Esculentin-1a derived peptides kill Pseudomonas aeruginosa biofilm on soft contact lenses and retain antibacterial activity upon immobilization to the lens surface.

Contact lens (CL) wear is a risk factor for development of microbial keratitis, a vision threatening infection of the eye. Adverse events associated with colonization of lenses, especially by the multi-drug resistant and biofilm forming bacterium Pseudomonas aeruginosa remain a major safety issue. Therefore, novel strategies and compounds to reduce the onset of CL-associated ocular infections are needed. Recently, the activity of the frog skin-derived antimicrobial peptide Esc(1-21) and its diastereomer Esc(1-21)-1c was evaluated against both planktonic and sessile forms of this pathogen. Furthermore, Esc(1-21) was found to significantly reduce the severity of P. aeruginosa keratitis in a mouse model and preserve antipseudomonal activity in the presence of human basal tears. Here, we have analyzed the activity of the peptides on P. aeruginosa biofilm formed on soft CLs. Microbiological assays and scanning electron microscopy analysis indicated that the peptides were able to disrupt the bacterial biofilm, with the diastereomer having the greater efficacy (up to 85% killing vs no killing at 4 µM for some strains). Furthermore, upon covalent immobilization to the CL, the two peptides were found to cause more than four log reduction in the number of bacterial cells within 20 minutes and to reduce bacterial adhesion to the CL surface (77%-97% reduction) in 24 hours. Importantly, peptide immobilization was not toxic to mammalian cells and did not affect the lens characteristics. Overall, our data suggest that both peptides have great potential to be developed as novel pharmaceuticals for prevention and treatment of CL-associated P. aeruginosa keratitis.

Antimicrobial peptides and wound healing: biological and therapeutic considerations.

Repair of tissue wounds is a fundamental process to re-establish tissue integrity and regular function. Importantly, infection is a major factor that hinders wound healing. Multicellular organisms have evolved an arsenal of host-defense molecules, including antimicrobial peptides (AMPs), aimed at controlling microbial proliferation and at modulating the host's immune response to a variety of biological or physical insults. In this brief review, we provide the evidence for a role of AMPs as endogenous mediators of wound healing and their promising therapeutic potential for the treatment of non-life-threatening skin and other epithelial injuries.

Esculentin-1a(1-21)NH2: a frog skin-derived peptide for microbial keratitis.

Pseudomonas aeruginosa is the primary bacterial pathogen causing contact lens related keratitis. Available ophthalmic agents have reduced efficacy and antimicrobial peptides (AMPs) hold promise as future antibiotics. Here we investigated the in vitro and in vivo anti-Pseudomonal activity of esculentin-1a(1-21)NH2, derived from a frog skin AMP. The data revealed a minimum inhibitory concentration between 2 and 16 µM against reference strains or drug-resistant clinical isolates of P. aeruginosa without showing toxicity to human corneal epithelial cells up to 50 µM. At 1 µM the peptide rapidly killed bacterial cells and this activity was fully retained in 150 mM sodium chloride and 70 % (v/v) human basal tears, particularly against the virulent ATCC 19660 strain. Furthermore, its dropwise administration at 40 µM to the ocular surface in a murine model of P. aeruginosa keratitis (three times daily, for 5 days post-infection) resulted in a significant reduction of infection. The mean clinical score was 2.89 ± 0.26 compared to 3.92 ± 0.08 for the vehicle control. In addition, the corneal level of viable bacteria in the peptide treated animals was significantly lower with a difference of 4 log10 colony counts, compared to 7.7 log10 cells recovered in the control. In parallel, recruitment of inflammatory cells was reduced by half compared to that found in the untreated eyes. Similar results were obtained when esculentin-1a(1-21)NH2 was applied prior to induction of keratitis. Overall, our findings highlight esculentin-1a(1-21)NH2 as an attractive candidate for the development of novel topical pharmaceuticals against Pseudomonas keratitis.

