January 2009
Volume 50, Issue 1
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Immunology and Microbiology  |   January 2009
Nona-D-Arginine Therapy for Pseudomonas aeruginosa Keratitis
Author Affiliations
  • Priyanka Karicherla
    From the Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana.
  • Jeffery A. Hobden
    From the Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, Louisiana.
Investigative Ophthalmology & Visual Science January 2009, Vol.50, 256-262. doi:10.1167/iovs.08-2344
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      Priyanka Karicherla, Jeffery A. Hobden; Nona-D-Arginine Therapy for Pseudomonas aeruginosa Keratitis. Invest. Ophthalmol. Vis. Sci. 2009;50(1):256-262. doi: 10.1167/iovs.08-2344.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Nona-D-arginine amide (D9R) was evaluated as treatment for Pseudomonas aeruginosa keratitis when administered alone and with ciprofloxacin.

methods. Mouse corneas were infected with P. aeruginosa. Immediately after infection and hourly for 5 hours, eyes received 5 μL of either Dulbecco phosphate-buffered saline (D-PBS), 10 μM D9R, or 100 μM D9R. At 16 hours postinfection (PI) and then hourly for 5 hours, eyes treated with D9R or D-PBS then received 5 μL ciprofloxacin (0.08%) or deionized water. On days 1, 7, and 14 PI, eyes were scored on a scale of 0 (normal eye) to +4 (corneal perforation). On day 1 PI, mice were euthanatized and eyes were harvested for histopathology or colony-forming unit (CFU) determination. At 6, 12, and 24 hours PI, corneas treated with 100 μM D9R or D-PBS alone were harvested for the determination of IL-1β cytokine concentrations.

results. Eyes treated with 10 or 100 μM D9R and ciprofloxacin had significantly less disease than eyes treated with D-PBS and ciprofloxacin. Fewer than 30 CFUs were recovered from any eye treated with ciprofloxacin. Eyes treated with D9R alone had significantly less disease and lower IL-1β cytokine concentrations than D-PBS–treated eyes; however, there were no significant differences in CFU (≥4 log10) between these groups.

conclusions. Administration of 10 or 100 μM D9R effectively reduced the abnormality associated with P. aeruginosa keratitis. Treatment with D9R and ciprofloxacin was superior to treatment with antibiotic alone by reducing ocular disease through suppression of the proinflammatory cytokine IL-1β and eradicating viable bacteria from the eye.

