Flandrin Orneika, Yang Yujia, Abboud Sarah, Kumar Naren G, Datta Ananya, Bautista Diana M, Evans David J, Fleiszig Suzanne M J
bioRxiv. 2025 Apr 12:2025.04.12.648450. doi: 10.1101/2025.04.12.648450.
Previously we showed that transient receptor potential ion channels TRPA1 and TRPV1 selectively protect the cornea against bacterial adhesion, with TRPA1 countering the Gram-negative pathogen Pseudomonas aeruginosa and TRPV1 countering environmental bacteria. Here, we explored parameters of this specificity using a Gram-positive pathogen Staphylococcus aureus.
Healthy corneas of C57BL/6J wild-type (WT), TRPA1 (-/-) or TRPV1 (-/-) mice were challenged with S. aureus for 4 or 6 hours. Some experiments instead/also used resiniferatoxin (RTX) to deplete TRPV1-expressing nerves, JNJ-17203212 to selectively antagonize TRPV1, or the anesthetic bupivacaine to inhibit nerve firing. Adherent bacteria were quantified using FISH labeling (16S rRNA-targeted probe). Lyz2+, CD11c+ and CD45+ cells were visualized/quantified using hybrid mT/mG + LysMcre mice (red cell membranes; Lyz2+-GFP), CD11c+-YFP mice, and anti-CD45-antibody respectively.
Corneas of TRPV1 (-/-) not TRPA1 (-/-) mice were found more susceptible to S. aureus adhesion compared to WT. Accordingly, either ablation of TRPV1-expressing nerves or TRPV1 antagonism increased adhesion. Defense against S. aureus adhesion did not depend on nerve firing. Despite having no significant impact on CD11c+ or Lyz2+ cell numbers, S. aureus challenge increased CD45+ cell counts, also dependent on TRPV1-expressing nerves, and it increased Lyz2+ cell sphericity and volume.
Healthy corneas utilize TRPV1 to protect against S. aureus adhesion independently of sensory nerve firing. This contrasts with defense against P. aeruginosa adhesion which requires TRPA1 and nerve firing. How the differential immune cell responses to these two pathogens relate to TRP-dependent defense against adhesion remains to be determined.
此前我们发现，瞬时受体电位离子通道TRPA1和TRPV1可选择性地保护角膜免受细菌黏附，其中TRPA1可对抗革兰氏阴性病原体铜绿假单胞菌，而TRPV1可对抗环境细菌。在此，我们使用革兰氏阳性病原体金黄色葡萄球菌探究了这种特异性的参数。
用金黄色葡萄球菌对C57BL/6J野生型（WT）、TRPA1（-/-）或TRPV1（-/-）小鼠的健康角膜进行4或6小时的攻击。一些实验还使用树脂毒素（RTX）来耗尽表达TRPV1的神经，使用JNJ-17203212选择性拮抗TRPV1，或使用麻醉剂布比卡因抑制神经放电。使用FISH标记（靶向16S rRNA的探针）对黏附细菌进行定量。分别使用杂交mT/mG + LysMcre小鼠（红细胞膜；Lyz2+-绿色荧光蛋白）、CD11c+-黄色荧光蛋白小鼠和抗CD45抗体对Lyz2+、CD11c+和CD45+细胞进行可视化/定量分析。
与野生型相比，发现TRPV1（-/-）而非TRPA1（-/-）小鼠的角膜更容易受到金黄色葡萄球菌的黏附。因此，表达TRPV1的神经的消融或TRPV1的拮抗作用均会增加黏附。对金黄色葡萄球菌黏附的防御不依赖于神经放电。尽管对CD11c+或Lyz2+细胞数量没有显著影响，但金黄色葡萄球菌攻击会增加CD45+细胞计数，这也依赖于表达TRPV1的神经，并且会增加Lyz2+细胞的球形度和体积。
健康角膜利用TRPV来保护自身免受金黄色葡萄球菌的黏附，且与感觉神经放电无关。这与对抗铜绿假单胞菌黏附的防御不同，后者需要TRPA1和神经放电。这两种病原体的不同免疫细胞反应与TRP依赖性黏附防御之间的关系仍有待确定。
