Mechanisms of PM10 Disruption of the Nrf2 Pathway in Cornea.

We have previously shown that PM10 exposure causes oxidative stress and reduces Nrf2 protein levels, and SKQ1 pre-treatment protects against this damage in human corneal epithelial cells (HCE-2). The current study focuses on uncovering the mechanisms underlying acute PM10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 µg/mL PM10. Cell viability, oxidative stress markers, programmed cell death, DNA damage, senescence markers, and pro-inflammatory cytokines were analyzed. Nrf2 cellular location and its transcriptional activity were determined. Effects of the Nrf2 inhibitor ML385 were similarly evaluated. Data showed that PM10 decreased cell viability, Nrf2 transcriptional activity, and mRNA levels of antioxidant enzymes, but increased p-PI3K, p-NFκB, COX-2, and iNOS proteins levels. Additionally, PM10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and ß-galactosidase (ß-gal) staining, which confirmed the senescence. SKQ1 pre-treatment reversed these effects. ML385 lowered the Nrf2 protein levels and mRNA levels of its downstream targets. ML385 also abrogated the protective effects of SKQ1 against PM10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.

Airborne Exposure of the Cornea to PM10 Induces Oxidative Stress and Disrupts Nrf2 Mediated Anti-Oxidant Defenses.

The purpose of this study is to test the effects of whole-body animal exposure to airborne particulate matter (PM) with an aerodynamic diameter of <10 µm (PM10) in the mouse cornea and in vitro. C57BL/6 mice were exposed to control or 500 µg/m3 PM10 for 2 weeks. In vivo, reduced glutathione (GSH) and malondialdehyde (MDA) were analyzed. RT-PCR and ELISA evaluated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and inflammatory markers. SKQ1, a novel mitochondrial antioxidant, was applied topically and GSH, MDA and Nrf2 levels were tested. In vitro, cells were treated with PM10 ± SKQ1 and cell viability, MDA, mitochondrial ROS, ATP and Nrf2 protein were tested. In vivo, PM10 vs. control exposure significantly reduced GSH, corneal thickness and increased MDA levels. PM10-exposed corneas showed significantly higher mRNA levels for downstream targets, pro-inflammatory molecules and reduced Nrf2 protein. In PM10-exposed corneas, SKQ1 restored GSH and Nrf2 levels and lowered MDA. In vitro, PM10 reduced cell viability, Nrf2 protein, and ATP, and increased MDA, and mitochondrial ROS; while SKQ1 reversed these effects. Whole-body PM10 exposure triggers oxidative stress, disrupting the Nrf2 pathway. SKQ1 reverses these deleterious effects in vivo and in vitro, suggesting applicability to humans.

HIF-1α is essential for effective PMN bacterial killing, antimicrobial peptide production and apoptosis in Pseudomonas aeruginosa keratitis.

Hypoxia-inducible factor (HIF)-1α, is a transcription factor that controls energy metabolism and angiogenesis under hypoxic conditions, and a potent regulator of innate immunity. The studies described herein examined the role of HIF-1α in disease resolution in BALB/c (resistant, cornea heals) mice after ocular infection with Pseudomonas (P.) aeruginosa. Furthermore, the current studies focused on the neutrophil (PMN), the predominant cell infiltrate in keratitis. Using both siRNA and an antagonist (17-DMAG), the role of HIF-1α was assessed in P. aeruginosa-infected BALB/c mice. Clinical score and slit lamp photography indicated HIF-1α inhibition exacerbated disease and corneal destruction. Real time RT-PCR, immunohistochemistry, ELISA, Greiss and MPO assays, bacterial load, intracellular killing, phagocytosis and apoptosis assays further tested the regulatory role of HIF-1α. Despite increased pro-inflammatory cytokine expression and increased MPO levels after knocking down HIF-1α expression, in vivo studies revealed a decrease in NO production and higher bacterial load. In vitro studies using PMN provided evidence that although inhibition of HIF-1α did not affect phagocytosis, both bacterial killing and apoptosis were significantly affected, as was production of antimicrobial peptides. Overall, data provide evidence that inhibition of HIF-1α converts a normally resistant disease response to susceptible (corneal thinning and perforation) after induction of bacterial keratitis. Although this inhibition does not appear to affect PMN transmigration or phagocytosis, both in vivo and in vitro approaches indicate that the transcriptional factor is essential for effective bacterial killing, apoptosis and antimicrobial peptide production.

