A Novel Combination Therapy Tß4/VIP Protects against Hyperglycemia-Induced Changes in Human Corneal Epithelial Cells.

Despite the prevalence of diabetic retinopathy, the majority of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, there is limited treatment for diabetes-induced corneal damage. The current project investigates a novel, peptide-based combination therapy, thymosin beta-4 and vasoactive intestinal peptide (Tß4/VIP), against high-glucose-induced damage to the corneal epithelium. Electric cell-substrate impedance sensing (ECIS) was used for real-time monitoring of barrier function and wound healing of human corneal epithelial cells maintained in either normal glucose (5 mM) or high glucose (25 mM) ± Tß4 (0.1%) and VIP (5 nM). Barrier integrity was assessed by resistance, impedance, and capacitance measurements. For the wound healing assay, cell migration was also monitored. Corneal epithelial tight junction proteins (ZO-1, ZO-2, occludin, and claudin-1) were assessed to confirm our findings. Barrier integrity and wound healing were significantly impaired under high-glucose conditions. However, barrier function and cell migration significantly improved with Tß4/VIP treatment. These findings were supported by high-glucose-induced downregulation of tight junction proteins that were effectively maintained similar to normal levels when treated with Tß4/VIP. These results strongly support the premise that Tß4 and VIP work synergistically to protect corneal epithelial cells against hyperglycemia-induced damage. In addition, this work highlights the potential for significant translational impact regarding the treatment of diabetic patients and associated complications of the cornea.

Functional optimization of electric cell-substrate impedance sensing (ECIS) using human corneal epithelial cells.

An intact epithelium is key to maintaining corneal integrity and barrier function which can lead to impaired ocular defense and sight-threatening opacity when compromised. Electrical cell-substrate impedance sensing or ECIS is a non-invasive method to measure real-time cellular behaviors including barrier function and cell migration. The current study uses ECIS technology to assess and optimize human telomerase-immortalized corneal epithelial cells to generate quantifiable measurements that accurately reflect changes in cell behavior in vitro. Five cell densities were assessed in two different media to determine the optimal conditions for monitoring of cellular behavior over time. Parameters of evaluation included: overall impedance (Z), barrier resistance (R), cell capacitance (C), and mathematical modeling of the R data to further generate Rb (the electrical resistance between HUCLs), α (the resistance between the HUCLs and the substrate), and Cm (the capacitance of the cell membrane) measurements. All parameters of assessment strongly indicated DMEM/F12 at 60,000 cells as the optimal condition for ECIS assessment of HUCLs. Furthermore, this work highlights the ability of the sensitive ECIS biosensor technology to comprehensively and quantitatively assess corneal epithelial cell structure and function and the importance of optimizing not only cell density, but choice of media used for in vitro culturing.

A Dual Role for Cysteinyl Leukotriene Receptors in the Pathogenesis of Corneal Infection.

Cysteinyl leukotrienes (CysLTs) have been defined as central mediators of inflammation. Despite our extensive understanding of these bioactive lipid mediators in the pathogenesis of diseases such as asthma, allergic rhinitis, and even neurological disorders, information regarding the eye is markedly lacking. As a result, this study examined the expression profiles of two major CysLT receptors, CysLT1 and CysLT2, in the cornea using experimental mouse models of Pseudomonas aeruginosa-induced keratitis with contrasting outcomes: susceptible C57BL/6 (B6) and resistant BALB/c. Postinfection, disparate levels of CysLT receptors were accompanied by distinct expression profiles for select proinflammatory and anti-inflammatory cell surface markers detected on macrophages and polymorphonuclear neutrophils between the two strains. Further, inhibition of either CysLT receptor converted the disease response of both strains, where corneal perforation was prevented in B6 mice, and BALB/c mice fared significantly worse. In addition, receptor antagonist studies revealed changes in inflammatory cell infiltrate phenotypes and an influence on downstream CysLT receptor signaling pathways. Although the B6 mouse model highlights the established proinflammatory activities related to CysLT receptor activation, results generated from BALB/c mice indicate a protective mechanism that may be essential to disease resolution. Further, basal expression levels of CysLT1 and CysLT2 were significantly higher in uninfected corneas of both mouse strains as opposed to during infection, suggestive of a novel role in homeostatic maintenance within the eye. In light of these findings, therapeutic targeting of CysLT receptors extends beyond inhibition of proinflammatory activities and may impact inflammation resolution, as well as corneal surface homeostasis.