A severe ocular infection, Pseudomonas aeruginosa keratitis can progress rapidly, resulting in intense inflammation, irreversible stromal scarring, and probable loss of vision. 1 The prognosis for patients with P. aeruginosa corneal disease is often poor. Even if the infected cornea is rendered sterile with antibacterial chemotherapy, the resultant pathologic condition may range from restoration of visual acuity with minimal corneal scarring to extensive tissue destruction and the need for corneal transplantation. 
Damage to ocular tissue is caused by an acute inflammatory response to infection, primarily because of an influx of neutrophils precipitated by the release of proinflammatory cytokines such as IL-1β, TNFα, and IFN-γ and by toxic products secreted from the bacteria. Several studies 2 3 4 5 have shown that exotoxin A (ToxA) is an important virulence factor produced by the bacterium. Mutants deficient in ToxA production are significantly less virulent in the eye than wild-type parent strains. 6 Complementation of these mutants with a functional toxA gene fully restored ocular virulence. 
ToxA is an A-B type toxin that is not toxic until activated in vivo by furin, a host endoprotease that cleaves the toxin and releases the enzymatically active A subunit into the cytoplasm. 5 6 Furin is the only host protease capable of ToxA activation. 7 8 Once inside the cytoplasm, the A subunit catalyzes the NAD-ribosylation of elongation factor 2, thereby inactivating protein synthesis and killing the cell. ToxA, with an LD50 of 0.2 μg/kg, is considered the most potent toxin produced by P. aeruginosa. 9  
Recent studies have shown that polyarginine peptides can function as potent furin inhibitors in vitro and in vivo, thus serving as effective agents in preventing microbial toxin-mediated disease. 10 Hexa-D-arginine amide significantly improved survival rates in mice injected intraperitoneally with lethal doses of ToxA. 11 Further studies indicated that nona-D-arginine amide (D9R) was even more potent in abrogating the effects of anthrax toxin in RAW cells. 12  
High stability, low toxicity, small molecular weight, and extremely low Ki (1.3 nM) against furin make D9R a promising therapeutic agent in preventing bacterial toxemia. 13 Given that ToxA is an important virulence factor for P. aeruginosa corneal disease, it was hypothesized that inhibition of its activation by D9R should be effective in reducing the severity associated with these infections. Thus, in this study, the therapeutic potential of D9R in treating P. aeruginosa keratitis was evaluated when administered alone and in combination with the antibiotic ciprofloxacin. When topically applied immediately after P. aeruginosa corneal infection, D9R significantly decreased ocular disease compared with untreated or vehicle-treated eyes. Furthermore, D9R treatment significantly improved the clinical outcome of P. aeruginosa–infected eyes treated with ciprofloxacin, as demonstrated by the elimination of viable bacteria from infected tissue coupled with a noticeable decrease in ocular disease. 
Materials and Methods
Mice
Female 6- to 8-week-old Swiss black (SWBLK) mice were purchased from Taconic Farms, Inc. (Germantown, NY). Mice were maintained in accordance with institutional guidelines and the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Preparation of Inoculum
Wild-type P. aeruginosa strain PAO1 was used for all infections. A bacterial colony was picked from an LB (Sigma-Aldrich, St. Louis, MO) agar plate and was inoculated into 10 mL LB broth (Sigma-Aldrich). Cultures were grown at 37°C on a rotary shaker at 160 rpm for 18 hours. Overnight cultures were then subcultured into 10 mL fresh LB broth, and the bacteria were grown to logarithmic phase to an optical density of 0.2 (approximately 2 × 108 CFU/mL) at 600 nm. The inoculum was centrifuged at 5000g for 15 minutes and resuspended in Dulbecco phosphate-buffered saline with 1 mM calcium and 0.5 mM magnesium (D-PBS, pH 7.4; Gibco Invitrogen, Grand Island, NY) to a concentration of 2 × 108 CFU/mL. 
Preparation of Treatments
D9R (Pepceuticals [Nottingham, UK] and American Peptide [Sunnyvale, CA]) was dissolved in sterile distilled, deionized water at a stock concentration of 1 mM and was stored at −80°C. Ophthalmic drops (0.3% ciprofloxacin; Ciloxan; Alcon Laboratories, Fort Worth, TX) were diluted with sterile distilled, deionized water to a final concentration of 0.08%. The concentration of ciprofloxacin used in treating a mouse eye in this study (0.08%) was proportional to the concentration of ciprofloxacin a human eye would receive in a 50-μL drop of a 0.3% solution based on the total surface area of the cornea exposed to the drug. 
Concentrations of D9R used in this study (10 or 100 μM) were determined empirically based on toxicity studies performed in vitro with immortalized human corneal epithelial cells (HCECs). 14 HCECs were seeded in 96-well microtiter plates (170–1700 cells/well) in hexaplicate and cultivated overnight in serum-free keratinocyte growth medium (KGM; Lonza Rockland, Rock Island, ME). Cells were then left untreated or incubated with 5 μL D-PBS or D9R (10 or 100 μM) for 24 hours at 37°C. Cell viability was measured using a colorimetric MTS tetrazolium dye assay (CellTiter 96 AQueous One Solution Cell Proliferation Assa; Promega, Madison, WI). As shown in Figure 1 , the growth rate of HCECs was similar among all groups with no significant (P > 0.05) differences between the absorbance values, suggesting that D9R is nontoxic and does not interfere with cell proliferation. 
