Inactivation of the miR-183/96/182 Cluster Decreases the Severity of Pseudomonas aeruginosa-Induced Keratitis

Abstract

Purpose: The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions.

Methods: Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays.

Results: MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease.

Conclusions: MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

Figure 1

MicroRNA-183/96/182 is expressed in the cornea in both mice and humans and is inactivated in the ko mice. (A, B) Quantitative RT-PCR analysis in mouse (A) and human (B) cornea. n = 3 for each group. wt, wild-type (miR-183C^+/+); het, heterozygous (miR-183C^GT/+); ko, knockout (miR-183C^GT/GT). (C–F) X-gal staining of wt (C, E) and heterozygous mouse cornea (D, F). *P < 0.05; ****P < 0.0001. Ep, epithelium; Bl, Bowman's layer; St, stroma; Dm, Descemet's membrane; En, endothelium.

Figure 2

(A–C) MicroRNA-183/96/182 is expressed in the trigeminal ganglion. (A, B) X-gal staining in the brain of a 6-week-old miR-183C^GT/+ mouse. CNV, cranial nerve V; TG, trigeminal ganglia. (C) Quantitative RT-PCR analysis. (D–H) Inactivation of miR-183/96/182 had significant impact on subbasal plexus of corneal nerves and neuropeptide expression in the cornea. (D) Immunofluorescence of flat-mount cornea with anti-β-III-tubulin antibody showing decreased corneal nerve density in the subbasal plexus. Images in the bottom row are under higher magnification of the same corneas as the top row. (E–H) Quantitative RT-PCR analysis on Tac1, CGRP, Cx3cl1, and TRPV1 in the cornea of noninfected (N) and PA-infected (5) wild-type (open bars) and miR-183/96/182 ko animals (filled bars) at 5 dpi. *P < 0.05.

Figure 3

Inactivation of miR-183/96/182 resulted in decreased expression of proinflammatory cytokines and PA-induced keratitis. (A) Clinical scores at 1, 3, and 5 dpi. n = 8 for wt; n = 10 for ko mice. Horizontal bars represent the mean values. (B) Examples of slit-lamp photography of eyes of wt and ko animals at 5 dpi. (C–J) Inactivation of miR-183/96/182 resulted in decreased expression of proinflammatory cytokines and related receptors in corneas of noninfected (N) and PA-infected (5) wt (open bars) and ko animals (filled bars) at 5 dpi. n = 5 for both genotypes. *P < 0.05.

Figure 4

(A, B) Inactivation of miR-183/96/182 decreased inflammatory cytokines, IL-1β (A) and MIP-2 (B) at protein levels in the infected cornea by ELISA. (C) Myeloperoxidase levels were decreased in the cornea of ko mice. (D) Bacterial load was significantly increased in the cornea of ko mice at 1 dpi, but decreased at 5 dpi.

Figure 5

MicroRNA-183/96/182 is expressed in mouse Mϕ and PMNs (A, B) and in human PBMCs and PMNs (C, D). It is inactivated in the miR-183/96/182 ko mice (A, B). n = 3 for each group. ****P < 0.0001.

Figure 6

MicroRNA-183/96/182 modulates phagocytosis and intracellular killing capacities of Mϕ and PMNs. (A–D) In vitro overexpression (A, B) and knockdown (C, D) of miR-183/96/182 in Mϕ cell line, Raw264.7. (E, F) Ex vivo phagocytosis and intracellular killing assays in PMN from miR-183/96/182 ko and wt mice. n = 4 for each group. *P < 0.05; **P < 0.01.

Figure 7

Schematic illustration of the roles of miR-183/96/182 in the corneal response to PA infection. In wt animals, miR-183/96/182 promotes the integrity of sensory innervation and proinflammatory neuropeptide release, including sP and Cx3cl1; in innate immune system, miR-183/96/182 modulates extravasation of PMN and Mϕ and their capacity of phagocytosis and intracellular killing of bacteria. When miR-183/96/182 is inactivated, sensory innervation to the cornea is decreased, which leads to decreased release of proinflammatory neuropeptide on PA infection, which leads to decreased activation of resident Mϕ and decreased chemoattractant cytokines, decreased infiltration of PMN, and possibly Mϕ, to the infected cornea; meanwhile, inactivation of miR-183/96/182 potentiates PMNs and Mϕ to increase their capacity of phagocytosis and intracellular killing of bacteria, leading to efficient removal of invading PA with less collateral damage to corneal tissue, and therefore, decreased keratitis. , sensory nerve ending; , PMN; , Mϕ; , PA; , neuropeptides and cytokines.