Adjunctive Thymosin Beta-4 Treatment Influences PMN Effector Cell Function during Pseudomonas aeruginosa-Induced Corneal Infection.

Previous work examining the therapeutic efficacy of adjunct thymosin beta 4 (Tß4) to ciprofloxacin for ocular infectious disease has revealed markedly reduced inflammation (inflammatory mediators and innate immune cells) with increased activation of wound healing pathways. Understanding the therapeutic mechanisms of action have further revealed a synergistic effect with ciprofloxacin to enhance bacterial killing along with a regulatory influence over macrophage effector cell function. As a natural extension of the aforementioned work, the current study uses an experimental model of P. aeruginosa-induced keratitis to examine the influence of Tß4 regarding polymorphonuclear leukocyte (PMN/neutrophil) cellular function, contributing to improved disease response. Flow cytometry was utilized to phenotypically profile infiltrating PMNs after infection. The generation of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and PMN apoptosis were investigated to assess the functional activities of PMNs in response to Tß4 therapy. In vitro work using peritoneal-derived PMNs was similarly carried out to verify and extend our in vivo findings. The results indicate that the numbers of infiltrated PMNs into infected corneas were significantly reduced with adjunctive Tß4 treatment. This was paired with the downregulated expression of proinflammatory markers on these cells, as well. Data generated from PMN functional studies suggested that the corneas of adjunctive Tß4 treated B6 mice exhibit a well-regulated production of ROS, NETs, and limited PMN apoptosis. In addition to confirming the in vivo results, the in vitro findings also demonstrated that neutrophil elastase (NE) was unnecessary for NETosis. Collectively, these data provide additional evidence that adjunctive Tß4 + ciprofloxacin treatment is a promising option for bacterial keratitis that addresses both the infectious pathogen and cellular-mediated immune response, as revealed by the current study.

Adjunctive Thymosin Beta-4 Treatment Influences MΦ Effector Cell Function to Improve Disease Outcome in Pseudomonas aeruginosa-Induced Keratitis.

Our previous work has shown that topical thymosin beta 4 (Tß4) as an adjunct to ciprofloxacin treatment reduces inflammatory mediators and inflammatory cell infiltrates (neutrophils/PMN and macrophages/MΦ) while enhancing bacterial killing and wound healing pathway activation in an experimental model of P. aeruginosa-induced keratitis. This study aimed to mechanistically examine how Tß4 influences MΦ function in particular, leading to reduced inflammation and enhanced host defense following P. aeruginosa-induced infection of the cornea. Flow cytometry was conducted to profile the phenotype of infiltrating MΦ after infection, while generation of reactive nitrogen species and markers of efferocytosis were detected to assess functional activity. In vitro studies were performed utilizing RAW 264.7 cells to verify and extend the in vivo findings. Tß4 treatment decreases MΦ infiltration and regulates the activation state in response to infected corneas. MΦ functional data demonstrated that the adjunctive Tß4 treatment group significantly downregulated reactive nitrogen species (RNS) production and efferocytotic activity. In addition, the in vitro studies showed that both Tß4 alone and adjunctive Tß4 treatment influenced MΦ cellular function following LPS stimulation. Collectively, these data provide further evidence that adjunctive Tß4 + ciprofloxacin treatment offers a more efficacious option for treating bacterial keratitis. Not only does the adjunctive therapy address both the infectious pathogen and corneal wound healing response, but it also influences MΦ infiltration, activation, and function, as revealed by the current study.

