A2E Induces IL-1ß Production in Retinal Pigment Epithelial Cells via the NLRP3 Inflammasome

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.

Beta cyclodextrins bind, stabilize, and remove lipofuscin bisretinoids from retinal pigment epithelium

“…Accumulation of A2E leads to plasma membrane destabilization (17,19,37), increased lysosomal pH (38)(39)(40), decreased lysosomal enzyme activity (39), and derailing of cholesterol trafficking with the consequent buildup of free cholesterol in lysosomes (41). Moreover, accumulation of LBs in RPE is known to induce complement activation (42,43) and secretion of proinflammatory cytokines, including IL-1β, IL-8, IL-6, VEGFα, TNFa, and chemokines (44)(45)(46) responsible, at least in part, for the perpetuation of the chronic inflammatory milieu linked with AMD (47,48). Hence, irrespectively of how much atRAL or LBs contribute to the retinal degenerative process in the young, we expect that the steady and prolonged accumulation of LBs will progressively weigh more on this balance and therefore, the removal of LBs should have a beneficial impact, particularly, for the elderly retinas.…”

Interleukin-17A Induces IL-1β Secretion From RPE Cells Via the NLRP3 Inflammasome

“…4 Recent efforts have drawn attention to the inflammasome machinery, particularly its product IL-1b, as an inciting factor in AMD. [5][6][7][8] It has been well documented that RPE cells can be triggered to secrete a number of inflammatory cytokines and chemokines. 9 Among these, IL-1b is one of the initial cytokines produced following RPE activation.…”

“…At the posttranscriptional level, generation of mature IL-1b is regulated by inflammasomes, which is formed by NACHT, LRR, and PYD domains-containing (NLRP) proteins, the most studied member of which is NLRP3. 12 More recently, the role of the NLRP3 inflammasome in AMD pathogenesis has been investigated extensively using AMD-related stimuli, such as A2E, 8 Alu RNA, 13 lysosomal activation, 14 and oxidative stress. 15 However, it still is not clear whether this component of innate immunity could be activated or regulated by the local inflammatory cytokine milieu generated during adaptive immune responses.…”

A2E Suppresses Regulatory Function of RPE Cells in Th1 Cell Differentiation Via Production of IL-1β and Inhibition of PGE2

“…These dimers were the main triggers associated with RPE cell oxidative stress and degeneration in AMD. [25][26][27] One of the essential dimers was A2E, which not only induced inflammatory status of RPE cells, 28 but also induced AMD-like lesions in animal models. 29 However, how A2E impaired the immunoregulatory function of RPE cells in T-cell subset differentiation and triggered inflammation in AMD development should be elucidated.…”