Vitamin D: Implications for ocular disease and therapeutic potential.

Vitamin D is a multifunctional hormone that is now known to play a significant role in a variety of biological functions in addition to its traditional role in regulating calcium homeostasis. There are a large number of studies demonstrating that adequate vitamin D levels are important in maintaining health and show that vitamin D is able to be utilized at local tissue sites. In the eye, we have increasing evidence of the association between disease and vitamin D. In this narrative review, we summarize recent findings on vitamin D and its relationship to various ocular pathologies and the therapeutic potential for some of these, as well as examine the basic science studies that demonstrate that vitamin D is biologically relevant in the eye.

Dry eye modulates the expression of toll-like receptors on the ocular surface.

We aimed to determine if toll-like receptor (TLR) expression is modulated in response to dry eye-associated conditions and in dry eye syndrome (DES). Primary human corneal epithelial cells (HCEC), an SV40 HCEC cell line or a normal human conjunctival epithelial cell line (IOBA-NHC) were cultured under hyperosmolar stress (HOS) (400-500 mOsm/kg) or with DES associated cytokines (IL-1α/ß, TNFα or TGFß) at concentrations ranging from 1 to 1000 ng/ml for up to 24 h. Epithelial cells were harvested from a human cornea organ culture model following 24 h of desiccation. Conjunctival impression cytology samples were harvested from subjects with DES and age and gender-matched normal subjects. TLR4, TLR5 or TLR9 mRNA or protein was examined by quantitative RT-PCR, western blotting or flow cytometry. TLR functionality was evaluated in terms of addition of TLR agonists and quantitation of secreted inflammatory cytokines by the use of ELISA and Luminex assays. In SV40 HCEC, HOS significantly increased TLR4 by 8.18 fold, decreased TLR9 by 0.58 fold, but had no effect on TLR5 mRNA expression. TLR4 and TLR9 protein were decreased by 67.7% and 72% respectively. TLR4 mRNA was also significantly up-regulated by up to 9.70 and 3.36 fold in primary HCEC and IOBA-NHC respectively. DES associated cytokines had no effect on TLR4, TLR5 and TLR9 expression. In response to desiccation, TLR4 and TLR5 mRNA were significantly up-regulated by 4.81 and 2.51 fold respectively, while TLR9 mRNA was down-regulated by 0.86 fold in HCEC. A similar trend for TLR4 and TLR9 protein was observed. TLR9 mRNA was significantly down-regulated by almost 59.5% in DES subjects. In conclusion, changes in TLR expression occur in dry eye and could have an important role in ocular surface susceptibility to inflammation and infection.

Protective role of murine ß-defensins 3 and 4 and cathelin-related antimicrobial peptide in Fusarium solani keratitis.

Antimicrobial peptides (AMPs), such as ß-defensins and cathelicidins, are essential components of innate and adaptive immunity owing to their extensive multifunctional activities. However, their role in fungal infection in vivo remains elusive. In this study, we investigated the protective effect of murine ß-defensin 3 (mBD3), mBD4, and the cathelicidin cathelin-related antimicrobial peptide (CRAMP) in a murine model of Fusarium solani keratitis. C57BL/6 mice showed significant corneal disease 1 and 3 days after infection, which was accompanied by enhanced expression of ß-defensins and CRAMP. Disease severity was significantly improved 7 days after infection, at which time AMP expression was returning to baseline. Mice deficient in mBD3 (genetic knockout), mBD4 (short interfering RNA knockdown), or CRAMP (genetic knockout) exhibited enhanced disease severity and progression, increased neutrophil recruitment, and delayed pathogen elimination compared to controls. Taken together, these data suggest a vital role for AMPs in defense against F. solani keratitis, a potentially blinding corneal disease.

Antimicrobial compounds in tears.

