Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy Improves Pseudomonas aeruginosa-Induced Keratitis

With increasing multidrug resistance and contraindication for corticosteroid use, the goal of this study was to develop thymosin beta-4 (Tβ4) as an adjunctive therapy to antibiotics for the treatment of bacterial keratitis that effectively promotes enhanced wound healing, host defense, and inflammation resolution. Disease outcome was assessed by clinical score, slit lamp photography, and histopathology. Cytokine profile, bacterial load, PMN infiltration, and Griess and reactive oxygen species (ROS) levels were determined. Adjunct Tβ4 treatment resulted in a significant improvement compared to PBS, Tβ4, and most remarkably, ciprofloxacin, correlating with changes in mediators of inflammation and wound healing. Collectively, these data provide evidence that wound healing is an essential aspect in the development of new therapies to treat corneal infection. Use of adjunctive Tβ4 provides a more efficacious approach for bacterial keratitis by addressing both the infectious pathogen and deleterious host response.


Background
Microbial keratitis is a rapidly progressing, visually debilitating infection of the cornea that can lead to corneal scarring, endophthalmitis, and perforation. Corneal opacification, a complication of keratitis, is among the leading causes of legal blindness worldwide, second to cataracts. Pseudomonas aeruginosa and Staphylococcus aureus are the two bacteria most commonly associated with this type of infection [1][2][3][4]. In addition, reports indicate that up to 38% of fungal keratitis cases are co-infected with bacteria [5]. Risk factors include patients who are immunocompromised, those who have undergone refractive corneal surgery, and those with prior penetrating keratoplasty, as well as extended wear contact lens users [6]. Current treatment of microbial keratitis primarily addresses the pathogen using antibiotics. Bacterial clearance is of utmost importance, yet does not guarantee good visual outcome. Fortified antibiotic use has been shown to be toxic to the corneal epithelium and inhibit the healing process necessary for restoring visual acuity [7,8]. As resistance to antibiotics gains prevalence, microbial keratitis has become an important global healthcare issue [1,2]. Clinicians are often left to rely upon the eye's innate ability to heal itself, as there are limited options beyond antibiotics and corticosteroids for treating patients with corneal infection. Beyond antibiotics, agents in use, such as lubricating ointments, artificial tears, and anti-inflammatory drops, do not fully accommodate clinical needs and have many harmful complications. In particular, topical corticosteroids are often used to reduce the host inflammatory response. Yet, controversy remains regarding the benefits and risks associated with this immunosuppressive therapy [9,10]. To this end,

Experimental Animal Protocol
The left central cornea of 8-week-old C57BL6 (B6) female mice (The Jackson Laboratory, Bar Harbor, ME, USA) were scarified as previously described [22]. A 5 µL aliquot containing 10 6 CFU/mL of the cytotoxic Pseudomonas aeruginosa strain ATCC 19660 was applied topically to the wounded corneal surface. Mice were randomized into four different treatment groups consisting of either PBS as controls, Tβ4 (0.1%), ciprofloxacin (0.3%), or Tβ4 + ciprofloxacin, administered topically (5 µL) 3× per day beginning 24 h after infection. Uninfected, normal corneas were appropriately used as an additional control where noted. All animals were treated in a manner authorized by Wayne State University Institutional Animal Care and Use committee (protocol 16-090) and conformed to the Association for Research in Vision and Ophthalmology's statement on the Use of Animals in Ophthalmic and Vision Research.

Ocular Response to Bacterial Infection
Infected eyes were observed daily in a blinded fashion and disease response was graded using an established grading scale [23] for statistical comparison. Mice were examined and a clinical score was calculated for 3 and 5 days postinfection (p.i.) for each group to express disease severity as mean clinical score ± SEM. Slit-lamp photography was used to illustrate disease response at corresponding time points.

Bacterial Load
Corneas from B6 animals treated with PBS, Tβ4, ciprofloxacin, or Tβ4 + ciprofloxacin were collected at 5 days p.i. and the number of viable bacteria was quantitated. Individual corneas were Cells 2018, 7, 145 3 of 15 homogenized in sterile 0.9% saline containing 0.25% BSA. Serial 10-fold dilutions of each sample were plated on Pseudomonas isolation agar in triplicate and plates were incubated overnight at 37 • C. Results are reported as CFU/cornea ± SEM.