As determined by broth tube dilution, 15 the minimum inhibitory concentration (MIC) of ciprofloxacin for the strain of P. aeruginosa PAO1 used in these studies was 0.75 μg/mL. When similarly tested, D9R dissolved in D-PBS had no inhibitory activity against P. aeruginosa PAO1, even at 1 mM. Potential interactions between ciprofloxacin and D9R were assessed with a checkerboard array, as described by Pillai et al. 16 The addition of D9R (at concentrations used in this study) to the fluoroquinolone was neither additive, synergistic, nor antagonistic with regard to the bactericidal activity of the fluoroquinolone (data not shown). 
Mouse Model of Keratitis and Treatment Regimen
Mice were infected essentially as described by Kwon and Hazlett. 17 In brief, mice were anesthetized with ketamine (200 mg/kg; Vedco, St. Joseph, MO) and xylazine (10 mg/kg; NLS Animal Health, Pittsburg, PA), and the corneas of right eyes were scarified three times with a 21-gauge needle with three 1-mm incisions. Wounded corneas were then topically inoculated with 5 μL (1 × 106 CFU) P. aeruginosa PAO1 in D-PBS. Eyes were left untreated or received a 5-μL drop of D-PBS, 10 μM D9R, or 100 μM D9R immediately after infection. Application of D-PBS or D9R continued hourly for 5 hours (total of 6 drops/infected eye). 
At 16 hours postinfection (PI) and then hourly for 5 hours, eyes treated with D9R or PBS then received a 5-μL drop of ciprofloxacin (0.08%) or deionized water. At least five mice were infected per treatment group per experiment, and all experiments were repeated at least three times. The frequency of dosing (every hour) was chosen based on the dosing regimen typically used with antibiotics to treat bacterial keratitis. 
Slit Lamp Examination of Corneas
On days 1, 7, and 14 PI, the ocular response to bacterial challenge and treatment was examined and photographed with a slit lamp microscope (Biomicroscope SL-D7; Topcon, Tokyo, Japan). Disease was scored in a masked fashion. The scoring system used was essentially that described by Hazlett et al. 18 with some minor modification: 0 = clear or normal cornea; +1 = light to dense opacity at wound site; +2 = dense opacity fully covering the pupil; +3 = dense opacity fully covering the anterior segment; and +4 = corneal perforation or phthisis bulbi (i.e., shrunken eye). Mean scores between treatment groups were compared using one-way analysis of variance (ANOVA; GraphPad Prism version 4.0 [GraphPad Software, San Diego, CA]). P ≤ 0.05 was considered statistically significant. 
Quantitation of Viable Bacteria per Eye
Before treatment with ciprofloxacin at 16 hours PI and after SLE at 24 hours PI, mice were euthanatized, and the eyes were harvested for CFU determination. Individual eyes were homogenized in 1 mL sterile D-PBS. Serial 10-fold dilutions of the samples were plated onto Pseudomonas isolation agar (BD Diagnostics, Franklin Lakes, NJ) in duplicate, and the plates were incubated for 24 hours at 37°C. The lower limit of detection of this technique is 10 CFUs/eye. For plates that showed no bacterial growth, a value of 10 CFUs/eye was assigned to calculate the mean number of CFUs. CFU per eye is expressed as a log10 value. The numbers of viable bacteria in the eyes are expressed as the mean CFU of bacteria ± SEM. Mean CFU between treatment groups was compared using an unpaired Student’s t-test (GraphPad Prism). P ≤ 0.05 was considered statistically significant. 
Histopathology
At 24 hours PI, mice were euthanatized, and the eyes were enucleated. Eyes were fixed in neutral phosphate-buffered formalin (10%) for at least 24 hours and were processed for routine light microscopy (LSU Health Sciences Center Morphology and Imaging Core Laboratory, New Orleans, LA). Serial sections (5 μm) were cut through the visual axis and were stained with hematoxylin and eosin for light microscopic histopathologic evaluation. Stained sections were examined in a masked fashion for epithelial and endothelial integrity and the presence of stromal edema and infiltrate. The anterior chamber was examined for the presence of cellular infiltrate. 
D9R and Production of IL-1β In Vivo and In Vitro
Short, cationic peptides similar to D9R are important constituents of innate immunity and are known to have immunomodulatory activities. 19 Therefore, the effect of D9R in modulating the production of IL-1β, a key proinflammatory cytokine in the development of P. aeruginosa–induced corneal disease, 20 21 22 was examined in vivo in infected mouse corneas and in vitro by HCECs. 
For the in vivo experiment, mice were infected and treated with D9R or D-PBS, as previously described. Corneas were harvested at 6, 12, and 24 hours PI and were individually frozen at −80°C. Five mice were infected per treatment group per time point, and all experiments were repeated at least twice. Excised corneas were thawed on ice and individually homogenized in 60 μL PBS with 1% Triton X-100 (Sigma) using sterile tissue homogenizers. Homogenates were centrifuged at 5000g at 4°C for 15 minutes. Supernatants were immediately frozen at −80°C until assayed. IL-1β in the homogenates was quantified using assay (Bio-Plex; Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. The sensitivity of the assay was 10 pg/mL. A value of 0 was assigned to samples that were below the range of detection 
For the in vitro assay, HCECs were seeded in 96-well plates and maintained in serum-free KGM (Lonza Rockland). After reaching approximately 70% confluence, cells were preincubated with 200 ng/mL human LBP (R&D Systems, Minneapolis, MN) for 2 hours at 37°C. Heat-killed P. aeruginosa PAO1 was added at a concentration of 2 × 108 CFUs/mL to stimulate cytokine expression. Immediately after the addition of the heat-killed bacteria, 5 μL D-PBS, 100 μM D9R, or KGM (untreated control) was added to the cells. Cultures were then maintained for 24 hours, after which spent media were harvested, centrifuged to remove cellular debris, and frozen at −70°C until further analysis. Culture supernatants were analyzed for IL-1β production with a commercially available ELISA kit (R&D Systems). The sensitivities of the IL-1β assay were less than 1.0 pg/mL. All assays were performed in hexplicate, and the experiment was repeated at least three times. IL-1β concentrations are represented as mean ± SEM. 
