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2023 OMIG Abstract
Purpose: The healthy cornea is remarkably resistant to bacterial adhesion. Previously, we discovered a role for Transient Receptor Potential (TRP) Vanilloid 1 (TRPV1) ion channels in preventing the adhesion of environmental bacteria to the healthy murine cornea, and for Ankyrin 1 (TRPA1) ion channels in preventing corneal adhesion by deliberately inoculated Pseudomonas aeruginosa, a Gram-negative bacterium and a leading cause of corneal infection. Here, we explored the role of TRPV1 and TRPA1 in corneal defense against the adhesion of inoculated S. aureus, a Gram-positive pathogen of the cornea.
Methods: Healthy corneas of anesthetized C57BL/6J wild-type (WT), TRPA1 (-/-) or TRPV1 (-/-) mice were inoculated with ~1 x 1011 CFU bacteria every hour for 4 h with a clinical isolate of Staphylococcus aureus strain S33 or P. aeruginosa strain PAO1. In some experiments, before inoculation, WT mice were treated with bupivacaine to block sensory nerve function or resiniferatoxin (RTX) to ablate TRPV1-expressing sensory nerves. Mice were euthanized at 4 h and enucleated eyes were fixed overnight in paraformaldehyde (2%). A universal bacterial 16S rRNA FISH probe was used to label adherent bacteria which were quantified using confocal microscopy and ImageJ. Two-way ANOVA was used for statistical analysis. P values < 0.05 were considered significant.
Results: Healthy TRPV1 (-/-) corneas were significantly more susceptible to adhesion of S. aureus while TRPA1 (-/-) corneas retained a resistance similar to WT; TRPV1 (-/-) 19 ± 9, TRPA1 (-/-) 5 ± 2, WT 4 ± 1 bacteria/field of view (P < 0.05). RTX treatment aligned with this phenotype; RTX treated 19 ± 1 vs. control 7 ± 3 bacteria/field of view (P < 0.05). As expected, bupivacaine treatment increased P. aeruginosa adhesion to healthy corneas vs. controls (by ~4-fold, P < 0.0001). However, bupivacaine treatment did not compromise corneal resistance to the adhesion of S. aureus S33 (P > 0.05).
Conclusions: TRPV1, but not TRPA1, helps defend the healthy murine cornea against the adhesion of inoculated S. aureus, contrasting with TRPA1-mediated defense vs. P. aeruginosa. Data suggest that TRPV1- and TRPA1-mediated defense mechanisms are distinct with TRPV1-mediated defense(s) being unaffected by local anesthetic block of sensory nerve firing. Ongoing studies will decipher the role of resident and/or infiltrating immune cells, secreted molecular factors, and bacterial ligands in these TRP-mediated corneal defenses.
Disclosure:
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