Myeloperoxidase (MPO) Assay
An MPO assay was performed to correlate the number of PMN in infected corneas of B6 mice treated with PBS, Tβ4, ciprofloxacin, or Tβ4 + ciprofloxacin. Corneas were removed at 5 days p.i. and homogenized in 1.0 mL of 50 mM phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethyl-ammonium (HTAB). Samples were processed as previously described [26]. The change in absorbency (460 nm) was monitored for 5 min at 30 s intervals. The slope of the line was determined for each sample and used to calculate units of MPO/cornea. One unit of MPO activity is equivalent to~2 × 10 5 PMN [27] with a sensitivity of 0.90 units.

Real-Time RT-PCR
Total RNA was isolated from individual whole corneas at 3 and 5 days p.i. for gene expression analysis using RNA-STAT 60 (Tel-Test, Friendswood, TX, USA) according to the manufacturer's recommendations and quantified by spectrophotometric determination (260 nm). 100 ng of total RNA was reverse transcribed and used to produce a cDNA template and amplified as previously described [29]. All primers for the PCR reaction were designed using Primer3 (Table 1). Semiquantitative real-time RT-PCR was performed using the CFX Connect Real-Time RT-PCR Detection System (BioRad, Hercules, CA, USA). PCR amplification conditions were determined using routine methods [30]. Relative transcript levels were determined using the relative standard curve method comparing the amount of target normalized to an endogenous reference, β-actin. Data are shown as the mean ± SD for relative transcript levels normalized to β-actin and relative to the expression of uninfected (normal) controls. Table 1. Nucleotide sequence of the specific primers used for PCR amplification.

ELISA Analysis
Normal (uninfected) and infected corneas were removed at 3 and 5 days p.i. from B6 mice treated with PBS, Tβ4, ciprofloxacin, or Tβ4 + ciprofloxacin. Individual corneas were homogenized in 1 mL RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) with a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). An aliquot of each supernatant was assayed in duplicate for IL-1β and MIP-2 per the manufacturer's instruction (R&D Systems, St. Paul, MN, USA). The reported sensitivity of these assays was 4.8 pg/mL for IL-1β and 1.5 pg/mL for MIP-2. Results are expressed as average ng of each cytokine/mL ± SEM.

Statistical Analysis
Sample sizes were determined statistically prior to experimentation based on previous work [26,28,29,31], which includes a <5% mortality rate. Using a power analysis, we assume a mean difference = 2, standard deviation = 1, α = 0.05, power = 0.8, and a sample size ratio = 1. The difference in clinical scores between two groups at each time point was tested by the Mann-Whitney U test (GraphPad Prism; GraphPad Software, La Jolla, CA, USA). For all other experiments, a one-way ANOVA followed by Bonferroni's multiple comparison test (GraphPad Prism) was used for analysis of 3+ groups. An unpaired Student's t test was used for comparison between 2 groups. Data were considered significant at P < 0.05. A minimum of N = 5/group/time point were used unless otherwise stated. All experiments and/or measurements were carried out in triplicate and representative data from a typical experiment is shown.

Tβ4 Adjunct Therapy Significantly Improves Disease Response
Clinical scores, accompanied by representative photographs, were recorded ( Figure 1) to initially assess the disease response. Tβ4 treatment was similar to PBS controls, ranging from dense opacity covering the entire anterior segment (+3) at 3 days to corneal melting and perforation (+4) by 5 days p.i. Disease response was significantly improved in ciprofloxacin-treated mice, demonstrating dense opacity, partially or fully covering the pupil (+2) at 3 days with increasing progress at 5 days p.i. Infected corneas treated with Tβ4 + ciprofloxacin exhibited the most improvement in disease severity (+1) with only moderate opacity covering the pupil at 3 days p.i., which was further reduced to slight central opacity at 5 days and significantly improved over all three treatment groups.
expressed as average ng of each cytokine/mL ± SEM.