The statistical significance of differences between two or more means was compared by Student’s t-test (GraphPad Prism). P ≤ 0.05 was considered significant. 
Results
Ocular Response to Infection
Photographs of SWBLK mouse eyes after treatment are shown in Figure 2(magnification, ×40), and the mean slit-lamp examination (SLE) disease scores (±SEM) of these mice are shown in Figure 3A . All eyes were normal before infection (Fig. 2A) . At 24 hours PI, no significant differences in disease scores (P ≥ 0.05) were observed between the eyes of mice that were left untreated (Fig. 2B) , were treated with D-PBS (Fig. 2C) , or were treated with 0.08% ciprofloxacin (Fig. 2D) . Dense corneal opacity obscured anterior chamber details in all three groups. Frank corneal ulcers were observed in untreated and D-PBS–treated eyes but not in eyes treated with ciprofloxacin. 
Eyes treated with 10 or 100 μM D9R, followed by treatment with 0.08% ciprofloxacin (Figs. 2E 2F , respectively), had significantly lower disease scores (P ≤ 0.001) than untreated eyes, eyes treated with PBS, or eyes treated with 0.08% ciprofloxacin (Fig. 2) . Corneal opacities in these eyes were essentially limited to wound sites, and the anterior chamber responses (as indicated by the presence of cells and flare) were mild. Increasing the concentration of D9R from 10 to 100 μM significantly reduced disease scores (P ≤ 0.01) from a mean SLE score of 2.02 ± 0.12 to 1.32 ± 0.23 (Fig. 3A)
Because 100 μM D9R was the most efficacious dosage tested (with no apparent cytotoxicity in HCECs), the experiment was repeated, and infection was allowed to progress until day 14 PI. Mean SLE scores on days 1, 7, and 14 PI are shown in Figure 3B . Eyes treated with a combination of 100 μM D9R and ciprofloxacin had the best clinical outcome on all days examined when compared with other treatment groups. Eyes receiving combination therapy maintained significantly lower disease scores than PBS-treated eyes (P ≤ 0.001) or eyes treated with ciprofloxacin alone (P ≤ 0.05) on days 7 and 14 PI. SLE scores for the eyes receiving combination therapy were lower than those treated with D9R alone, but this difference was not significant (P ≥ 0.05). Eyes treated with D9R alone also showed significantly less ocular disease (P ≤ 0.001) than PBS-treated or untreated eyes. 
Viable Bacteria per Eye
At 16 hours PI, there were 5.66 ± 0.19 log10 CFUs of P. aeruginosa in an untreated eye. Mean log10 numbers of viable bacteria per eye at 24 hours PI are shown in Figure 4 . There was essentially no significant increase in the number of CFUs from 16 to 24 hours PI in the untreated eye (P ≥ 0.05). All eyes treated with ciprofloxacin, regardless of previous treatment with or without D9R, had significantly fewer bacteria (fewer than 30 CFUs/eye; P ≤ 0.003) than untreated or D-PBS–treated eyes. No significant differences in CFU numbers were observed between eyes treated with 10 or 100 μM D9R or with D-PBS (P ≥ 0.05). 
Histopathology
Histologic sections from normal and P. aeruginosa–infected eyes (treated and untreated) were examined in a masked fashion and qualitatively compared. Representative sections of the central cornea (directly over the pupillary axis) are shown in Figure 5(magnification, ×100). 
An uninfected, untreated cornea is shown in Figure 5A . As expected, the multilayered corneal epithelium and endothelial monolayer are intact. The only cells present in the neatly arrayed stroma are keratocytes. The anterior chamber is free of any cellular infiltrate. Twenty-four hours after infection with P. aeruginosa, the untreated cornea (Fig. 5B)is highly edematous, likely because of a damaged endothelial layer. The stromal layer is engorged with neutrophils, and the epithelium is desquamating. The anterior chamber is full of proteinaceous exudate and neutrophils. Corneas treated with D-PBS appeared similar to untreated corneas (Fig. 5C)
Corneas treated with 0.08% ciprofloxacin (Fig. 5D)were less edematous than untreated (Fig. 5B)or D-PBS–treated (Fig. 5C)corneas but were still inflamed compared with an uninfected cornea (Fig. 5A) . Neutrophils were present in the stroma and anterior chamber, but to a lesser extent than in untreated or D-PBS–treated corneas. The corneal epithelium essentially appeared intact, though the superficial layer of cells looked as if they were beginning to desquamate. Corneal endothelial cells seemed enlarged and rounded compared with the flattened appearance of normal endothelial cells. Furthermore, the presence of central guttae (focal thickening of Descemet membrane) suggested endothelial cell dysfunction. 
Corneas treated with 10 or 100 μM D9R and 0.08% ciprofloxacin are shown in Figures 5E and 5F , respectively. These corneas were significantly less inflamed than untreated P. aeruginosa–infected corneas or corneas treated with D-PBS or ciprofloxacin. The corneal epithelium and endothelium looked like an uninfected cornea, and few neutrophils were observed in either the stroma or the anterior chamber. The histopathology of corneas treated with 10 or 100 μM D9R without subsequent treatment with ciprofloxacin was essentially the same as of corneas treated with ciprofloxacin—that is, significantly reduced inflammation, few neutrophils in the stroma and anterior chamber, and relatively normal corneal epithelium and endothelium (data not shown). 