The tear film coats the cornea and conjunctiva and serves several important functions. It provides lubrication, prevents drying of the ocular surface epithelia, helps provide a smooth surface for refracting light, supplies oxygen and is an important component of the innate defense system of the eye providing protection against a range of potential pathogens. This review describes both classic antimicrobial compounds found in tears such as lysozyme and some more recently identified such as members of the cationic antimicrobial peptide family and surfactant protein-D as well as potential new candidate molecules that may contribute to antimicrobial protection. As is readily evident from the literature review herein, tears, like all mucosal fluids, contain a plethora of molecules with known antimicrobial effects. That all of these are active in vivo is debatable as many are present in low concentrations, may be influenced by other tear components such as the ionic environment, and antimicrobial action may be only one of several activities ascribed to the molecule. However, there are many studies showing synergistic/additive interactions between several of the tear antimicrobials and it is highly likely that cooperativity between molecules is the primary way tears are able to afford significant antimicrobial protection to the ocular surface in vivo. In addition to effects on pathogen growth and survival some tear components prevent epithelial cell invasion and promote the epithelial expression of innate defense molecules. Given the protective role of tears a number of scenarios can be envisaged that may affect the amount and/or activity of tear antimicrobials and hence compromise tear immunity. Two such situations, dry eye disease and contact lens wear, are discussed here.

Dry eye disease and microbial keratitis: is there a connection?

Dry eye is a common ocular surface disease of multifactorial etiology characterized by elevated tear osmolality and inflammation leading to a disrupted ocular surface. The latter is a risk factor for ocular surface infection, yet overt infection is not commonly seen clinically in the typical dry eye patient. This suggests that important innate mechanisms operate to protect the dry eye from invading pathogens. This article reviews the current literature on epidemiology of ocular surface infection in dry eye patients and laboratory-based studies on innate immune mechanisms operating at the ocular surface and their alterations in human dry eye and animal models. The review highlights current understanding of innate immunity in dry eye and identifies gaps in our knowledge to help direct future studies to further unravel the complexities of dry eye disease and its sequelae.

Novel Peptides with Dual Properties for Treating Pseudomonas aeruginosa Keratitis: Antibacterial and Corneal Wound Healing.

The corneal epithelium is a layer in the anterior part of eye that contributes to light refraction onto the retina and to the ocular immune defense. Although an intact corneal epithelium is an excellent barrier against microbial pathogens and injuries, corneal abrasions can lead to devastating eye infections. Among them, Pseudomonas aeruginosa-associated keratitis often results in severe deterioration of the corneal tissue and even blindness. Hence, the discovery of new drugs able not only to eradicate ocular infections, which are often resistant to antibiotics, but also to elicit corneal wound repair is highly demanded. Recently, we demonstrated the potent antipseudomonal activity of two peptides, Esc(1-21) and its diastereomer Esc(1-21)-1c. In this study, by means of a mouse model of P. aeruginosa keratitis and an in vivo corneal debridement wound, we discovered the efficacy of these peptides, particularly Esc(1-21)-1c, to cure keratitis and to promote corneal wound healing. This latter property was also supported by in vitro cell scratch and ELISA assays. Overall, the current study highlights Esc peptides as novel ophthalmic agents for treating corneal infection and injury, being able to display a dual function, antimicrobial and wound healing, rarely identified in a single peptide at the same micromolar concentration range.

Role of host-defence peptides in eye diseases.

The eye and its associated tissues including the lacrimal system and lids have evolved several defence mechanisms to prevent microbial invasion. Included among this armory are several host-defence peptides. These multifunctional molecules are being studied not only for their endogenous antimicrobial properties but also for their potential therapeutic effects. Here the current knowledge of host-defence peptide expression in the eye will be summarised. The role of these peptides in eye disease will be discussed with the primary focus being on infectious keratitis, inflammatory conditions including dry eye and wound healing. Finally the potential of using host-defence peptides and their mimetics/derivatives for the treatment and prevention of eye diseases is addressed.

