Inactivation of miR-183/96/182 Reduces Severity of Pseudomonas aeruginosa Infection of the Cornea

Abstract

Purpose: The miR-183/96/182 cluster (referred to as miR-183/96/182) is highly expressed in all sensory organs. The cornea is densely innervated by sensory neurons. The purpose of the current study is to test the role of miR-183/96/182 in the murine response to corneal infection with Pseudomonas aeruginosa (PA).

Methods: miR-183/96/182 knockout (ko), miR-183C^GT/GT, and their wild type control (wt) mice were employed. The central corneas of anesthetized mice were scarred and 5 ml of 1X10⁶ CFU/ml of PA (strain 19660; ATCC) was topically applied. Corneal disease was graded at 1, 3, and 5 days post infection (dpi).Corneal RNA was harvested at 5 dpi. X-gal staining and immunofluorescence were performed on corneal flat-mounts and cryosections. Peritoneal polymorphonuclear neutrophils (PMNs) and macrophages (MΦ) were isolated for RNA preparation and quantitative (q)RT-PCR, or phagocytosis and intracellular bacterial killing assays.

Results: 1. miR-183/96/182 is highly expressed in corneal epithelium and in trigeminal ganglia of wild type (wt) mice; while completely inactivated in ko animals.<br /> 2. Compared to wt, ko animals showed decreased disease upon PA infection, as well as decreased expression of inflammation-related cytokines and their receptors in the cornea.<br /> 3. Inactivation of miR-183/96/182 results in decreased corneal nerve density, branching and number of beaded nerves, and, consistently, expression of pain receptor, TRPV1, and several key neuropeptides.<br /> 4. miR-183/96/182 is highly expressed in peritoneal MΦ. Its inactivation resulted in significant changes in their cytokine expression profile.<br /> 5. miR-183 and miR-182 are expressed in PMNs at levels lower than in MΦ. In vitro assays suggested changes of PMN phagocytosis and bacterial killing capacity in ko mice.

Conclusions: Inactivation of miR-183/96/182 resulted in significantly reduced disease in PA-induced keratitis. Loss of miR-183/96/182 leads to changes in corneal innervation and innate immune responses, as well as neuro-immune interactions, contributing to decreased corneal disease. Since sensory innervation and innate immunity also play an important role in other forms of microbial keratitis, corneal wound healing, diabetic neuropathy, and dry eye syndrome, miR-183/96/182 ko mice may provide a new opportunity to study the roles of miR-183/96/182 in various physiological and pathological processes.<br />