IL-1β Expression with D9R
IL-1β was not constitutively expressed by uninfected mouse corneas. Corneal concentrations of IL-1β after infection with P. aeruginosa are shown in Figure 6A . Treatment with 100 μM D9R significantly reduced IL-1β expression at all time points tested (P ≤ 0.001; n = 10 per time point). As with mouse corneas, HCECs did not constitutively express IL-1β. When stimulated with heat-killed P. aeruginosa, HCECs produced 7.6 ± 1.0 pg/mL IL-1β (Fig. 6B) . The addition of PBS had no effect on the expression of IL-1β by stimulated HCECs (P ≥ 0.50), whereas 100 μM D9R significantly reduced IL-1β expression by approximately 40% (P ≤ 0.05; n = 17). 
Discussion
The fulminant destruction of the cornea that typifies P. aeruginosa ocular infection is a result of bacterial toxic products and an exuberant inflammatory response (primarily an influx of neutrophils) by the host. 23 24 25 Thus, antibiotics can eliminate viable bacteria from the cornea but do little to prevent the subsequent damage from released bacterial and neutrophil-derived proteases and toxins. Concomitant treatment with corticosteroids improves the clinical outcome by dampening the host inflammatory response to infection, but their use in treating bacterial keratitis remains controversial. 26 27  
Previous efforts have been made with varying degrees of success to minimize the contribution of the P. aeruginosa metalloproteinase pseudolysin by treating infected eyes with nonspecific inhibitors of this enzyme. 28 29 30 31 In this study, the activation of ToxA (a demonstrated virulence factor for ocular infections) 6 was presumably blocked by inhibiting the enzyme responsible for its activation (furin) with topically applied D9R. Applications of 10 or 100 μM D9R significantly reduced disease in P. aeruginosa–infected eyes. Furthermore, treatment with D9R was therapeutically synergistic with conventional antibiotic therapy (topical ciprofloxacin), resulting in clinical cure (elimination of viable bacteria from tissue with minimal tissue damage). 
The efficacy of D9R in reducing disease in a P. aeruginosa–infected cornea cannot likely be fully attributed to blocking ToxA activation given that its primary target is an endoprotease with multiple roles in homeostasis and disease. 32 Furin is 1 of 7 endoproteases that function as proprotein convertases (PCs). 33 With the exception of PC7, the expression of furin and the other PCs appears to be essential in normal embryogenesis. However, PCs can apparently compensate for each other in the physiological process of postpartum because of similarities in the active sites of these enzymes. 34 35 Indeed, D9R is capable of inhibiting PC5/6 and PC7 (Ki, 19 nM and 81 nM, respectively) and furin (Ki, 1.3 nM). 36  
Inhibition of furin and other PCs by D9R might have a beneficial effect on the outcome of P. aeruginosa corneal disease by preventing the activation of endogenous corneal proteases. Furin activates six pro–membrane-type matrix metalloproteinases (MT-MMPs) 37 : MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), MT4-MMP (MMP-17), MT5-MMP (MMP-24), and MT6-MMP (leukolysin). Peak expression of all six enzymes showed a good correlation with the inflammatory response observed in P. aeruginosa–infected corneas. 38 39 MT-MMPs and other furin-like PCs could also activate other pro–matrix metalloproteinases (MMPs) such as MMP-9 (gelatinase B), an important contributor to P. aeruginosa corneal disease. 40 41 Inhibition of furin and other PCs by D9R could prevent the activation of pro–MT-MMPs and pro–MMP-9, thus leading to a tamer inflammatory response with less tissue damage to the cornea. 
As a short cationic peptide, D9R shares many properties of other cationic host-defense antimicrobial peptides (AMPs) found in the eye. 42 These AMPs bridge innate and acquired immunity and have anti-infective and immunomodulatory activities. 19 The antibacterial activity of many AMPs is antagonized by the presence of divalent cations such as Ca2+ and Mg2+. In this study, the D-PBS vehicle for D9R was supplemented with these cations, perhaps explaining why no significant killing of P. aeruginosa by D9R was observed in vivo or in vitro. 
D9R did have immunomodulatory activity in that it significantly reduced IL-1β expression by HCECs stimulated by heat-killed P. aeruginosa and in vivo in P. aeruginosa–infected corneas. IL-1β plays a major role in the pathogenesis of P. aeruginosa corneal disease. Rudner et al. 20 showed that protracted IL-1β expression in P. aeruginosa–infected mouse corneas resulted in increased macrophage-inflammatory protein (MIP)-2 expression, PMN persistence, and corneal perforation. Mice deficient in IL-1β converting enzyme expression had significantly reduced corneal inflammation after P. aeruginosa infection than did wild-type mice. 22 Administration of neutralizing anti–IL-1β antibody during P. aeruginosa corneal infection significantly reduced MMP-9 expression and promoted the production of tissue inhibitor of matrix metalloproteinase (TIMP)-1 (an antagonist of MMP-9), thereby resulting in reduced corneal damage. 21  
In conclusion, D9R could be a useful addition to conventional antibiotic therapy of P. aeruginosa corneal disease. Similar to a corticosteroid, it markedly reduces the amount of corneal disease associated with the hyperinflammatory response to P. aeruginosa infection (through the suppression of IL-1β), and, in conjunction with a bactericidal antibiotic such as ciprofloxacin, a genuine clinical cure is achieved. D9R likely has several mechanisms of action relating to its anti-inflammatory activity, but more studies are needed to define these mechanisms. 
 
