Abstract
Purpose :
Healthy corneas resist formation of a bacterial microbiome, unlike the adjacent conjunctiva. Previously, we discovered a role for Transient Receptor Potential (TRP) Vanilloid 1 (TRPV1) ion channels in preventing adhesion of environmental bacteria to the murine cornea, and for Ankyrin 1 (TRPA1) ion channels in preventing corneal adhesion by Pseudomonas aeruginosa, an opportunistic pathogen. Here we explored if TRPA1/V1 prevention of adhesion involves a differentiation between commensal versus pathogenic bacteria.
Methods :
Anesthetized C57BL/6 wild type (WT), TRPA1V1 (-/-), TRPA1 (-/-) and TRPV1 (-/-) mice were inoculated with ~1 x 1011 CFU every hour for 4 h with the following bacteria: a murine eyelid commensal (Macrococcus spp.), conjunctival commensal Corynebacterium mastitidis, Staphylococcus aureus S33 and P. aeruginosa PAO1. In some experiments, before inoculation, WT mice were treated with bupivacaine to block all sensory nerve function or resiniferatoxin (RTX) to ablate TRPV1-expressing sensory nerves. Mice were euthanized at 4 h and enucleated eyes fixed overnight in paraformaldehyde (2%). A universal bacterial 16S rRNA FISH probe was used to label adhered bacteria which were quantified using confocal microscopy and ImageJ. Two-way ANOVA was used for statistical analysis, P < 0.05 was considered significant.
Results :
TRPA1 (-/-) and TRPV1 (-/-) corneas were more susceptible to adhesion of the murine commensal; TRPA1 (-/-) 14 ± 5 or TRPV1 (-/-) 15 ± 6 vs. WT 4 ± 1 bacteria/field of view (P < 0.01). RTX treatment (TRPV1 ablation) aligned with this phenotype; 18 ± 3 vs 6 ± 2 bacteria/field of view for RTX and control corneas, respectively (P < 0.01). Bupivacaine treatment significantly increased P. aeruginosa adhesion vs. controls (~ 4-fold increase, P < 0.0001), but had no effect on adhesion of the murine commensal (P > 0.05). S. aureus S33 adhesion depended only on TRPV1; TRPA1 (-/-) 5 ± 2, TRPV1 (-/-) 19 ± 9 vs. WT 4 ± 1 bacteria/field of view (P < 0.05). Adhesion of C. mastitidis was not significantly impacted by the absence of TRPA1/V1 (P > 0.05).
Conclusions :
The roles of TRPV1 and TRPA1 ion channels in defending the murine cornea against bacterial adhesion do not necessarily correlate with bacterial status as a commensal or a pathogen. Furthermore, data suggest that the roles played by these ion channels to prevent bacterial adhesion can be independent of sensory nerve activation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.