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Updated: Oct 9, 2025
Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis
Published on: May 12, 2020
Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa-Induced Keratitis
Sharon McClellan1, Ahalya Pitchaikannu1, Robert Wright1
1Department of Ophthalmology, Visual and Anatomical Sciences, School of Medicine, Wayne State University, Detroit, Michigan, United States.
Purpose:
Previously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential.
Methods:
Eight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid-modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-β-III tubulin antibody.
Results:
Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1β at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII+ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression.
Conclusions:
Prophylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.
Insights
Prophylactic knockdown of the miR-183/96/182 cluster (miR-183C) reduces Pseudomonas aeruginosa keratitis severity. This therapeutic strategy modulates innate immunity and corneal nerve density for protection.
Area of Science:
- Ophthalmology
- Microbiology
- Molecular Biology
Background:
- Pseudomonas aeruginosa (PA) keratitis is a severe eye infection.
- The miR-183/96/182 cluster (miR-183C) has been implicated in modulating the severity of PA-induced keratitis.
Purpose of the Study:
- To test the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis.
- To evaluate the therapeutic potential of targeting miR-183C for PA keratitis.
Main Methods:
- Mice lacking the miR-183C cluster were used.
- Anti-miR-183C oligoribonucleotides were administered prophylactically via subconjunctival injection and topical application.
- Corneal disease severity, bacterial load, immune cell infiltration, and corneal nerve density were assessed.
Main Results:
- Prophylactic anti-miR-183C treatment reduced PA keratitis severity and bacterial load.
- Treatment modulated innate immune responses, including decreased polymorphonuclear leukocytes and increased macrophage/dendritic cell activation.
- A transient reduction in corneal nerve density was observed, with full recovery post-treatment, potentially mediated by Neuropilin 1 downregulation.
Conclusions:
- Prophylactic miR-183C knockdown demonstrates a protective effect against PA keratitis.
- The protective mechanism involves the regulation of innate immunity and neuroimmune interactions within the cornea.
- Targeting miR-183C represents a potential therapeutic strategy for bacterial keratitis.
