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

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.

Microbial keratitis is a potentially blinding disease, costing an estimated $175 million in direct health care expenditures in the United States. Pseudomonas aeruginosa (PA) is a gramnegative opportunistic pathogen capable of inducing keratitis and causes one of the most rapidly developing and destructive diseases of the cornea; it remains the most commonly recovered causative organism in contact lens-related disease. 34,35 Currently, bacterial keratitis is mainly treated by topical administration of antibiotics; however, frequent emergence of antibiotic-resistant bacteria poses serious challenges for effective management. 36 Development of alternative treatment depends on discoveries of new molecular mechanisms and therapeutic targets. However, the roles of miRNAs in PA-induced keratitis are still largely unknown. Recently, Yang et al. 37 reported that miR-155 suppresses macrophage (M/)mediated bacterial phagocytosis and intracellular killing; inactivation of miR-155 in M/ mice increased their resistance to PA-induced keratitis, 37 supporting that miRNA plays a role in bacterial keratitis through its modulation of innate immune functions.

Mice
All animals were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The miR-183C GT/GT mice are on a 129S2/BL6-mixed background 19 and were originally derived from a gene-trap ESC clone. 19,24,25 Male and female, young adult (8-week-old) miR-183C GT/GT mice, their heterozygous (miR-183C GT/þ ) and wildtype (wt) littermates (miR-183C þ/þ ) were used in this study.

Nerve Staining
Corneas were excised posterior to the limbal region and flattened by four evenly spaced cuts from the periphery toward the center. Corneas were fixed in 2% paraformaldehyde (PFA) in 0.1M phosphate buffer (PB), pH 7.4 overnight (O/N) at 48C; then, permeabilized with 0.3% Triton X-100 in PBS for 1 hour at room temperature (RT) and blocked with PBS with 0.3% Triton X-100, 2.5% BSA, and 5% donkey serum (Covance, Battle Creek, MI, USA) for 2 hours. Then, corneas were incubated with rabbit anti-bIII-tubulin antibody (Covance) for 2 days at 48C. After four rinses with PBS plus 0.3% Triton X-100 and 0.05% Tween-20, corneas were incubated with Alexafluor-488-conjugated donkey anti-rabbit secondary antibody (Ther-moFisher, Grand Island, NY, USA) O/N at 48C. Excess antibody was removed with rinses in PBS. Then, the corneas were flattened onto poly-L-lysine-coated slides and coverslipped using Vectashield mounting media (Vector Labs, Burlingame, CA, USA). Controls were treated similarly with omission of primary antibody or addition of nonspecific IgG.

Corneal Infection and Grading
P. aeruginosa infection of the cornea was performed as described before. 58 Briefly, mice were anesthetized with ethyl ether in a well-ventilated hood. The cornea of the left eye was wounded; 5.0 3 10 6 colony forming units (CFU) of PA (strain 19660; ATCC, Manassas, VA, USA) in a 5 lL volume was topically delivered. Corneal disease was graded at 1, 3, and 5 dpi using an established scale 58 : 0, clear or slight opacity, partially or fully covering the pupil; þ1, slight opacity, covering the anterior segment; þ2, dense opacity, partially or fully covering the pupil; þ3, dense opacity, covering the entire anterior segment; and þ4, corneal perforation. Photography with a slit lamp was used to illustrate disease.

RNA Preparation and Real-Time RT-PCR
Total RNA was prepared using the miRVana miRNA isolation kit (Life Technologies, Foster City, CA, USA) for miRNA studies, or the RNeasy (Qiagen, Frederick, MD, USA) and RNA STAT-60 kit (Tel-Test, Friendswood, TX, USA) for mRNA studies as described previously. 17,52,60 Quantitative (q)RT-PCR for miR-NAs was performed using Taqman miRNA primers and RT-PCR kit (Life Technologies) on a CFX Connect Real-time System (Bio-Rad, Hercules, CA, USA) with snRNA U6 as an endogenous control as described before. 17,60 Quantitative RT-PCR assays of protein-coding genes were performed using QuantiFast SYBR Green RT-PCR kit and QuantiTect primers (Qiagen) with 18s rRNA and b-actin as endogenous controls. 17,19,60