Statistical Analysis
Sample sizes were determined statistically prior to experimentation based on previous work [26,28,29,31], which includes a <5% mortality rate. Using a power analysis, we assume a mean difference = 2, standard deviation = 1, α = 0.05, power = 0.8, and a sample size ratio = 1. The difference in clinical scores between two groups at each time point was tested by the Mann-Whitney U test (GraphPad Prism; GraphPad Software, La Jolla, CA, USA). For all other experiments, a one-way ANOVA followed by Bonferroni's multiple comparison test (GraphPad Prism) was used for analysis of 3+ groups. An unpaired Student's t test was used for comparison between 2 groups. Data were considered significant at P < 0.05. A minimum of N = 5/group/time point were used unless otherwise stated. All experiments and/or measurements were carried out in triplicate and representative data from a typical experiment is shown.

Tβ4 Adjunct Therapy Significantly Improves Disease Response
Clinical scores, accompanied by representative photographs, were recorded ( Figure 1) to initially assess the disease response. Tβ4 treatment was similar to PBS controls, ranging from dense opacity covering the entire anterior segment (+3) at 3 days to corneal melting and perforation (+4) by 5 days p.i. Disease response was significantly improved in ciprofloxacin-treated mice, demonstrating dense opacity, partially or fully covering the pupil (+2) at 3 days with increasing progress at 5 days p.i. Infected corneas treated with Tβ4 + ciprofloxacin exhibited the most improvement in disease severity (+1) with only moderate opacity covering the pupil at 3 days p.i., which was further reduced to slight central opacity at 5 days and significantly improved over all three treatment groups. Histopathological sections from each treatment group ( Figure 2) further confirm the observed disease response. Staining of PBS-treated controls ( Figure 2A) revealed a completely denuded epithelium, massive edema, and excessive stromal degradation accompanied by heavy cellular infiltrate throughout the anterior chamber. Tβ4 treatment ( Figure 2B) appeared very slightly improved with a somewhat less stromal degradation comparatively, but still edematous with a heavy cellular infiltrate throughout the stroma and anterior chamber. While the bacteria appeared to be cleared, ciprofloxacin treatment ( Figure 2C) exhibited stromal edema, infiltrating cells within the anterior chamber and a disrupted, detached epithelium. The combination treatment ( Figure 2D) revealed little to no edema with markedly reduced inflammatory cell infiltrate and a mostly intact epithelium indicative of activated wound healing.
Histopathological sections from each treatment group ( Figure 2) further confirm the observed disease response. Staining of PBS-treated controls ( Figure 2A) revealed a completely denuded epithelium, massive edema, and excessive stromal degradation accompanied by heavy cellular infiltrate throughout the anterior chamber. Tβ4 treatment ( Figure 2B) appeared very slightly improved with a somewhat less stromal degradation comparatively, but still edematous with a heavy cellular infiltrate throughout the stroma and anterior chamber. While the bacteria appeared to be cleared, ciprofloxacin treatment ( Figure 2C) exhibited stromal edema, infiltrating cells within the anterior chamber and a disrupted, detached epithelium. The combination treatment ( Figure 2D) revealed little to no edema with markedly reduced inflammatory cell infiltrate and a mostly intact epithelium indicative of activated wound healing.

Tβ4 Adjunct Treatment Significantly Reduces Bacterial Load, ROS, MPO, and Nitrite Levels
Viable bacteria were determined by direct plate counts from infected corneas at 5 days p.i., as shown in Figure 3A. No differences were detected between PBS and Tβ4 treatment groups. As expected, ciprofloxacin significantly reduced the bacterial burden by ~350-fold. However, Tβ4 + ciprofloxacin significantly decreased the bacterial load even further compared to all three treatment groups and, in fact, was undetectable. MPO assay ( Figure 3B) revealed that PMN infiltration into infected corneas was higher in Tβ4 versus PBS treatment groups at 5 days. In contrast, MPO activity was significantly decreased with ciprofloxacin treatment, while Tβ4 + ciprofloxacin treatment resulted in further reduction in MPO levels compared to all three treatment groups, suggesting that

