Abstract
Purpose :
Retinal pigment epithelial (RPE) cells form the outer blood-retinal barrier, protecting the neuroretina from invading pathogens. Antimicrobial peptides (AMPs) are host defense peptides displaying direct antimicrobial activity. This study set out to determine the AMP expression pattern in RPE cells and their regulation under pathophysiological conditions.
Methods :
The online platform Spectacle (https://singlecell-eye.org) was used to analysis the expression of AMP genes, including amyloid beta precursor protein (APP), RARRES2, FAM3A, PI3, GNLY, Camp, and Hamp in human RPE cells . Semi-quantitative PCR and
immunohistochemistry were employed to validate AMPs expression in human and mouse RPE cells and in human donor eyes. RPE cells were exposed to Toll-like receptor (TLR) agonists (Pam3csk, LPS, PGN-SA, poly(I:C), and poly(dA:dT)) to induce activation or doxorubicin, H2O2, and TNF-α to induce senescence. The alterations in the expression of AMPs, the APP-cleaving enzymes including ADAM9, ADAM17, ADAM19, BACE1, and PS1, and the APP products Aβ40 and Aβ42 following the treatment were examined by qPCR and Western blotting or ELISA.
Results :
Single-cell sequencing along with semi-quantitative PCR and immunofluorescence assays revealed that RPE cells express a variety of AMPs, including APP, RARRES2, FAM3A, PI3, GNLY, Camp, and Hamp. Among these genes, APP, RARRES2, and FAM3A were highly expressed under physiological conditions, and the expression levels of APP and RARRES2 were comparable to Actb. The TLR3 agonist Poly(I:C) significantly increased the expression of Camp and Hamp in RPE cells. Doxorubicin, H2O2 and TNFα can all induce RPE senescence. Senescent RPE cells had impaired bactericidal capacity and showed decreased APP and RARRES2, but increased FAM3A, PI3, GNLY, Camp, Hamp. Interestingly, senescent RPE expressed higher levels of APP-cleaving enzymes BACE1 and PS1, and produced higher levels of APP products Aβ40 and Aβ42 peptides, as well as increased ratio of intracellular Aβ42/Aβ40. Immunostaining of donor human eyes revealed an age-dependent accumulation of Aβ42 in RPE cells.
Conclusions :
RPE cells expressed a variety of AMPs, possibly to protect the neuroretina from blood-borne pathogens. Aging disrupts the expression of AMPs, leading to reduced antibacterial capacity and increased release and deposition of the neurotoxic peptide Aβ42 in RPE cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.