Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa-Induced Keratitis.

Abstract

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.

• Authors: Sharon McClellan, Ahalya Pitchaikannu, Robert Wright, Denise Bessert, Mason Iulianelli, Linda D Hazlett, Shunbin Xu
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 62 Issue 15 Pg. 14 (12 01 2021) ISSN: 1552-5783 [Electronic] United States
• PMID: 34919120 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Cytokines
• MicroRNAs
• Mirn182 microRNA, mouse
• Mirn183 microRNA, mouse
• Mirn96 microRNA, mouse

Topics

• Animals
• Corneal Ulcer (genetics, metabolism, prevention & control)
• Cytokines (metabolism)
• Enzyme-Linked Immunosorbent Assay
• Eye Infections, Bacterial (genetics, metabolism, prevention & control)
• Female
• Flow Cytometry
• Gene Expression Regulation (physiology)
• Gene Knockdown Techniques
• Immunity, Innate
• Mice
• Mice, Inbred C57BL
• Mice, Knockout
• MicroRNAs (genetics)
• Microscopy, Fluorescence
• Neutrophils (physiology)
• Pseudomonas Infections (genetics, metabolism, prevention & control)
• Real-Time Polymerase Chain Reaction
• Transfection