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Louis Scott, Emma E Vincent, Natalie Hudson, David Copland, Kate Heesom, Matthew Campbell, Andrew Halestrap, Andrew D Dick, Sofia Theodoropoulou; Mapping interleukin-33 to metabolic function in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2019;60(9):1685.
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© ARVO (1962-2015); The Authors (2016-present)
Interleukin-33 (IL-33), is an IL-1 family cytokine that is constitutively expressed in the inner retina, RPE and choroid. IL-33 functions as an “alarmin” molecule released from barrier cells following cellular stress. We have previously demonstrated a role for IL-33 in the homeostatic regulation and maintenance of mitochondrial metabolism. The aim of this study was to further determine the role of IL-33 in the regulation of RPE metabolism, utilizing a proteomics approach to map the molecular interactions (interactome) of IL-33.
Using retinal pigment epithelium (RPE) whole cell lysates derived from both human (ARPE-19) and mouse (primary isolated RPE), immunoprecipitation of IL-33 was performed. Proteomic analysis of the IL-33 “pull-down” was assessed using tandem mass-tagging (mass spectrometry-based quantification). Interactions were mapped to human or murine UniProt databases, and ranked analysis performed using STRING (https://string-db.org/).The role of cellular IL-33 was further assessed in vitro using both knock-down (siRNA) and over-expression (CRISPRcas9) approaches. Cellular metabolism was assessed using seahorse extracellular flux, C13 glucose pathway tracing and immunoblotting/RT-PCR quantification of metabolic pathway intermediates.
Pathway analysis of the IL-33 interactome demonstrated an enrichment of targets involved with protein translation, RNA splicing, metabolism and innate immune regulation. Importantly for cellular metabolism and regulation of RPE glycolytic potential, IL-33 interacts with the splicing factors heterogeneous nuclear ribonucleoproteins A1 + A2 (hnRNPA1/2) and serine/arginine-rich splicing factor 3 (SRSF3) which regulate alternative splicing of the pyruvate kinase gene (Pkm).In IL-33 deficient RPE, altered ratios of Pkm splice variants are observed, alongside with reduced mitochondrial function and structural and morphological differences. Overexpression of IL-33 lead to increased glycolytic flux and mitochondrial activity, dependent on pyruvate import into the mitochondria.
These findings highlight a role for intracellular IL-33 in the regulation of metabolic enzyme expression, serving as a molecular check-point to license mitochondrial respiration. This has implications for a degenerative retina, where the strain on mitochondrial function with age may subvert the ability of RPE to respond appropriately to the metabolic demands of inflammation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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