Antimicrobial Effects of Thymosin Beta-4 and Ciprofloxacin Adjunctive Therapy in Pseudomonas aeruginosa Induced Keratitis.

Prior work has indicated that thymosin beta 4 (Tß4) administered with ciprofloxacin markedly improves disease outcome for Pseudomonas aeruginosa (PA)-induced keratitis. As a result, the goal of the current study was to elucidate mechanisms by which Tß4 mitigates the corneal response; specifically, regarding its bactericidal influence and potential synergy with ciprofloxacin. An in vitro approach was carried out using minimum inhibitory concentration (MIC) assays to assess bactericidal activity against PA. In addition, antimicrobial peptide (AMP) production was evaluated at the mRNA levels using human corneal epithelial cells in response to lipopolysaccharide (LPS) challenge. The results of the MIC assays did not show direct bactericidal activity with Tß4 alone, although ciprofloxacin exhibited significant killing at concentrations far lower than clinically dosed. Tß4, however, displayed an indirect effect on bacterial killing, as shown by an upregulation of AMPs and related molecules. The cumulative data from this study indicate an indirect bactericidal role of Tß4, as well as a synergistic relationship with ciprofloxacin. Furthermore, ciprofloxacin alone was found to influence cellular functions that otherwise have yet to be reported. These results highlight a mechanism of intracellular communication for Tß4 and further strengthen its development as an adjunct therapy with antibiotics for corneal infections.

Application of a Flow-Based Hollow-Fiber Co-Culture System to Study Cellular Influences under Hyperglycemic Conditions.

Elucidation of the basic mechanisms underlying human disease pathogenesis depends on the findings afforded to us through in vivo and in vitro approaches. While there are inherent limitations in any model system, 2D in vitro culture systems tend to be particularly restricted due to their static nature. Here, we adapted a flow-based hollow-fiber cartridge system to better understand the cellular influences of human retinal microvascular endothelial cells and mouse-derived neutrophils under high glucose conditions similar to those observed in diabetes. Analyses by western blot and flow cytometry indicate that pro-inflammatory molecules known to be associated with the pathogenesis of diabetic retinopathy were significantly elevated following high glucose exposure, including VEGF, ICAM-1, and ROS. Changes in mitochondrial potential were also observed. Further, we demonstrate that this innovative system allows for cross-species co-culture as well as long-term culturing conditions. This in vitro modeling system not only mimics the retinal microvasculature, it also allows for the examination of cellular interactions and mechanisms that contribute to diabetic retinopathy, a visually debilitating complication of diabetes.

VIP modulates the ALX/FPR2 receptor axis toward inflammation resolution in a mouse model of bacterial keratitis.

Vasoactive intestinal peptide (VIP) has been shown to regulate corneal inflammation. Formyl peptide receptor 2 (FPR2) is a transmembrane protein belonging to the GPCR family. Ligands include pro-resolving lipids, lipoxin A4 (LXA4) and resolvin D1 (RvD1). The current study focuses on the effect of VIP regarding the FPR2 receptor axis in improving disease outcome in a mouse model of bacterial keratitis. Infection was induced in C57BL/6 (B6) mice using P. aeruginosa (PA) ATCC 19660. Mice received topical treatment (VIP or PBS) 3× daily after infection. Mean clinical scores, bacterial plate counts, Griess and myeloperoxidase (MPO) assays indicate that topical VIP effectively abrogates the disease response. Findings also reveal that VIP influences FPR2 pathway activation independent of archetypal VIP receptors. Exploring the immunoresolving role of FPR2, its ligand RvD1 and related enzymes (5-LOX, 12/15-LOX), our results suggest a mechanism by which VIP treatment influences the disease response in bacterial keratitis, which could offer a therapeutic point of intervention for enhancing this pro-resolving circuit.

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 (Tß4) 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 Tß4 treatment resulted in a significant improvement compared to PBS, Tß4, 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 Tß4 provides a more efficacious approach for bacterial keratitis by addressing both the infectious pathogen and deleterious host response.

Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

Although autacoids primarily derived from the cyclooxygenase-2 and 5-lipoxygenase (LOX) pathways are essential mediators of inflammation, endogenous specialized proresolving mediators (SPMs) act as robust agonists of resolution. SPM biosynthesis is initiated by the conversion of arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid primarily via the 12/15-LOX pathway. Although 12/15-LOX activity is prominent in the cornea, the role of SPM pathway activation during infection remains largely unknown and is the focus of the current study. Pseudomonas keratitis was induced in resistant BALB/c and susceptible C57BL/6 (B6) mice. Biosynthetic pathways for proinflammatory autacoids and SPMs were assessed. Divergent lipid mediator profiles demonstrate the importance of 15-LOX pathways in the pathogenesis of ocular infectious disease. Results indicate that an imbalance of LOX enzymatic pathways contributes to susceptibility observed in B6 mice where deficient activation of SPM circuits, as indicated by reduced 15-hydroxy-eicosatetraenoic acid and 17-hydroxydocosahexaenoic acid levels, prevented transition toward resolution and led to chronic inflammation. In sharp contrast, BALB/c mice demonstrated a well-balanced axis of 5-LOX/12-LOX/15-LOX pathways, resulting in sufficient proresolving bioactive metabolite formation and immune homeostasis. Furthermore, a novel immunoregulatory role for 15-LOX was revealed in inflammatory cells (polymorphonuclear leukocytes and macrophages), which influenced phagocytic activity. These data provide evidence that SPM circuits are essential for host defense during bacterial keratitis.-Carion, T. W., Greenwood, M., Ebrahim, A. S., Jerome, A., Suvas, S., Gronert, K., Berger, E. A. Immunoregulatory role of 15-lipoxygenase in the pathogenesis of bacterial keratitis.

A regulatory role for ß-adrenergic receptors regarding the resolvin D1 (RvD1) pathway in the diabetic retina.

Diabetic retinopathy is a visually debilitating disease with limited treatment options available. Compound 49b, a ß-adrenergic receptor agonist, has been demonstrated to effectively reduce disease pathogenesis associated with diabetic retinopathy. While the exact mechanisms are not fully understood, previous studies have determined that it reduces the pro-inflammatory cytokine, TNF-α, and inhibits apoptosis of the retinal microvasculture. As inflammation becomes more recognized in driving disease pathogenesis, so does the regulation by pro-resolving pathways as therapeutic points of intervention. The current study sought to explore whether Compound 49b had any influence on pro-resolving pathways, thus contributing to improved disease outcome. Using in vivo (animal model of type 1 diabetes) and in vitro (retinal endothelial cells, Müller cells, neutrophils/PMN) techniques, it was determined that high glucose lowers pro-resolving lipid mediator, resolvin D1 (RvD1) levels and differentially alters required enzymes, 5-lipoxygenase (5-LOX), 15-LOX-1 and 15-LOX-2. RvD1 receptors formyl peptide receptor 2 (ALX/FPR2) and G-protein coupled receptor 32 (GPR32) were also downregulated in response to hyperglycemic conditions. Moreover, it was observed that ß-adrenergic receptor activation restored high glucose-induced decreases in both enzyme activity and RvD1 levels observed in vivo and in vitro. The current study is the first to describe a regulatory role for ß-adrenergic receptors on pro-resolving pathways.