Tβ4 Adjunct Treatment Significantly Reduces Bacterial Load, ROS, MPO, and Nitrite Levels
Viable bacteria were determined by direct plate counts from infected corneas at 5 days p.i., as shown in Figure 3A. No differences were detected between PBS and Tβ4 treatment groups. As expected, ciprofloxacin significantly reduced the bacterial burden by~350-fold. However, Tβ4 + ciprofloxacin significantly decreased the bacterial load even further compared to all three treatment groups and, in fact, was undetectable. MPO assay ( Figure 3B) revealed that PMN infiltration into infected corneas was higher in Tβ4 versus PBS treatment groups at 5 days. In contrast, MPO activity was significantly decreased with ciprofloxacin treatment, while Tβ4 + ciprofloxacin treatment resulted in further reduction in MPO levels compared to all three treatment groups, suggesting that the combination treatment had the strongest influence on reducing PMN infiltration into the infected corneas. Levels of ROS ( Figure 3C), key signaling molecules known to play a major role during an inflammatory response, were significantly elevated in Tβ4-compared to PBS-treated corneas at 5 days after infection, but significantly decreased following ciprofloxacin treatment. Again, Tβ4 + ciprofloxacin further reduced ROS levels, as detected by DCFH-DA. Nitric oxide (NO) is a reactive free radical produced predominately by macrophages during inflammation. To correlate macrophage activation in response to infection, nitrite (a stable oxidized product of NO) was measured at 5 days p.i. for each treatment group ( Figure 3D). Corneas of ciprofloxacin-treated mice showed similar nitrite levels when compared to PBS controls. Only Tβ4 and Tβ4 + ciprofloxacin treatment groups resulted in significantly reduced nitrite levels following infection. ciprofloxacin further reduced ROS levels, as detected by DCFH-DA. Nitric oxide (NO) is a reactive free radical produced predominately by macrophages during inflammation. To correlate macrophage activation in response to infection, nitrite (a stable oxidized product of NO) was measured at 5 days p.i. for each treatment group ( Figure 3D). Corneas of ciprofloxacin-treated mice showed similar nitrite levels when compared to PBS controls. Only Tβ4 and Tβ4 + ciprofloxacin treatment groups resulted in significantly reduced nitrite levels following infection.

Tβ4 Adjunct Treatment Modulates Key Proinflammatory Mediators
Select proinflammatory mediators, known to play a role in bacterial keratitis [26], were investigated to determine the immunoregulatory influence of Tβ4. As presented in Figure 4, transcript levels of TNF-α ( Figure 4A), IL-1β ( Figure 4B), iNOS ( Figure 4C), and MIP-2 ( Figure 4D) were analyzed in whole corneal lysates of the four treatment groups at 3 and 5 days p.i. Results indicated that corneal mRNA expression of proinflammatory cytokines/chemokines were all upregulated in PBS-treated animals at both time points. Compared to controls, Tβ4 treatment lowered these same gene transcripts at 3 days p.i., but then comparatively increased at 5 days.

Tβ4 Adjunct Treatment Modulates Key Proinflammatory Mediators
Select proinflammatory mediators, known to play a role in bacterial keratitis [26], were investigated to determine the immunoregulatory influence of Tβ4. As presented in Figure 4, transcript levels of TNF-α ( Figure 4A), IL-1β ( Figure 4B), iNOS ( Figure 4C), and MIP-2 ( Figure 4D) were analyzed in whole corneal lysates of the four treatment groups at 3 and 5 days p.i. Results indicated that corneal mRNA expression of proinflammatory cytokines/chemokines were all upregulated in PBS-treated animals at both time points. Compared to controls, Tβ4 treatment lowered these same gene transcripts at 3 days p.i., but then comparatively increased at 5 days. Ciprofloxacin and Tβ4 + ciprofloxacin treatment significantly reduced levels of proinflammatory mediators. As shown in Figure 5, when selectively confirmed at the protein level, IL-1β ( Figure 5A) and MIP-2 ( Figure 5B) followed similar trends, where PBS and Tβ4 treatment groups exhibited sustained production of both molecules over time. Ciprofloxacin significantly decreased IL-1β and MIP-2, but levels were even further reduced with the Tβ4 combination treatment. Overall, it appears that improved disease outcome subsequent to the adjunct treatment is due, in part, to a decreased proinflammatory cytokine/chemokine profile. mediators. As shown in Figure 5, when selectively confirmed at the protein level, IL-1β ( Figure 5A) and MIP-2 ( Figure 5B) followed similar trends, where PBS and Tβ4 treatment groups exhibited sustained production of both molecules over time. Ciprofloxacin significantly decreased IL-1β and MIP-2, but levels were even further reduced with the Tβ4 combination treatment. Overall, it appears that improved disease outcome subsequent to the adjunct treatment is due, in part, to a decreased proinflammatory cytokine/chemokine profile.   and MIP-2 ( Figure 5B) followed similar trends, where PBS and Tβ4 treatment groups exhibited sustained production of both molecules over time. Ciprofloxacin significantly decreased IL-1β and MIP-2, but levels were even further reduced with the Tβ4 combination treatment. Overall, it appears that improved disease outcome subsequent to the adjunct treatment is due, in part, to a decreased proinflammatory cytokine/chemokine profile.