Figure 1.
 
D9R is nontoxic to human corneal epithelial cells. Cell viability was measured using a colorimetric MTS tetrazolium dye assay. The growth rate of HCECs was similar among all groups with no significant (P ≥ 0.05) differences between the absorbance values, suggesting that D9R at concentrations of 10 μM and 100 μM is nontoxic and does not interfere with cell proliferation.
Figure 1.
 
D9R is nontoxic to human corneal epithelial cells. Cell viability was measured using a colorimetric MTS tetrazolium dye assay. The growth rate of HCECs was similar among all groups with no significant (P ≥ 0.05) differences between the absorbance values, suggesting that D9R at concentrations of 10 μM and 100 μM is nontoxic and does not interfere with cell proliferation.
Figure 2.
 
SLE photographs of mouse eyes 24 hours PI. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with D-PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×40.
Figure 2.
 
SLE photographs of mouse eyes 24 hours PI. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with D-PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×40.
Figure 3.
 
Mean disease scores (±SEM) in SWBLK mice 24 hours PI. (A) Mean disease scores (±SEM) in SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (n = 50) supplemented with calcium and magnesium, 0.08% ciprofloxacin (n = 30), 10 μM D9R (D9R-10 μM; n =30), 100 μM D9R (D9R-100 μM; n =20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 30), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 20). Eyes treated with a combination of 10 or 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than D-PBS–treated eyes or eyes treated with ciprofloxacin alone. No differences were observed between eyes treated with D-PBS and eyes treated with ciprofloxacin (P ≥ 0.05). *P ≤ 0.001 compared with D-PBS or ciprofloxacin-treated groups. (B) Mean disease scores (±SEM) in SWBLK mice 1, 7, and 14 days PI. Untreated (n =15) or treated with PBS, pH 7.4 (n =15), 0.08% ciprofloxacin (n =15), 100 μM D9R (D9R-100 μM; n = 15), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 15). Eyes treated with a combination of 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than PBS-treated eyes or eyes treated with ciprofloxacin alone on day 1 PI. No differences were observed between eyes treated with PBS and those treated with ciprofloxacin (P ≥ 0.05) at this time point. Eyes receiving the combination therapy maintained significantly lower disease scores than PBS-treated eyes (P < 0.001) or eyes treated with ciprofloxacin alone (P < 0.05) on days 7 and 14 PI. *P ≤ 0.001 compared with PBS-treated group. + P ≤ 0.05 compared with ciprofloxacin-treated group.
Figure 3.
 
Mean disease scores (±SEM) in SWBLK mice 24 hours PI. (A) Mean disease scores (±SEM) in SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (n = 50) supplemented with calcium and magnesium, 0.08% ciprofloxacin (n = 30), 10 μM D9R (D9R-10 μM; n =30), 100 μM D9R (D9R-100 μM; n =20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 30), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 20). Eyes treated with a combination of 10 or 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than D-PBS–treated eyes or eyes treated with ciprofloxacin alone. No differences were observed between eyes treated with D-PBS and eyes treated with ciprofloxacin (P ≥ 0.05). *P ≤ 0.001 compared with D-PBS or ciprofloxacin-treated groups. (B) Mean disease scores (±SEM) in SWBLK mice 1, 7, and 14 days PI. Untreated (n =15) or treated with PBS, pH 7.4 (n =15), 0.08% ciprofloxacin (n =15), 100 μM D9R (D9R-100 μM; n = 15), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 15). Eyes treated with a combination of 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than PBS-treated eyes or eyes treated with ciprofloxacin alone on day 1 PI. No differences were observed between eyes treated with PBS and those treated with ciprofloxacin (P ≥ 0.05) at this time point. Eyes receiving the combination therapy maintained significantly lower disease scores than PBS-treated eyes (P < 0.001) or eyes treated with ciprofloxacin alone (P < 0.05) on days 7 and 14 PI. *P ≤ 0.001 compared with PBS-treated group. + P ≤ 0.05 compared with ciprofloxacin-treated group.
Figure 4.
 
Viable bacteria in eyes of SWBLK mice 24 hours PI. Viable bacteria in eyes of SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (D-PBS; n = 30), 10 μM D9R (D9R-10 μM; n = 20), 100 μM D9R (D9R-100 μM; n = 20), 0.08% ciprofloxacin (Ciprofloxacin; n = 20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 25); 100 μM D9R and 0.08% ciprofloxacin (D 100+C; n = 20).There was significant reduction (P ≤ 0.001) in log10 CFU in eyes treated with ciprofloxacin (with or without D9R) compared with all other groups. Eyes treated with 10 or 100 μM D9R (0–5 hours PI) alone had significantly less (P ≤ 0.001) disease than PBS-treated eyes; however, there were no significant differences in CFU among these three groups (P ≥ 0.100). *P ≤ 0.001 compared with D-PBS–treated group.
Figure 4.
 