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

PURPOSE: The microRNA-183/96/182 cluster (miR-183/96/182) plays important roles in sensory organs. Because the cornea is replete with sensory innervation, we hypothesized that miR-183/96/182 modulates the corneal response to bacterial infection through regulation of neuroimmune interactions. METHODS: Eight-week-old miR-183/96/182 knockout (ko) mice and their wild-type littermates (wt) were used. The central cornea of anesthetized mice was scarred and infected with Pseudomonas aeruginosa (PA), strain 19660. Corneal disease was graded at 1, 3, and 5 days postinfection (dpi). Corneal RNA was harvested for quantitative RT-PCR. Polymorphonuclear neutrophils (PMN) were enumerated by myeloperoxidase assays; the number of viable bacteria was determined by plate counts, and ELISA assays were performed to determine cytokine protein levels. A macrophage (Mϕ) cell line and elicited peritoneal PMN were used for in vitro functional assays. RESULTS: MicroRNA-183/96/182 is expressed in the cornea, and in Mϕ and PMN of both mice and humans. Inactivation of miR-183/96/182 resulted in decreased corneal nerve density compared with wt mice. Overexpression of miR-183/96/182 in Mϕ decreased, whereas knockdown or inactivation of miR-183/96/182 in Mϕ and PMN increased their capacity for phagocytosis and intracellular killing of PA. In PA-infected corneas, ko mice showed decreased proinflammatory neuropeptides such as substance P and chemoattractant molecules, MIP-2, MCP1, and ICAM1; decreased number of PMN at 1 and 5 dpi; increased viable bacterial load at 1 dpi, but decreased at 5 dpi; and markedly decreased corneal disease. CONCLUSIONS: MicroRNA-183/96/182 modulates the corneal response to bacterial infection through its regulation of corneal innervation and innate immunity.

VIP protects human retinal microvascular endothelial cells against high glucose-induced increases in TNF-α and enhances RvD1.

PURPOSE: The purpose of our study was to evaluate the therapeutic effect of VIP on human retinal endothelial cells (HREC) under high glucose conditions. Diabetes affects almost 250 million people worldwide. Over 40% of diabetics are expected to develop diabetic retinopathy, which remains the leading cause of visual impairment/blindness. Currently, treatment is limited to late stages of retinopathy with no options available for early stages. To this end, the purpose of the current study is to evaluate the therapeutic effect of vasoactive intestinal peptide (VIP) on HREC under high glucose conditions. METHODS: Primary HREC were cultured in normal (5mM) or high (25mM) glucose medium +/- VIP treatment. Protein levels of TNF-α, resolvin D1 (RvD1), formyl peptide receptor 2 (FPR2), G protein-coupled receptor 32 (GPR32), VEGF, and VIP receptors, VPAC1 and VPAC2 were measured. RESULTS: High glucose-induced changes in TNF-α and RvD1 were restored to control levels with VIP treatment. RvD1 receptors, ALX/FPR2 and GPR32, were partially rescued with VIP treatment. VPAC2 expression appeared to be the major receptor involved in VIP signaling in HREC, as VPAC1 receptor was not detected. In addition, VIP did not induce HREC secretion of VEGF under high glucose conditions. CONCLUSIONS: Our results demonstrate that VIP's therapeutic effect on HREC, occurs in part, through the balance between the pro-inflammatory cytokine, TNF-α, and the pro-resolving mediator, RvD1. Although VPAC1 is considered the major VIP receptor, VPAC2 is predominantly expressed on HREC under both normal and high glucose conditions.

ß-Adrenergic receptor agonist, compound 49b, inhibits TLR4 signaling pathway in diabetic retina.

Diabetic retinopathy has recently become associated with complications similar to chronic inflammatory diseases. Although it is clear that tumor necrosis factor-α is increased in diabetes, the role of innate immunity is only recently being investigated. As such, we hypothesized that diabetes would increase Toll-like receptor 4 (TLR4) signaling, which could be inhibited by a ß-adrenergic receptor agonist (Compound 49b) previously shown to have anti-inflammatory actions. In order to investigate ß-adrenergic receptor signaling and TLR4 in the diabetic retina, streptozotocin-injected diabetic mice, as well as human primary retinal endothelial cells (RECs) and rat retinal Müller cells (rMC-1) exposed to high glucose (25 mM), were treated with a novel ß-adrenergic receptor agonist, Compound 49b (50 nM), or phosphate-buffered saline (control). TLR4 and its downstream signaling partners (MyD88, IL-1 receptor-associated kinase 1, TNF receptor-associated factor 6 and total and phosphorylated nuclear factor-κB) were examined. In addition, we assessed high-mobility group box 1 (HMGB1) protein levels. Our data showed that diabetes or high-glucose culture conditions significantly increased TLR4 and downstream signaling partners. Compound 49b was able to significantly reduce TLR4 and related molecules in the diabetic animal and retinal cells. HMGB1 was significantly increased in RECs and Müller cells grown in high-glucose culture conditions, which was subsequently reduced with Compound 49b treatment. Our findings suggest that high glucose may increase HMGB1 levels that lead to increased TLR4 signaling. Compound 49b significantly inhibited this pathway, providing a potential mechanism for its protective actions.