X-Gal Staining
Staining with X-gal was performed as described before. 19 6 , 1 mg X-gal/mL) at 378C O/N. After washing with PBS, corneas were observed and photographed under a dissection scope (Accu-scope, Inc., Comack, NY, USA). Then, for whole-mount microscopy, the cornea was flattened by four evenly spaced cuts from the periphery toward the center corneas and mounted on Superfrost plus slides (ThermoFisher); for cross-sections, the cornea was soaked in 20% sucrose in 0.1M PB pH 7.4 O/N and mounted in optimum cutting temperature (OCT; Fisher Healthcare, Houston, TX, USA), 12-lm cryoprotected cross-sections were made and examined using light microscopy (Leica DM4000B; Leica Microsystem, Inc., Buffalo Grove, IL, USA).
For TG X-staining, the whole head of a heterozygous mouse was fixed in 4% PFA in 0.1 M PB, pH 7.4 for 2 hours after the skull was opened. Then the head was sequentially soaked in Buffers A and B, stained in Buffer C and postfixed with 4% PFA as described above. Then, the stained head was carefully dissected under a dissection scope to expose the TG.

Enzyme-Linked Immunosorbent Assay
Protein levels for IL-1b and MIP-2 were tested using ELISA kits (R&D Systems, Minneapolis, MN, USA) as described before. 53,63 Briefly, corneas were homogenized in 1 mL PBS with 0.1% Tween-20 and protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA). An aliquot of each supernatant was assayed in duplicate per the manufacturer's instructions. Sensitivities of the ELISA assays were less than 2.31 pg/mL for IL-1b, and less than 1.5 pg/mL for MIP-2.

Quantitation of Corneal PMN
A myeloperoxidase (MPO) assay was performed as described before. [64][65][66][67] Briefly, corneas were harvested, homogenized in 1 mL potassium phosphate buffer (50 mM, pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (Sigma-Aldrich Corp., St. Louis, MO, USA) using glass microtissue grinders. The samples were freeze-thawed four times and centrifuged at 21,000g for 10 minutes. To measure MPO, a 100-lL supernatant was added to fresh 2.9 mL o-dianisidine dihydrochloride substrate buffer (16.7 mg/mL; Sigma-Aldrich Corp.) with 0.0005% hydrogen peroxide. The change in absorbance at 460 nm was read every 30 seconds for 5 minutes on a Hekios a spectrophotometer (ThermoFisher). Afterward, units of MPO per cornea were calculated: 1 unit of MPO activity is equivalent to approximately 2 3 10 5 PMN. 68 Quantitation of Viable Bacteria Bacteria were quantitated as described before. [64][65][66][67] Briefly, each cornea was homogenized in 1.0 mL sterile saline containing 0.25% BSA; 0.1 mL corneal homogenate was serially diluted 1:10 in the same solution and selected dilutions were plated in triplicate on Pseudomonas isolation agar (Becton Dickinson, Sparks, MD, USA). Plates were incubated O/N at 378C and the number of viable bacteria counted. Results are reported as log 10 number of CFU/cornea 6 SEM.

In Vitro Transfection and Phagocytosis and Intracellular Killing Assays
A total of 4 3 10 5 RAW264.7 M/ (a cell line derived from the mouse; ATCC) were plated in a 48-well plate and transfected with anti-miR-183/anti-miR-96/anti-miR-182 (10 nM for each) or negative control anti-miR oligonucleotides with scrambled sequences (30 nM; Exiqon, Vedbaek, Denmark) and miRNA mimics of miR-183, miR-96, and miR-182 (10 nM for each) or negative control mimics (30 nM; ThermoFisher) using RNAi-Max Lipofectamine (ThermoFisher) as described before. 69 All experiments were conducted with four replicate wells. Fortyeight hours after transfection, 1 3 10 7 CFU PA (strain 19660)/ well was added. One hour after coculture at 378C, gentamicin (300 lg/mL) was added to kill extracellular bacteria. Thirty minutes after gentamicin treatment, cells were washed with 250 lL Hanks' balanced salt solution (HBSS) twice to remove gentamicin and dead extracellular bacteria. For the phagocytosis assay, cells were lysed in 250 lL ice-cold sterile water. The cell lysate was plated on PA isolation agar plates (Becton Dickinson) and incubated at 378C for 24 hours as described before. 64 Colonies were manually counted; CFU/mL of cell lysate was calculated based on dilution factors. For the intracellular killing assay, fresh media was added to the wells after washing with HBSS to remove gentamicin and dead extracellular bacteria, and then cells were incubated for an additional hour before being washed, lysed, and plated. Killing efficiency was calculated as ([CFU/mL at 1 hour] À [CFU/mL at 2 hour])/(CFU/mL at 1 hour).