Anti-inflammatory and wound healing potential of medicinal maggot excretions/secretions at the ocular surface.

PURPOSE: In the skin, Lucilia sericata maggot excretions/secretions (ES) accelerate wound healing and limit inflammation. This study aimed to determine whether ES have similar beneficial effects at the ocular surface. METHODS: Human corneal epithelial cells (HCEC) were cultured with ES and cell viability was determined by the MTT assay. Additionally, mRNA expression of growth factors, antimicrobial peptides (AMPs) and cytokines was assessed by qPCR. ES ability to modulate TLR-induced IL-6 and IL-8 expression was determined by qPCR and ELISA. ES potential to promote corneal healing was evaluated in vitro by a migration assay in HCEC, and in vivo using a mouse model. RESULTS: ES did not impair HCEC viability up to 25 µg/ml. Among the factors evaluated, only hBD-2 was upregulated (2.5-fold) by 1.5 µg/ml ES after 6 hrs (P = 0.04). In HCEC, ES reduced Poly I:C-induced IL-6 and IL-8 mRNA (P ≤ 0.001) and protein (P ≤ 0.0001) expression. A similar effect was observed with Flagellin (TLR5 agonist) but it was less robust for FSL-1 (TLR2/6 agonist) and Pam3CSK4 (TLR1/2 agonist). The greatest in vitro migration effect was observed with 6.2 µg/ml ES after 44 hrs where gap area compared to vehicle was 53.3 ± 3.7% vs. 72.6 ± 5.4% (P = 0.001). In the mouse model, the maximum healing effect was present with 1.5 µg/ml ES after 12 hrs with a wound area of 19.0 ± 2.7% vs. 60.1 ± 21.6% (P = 0.003) or 77% reduction of the wound area compared to the negative control. CONCLUSIONS: ES significantly reduce in vitro TLR-induced production of inflammatory cytokines and promote corneal wound healing.

"Click" immobilization on alkylated silicon substrates: model for the study of surface bound antimicrobial peptides.

We describe an effective approach for the covalent immobilization of antimicrobial peptides (AMPs) to bioinert substrates via Cu(I) -catalyzed azide-alkyne cycloaddition (CuAAC). The bioinert substrates were prepared by surface hydrosilylation of oligo(ethylene glycol) (OEG) terminated alkenes on hydrogen-terminated silicon surfaces. To render the OEG monolayers "clickable", mixed monolayers were prepared using OEG-alkenes with and without a terminal alkyne protected by a trimethylgermanyl (TMG) group. The mixed monolayers were characterized by X-ray photoelectron spectroscopy (XPS), elliposometry and contact angle measurement. The TMG protecting group can be readily removed to yield a free terminal alkyne by catalytic amounts of Cu(I) in an aqueous media. This step can then be combined with the subsequent CuAAC reaction. Thus, the immobilization of an azide modified AMP (N3-IG-25) was achieved in a one-pot deprotection/coupling reaction. Varying the ratio of the two alkenes in the deposition mixture allowed for control over the density of the alkynyl groups in the mixed monolayer, and subsequently the coverage of the AMPs on the monolayer. These samples allowed for study of the dependence of antimicrobial activities on the AMP density. The results show that a relative low coverage of AMPs (∼1.6×10(13) molecule per cm(2)) is sufficient to significantly suppress the viability of Pseudomonas aeruginosa, while the surface presenting the highest density of AMPs (∼2.8×10(13) molecule per cm(2)) is still cyto-compatible. The remarkable antibacterial activity is attributed to the long and flexible linker and the site-specific "click" immobilization, which may facilitate the covalently attached peptides to interact with and disrupt the bacterial membranes.

Toll-like receptor activation modulates antimicrobial peptide expression by ocular surface cells.