Mammalian target of rapamycin regulates IL-10 and resistance to Pseudomonas aeruginosa corneal infection.

IL-10 is important in the resistance response of BALB/c mice to experimental Pseudomonas aeruginosa corneal infection. However, the cellular mechanisms by which this anti-inflammatory cytokine is regulated remain unknown. Because the mammalian target of rapamycin (mTOR) regulates IL-10 in other disease models, the present study tested its role in bacterial keratitis. After infection, corneas of rapamycin versus control-treated BALB/c mice showed worsened disease, and real-time RT-PCR confirmed that mTOR mRNA levels were significantly decreased. Rapamycin treatment also increased clinical score, polymorphonuclear neutrophil (PMN) infiltration (determined by myeloperoxidase assay), and bacterial load, but it diminished PMN bactericidal activity. Inhibition of mTOR also led to elevated mRNA and protein levels of IL-12p40, matrix metalloproteinase 9, and inducible NO synthase, whereas mRNA and protein levels of IL-10, its regulator/effector STAT-3, and suppressor of cytokine signaling 3 (a proinflammatory cytokine regulator) were decreased. Furthermore, mTOR inhibition reduced levels of proapoptotic caspase-3 and increased levels of B cell lymphoma-2 (antiapoptotic), indicative of delayed apoptosis. mTOR inhibition also altered genes related to TLR signaling, including elevation of TLR4, TLR5, and IL-1R1, with decreases in IL-1R-associated kinase 1 and an inhibitor of NF-κB, NF-κB inhibitor-like 1. Rapamycin treatment also increased levels of IFN-γ and CCAAT/enhancer binding protein, ß, a gene that regulates expression of preprotachykinin-A (the precursor of substance P). Collectively, these data, as well as a rescue experiment using rIL-10 together with rapamycin, which decreased PMN in cornea, provide concrete evidence that mTOR regulates IL-10 in P. aeruginosa-induced bacterial keratitis and is critical to balancing pro- and anti-inflammatory events, resulting in better disease outcome.

Vasoactive intestinal peptide downregulates proinflammatory TLRs while upregulating anti-inflammatory TLRs in the infected cornea.

TLRs recognize microbial pathogens and trigger an immune response, but their regulation by neuropeptides, such as vasoactive intestinal peptide (VIP), during Pseudomonas aeruginosa corneal infection remains unexplored. Therefore, C57BL/6 (B6) mice were injected i.p. with VIP, and mRNA, protein, and immunostaining assays were performed. After VIP treatment, PCR array and real-time RT-PCR demonstrated that proinflammatory TLRs (conserved helix-loop-helix ubiquitous kinase, IRAK1, TLR1, TLR4, TLR6, TLR8, TLR9, and TNFR-associated factor 6) were downregulated, whereas anti-inflammatory TLRs (single Ig IL-1-related receptor [SIGIRR] and ST2) were upregulated. ELISA showed that VIP modestly downregulated phosphorylated inhibitor of NF-κB kinase subunit α but upregulated ST2 ~2-fold. SIGIRR was also upregulated, whereas TLR4 immunostaining was reduced in cornea; all confirmed the mRNA data. To determine whether VIP effects were cAMP dependent, mice were injected with small interfering RNA for type 7 adenylate cyclase (AC7), with or without VIP treatment. After silencing AC7, changes in mRNA levels of TLR1, TNFR-associated factor 6, and ST2 were seen and unchanged with addition of VIP, indicating that their regulation was cAMP dependent. In contrast, changes were seen in mRNA levels of conserved helix-loop-helix ubiquitous kinase, IRAK1, 2, TLR4, 9 and SIGIRR following AC7 silencing alone; these were modified by VIP addition, indicating their cAMP independence. In vitro studies assessed the effects of VIP on TLR regulation in macrophages and Langerhans cells. VIP downregulated mRNA expression of proinflammatory TLRs while upregulating anti-inflammatory TLRs in both cell types. Collectively, the data provide evidence that VIP downregulates proinflammatory TLRs and upregulates anti-inflammatory TLRs and that this regulation is both cAMP dependent and independent and involves immune cell types found in the infected cornea.

