Abstract
Purpose :
Alternate complement signaling via NF-kB activation is known to regulate subRPE drusen and intracellular lipid deposition in iPSC-derived RPE (iRPE) models of age-related macular degeneration (AMD). Although toll-like receptor (TLR) stimulation is known to culminate in NF-kB activation, whether it is responsible for triggering complement-induced AMD phenotype is less clear. Here, we explore the role of TLR3 and TLR4 in complement-induced cellular responses in AMD pathogenesis.
Methods :
Mature iRPE were pretreated with TLR3 or TLR4 inhibitors followed by treatment with either no serum, complement competent human serum (CC-HS) with activated anaphylatoxins, or complement incompetent human serum (CI-HS) with inhibited anaphylatoxin activity as previously described (Sharma et al., 2021). ELISA assays were performed on apical and basal media of cells to quantify cytokine IL-8. Target TLRs were located using immunofluorescent staining and intracellular lipid was detected by BODIPY dye. Additionally, iRPE were treated with TLR3 activator Poly (I:C) and stained for BODIPY and ubiquitin ligase TRIM3 known to mediate polyubiquitination of TLR3. Cells were visualized by fluorescence microscopy.
Results :
Inhibitors for TLR3 and TLR4 decreased complement-induced lipid deposition exhibited by reduced BODIPY levels. TLR 4 inhibitor decreased human complement-induced IL-8 secretion in apical media and TLR3 inhibitor decreased IL-8 secretion in basal media. No significant difference in IL-8 secretion was found between CC-HS controls and TLR 4 inhibited cells in basal media or TLR3 inhibited cells in apical media. In iRPE treated with Poly (I:C) at concentrations of 10, 50, and 100 ug/ml, BODIPY and TRIM3 levels increased in a concentration-dependent manner.
Conclusions :
In iRPE, complement-induced lipid deposition and activation of NF-kB leading to elevated IL-8 secretion characteristic of inflammation and AMD are mediated by TLR 3 and TLR 4. The regulatory role of these receptors in AMD pathogenesis reflects a possible upstream presence of aberrant RNA and viruses or bacterial endotoxins that trigger the activation of TLR3 and TLR4 respectively.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.