The ability of the ocular surface to respond to pathogens is in part attributed to toll-like receptors (TLRs) that recognize conserved motifs on various microbes. This study examines TLR expression on various ocular surface cells, if TLR agonists can modulate the expression of antimicrobial peptides (AMPs), human beta defensins (hBD) and cathelicidin (hCAP-18/LL-37) which maybe functionally active against Pseudomonas aeruginosa (PA) and if TLR agonists or AMPs can modulate TLR mRNA expression. TLR1-10 mRNA expression was examined in corneal epithelial, corneal stromal cells and conjunctival epithelial cells by RT-PCR. To confirm protein expression flow cytometry or immunostaining was performed for selected TLRs on some cell cultures. Ocular surface cells were cultured with a range of TLR agonists and then hBD-1, 2, 3, or hCAP-18 mRNA and protein expression was determined by RT-PCR and immunoblotting. In some experiments, cells were cultured with a cocktail of agonists for TLR3, 5 and 6/2 and the antimicrobial activity of the culture media was tested against PA. TLR mRNA expression was also examined in primary human corneal epithelial cells (HCEC) treated with either 3 µg/ml of hBD-2, 5 µg/ml of LL-37 or TLR4, 5 and 9 agonists. Overall, the ocular surface cells expressed mRNA for most of the TLRs but some differences were found. TLR2 was not detected in corneal fibroblasts, TLR4 was not detected in primary cultured or freshly isolated HCEC, TLR5 was not detected in conjunctival epithelial cells (IOBA-NHC) and corneal fibroblasts, TLR7 was not detected in freshly isolated HCEC and TLR10 was not detected in HCEC and IOBA-NHC. TLR8 mRNA was not expressed by any of the samples tested. Immunostaining of cadaver corneas revealed TLR5 and 9 expression throughout the cornea while TLR3 was significantly expressed only in the epithelium. Flow cytometry and immunostaining revealed cultured fibroblasts expressed TLR9 but had no significant TLR3 expression. hBD-2 expression was upregulated by TLR1/2, 3, 4, 5 and 6/2 agonists depending on the cell type, whereas only the TLR3 agonist upregulated the expression of hCAP-18 in primary HCEC. The combination of TLR3, 5 and 6/2 agonists in primary HCEC, upregulated hBD-2 and hCAP-18 mRNA and peptide expression and secretion into the culture media, which significantly killed PA. This antimicrobial activity was primarily attributed to LL-37. TLR agonists did not modulate TLR expression itself, however, LL-37 or hBD-2 downregulated TLR5, 7 and/or 9 mRNA depending on the cell type. TLRs are expressed on the ocular surface and TLR agonists trigger the production of LL-37 and hBD-2, with LL-37 being particularly important for protecting the ocular surface against PA infection.

Toll-like receptors in ocular surface disease.

The ability of the ocular surface to mount an immune response is in part attributed to a family of proteins called toll-like receptors (TLRs). The latter are evolutionary conserved receptors that recognize and respond to various microbes and endogenous ligands. In addition to their recognition function, TLR activation triggers a complex signal transduction cascade that induces the production of inflammatory cytokines and co-stimulatory molecules, thus initiating innate and adaptive immunity. Toll-like receptor expression at the ocular surface is modulated during infection (e.g. Herpes simplex, bacterial keratitis and fungal keratitis) as well as during various inflammatory conditions (allergic conjunctivitis and dry-eye syndrome). Here recent findings regarding TLR expression and their involvement in various ocular surface diseases are discussed.

The effect of acute exercise on environmentally induced symptoms of dry eye.

The purpose of this study was to investigate the effects of acute exercise on environmentally induced symptoms of dry eye. Twelve participants without dry eye disease volunteered to complete three experimental visits in a randomized order; (1) control condition seated for 1 h at a relative humidity (RH) of 40% (CONT), (2) dry condition seated for 1 h at a RH of 20% (DRY), and (3) exercise condition seated for 40 min followed by 20 min of cycling exercise at a RH of 20% (EXER). Tear volume, tear matrix metalloproteinase 9 (MMP-9), perception of dry eye symptoms (frequency and severity), core temperature, and ocular surface temperature (OST) were measured at the end of each exposure. The perception of dry eye frequency and MMP-9 concentration were significantly higher in DRY compared to CONT (P < 0.012), with no differences in EXER compared to CONT. The results suggest that an acute bout of exercise may attenuate symptoms of environmentally induced dry eye, and warrant further research.