Impact of Airborne Exposure to PM10 Increases Susceptibility to P. aeruginosa Infection.

The effects of exposure to airborne particulate matter with a size of 10 µm or less (PM10) on C57BL/6 mouse corneas, their response to Pseudomonas aeruginosa (PA) infection, and the protective effects of SKQ1 were determined. C57BL/6 mouse corneas receiving PBS or SKQ1 were exposed to control (air) or PM10 for 2 weeks, infected, and the disease was documented by clinical score, PMN quantitation, bacterial plate count, RT-PCR and Western blot. PBS-treated, PM10-exposed corneas did not differ at 1 day postinfection (dpi), but exhibited earlier (3 dpi) corneal thinning compared to controls. By 3 dpi, PM10 significantly increased corneal mRNA levels of several pro-inflammatory cytokines, but decreased IL-10, NQO1, GR1, GPX4, and Nrf2 over control. SKQ1 reversed these effects and Western blot selectively confirmed the RT-PCR results. PM10 resulted in higher viable bacterial plate counts at 1 and 3 dpi, but SKQ1 reduced them at 3 dpi. PM10 significantly increased MPO in the cornea at 3 dpi and was reduced by SKQ1. SKQ1, used as an adjunctive treatment to moxifloxacin, was not significantly different from moxifloxacin alone. Exposure to PM10 increased the susceptibility of C57BL/6 to PA infection; SKQ1 significantly reversed these effects, but was not effective as an adjunctive treatment.

Host-microbe interactions in cornea.

Corneal infections result through interaction between microbes and host innate immune receptors. Damage to the cornea occurs as a result of microbial virulence factors and is often exacerbated by lack of a controlled host immune response; the latter contributing to bystander damage to corneal structure. Understanding mechanisms involved in host microbial interactions is critical to development of novel therapeutic targets, ultimate control of microbial pathogenesis, and restoration of tissue homeostasis. Studies on these interactions continue to provide exciting findings directly related to this ultimate goal.

PM10 and Pseudomonas aeruginosa: effects on corneal epithelium.

Purpose: In vivo data indicate that mouse corneas exposed to PM10 showed early perforation and thinning after infection with Pseudomonas aeruginosa. To understand the mechanisms underlying this finding, we tested the effects of PM10 and the mitochondria targeted anti-oxidant SKQ1 in immortalized human corneal epithelial cells (HCET) that were challenged with Pseudomonas aeruginosa strain 19660. Methods: Mouse corneas were infected with strain 19660 after a 2 week whole-body exposure to PM10 or control air and assessed by clinical scores, slit lamp photography and western blot. HCET were exposed to 100µg/ml PM10 for 24h before challenge with strain 19660 (MOI 20). A subset of cells were pre-treated with 50nM SKQ1 for 1h before PM10 exposure. Phase contrast microscopy was used to study cell morphology, cell viability was measured by an MTT assay, and ROS by DCFH-DA. Levels of pro-inflammatory markers and anti-oxidant enzymes were evaluated by RT-PCR, western blot and ELISA. Reduced glutathione (GSH) and malondialdehyde (MDA) levels were evaluated by assay kits. Results: In vivo, whole body exposure to PM10 vs. control air exposed mouse corneas showed early perforation and/or corneal thinning at 3 days post infection, accompanied by increased TNF-α and decreased SOD2 protein levels. In vitro, PM10 induced a dose dependent reduction in cell viability of HCET and significantly increased mRNA levels of pro-inflammatory molecules compared to control. Exposure to PM10 before bacterial challenge further amplified the reduction in cell viability and GSH levels. Furthermore, PM10 exposure also exacerbated the increase in MDA and ROS levels and phase contrast microscopy revealed more rounded cells after strain 19660 challenge. PM10 exposure also further increased the mRNA and protein levels of pro-inflammatory molecules, while anti-inflammatory IL-10 was decreased. SKQ1 reversed the rounded cell morphology observed by phase contrast microscopy, increased levels of MDA, ROS and pro-inflammatory molecules, and restored IL-10. Conclusions: PM10 induces decreased cell viability, oxidative stress and inflammation in HCET and has an additive effect upon bacterial challenge. SKQ1 protects against oxidative stress and inflammation induced by PM10 after bacterial challenge by reversing these effects. The findings provide insight into mechanisms underlying early perforation and thinning observed in infected corneas of PM10 exposed mice.