Viable bacteria in eyes of SWBLK mice 24 hours PI. Viable bacteria in eyes of SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (D-PBS; n = 30), 10 μM D9R (D9R-10 μM; n = 20), 100 μM D9R (D9R-100 μM; n = 20), 0.08% ciprofloxacin (Ciprofloxacin; n = 20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 25); 100 μM D9R and 0.08% ciprofloxacin (D 100+C; n = 20).There was significant reduction (P ≤ 0.001) in log10 CFU in eyes treated with ciprofloxacin (with or without D9R) compared with all other groups. Eyes treated with 10 or 100 μM D9R (0–5 hours PI) alone had significantly less (P ≤ 0.001) disease than PBS-treated eyes; however, there were no significant differences in CFU among these three groups (P ≥ 0.100). *P ≤ 0.001 compared with D-PBS–treated group.
Figure 5.
 
Histopathology of SWBLK mouse corneas. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×100.
Figure 5.
 
Histopathology of SWBLK mouse corneas. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×100.
Figure 6.
 
(A) IL-1β concentrations in mouse corneas. Treatment with 100 μM D9R significantly reduced the levels of IL-1β in P. aeruginosa–infected mouse corneas. *P ≤ 0.001 compared with PBS-treated group. (B) Addition of 100 μM D9R significantly suppressed IL-1β expression in HCECs stimulated with heat-killed P. aeruginosa. *P ≤ 0.05 compared to untreated group.
Figure 6.
 
(A) IL-1β concentrations in mouse corneas. Treatment with 100 μM D9R significantly reduced the levels of IL-1β in P. aeruginosa–infected mouse corneas. *P ≤ 0.001 compared with PBS-treated group. (B) Addition of 100 μM D9R significantly suppressed IL-1β expression in HCECs stimulated with heat-killed P. aeruginosa. *P ≤ 0.05 compared to untreated group.
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Figure 1.
 
D9R is nontoxic to human corneal epithelial cells. Cell viability was measured using a colorimetric MTS tetrazolium dye assay. The growth rate of HCECs was similar among all groups with no significant (P ≥ 0.05) differences between the absorbance values, suggesting that D9R at concentrations of 10 μM and 100 μM is nontoxic and does not interfere with cell proliferation.
Figure 1.
 
D9R is nontoxic to human corneal epithelial cells. Cell viability was measured using a colorimetric MTS tetrazolium dye assay. The growth rate of HCECs was similar among all groups with no significant (P ≥ 0.05) differences between the absorbance values, suggesting that D9R at concentrations of 10 μM and 100 μM is nontoxic and does not interfere with cell proliferation.
Figure 2.
 
SLE photographs of mouse eyes 24 hours PI. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with D-PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×40.
Figure 2.
 
SLE photographs of mouse eyes 24 hours PI. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with D-PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×40.
Figure 3.
 
Mean disease scores (±SEM) in SWBLK mice 24 hours PI. (A) Mean disease scores (±SEM) in SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (n = 50) supplemented with calcium and magnesium, 0.08% ciprofloxacin (n = 30), 10 μM D9R (D9R-10 μM; n =30), 100 μM D9R (D9R-100 μM; n =20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 30), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 20). Eyes treated with a combination of 10 or 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than D-PBS–treated eyes or eyes treated with ciprofloxacin alone. No differences were observed between eyes treated with D-PBS and eyes treated with ciprofloxacin (P ≥ 0.05). *P ≤ 0.001 compared with D-PBS or ciprofloxacin-treated groups. (B) Mean disease scores (±SEM) in SWBLK mice 1, 7, and 14 days PI. Untreated (n =15) or treated with PBS, pH 7.4 (n =15), 0.08% ciprofloxacin (n =15), 100 μM D9R (D9R-100 μM; n = 15), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 15). Eyes treated with a combination of 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than PBS-treated eyes or eyes treated with ciprofloxacin alone on day 1 PI. No differences were observed between eyes treated with PBS and those treated with ciprofloxacin (P ≥ 0.05) at this time point. Eyes receiving the combination therapy maintained significantly lower disease scores than PBS-treated eyes (P < 0.001) or eyes treated with ciprofloxacin alone (P < 0.05) on days 7 and 14 PI. *P ≤ 0.001 compared with PBS-treated group. + P ≤ 0.05 compared with ciprofloxacin-treated group.
Figure 3.
 
