Abstract
AimsWith ageing extracellular material is deposited in Bruch’s membrane, as drusen. Lipofuscin is deposited in retinal pigment epithelial cells. Both of these changes are associated with age related macular degeneration, a disease now believed to involve chronic inflammation at the retinal-choroidal interface. We hypothesise that these molecules may act as danger signals, causing the production of inflammatory chemokines and cytokines by the retinal pigment epithelium, via activation of pattern recognition receptors.MethodsARPE-19 cells were stimulated in vitro with the following reported components of drusen: amyloid-ß (1-42), Carboxyethylpyrrole (CEP) modified proteins (CEP-HSA), Nε-(Carboxymethyl)lysine (CML) modified proteins and aggregated vitronectin. The cells were also stimulated with the major fluorophore of lipofuscin: N-retinylidene-N-retinylethanolamine (A2E). Inflammatory chemokine and cytokine production was assessed using Multiplex assays and ELISA. The mechanistic evaluation of the NLRP3 inflammasome pathway was assessed in a stepwise fashion.ResultsOf all the molecules tested only A2E induced inflammatory chemokine and cytokine production. 25 µM A2E induced the production of significantly increased levels of the chemokines IL-8, MCP-1, MCG and MIP-1α, the cytokines IL-1ß, IL-2, IL-6, and TNF-α, and the protein VEGF-A. The release of IL-1ß was studied further, and was determined to be due to NLRP3 inflammasome activation. The pathway of activation involved endocytosis of A2E, and the three inflammasome components NLRP3, ASC and activated caspase-1. Immunohistochemical staining of ABCA4 knockout mice, which show progressive accumulation of A2E levels with age, showed increased amounts of IL-1ß proximal to the retinal pigment epithelium.ConclusionsA2E has the ability to stimulate inflammatory chemokine and cytokine production by RPE cells. The pattern recognition receptor NLRP3 is involved in this process. This provides further evidence for the link between A2E, inflammation, and the pathogenesis of AMD. It also supports the recent discovery of NLRP3 inflammasome activation in AMD.
Highlights
In the western world age related macular degeneration (AMD) is the leading cause of blindness in the elderly population. [1,2] AMD can be classified into two groups: ‘dry’ and ‘wet’ AMD
Of all the molecules tested only A2E induced inflammatory chemokine and cytokine production. 25 mM A2E induced the production of significantly increased levels of the chemokines IL-8, MCP-1, MCG and MIP-1a, the cytokines IL1ß, IL-2, IL-6, and tumor necrosis factor alpha (TNF-a), and the protein Vascular endothelial growth factor (VEGF)-A
The pattern recognition receptor NOD-like receptor protein P3 (NLRP3) is involved in this process
Summary
In the western world age related macular degeneration (AMD) is the leading cause of blindness in the elderly population. [1,2] AMD can be classified into two groups: ‘dry’ (atrophic) and ‘wet’ (neovascular) AMD. In the western world age related macular degeneration (AMD) is the leading cause of blindness in the elderly population. [1,2] AMD can be classified into two groups: ‘dry’ (atrophic) and ‘wet’ (neovascular) AMD. Dry AMD accounts for approximately 90% of cases of AMD, [3] and is characterized by primary loss of the retinal pigment epithelium (RPE) with secondary atrophy of the overlying photoreceptors and underlying choriocapillaris. Vascular endothelial growth factor (VEGF) inhibitors have provided a breakthrough in the treatment of wet AMD. [4] there is currently no effective treatment for dry AMD. Greater understanding of the pathogenesis of AMD may provide new treatment strategies for this blinding disease. One hallmark of AMD is the presence of drusen. The deposition of extracellular material as drusen, at the level of
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