title: A2E induces IL-1ß production in retinal pigment epithelial cells via the NLRP3 inflammasome.
abstract: OBJECTIVE
  With 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.
  
  
  METHODS
  ARPE-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.
  
  
  RESULTS
  Of 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.
  
  
  CONCLUSIONS
  A2E 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.
authors.affiliation: UCL Institute of Ophthalmology, Ocular Biology and Therapeutics, London, United Kingdom.
authors.forename: Owen A
authors.initials: OA
authors.lastname: Anderson
authors.forename: Arthur
authors.initials: A
authors.lastname: Finkelstein
authors.forename: David T
authors.initials: DT
authors.lastname: Shima
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000172:deficiency / Q000235:genetics; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D057092:Geographic Atrophy / Q000276:immunology / Q000473:pathology; D006801:Humans; D058847:Inflammasomes / Q000378:metabolism*; D018836:Inflammation Mediators / Q000378:metabolism; D053583:Interleukin-1beta / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D057507:Mice, 129 Strain; D018345:Mice, Knockout; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D012176:Retinoids / Q000502:physiology*; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: C112040:A2-E (N-retinylidene-N-retinylethanolamine); D018528:ATP-Binding Cassette Transporters; C512841:Abca4 protein, mouse; D002352:Carrier Proteins; D058847:Inflammasomes; D018836:Inflammation Mediators; D053583:Interleukin-1beta; D000071199:NLR Family, Pyrin Domain-Containing 3 Protein; C453881:NLRP3 protein, human; D011498:Protein Precursors; D034741:RNA, Small Interfering; D012176:Retinoids