Abstract
Purpose :
Bacterial keratitis is a prevalent eye infection that causes corneal opacification and purulent discharge, especially among contact lens wearers. Such infections recruit innate immune cells into the cornea, predominately neutrophils (PMN). CD177 is a GPI-anchored protein expressed on ~50% of circulating PMN. Proteinase-3 (PR3) is a serine protease that binds CD177 and is shown to be released from PMN granules upon activation or expressed on the plasma membrane (mPR3). CD177+ PMN can be protective or pathogenic in various diseases ranging from IBD to COVID-19. On the other hand, elevated PR3 is found in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated systemic vasculitis. With little known regarding the eye, this study investigates the expression and role(s) of CD177 and PR3 in the cornea following bacterial keratitis.
Methods :
This work uses an experimental model of bacterial keratitis carried out in 8-week-old, female susceptible C57BL/6 (B6) and resistant BALB/c mice. The left eye of each mouse was scarified then infected with P. aeruginosa ATCC strain 19660 (5 μL of 1 x 106 CFU). Corneas from naïve, uninfected mice from both strains served as controls. Corneas were harvested at 1, 3, and 5 days post-infection (p.i.). Levels of CD177 and PR3 were determined at the protein level by Western blot and by phenotypic profiling using flow cytometry. In vitro assessment was carried out using HL-60, a human promyelocytic cell line, and siCD177 knockdown.
Results :
Results from the in vivo model showed no differences in protein levels at 1 day p.i., but significantly higher levels of both CD177 and PR3 in B6 vs. BALB/c at 3 and 5 days p.i. Flow cytometry data revealed CD177+ and PR3+ expression on both PMN and macrophages from B6 and BALB/c infected corneas with differential mean fluorescence intensities detected between the strains under normal conditions and following infection. In vitro results indicated that cell activation was altered following CD177 knockdown with differences in downstream signaling.
Conclusions :
As one of the first studies to explore the role of CD177 and PR3 in the pathogenesis of bacterial keratitis, our findings reveal strain-specific expression profiles for PMN that may contribute to resistance vs. susceptibility. In addition, we show the presence of CD177+PR3+ macrophages. Overall, these findings may uncover novel therapeutic targets to treat bacterial keratitis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.