Mouse Peritoneal PMN Isolation and Ex Vivo Functional Assay
To harvest peritoneal PMN, mice were injected intraperitoneally with 1 mL 9% casein solution 27 and 3 hours before harvesting; a standard peritoneal PMN isolation protocol was followed as described previously. 64,67-70 A total of 4 3 10 5 PMNs were plated on a 48-well plate and infected with 1 3 10 7 CFU PA (strain 19660). Two hours after infection, gentamicin (300 lg/mL) was added to kill extracellular bacteria. For the phagocytosis assay, 30 minutes after gentamicin treatment, cells were washed, lysed, and plated on PA isolation agar plates as described above. For the intracellular killing assay, cells were incubated for an additional 2 hours after washing; then, cells were lysed and plated. Killing efficiency was calculated as ([CFU/mL at 2 hour] À [CFU/mL at 4 hour])/(CFU/mL at 2 hour).

Statistical Analysis
The difference in clinical score between groups was tested by the Mann-Whitney U test. One-way ANOVA with Bonferroni test (GraphPad Prism; La Jolla, CA, USA) was used to analyze qRT-PCR data of proinflammatory neuropeptides and cytokines. An unpaired, two-tailed Student's t-test was used to determine the significance of qRT-PCR of miRNAs, ELISA, MPO, and plate count data 17,19,60 ; P < 0.05 was considered significant. Each experiment was repeated at least twice to ensure reproducibility and data from a representative experiment are shown. Quantitative data are expressed as the mean 6 SEM.

MicroRNA-183/96/182 is Expressed in the Cornea and TG and Is Inactivated in miR-183C GT/GT Mice
Quantitative RT-PCR analysis showed that all three members of miR-183/96/182 are expressed in the cornea of wt mice (Fig.  1A). Among them, miR-182 and miR-183 showed higher expression than miR-96 (~32-and 4-fold higher, respectively) (Fig. 1A), suggesting that miR-182 and miR-183 play major roles in the cornea. Consistently, miR-183/96/182 is also expressed in human cornea with a similar pattern as in the mouse (Fig.  1B).
In miR-183C GT/GT mice, miR-183/96/182 is inactivated in the cornea. In heterozygous mice, expression of both miR-182 and miR-183 were significantly decreased to approximately one-half of that in wt mice, suggesting a dosage effect because heterozygous mice carry only one wt allele of the miR-183/96/ 182 gene (Fig. 1A).
Because the b-gal cassette in the miR-183C GT allele follows the endogenous expression pattern of miR-183/96/182, 19 to test its spatial expression pattern, we performed X-gal staining in the cornea of miR-183C GT/þ mice. Our results showed that miR-183/96/182 is mainly expressed in the corneal epithelium (Figs. 1C-F). A punctate staining pattern in the corneal epithelium (Fig. 1F) suggested that the expression of miR-183/96/182 may be due to staining of corneal nerve endings.