Mean disease scores (±SEM) in SWBLK mice 24 hours PI. (A) Mean disease scores (±SEM) in SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (n = 50) supplemented with calcium and magnesium, 0.08% ciprofloxacin (n = 30), 10 μM D9R (D9R-10 μM; n =30), 100 μM D9R (D9R-100 μM; n =20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 30), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 20). Eyes treated with a combination of 10 or 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than D-PBS–treated eyes or eyes treated with ciprofloxacin alone. No differences were observed between eyes treated with D-PBS and eyes treated with ciprofloxacin (P ≥ 0.05). *P ≤ 0.001 compared with D-PBS or ciprofloxacin-treated groups. (B) Mean disease scores (±SEM) in SWBLK mice 1, 7, and 14 days PI. Untreated (n =15) or treated with PBS, pH 7.4 (n =15), 0.08% ciprofloxacin (n =15), 100 μM D9R (D9R-100 μM; n = 15), or 100 μM D9R and 0.08% ciprofloxacin (D100+C; n = 15). Eyes treated with a combination of 100 μM D9R and 0.08% ciprofloxacin had significantly lower disease scores (P ≤ 0.001) than PBS-treated eyes or eyes treated with ciprofloxacin alone on day 1 PI. No differences were observed between eyes treated with PBS and those treated with ciprofloxacin (P ≥ 0.05) at this time point. Eyes receiving the combination therapy maintained significantly lower disease scores than PBS-treated eyes (P < 0.001) or eyes treated with ciprofloxacin alone (P < 0.05) on days 7 and 14 PI. *P ≤ 0.001 compared with PBS-treated group. + P ≤ 0.05 compared with ciprofloxacin-treated group.
Figure 4.
 
Viable bacteria in eyes of SWBLK mice 24 hours PI. Viable bacteria in eyes of SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (D-PBS; n = 30), 10 μM D9R (D9R-10 μM; n = 20), 100 μM D9R (D9R-100 μM; n = 20), 0.08% ciprofloxacin (Ciprofloxacin; n = 20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 25); 100 μM D9R and 0.08% ciprofloxacin (D 100+C; n = 20).There was significant reduction (P ≤ 0.001) in log10 CFU in eyes treated with ciprofloxacin (with or without D9R) compared with all other groups. Eyes treated with 10 or 100 μM D9R (0–5 hours PI) alone had significantly less (P ≤ 0.001) disease than PBS-treated eyes; however, there were no significant differences in CFU among these three groups (P ≥ 0.100). *P ≤ 0.001 compared with D-PBS–treated group.
Figure 4.
 
Viable bacteria in eyes of SWBLK mice 24 hours PI. Viable bacteria in eyes of SWBLK mice 24 hours PI. Untreated (n = 15) or treated with D-PBS, pH 7.4 (D-PBS; n = 30), 10 μM D9R (D9R-10 μM; n = 20), 100 μM D9R (D9R-100 μM; n = 20), 0.08% ciprofloxacin (Ciprofloxacin; n = 20), 10 μM D9R and 0.08% ciprofloxacin (D10+C; n = 25); 100 μM D9R and 0.08% ciprofloxacin (D 100+C; n = 20).There was significant reduction (P ≤ 0.001) in log10 CFU in eyes treated with ciprofloxacin (with or without D9R) compared with all other groups. Eyes treated with 10 or 100 μM D9R (0–5 hours PI) alone had significantly less (P ≤ 0.001) disease than PBS-treated eyes; however, there were no significant differences in CFU among these three groups (P ≥ 0.100). *P ≤ 0.001 compared with D-PBS–treated group.
Figure 5.
 
Histopathology of SWBLK mouse corneas. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×100.
Figure 5.
 
Histopathology of SWBLK mouse corneas. (A) Uninfected eye. (B) P. aeruginosa–infected untreated eye. (C) P. aeruginosa–infected eye treated with PBS. (D) P. aeruginosa–infected eye treated with 0.08% ciprofloxacin. (E) P. aeruginosa–infected eye treated with 10 μM D9R and 0.08% ciprofloxacin. (F) P. aeruginosa–infected eye treated with 100 μM D9R and 0.08% ciprofloxacin. Original magnification, ×100.
Figure 6.
 
(A) IL-1β concentrations in mouse corneas. Treatment with 100 μM D9R significantly reduced the levels of IL-1β in P. aeruginosa–infected mouse corneas. *P ≤ 0.001 compared with PBS-treated group. (B) Addition of 100 μM D9R significantly suppressed IL-1β expression in HCECs stimulated with heat-killed P. aeruginosa. *P ≤ 0.05 compared to untreated group.
Figure 6.
 
(A) IL-1β concentrations in mouse corneas. Treatment with 100 μM D9R significantly reduced the levels of IL-1β in P. aeruginosa–infected mouse corneas. *P ≤ 0.001 compared with PBS-treated group. (B) Addition of 100 μM D9R significantly suppressed IL-1β expression in HCECs stimulated with heat-killed P. aeruginosa. *P ≤ 0.05 compared to untreated group.
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