The role of antimicrobial peptides at the ocular surface.

Antimicrobial peptides (AMPs) such as defensins and cathelicidins are small peptides with broad-spectrum activity against bacteria, fungi and viruses. In addition, several AMPs modulate mammalian cell behaviours including migration, proliferation and cytokine production. This review describes findings from recent studies showing the presence of various AMPs at the human ocular surface and discusses their mechanism of antimicrobial action and potential non-microbicidal roles. Corneal and conjunctival epithelial cells produce beta-defensins and the cathelicidin LL-37, whereas neutrophils, infiltrating in response to a specific stimulus, supply additional LL-37 as well as alpha-defensins. In vitro studies suggest that LL-37 and human beta-defensin-3 are the most likely to have significant independent antimicrobial activity, while other AMPs may act synergistically to help protect the ocular surface from invading pathogens. Current evidence also supports a role for some AMPs in modulating wound healing responses. Although yet to be brought to fruition, AMPs hold significant potential as therapeutic agents for the prophylaxis and treatment of infection, promotion of wound healing and immune modulation.

Elevated nerve growth factor in dry eye associated with established contact lens wear.

PURPOSE: Nerve growth factor (NGF) has been shown to be upregulated in conditions, which damage corneal nerves and to relieve dry eye. How NGF changes in nerve injury induced by established contact lens wear is not clear. The purpose of this study was to measure the subepithelial nerve plexus and tear NGF and transforming growth factor (TGF)-beta1 levels in patients with established contact lens associated with dry eye. METHODS: Non-contact lens wearers and subjects who had worn soft contact lenses for more than 1 year were recruited and were divided into three groups: (1) normal controls; (2) contact lens wearers without dry eye; (3) contact lens wearers with dry eye. Corneal sensitivity was measured with a Cochet-Bonnet aesthesiometer. Nerve density and branching in the subepithelial plexus were measured using in vivo confocal microscopy. Tear NGF and TGF-beta1 levels were measured with an enzyme immuno assay. RESULTS: There was a statistically significant decrease of corneal sensitivity in contact lens wearers compared with normal controls. The nerve density in the subepithelial plexus of contact lens wearers with dry eye was 538.8 +/- 39.3 microm/image (3.959 +/- 0.28 pm/microm(2)) and 537.1 +/- 30.9 microm/image (3.947 +/- 0.27 pm/microm(2)) in those without dry eye. Both of these values were significantly (P=0.032) lower than in the normal controls (4.412 +/- 0.21 pm/microm(2)). The concentration of tear NGF was increased in contact lens wearers with dry eye and was statistically significantly greater compared with contact lens wearers without dry eye. Transforming growth factor-beta1 levels were found to increase one fold in contact lens associated dry eye, and were significantly correlated to NGF. CONCLUSIONS: Corneal subepithelial nerve density was decreased in long-term contact lens wear but this change was not significantly correlated with tear film NGF concentration. Tear film NGF levels were elevated in contact lens related dry eye, likely in response to anti-inflammatory factors such as TGF-beta1.

Development of an in vitro model to study the biological effects of blinking.