Treatment Activates Corneal Wound Healing Pathways after Infection
mRNA levels of laminin α3 (LAMA3), laminin β3 (LAMB3), laminin γ2 (LAMC2), fibronectin, integrin α5 (ITGα5), ITGβ1, urokinase (uPA), urokinase receptor (uPAR), and TGF-β1 were measured at 3 and 5 days following infection in the four treatment groups, as shown in Figure 6A-I, respectively. The three subunits, α/β/γ, were examined given the prominent role of laminins as extracellular matrix glycoproteins required for cell adhesion and migration. α3 and γ2 chains were significantly increased in both Tβ4 treatment groups at 3 days p.i. compared to PBS and ciprofloxacin; levels remained Cells 2018, 7, 145 9 of 15 elevated at 5 days in the Tβ4 group only. Increased expression of β3 was limited to the adjunctive treatment at 3 days p.i. only. Given previous work regarding Tβ4 and laminin-332, we next focused on other components of wound healing. Fibronectin mRNA levels were up early in Tβ4-, ciprofloxacin-, and Tβ4 + ciprofloxacin-treated animals versus PBS, then the latter two groups became significantly decreased at 5 days compared to both PBS and Tβ4 treatments. This same trend was observed for ITGα5 and ITGβ1. ITGα5 binds to the major binding site of fibronectin, while ITGβ1 interacts with the minor binding site, further promoting cell adhesion. While the fibronectin:integrin interaction plays a central role in corneal epithelial cell wound healing and signals the cells to migrate, detachment of epithelial cells from the fibronectin extracellular matrix is also required for active movement. In this regard, we next looked at uPA and uPAR, which promote matrix remodeling and cell activation, and TGF-β1, a regulator of the uPA/uPAR pathway [32]. Both uPA and uPAR levels were significantly decreased in ciprofloxacin-treated animals and further reduced with the combination treatment during infection when compared to PBS. However, only the Tβ4 and combination treatments enhanced expression of the anti-inflammatory molecule, TGF-β1, at 5 days p.i. versus PBS, while the combination treatment significantly increased TGF-β1 at both time points compared to ciprofloxacin alone. mRNA levels of laminin α3 (LAMA3), laminin β3 (LAMB3), laminin γ2 (LAMC2), fibronectin, integrin α5 (ITGα5), ITGβ1, urokinase (uPA), urokinase receptor (uPAR), and TGF-β1 were measured at 3 and 5 days following infection in the four treatment groups, as shown in Figure 6A-I, respectively. The three subunits, α/β/γ, were examined given the prominent role of laminins as extracellular matrix glycoproteins required for cell adhesion and migration. α3 and γ2 chains were significantly increased in both Tβ4 treatment groups at 3 days p.i. compared to PBS and ciprofloxacin; levels remained elevated at 5 days in the Tβ4 group only. Increased expression of β3 was limited to the adjunctive treatment at 3 days p.i. only. Given previous work regarding Tβ4 and laminin-332, we next focused on other components of wound healing. Fibronectin mRNA levels were up early in Tβ4-, ciprofloxacin-, and Tβ4 + ciprofloxacin-treated animals versus PBS, then the latter two groups became significantly decreased at 5 days compared to both PBS and Tβ4 treatments. This same trend was observed for ITGα5 and ITGβ1. ITGα5 binds to the major binding site of fibronectin, while ITGβ1 interacts with the minor binding site, further promoting cell adhesion. While the fibronectin:integrin interaction plays a central role in corneal epithelial cell wound healing and signals the cells to migrate, detachment of epithelial cells from the fibronectin extracellular matrix is also required for active movement. In this regard, we next looked at uPA and uPAR, which promote matrix remodeling and cell activation, and TGF-β1, a regulator of the uPA/uPAR pathway [32]. Both uPA and uPAR levels were significantly decreased in ciprofloxacin-treated animals and further reduced with the combination treatment during infection when compared to PBS. However, only the Tβ4 and combination treatments enhanced expression of the anti-inflammatory molecule, TGF-β1, at 5 days p.i. versus PBS, while the combination treatment significantly increased TGF-β1 at both time points compared to ciprofloxacin alone.   Figure 7. Results showed that all three molecules were significantly reduced in corneas of Tβ4 + ciprofloxacin-treated mice compared to all other treatment groups. Ciprofloxacin treatment was similarly reduced compared to PBS and Tβ4 alone, but remained elevated compared to the adjunct therapy.
Protein levels of fibronectin ( Figure 7A), ITGα5 ( Figure 7B), and uPA ( Figure 7C) were selectively confirmed by ELISA at 3 and 5 days after infection, as shown in Figure 7. Results showed that all three molecules were significantly reduced in corneas of Tβ4 + ciprofloxacin-treated mice compared to all other treatment groups. Ciprofloxacin treatment was similarly reduced compared to PBS and Tβ4 alone, but remained elevated compared to the adjunct therapy.