Inactivation of miR-183/96/182 Resulted in Decreased Nerve Density and Decreased Expression of Proinflammatory Neuropeptides in the Cornea
Sensory innervation of the cornea is from the TG. [38][39][40] To test miR-183/96/182 expression in SN of the TG, we performed X-gal staining on miR-183C GT/þ mouse brain, and showed that miR-183/96/182 is highly expressed in the TG (Figs. 2A, 2B). Quantitative RT-PCR further confirmed its expression in TG (Fig. 2C). Similar to the cornea, miR-182 and miR-183 are predominately expressed in the TG, compared with miR-96 (~16-and 8-fold higher, respectively) (Fig. 2C). MicroRNA-183/96/182 is inactivated in the TG of miR-183C GT/GT mice. In the TG of heterozygous miR-183C GT/þ mice, miR-183 is significantly decreased to approximately one-half of that in wt mice; miR-182 expression is also reduced, although did not reach significance (Fig. 2C). Similarly as in the cornea, these data suggest that miR-183/ 96/182 expression in TG also has a dosage effect based on numbers of wt alleles (Fig. 2C).
To test whether inactivation of miR-183/96/182 has significant impact on corneal innervation, we performed immunofluorescence analysis using an anti-b-III tubulin antibody, a marker for neurons. Our results showed that, in ko animals, corneal nerve density is drastically decreased; the normal whorl-like pattern of the subbasal nerve plexus 39,71-77 is disrupted (Fig. 2D). Local axon enlargements or ''beads,'' which contain high densities of mitochondria and share similarities to nociceptor terminals, 38,39 are decreased in ko compared with wt cornea (Fig. 2D), suggesting that inactivation of miR-183/96/182 has significant consequences on corneal sensory innervation. Consistent with this hypothesis, qRT-PCR using corneal RNA of ko and wt mice showed decreased expression of nociceptor transient receptor potential vanilloid 1 (TRPV1) and SN-released proinflammatory neuropeptides Tac1, the precursor gene for substance P (sP),

Inactivation of miR-183/96/182 Resulted in Significant Decrease of Corneal Inflammatory Response to PA Infection and Severity of PA-Induced Keratitis
Because inactivation of miR-183/96/182 resulted in decreased nerve density and proinflammatory neuropeptides, we hypothesized that this disrupts neuroimmune cross-talk and modulates immune/inflammatory responses in the cornea. To test this hypothesis, the cornea of ko mice and their wt littermates were infected with PA. Our results showed that miR-183/96/182 ko mice had significantly decreased keratitis at 1, 3, and 5 dpi (Fig. 3A). Most of the ko mice had only slight corneal opacity; none of the corneas perforated (Figs. 3A, 3B). Quantitative RT-PCR analysis of the cornea at 5 dpi showed that many proinflammatory factors and their receptors, which have been shown to play important roles in PA-induced keratitis, were decreased in the infected cornea of ko mice (Figs. 3C-J). In wt animals, pattern-recognition receptor Toll-like receptor 4 (TLR4), IL-1b, IL-6, IL-12p40, chemokine (C-C motif) ligand 2 (CCL2) and its receptor CCR2, and intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) were increased in the infected eyes compared with noninfected contralateral eyes (Figs. 3C-J); whereas in ko mice, increased expression of these proinflammatory factors in infected eyes was mitigated (Figs. 3C-J). Expression levels of most of these inflammatory factors were decreased in the infected eyes of ko mice when compared with their wt littermates (Figs. 3C-J). Enzyme-linked immunosorbent assays demonstrated that, at the protein level, IL-1b was significantly decreased at 1 dpi, whereas macrophage inflammatory protein-2 (MIP-2) was decreased at both 1 and 5 dpi (Figs. 4A, 4B). These results suggest that inactivation of miR-183/96/182 resulted in a decreased inflammatory response in infected corneas and contributed to decreased severity of PA-induced keratitis.
Polymorphonuclear neutrophils are the predominant infiltrating immune cells in bacterial keratitis and play important roles in bacterial clearance. [53][54][55][56][57] To begin to understand the pathological processes involved, we first performed MPO assays to quantify PMN in the cornea. Our results showed that PMN in the infected cornea of ko animals were significantly decreased at 1 dpi and slightly more decreased at 5 dpi. This result is consistent with the observation of decreased expression of chemoattractant neuropeptides (e.g., sP) (Fig. 2E) and cytokines (e.g., CCL2 and MIP-2), and leukocyte adhesion molecules, ICAM1 and VCAM1 (Figs. 3G, 3I, 3J, 4B).
To gain further insight into the progression of pathological processes, we quantified viable bacteria in the infected cornea. Intriguingly, our results showed that bacterial load was increased in the infected cornea of ko compared with wt mice at 1 dpi. This observation is consistent with decreased PMN infiltration in ko animals at this time. However, at 5 dpi, bacterial load in the corneas of wt controls was further increased when compared with 1 dpi; whereas the bacterial load in ko animals was decreased at 5 dpi compared with 1 dpi, and was significantly lower than wt controls. These results suggest that, although they had a higher bacterial burden at 1 dpi, bacteria continued to be cleared more efficiently in ko mice than in wt controls.