PURPOSE: To develop a mechanical model in which a contact lens is swept over ocular surface cells under conditions that mimic the force and speed of the blink, and to investigate the resulting biological changes. METHODS: A computer controlled mechanical instrument was developed to hold a dish containing 3D cultured stratified human ocular surface epithelial cells, across which an arm bearing a contact lens was swept back and forth repeatedly at a speed and force mimicking the human blink. Cells were subjected to repeated sweep cycles for up to 1 h at a speed of 120 mm/s with or without an applied force of 19.6 mN (to mimic pressure exerted by upper eyelid), after which the cell layer thickness was measured, the cell layer integrity was investigated using fluorescent quantum dots (6 and 13 nm) and the phosphorylation levels of various protein kinases were analyzed by human phospho-kinase arrays. Data for selected kinases were further quantitated by enzyme immunoassays. RESULTS: The thickness of the cell layers did not change after exposure to sweep cycles with or without applied force. Quantum dots (6 and 13 nm) were able to penetrate the layers of cells exposed to sweep cycles but not layers of untreated control cells. The phosphorylation levels of HSP27 and JNK1/2/3 increased for cells exposed to sweep cycles with applied force compared to untreated control cells. CONCLUSIONS: The in vitro mechanical instrument is a useful tool to investigate the effects of blinking on the ocular surface.

Short Tandem Repeat (STR) Profiles of Commonly Used Human Ocular Surface Cell Lines.

PURPOSE: The purpose of this study is to establish the short tandem repeat (STR) profiles of several human cell lines commonly used in ocular surface research. MATERIALS AND METHODS: Independently DNA was extracted from multiple passages of three human corneal epithelial cell lines, two human conjunctival epithelial cell lines and one meibomian gland cell line, from different laboratories actively involved in ocular surface research. The samples were then subjected to STR analysis on a fee-for-service basis in an academic setting and the data compared against that in available databases. RESULTS: The STR profiles for the human corneal epithelial cells were different among the three cell lines studied and for each line the profiles were identical across the samples provided by three laboratories. Profiles for the human conjunctival epithelial cells were different among the two cell lines studied. Profiles for the meibomian gland cell line were identical across the samples provided by three laboratories. No samples were contaminated by elements of other cell lines such as HeLa. CONCLUSIONS: This comprehensive study provides verification of STR profiles for commonly used human ocular surface cell lines that can now be used as a reference by others in the field to authenticate the cell lines in use in their own laboratories.

Cathelicidin-deficient (Cnlp -/- ) mice show increased susceptibility to Pseudomonas aeruginosa keratitis.

PURPOSE: To examine the clinical progression and innate immune responses during Pseudomonas aeruginosa (PA) keratitis in cathelicidin-deficient (KO) mice. METHODS: PA (ATCC 19660) keratitis was induced in KO mice and wild-type (WT) littermates generated on a 129/SVJ background. Clinical score and histopathology were used to monitor the progression of infection at postinfection (PI) days 1, 3, 7, 14, and 21. Mouse corneas were harvested for viable bacteria quantitation, and myeloperoxidase (MPO) assays were performed to determine the number of infiltrating neutrophils. ELISA was used to quantitate interleukin (IL)-1beta, IL-6, macrophage inflammatory peptide (MIP)-2, keratinocyte-derived chemokine (KC), tumor necrosis factor (TNF)-alpha, and vascular endothelial growth factor (VEGF) levels in the corneas. RESULTS: WT mice were resistant (cornea healed), whereas KO mice showed increased susceptibility (corneas failed to recover by 21 days or perforated) to PA infection. Clinical scores were significantly elevated in the infected corneas of KO mice versus WT mice at 7, 14, and 21 days PI. Absence of cathelicidin resulted in significantly delayed clearance of PA in the cornea and an increased number of infiltrating neutrophils at 1, 3, 7, and 14 days PI. KO mice also exhibited differential expression of protein levels for IL-1beta, IL-6, MIP-2, KC, TNF-alpha, and VEGF up to day 21 PI compared with the WT mice. CONCLUSIONS: Cathelicidin-deficient mice showed considerable susceptibility to PA keratitis. The present study demonstrates direct in vivo evidence that endogenous expression of cathelicidin provides defense against corneal PA infection indicating its importance in host innate immunity at the ocular surface.