Tβ4 Influences a Lipid Mediator Circuit Known to Play a Role in Epithelial Wound Healing
To begin elucidating potential mechanisms by which Tβ4 exerts its proresolving effects on the inflammatory response, we assessed expression of lipid mediator biosynthetic cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, as shown in Figure 8. COX-2 ( Figure 8A), an enzyme that is rapidly induced during inflammation, and 5-LOX ( Figure 8B), an enzyme regarded as a marker of inflammatory leukocytes, were significantly decreased after infection in both ciprofloxacin and Tβ4 + ciprofloxacin treatment groups. In contrast, 12-LOX ( Figure 8C), an enzyme associated with the production of proresolving lipid mediators, and 12/15-LOX ( Figure 8D), a key marker of activated epithelial and mucosal proresolving pathways, were significantly enhanced in the Tβ4 + ciprofloxacin-treated animals compared to all other treatment groups; however, 12-LOX and 12/15-LOX were significantly increased compared to PBS controls after ciprofloxacin treatment at 3 days and 3 and 5 days p.i., respectively.

Tβ4 Influences a Lipid Mediator Circuit Known to Play a Role in Epithelial Wound Healing
To begin elucidating potential mechanisms by which Tβ4 exerts its proresolving effects on the inflammatory response, we assessed expression of lipid mediator biosynthetic cyclooxygenase (COX) and lipoxygenase (LOX) enzymes, as shown in Figure 8. COX-2 ( Figure 8A), an enzyme that is rapidly induced during inflammation, and 5-LOX ( Figure 8B), an enzyme regarded as a marker of inflammatory leukocytes, were significantly decreased after infection in both ciprofloxacin and Tβ4 + ciprofloxacin treatment groups. In contrast, 12-LOX ( Figure 8C), an enzyme associated with the production of proresolving lipid mediators, and 12/15-LOX ( Figure 8D), a key marker of activated epithelial and mucosal proresolving pathways, were significantly enhanced in the Tβ4 + ciprofloxacin-treated animals compared to all other treatment groups; however, 12-LOX and 12/15-LOX were significantly increased compared to PBS controls after ciprofloxacin treatment at 3 days and 3 and 5 days p.i., respectively. We next examined select lipid mediator end products, proresolving RvD1 and proinflammatory LTB4, along with their respective receptors, FPR2 and BLT1. As shown in Figure 9, RvD1 ( Figure 9A) protein levels were significantly enhanced in both Tβ4 treatment groups compared to PBS and ciprofloxacin alone at both time points following infection. Following this trend, mRNA levels of the RvD1 receptor, FPR2 ( Figure 9C), were significantly enhanced at both time points after Tβ4 We next examined select lipid mediator end products, proresolving RvD1 and proinflammatory LTB4, along with their respective receptors, FPR2 and BLT1. As shown in Figure 9, RvD1 ( Figure 9A) protein levels were significantly enhanced in both Tβ4 treatment groups compared to PBS and ciprofloxacin alone at both time points following infection. Following this trend, mRNA levels of the RvD1 receptor, FPR2 ( Figure 9C), were significantly enhanced at both time points after Tβ4 treatment-both alone and with ciprofloxacin. Consistent with a reduced inflammatory response, Tβ4 + ciprofloxacin-treated mice revealed a significant reduction in LTB4 ( Figure 9B) compared to all other treatment groups at 3 and 5 days following infection. Ciprofloxacin also reduced LTB4 compared to PBS and Tβ4 alone, but remained significantly higher than the adjunct therapy. BLT1 mRNA expression ( Figure 9D) was significantly reduced in both ciprofloxacin-and Tβ4 + ciprofloxacin-treated mice at both time points as well.

