Cleavage-activated transmembrane CDCP1 regulates host defense against bacterial keratitis

Abstract

Purpose : CUB domain-containing protein 1 (CDCP1) is a transmembrane protein originally found to be overexpressed on certain solid tumor cells. Cleavage of its extracellular domain initiates PCKδ-AKT pathway. Interestingly, CDCP1 is highly expressed on normal healthy corneal epithelial cells. Considering the important roles of these kinases in innate immunity, here we investigate the role of CDCP1 in regulation of host defense against bacterial keratitis.

Methods : Pseudomonas aeruginosa (PA) invasive strain 3346 was inoculated to scratched corneas of C57BL/6 WT and CDCP1-KO mice, as well as WT mice subconjunctivally injected with control or cleavage-blocking anti-CDCP1 IgG. Disease severity was scored every 24h; antimicrobial peptides (BD-1, BD-2, BD-3, CRAMP/LL-37, hepcidin) and bacterial burdens in infected corneas were quantified at 3h, 24h and 5 days post-inoculation. Human telomerase-immortalized corneal epithelial cells (hTCEpi) were treated with non-targeting/CDCP1 siRNA, control/cleavage-blocking anti-CDCP1 IgG, or vehicle/PKC and PI3K inhibitors prior to stimulation with 20% PA culture supernatants in culture media. Phospho -PKCδ and -PI3K were detected by flow cytometry after 1h. Secreted AMPs in culture media were quantified by ELISA after 24h and 48h, followed by evaluation of antimicrobial activity by transferring to fresh hTCEpi cells inoculated with PA3346, then quantification of extra- and intracellular bacteria after 1.5h.

Results : Infected CDCP1-KO mice developed more severe corneal pathology with increased bacterial burdens compared to WT mice. Cleavage-blocking CDCP1-treated mice showed that exacerbated keratitis was accompanied with reduced AMPs in the infected corneas. Knocking down CDCP1 or blocking CDCP1 cleavage on hTCEpi cells suppressed not only bacterial ligand-induced PKCδ and PI3K phosphorylation, but also AMP production. PKC and PI3K inhibition showed that AMP production was dependent on these signaling pathways. Fresh cells inoculated with PA3346 in conditioned media from CDCP1-KD or cleavage-blocked CDCP1 cells were found to have higher counts of intra- and extracellular bacteria.

Conclusions : Cleavage activation of CDCP1 on corneal epithelial cells was found to play a protective role in bacterial keratitis by promoting AMP production, which reduced bacterial burdens and corneal opacity. The data suggest that CDCP1 could be a new therapeutic target for bacterial keratitis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.