DISCUSSION
Bacterial keratitis is a potentially blinding corneal infectious disease and PA is the most commonly recovered causative organism in contact lens-related cases. PA-induced keratitis is also one of the most rapidly developing and destructive diseases of the cornea. 34,35 However, the roles of miRNAs in bacterial keratitis are still largely unknown. In this regard, Yang et al. 37 recently provided evidence that miR-155 modulates the   By X-gal staining and qRT-PCR analyses, we demonstrated that miR-183/96/182 is highly expressed in mouse cornea, with miR-182 and miR-183 predominately expressed, suggesting their major roles in the cornea (Fig. 1). Similar to mouse cornea, miR-183/96/182 is also expressed in human cornea, suggesting clinical relevance to the findings in mice. In the cornea, its expression is mostly in the epithelium with a unique punctate pattern, suggesting that its expression in the cornea is likely in the nerve endings (Figs. 1C-E). Consistently, miR-183/ 96/182 is highly expressed in the TG, where the SN to the cornea reside, with similar ratios among the three miRNAs in the cornea ( Figs. 2A-C). Inactivation of miR-183/96/182 resulted in a decrease of corneal nerve density and beaded nociceptor terminals (Fig. 2D) and decreased expression of TRPV1 and proinflammatory neuropeptides, including sP, CGRP, and Cx3cl1 (Figs. 2E-H). It has been shown that sPand CGRP-expressing SN coexpress TRPV1 40,78 ; activation of TRPV1 in sensory nerve endings by capsaicin results in calcium influx-dependent neuropeptide release, including sP and CGRP. 79,80 Therefore, our data suggest that decreased sensory innervation contributes to the decreased release of proinflammatory neuropeptides in the cornea and inflammatory responses. However, we do not exclude the possibility that decreased production of these neuropeptides from other sources (e.g., the corneal epithelium and tears) 38 also contributed to the overall decreased expression of proinflammatory neuropeptides.
Toll-like receptor 4 is a classic member of the pathogenrecognition receptor TLR family and functions as a primary sensor to detect gram-negative bacteria by binding to bacteriaderived lipopolysaccharides (LPS) expressed in bacterial cell walls. 81 As previously described, 82 PA infection induces TLR4 expression in the cornea of wt mice (Fig. 3D); however, intriguingly, in miR-183/96/182 ko mice, PA infection in the cornea did not result in upregulation of TLR4 (Fig. 3C); the level of TLR4 in the infected cornea of ko mice was significantly decreased compared with wt controls (Fig. 3C). Toll-like receptor 4 is expressed in TRPV1-containing trigeminal SN. 83 It has been shown that LPS binding sensitizes TRPV1 and increases neuropeptide release from SN. 84 Therefore, decreased expression of TLR4 in the cornea of miR-183/96/182 ko animals may contribute to decreased neuropeptide release.
The Cx3cl1, also known as fractalkine and a ligand for Cx3cr1, is a neuronally derived proinflammatory neuropeptide. 85 In the peripheral nervous system, dorsal root ganglionreleased Cx3cl1 promotes microglial production of inflammatory factors and contributes to chronic pain. [86][87][88][89][90] In cornea, Cx3cl1/Cx3cr1 signaling mediates direct physical association between corneal nerves and resident M/, which is suggested to act as an indicator of malfunctioning neuroimmune All data are relative expression levels normalized to snRNA U6. nd, not detected. * All data are presented as average (6 SEM). communication in diseases. 