Discussion
Microbial keratitis is one of the most common and destructive of corneal diseases in humans, ultimately culminating in blindness. However, even less severe cases of keratitis develop some level of corneal opacity and compromised visual acuity. Development of corneal opacity is not limited to infection, though, and can be caused by injury and other eye diseases as well. Edema, inflammatory cell infiltration, fibrosis, and vascularization can all contribute to corneal opacity. Development of a therapeutic agent that clears or limits corneal scarring and promotes corneal wound healing without adverse side effects continues to be a major hurdle in the clinical setting and is the focus of the current study. Herein, we examined the therapeutic potential of Tβ4 as a combination therapy with antibiotics, revealing strong clinical implications relevant to human treatment of bacterial keratitis and potentially other conditions that result in corneal opacity.
Using an established murine model of bacterial keratitis, we examined the therapeutic potential of Tβ4 as an adjunct therapy to ciprofloxacin. Although it is best known for its wound-healing capacity, Tβ4 has anti-inflammatory properties [33,34] and is described as a multifunctional "moonlighting" protein [35]. Tβ4 treatment has been shown to modulate proinflammatory mediators

Discussion
Microbial keratitis is one of the most common and destructive of corneal diseases in humans, ultimately culminating in blindness. However, even less severe cases of keratitis develop some level of corneal opacity and compromised visual acuity. Development of corneal opacity is not limited to infection, though, and can be caused by injury and other eye diseases as well. Edema, inflammatory cell infiltration, fibrosis, and vascularization can all contribute to corneal opacity. Development of a therapeutic agent that clears or limits corneal scarring and promotes corneal wound healing without adverse side effects continues to be a major hurdle in the clinical setting and is the focus of the current study. Herein, we examined the therapeutic potential of Tβ4 as a combination therapy with antibiotics, revealing strong clinical implications relevant to human treatment of bacterial keratitis and potentially other conditions that result in corneal opacity.
Using an established murine model of bacterial keratitis, we examined the therapeutic potential of Tβ4 as an adjunct therapy to ciprofloxacin. Although it is best known for its wound-healing capacity, Tβ4 has anti-inflammatory properties [33,34] and is described as a multifunctional "moonlighting" protein [35]. Tβ4 treatment has been shown to modulate proinflammatory mediators in the cornea, as demonstrated in serious wound and burn models [12,34]. However, the exact mechanisms whereby Tβ4 exerts its anti-inflammatory effects are only superficially understood. To this end, we provide evidence suggesting that Tβ4 may carry out its profound effects, in part, through specialized proresolving mediator (SPM) pathway activation. We have previously demonstrated the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease and that a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways are essential in inhibiting inflammation and promoting resolution and tissue restoration [29]. Regulating these key proresolving circuits is a critical element of restoring ocular immune homeostasis. Results indicate that Tβ4 influences 'resolution machinery'-including SPM enzymes (as observed with Tβ4 adjunctive therapy), receptors, and end products. A possible mechanism by which Tβ4 + ciprofloxacin is improving disease outcome compared to antibiotic alone may be through the activation of proresolving autacoid circuits. Though SPMs have been previously implicated in corneal wound healing [36], this is the first report indicating a potential, novel regulatory role for Tβ4 regarding these powerful pathways.
