Abstract
Purpose :
Toll-like receptors are intracellular and cell surface molecules that sense pathogen-associated molecular patterns and induce the activation of the innate immune system by the secretion of pro-inflammatory cytokines. Prior studies suggest Toll-like receptors (TLR) 3 and 4 may be involved in complement-induced AMD phenotype in iPSC-derived retinal pigment epithelium (iRPE) models and increase lipid deposition. However, the cellular localization of TLR3 and 4 receptors and their association with lipid accumulation hasn’t been fully worked out. This study aims to investigate TLR3, 4 localizations when altered by anaphylatoxin-induced signaling and their relationship to intracellular lipid accumulation and morphological changes in our iPSC-RPE model.
Methods :
Mature iRPE grown in transwell plates were treated with either complement competent human serum (CC-HS) that contained activated anaphylatoxins or complement incompetent human serum (CI-HS) with heat-inactivated anaphylatoxins (Sharma et al., 2021). RNAseq data was evaluated via eye integration to compare the expression of TLRs between CI-HS & and CC-HS iRPE. (McGaughey et al., 2019). Apical and basal media were collected for quantification of cytokines. TLRs, cell borders, and lipid deposits (LD) were detected by immunocytochemistry. LD quantification was completed by a custom program (Esh et al., 2023).
Results :
Although mRNA levels of TLRs are similar in CC-HS and CI-HS-treated iPSC-RPE, their protein expression is different. TLR-4 expression increases and on the basolateral side of cells, while TLR-3 accumulates perinuclearly in CC-HS treated RPE cells. LD tend to accumulate basolaterally in CC-HS treated iRPE. Additionally, when stained with phalloidin, iRPE cells display F-actin stress fibers and lost hexagonal morphology, indicating RPE degeneration. Secretion of pro-inflammatory cytokines IL-6 and IL-8 is elevated in injured cells.
Conclusions :
In our iRPE model of AMD, anaphylatoxin induced expression of TLR-4 and TLR-3 contributes to lipid accumulation and RPE atrophy. TLR presence on the basolateral side of iRPE may serve as an initiator for drusen formation. Lipid accumulation combined with known effects on cell morphology and inflammation indicates that there is an upstream regulator or a specific ligand binding to TLRs, contributing to AMD pathogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.