44 Decreased expression of Cx3cl1 (Fig. 2G) may have compromised neuroimmune communication in the cornea and contributed to the changes of the overall corneal response to PA infection (Fig. 3). Polymorphonuclear neutrophils are the predominant infiltrating cells in PA keratitis and are essential for microbial clearance. [53][54][55][56][57] Our data showed that the number of PMNs was significantly decreased (by~50%) in the infected cornea of ko mice at 1 dpi (Fig. 4C), resulting in decreased bacterial clearance and increased bacterial load (Fig. 4D). Substance P has been shown to be a potent chemoattractant and to induce leukocyte activation, NF-jB activation, cytokine production, and plasma extravasation. [91][92][93][94][95] Decreased expression of sP (Fig.  2E), together with decreased expression of proinflammatory cytokines, including multiple chemoattractant molecules (e.g., CCL2), also known as monocyte chemoattractant protein (MCP)-1 (Fig. 3G), ICAM1 (Fig. 3I), and MIP-2 (Fig. 4B), may have contributed to decreased recruitment of PMNs, and possibly M/, to the infected cornea. 53,56,[96][97][98] Intriguingly, at 5 dpi, bacterial load in the cornea of ko mice was significantly decreased compared with wt controls (Fig.  4D), whereas the number of PMNs showed no significant difference between ko and wt cornea (Fig. 4C). This cannot be explained by the decreased corneal nerve density and proinflammatory peptides. An alternative hypothesis is that inactivation of miR-183/96/182 has direct impact on PMN function and other components of the innate immune system. Supporting this hypothesis, our studies confirmed that miR-183/96/182 is expressed in M/ and PMNs in both mice and humans ( Fig. 5; Table). More importantly, our in vitro and ex vivo data showed that inactivation or knockdown of miR-183/ 96/182 in M/ and PMNs resulted in a significant increase in their capacity for phagocytosis and intracellular killing of PA ( Fig. 6). Therefore, in spite of the decreased number of PMNs and M/ in the infected cornea, bacteria were efficiently cleared without excessive collateral damage to the cornea, leading to decreased keratitis. Substance P has also been shown to be a potent anti-apoptosis regulator. 99,100 During PA infection, sP delays apoptosis of PMNs 58 ; persistence of PMNs is associated with increased corneal damage and perforation. [53][54][55][56][57][58] Therefore, decreased sP in the infected cornea of the ko animals could result in increased apoptosis of PMNs and contribute to decreased severity of keratitis.
In addition to the innate immune system, emerging evidence suggests that miR-183/96/182 is expressed in the adaptive immune system. [29][30][31] Although in vitro and ex vivo data suggest potential roles of miR-183/96/182 in modulating adaptive immune functions, including clonal expansion of activated Th cells 29 and Th2-induced Treg, 30 other reports 31 and our unpublished data (Xiao C, Xu S, written communication, 2016) do not support significant functions of miR-183/96/ 182 in adaptive immunity in vivo. Further investigation is needed to determine whether inactivation of miR-183/96/182 affects adaptive immunity and whether this impacts the corneal response to PA keratitis.
Overall, our data suggest that miR-183/96/182 is important in the corneal response to PA infection through its regulation of both sensory innervation of the cornea and innate immune functions at multiple levels in the pathogenesis of PA-induced keratitis (Fig. 7). The impact of inactivation of miR-183/96/182 on innate immune functions plays a major role in modulating the corneal response to PA infection and development of keratitis. It will be worthwhile to further tease out the effects of the developmental defects leading to a disrupted pattern of corneal innervation from the effects of gene ablation on innate immunity per se. To our knowledge, our data provide the first evidence that miR-183/96/182 plays an important role in innate immunity and modulates phagocytosis and bacterial killing capacities of both PMNs and M/. This finding has important implications to a wide range of diseases in which innate immunity has a critical role to disease outcome. It is also the first report that an miRNA cluster has a significant functional role in both sensory and innate immune systems. These results lead us to suggest that miR-183/96/182 represents a new therapeutic target for treatment of bacterial keratitis.