We also provide evidence that proper bacterial clearance is crucial to the anti-inflammatory and wound-healing effects carried out by Tβ4. Previous in vitro work has shown that Tβ4 exhibits moderate antimicrobial activity against P. aeruginosa [18]; however, the in vivo model indicated otherwise. Further, as observed in the Tβ4-only-treated animals, the persistence of bacteria appears to impede the ability of this molecule to shift cytokine production from pro-to anti-inflammatory and prevent Tβ4-induced activation of tissue restoration pathways critical for corneal wound healing, whereas the adjunct treatment indicates potential synergistic bactericidal effects between Tβ4 and ciprofloxacin. In light of these findings, it also suggests a regulatory mechanism of Tβ4 activity that requires removal of the infectious agent and offers an avenue of further investigation.
To develop the most effective treatment possible for corneal infectious diseases, we must focus on two major aspects of pathogenesis-clearance of the invading pathogen and tissue restoration/homeostasis. As mentioned, previous studies have shown that Tβ4 promotes corneal wound healing. Sosne et al. have shown improved wound healing with Tβ4 treatment using dry eye and alkali burn models [33,34]. It has also been reported that Tβ4 promotes laminin-332 synthesis [12], a migration factor for corneal epithelial cells. Although the exact mechanism is not known, Tβ4 has been shown to stabilize HIF-1, known to bind the promotor region of the laminin-332 α3 chain [37,38].We have expanded upon this work to reveal that Tβ4 influences multiple wound-healing pathways, including fibronectin:integrin and uPA:uPAR. During a healthy wound-healing response, the former interaction signals epithelial cells to migrate [39]. As the wound heals, fibronectin and ITGα5β1 expression is known to decrease correlating with what we observed with the adjunctive therapy. Active uPA triggers the degradation of fibronectin, allowing the epithelial cells to move along the extracellular matrix. However, sustained uPA upregulation leads to excessive stromal degradation and corneal inflammation [40], which was observed in PBS controls and even Tβ4-only treatment. IL-1 has also been shown to promote collagen breakdown by keratocytes [40]. Sustained activation of uPA and elevated IL-1β levels in these treatment groups prevents proper migration of epithelial cells and further exacerbates the inflammatory response. TGF-β1 regulates uPA protease activity to restrict excessive degradation of the basement membrane. Tβ4 + ciprofloxacin treatment demonstrates a better balanced combination of uPA and uPAR (and is decreased compared to ciprofloxacin alone) along with increased TGF-β1 during the course of infection. Collectively, these findings suggest that Tβ4 promotes multiple aspects of this intricate cascade of events leading to successful corneal wound healing as illustrated in Figure 10. Additionally, our findings indicate that Tβ4 adjunct therapy holds promise in overcoming limitations of current treatment modalities, namely the immunosuppressive effects associated with steroids. More importantly, we show that Tβ4 works synergistically with ciprofloxacin to enhance therapeutic action-a highly relevant aspect in light of increasing multidrug resistance in the major pathogens known to cause keratitis [1,2]. Taken together, these results strongly support further development of Tβ4 adjunct therapy, as it addresses critical aspects of corneal resolution and bacterial burden that together significantly improve disease outcome.

Conflicts of Interest:
The authors declare no conflict of interest. Additionally, our findings indicate that Tβ4 adjunct therapy holds promise in overcoming limitations of current treatment modalities, namely the immunosuppressive effects associated with steroids. More importantly, we show that Tβ4 works synergistically with ciprofloxacin to enhance therapeutic action-a highly relevant aspect in light of increasing multidrug resistance in the major pathogens known to cause keratitis [1,2]. Taken together, these results strongly support further development of Tβ4 adjunct therapy, as it addresses critical aspects of corneal resolution and bacterial burden that together significantly improve disease outcome.