strep FCS and 0.24% l-Glutamine and 0.34% Glucose. iPSdM cells were treated with 1 μg/ml E. coli 0111:B4 LPS for 24 h. The cell culture supernatants (conditioned media) from iPSdM cells were
LPS-conditioned microglia supernatants and LLOMe lead to cytoskeletal disorganization and accumulation of lipids in ARPE-19 cells
Our hypothesis was that activated microglia can prime inflammasome activation in RPE cells. Therefore, we first tested the effects of microglia-conditioned media together with LLOMe as lysosomal destabilization agent on RPE cytoskeletal morphology. ARPE-19 cells treated with either vehicle (DMSO), LLOMe alone, or conditioned media from non-activated microglia plus LLOMe did not show gros structural anomalies in the F-actin filament fibers as shown by phalloidin staining (Fig. 1A–C). In
Discussion
The aim of this study was to study paracrine signaling from activated microglia on retinal pigment epithelial cells with a special emphasis on NLRP3 inflammasome activation. The NLRP3 inflammasome seems to play an important role in the context of AMD [29]. This intracellular multimeric protein complex is a known regulator of mature IL-1β and IL-18 secretion [30] and inflammasome activation in the RPE was shown in patients with both atrophic and neovascular AMD [31]. Established triggers of
Conflict of interest
All authors declare no conflict of interests.
Acknowledgements
The work was supported by the Hans und Marlies Stock-Foundation, the Deutsche Forschungsgemeinschaft (FOR2240), and the ProRetina Foundation. We thank Dr. Sarah Hermann for critical reading of the manuscript and Prof. Harald Neumann for providing iPSdM cells.
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2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :The following were purchased; SV-40 immortalized human microglia cell line (ABM, Inc., Canada); human non-transformed RPE cell line, ARPE-19, (ATCC - CRL-2302; Rockville, MD, USA); TSPO ligand - XBD173 (Emapunil - APAC Pharmaceutical LLC, USA); TSPO ligands - PK11195 and Ro5-4864; LLOMe; Phorbol 12-myristate 13-acetate (PMA) and Zymosan A (Sigma-Aldrich, USA); Collagen I (ThermoFisher Scientific, USA); and DCFDA Cellular ROS Assay Kit (#ab113851) (Abcam, Cambridge, MA, USA). ARPE-19 cells were cultured as described previously [9]. For experiments, cells were grown to confluency in cell culture plates or for 1 month in 6-well plates with transwell inserts (Corning Costar, UK; Cat.
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