Efficacy of VIP as Treatment for Bacteria-Induced Keratitis Against Multiple Pseudomonas aeruginosa Strains.

PURPOSE: Previous studies have demonstrated the efficacy of vasoactive intestinal peptide (VIP) treatment in regulating inflammation following bacterial keratitis induced by the P. aeruginosa strain 19660. However, in the current study we assessed whether disease outcome is specific to 19660 or if VIP treatment is effective against multiple P. aeruginosa strains. METHODS: B6 mice received daily IP injections of VIP from -1 through 5 days post injection (p.i.). Control mice were similarly injected with PBS. Corneal infection was induced using PA 19660, PAO1 or KEI 1025. Disease response was documented and bacterial plate counts and myeloperoxidase assays were performed. Expression of select inflammatory mediators as well as enzymes associated with lipid mediator production was assessed after VIP treatment. KEI 1025 was characterized by cytotoxicity and invasion assays and then confirmed for ExoS/ExoU expression. RESULTS: VIP treatment converted the susceptible response to resistant for the three P. aeruginosa strains tested. Disease response was significantly reduced with no corneal perforation. Anti-inflammatory mediators were enhanced after VIP treatment, while pro-inflammatory molecules were reduced compared to controls. Furthermore, VIP reduced inflammatory cell persistence in the cornea after infection with each of the P. aeruginosa strains. CONCLUSIONS: VIP treatment is effective at ameliorating disease pathogenesis for multiple P. aeruginosa strains, both cytotoxic and invasive. This study is also the first to indicate a possible role for VIP regarding lipid mediator expression in the eye. In addition, the clinical isolate, KEI 1025, was characterized as an invasive strain. Overall, this study strengthens the preclinical development of VIP as a therapeutic agent for ocular infectious disease.

Functional analysis of the CpsA protein of Streptococcus agalactiae.

Streptococcal pathogens, such as the group B streptococcus (GBS) Streptococcus agalactiae, are an important cause of systemic disease, which is facilitated in part by the presence of a polysaccharide capsule. The CpsA protein is a putative transcriptional regulator of the capsule locus, but its exact contribution to regulation is unknown. To address the role of CpsA in regulation, full-length GBS CpsA and two truncated forms of the protein were purified and analyzed for DNA-binding ability. Assays demonstrated that CpsA is able to bind specifically to two putative promoters within the capsule operon with similar affinity, and full-length protein is required for specificity. Functional characterization of CpsA confirmed that the ΔcpsA strain produced less capsule than did the wild type and demonstrated that the production of full-length CpsA or the DNA-binding region of CpsA resulted in increased capsule levels. In contrast, the production of a truncated form of CpsA lacking the extracellular LytR domain (CpsA-245) in the wild-type background resulted in a dominant-negative decrease in capsule production. GBS expressing CpsA-245, but not the ΔcpsA strain, was attenuated in human whole blood. However, the ΔcpsA strain showed significant attenuation in a zebrafish infection model. Furthermore, chain length was observed to be variable in a CpsA-dependent manner, but could be restored to wild-type levels when grown with lysozyme. Taken together, these results suggest that CpsA is a modular protein influencing multiple regulatory functions that may include not only capsule synthesis but also cell wall associated factors.