Glycyrrhizin Interacts with TLR4 and TLR9 to Resolve P. aeruginosa Keratitis.

This study tests the mechanism(s) of glycyrrhizin (GLY) protection against P. aeruginosa keratitis. Female C57BL/6 (B6), TLR4 knockout (TLR4KO), myeloid specific TLR4KO (mTLR4KO), their wildtype (WT) littermates, and TLR9 knockout (TLR9KO) mice were infected with P. aeruginosa KEI 1025 and treated with GLY or PBS onto the cornea after infection. Clinical scores, photography with a slit lamp, RT-PCR and ELISA were used. GLY effects on macrophages (Mϕ) and polymorphonuclear neutrophils (PMN) isolated from WT and mTLR4KO and challenged with KEI 1025 were also tested. Comparing B6 and TLR4KO, GLY treatment reduced clinical scores and improved disease outcome after infection and decreased mRNA expression levels in cornea for TLR4, HMGB1, and RAGE in B6 mice. TLR9 mRNA expression was significantly reduced by GLY in both mouse strains after infection. GLY also significantly reduced HMGB1 (B6 only) and TLR9 protein (both B6 and TLR4KO). In TLR9KO mice, GLY did not significantly reduce clinical scores and only slightly improved disease outcome after infection. In these mice, GLY significantly reduced TLR4, but not HMGB1 or RAGE mRNA expression levels after infection. In contrast, in the mTLR4KO and their WT littermates, GLY significantly reduced corneal disease, TLR4, TLR9, HMGB1, and RAGE corneal mRNA expression after infection. GLY also significantly reduced TLR9 and HMGB1 corneal protein levels in both WT and mTLR4KO mice. In vitro, GLY significantly lowered mRNA expression levels for TLR9 in both Mϕ and PMN isolated from mTLR4KO or WT mice after incubation with KEI 1025. In conclusion, we provide evidence to show that GLY mediates its effects by blocking TLR4 and TLR9 signaling pathways and both are required to protect against disease.

Beta-defensin-2 promotes resistance against infection with P. aeruginosa.

Corneal infection with Pseudomonas aeruginosa results in corneal perforation in susceptible C57BL/6 (B6) mice, but not in resistant BALB/c mice. To explore the role of two important defensins, murine beta-defensin-1 (mBD1) and mBD2, in the ocular immune defense system, their mRNA and protein expression levels were tested by real-time RT-PCR and Western blot, respectively. mRNA, protein, and immunostaining data demonstrated that both mBD1 and mBD2 were constitutively expressed in normal BALB/c and B6 corneas, and they were disparately up-regulated in BALB/c (more) vs B6 (less) corneas after infection. To determine whether either defensin played a role in host resistance, BALB/c mice were treated with either mBD1 or mBD2 small interfering RNA by subconjunctival injection together with topical application. Increased corneal opacity and worsened disease were displayed after knockdown of mBD2 but not of mBD1. mBD2 silencing also increased bacterial counts and polymorphonuclear neutrophil infiltration in BALB/c corneas. Real-time RT-PCR data further demonstrated that mBD2, not mBD1, differentially modulated mRNA expression of proinflammatory cytokines/molecules such as IFN-gamma, MIP-2, IL-1beta, TNF-alpha, IL-6, and inducible NO synthase; TLR signaling molecules, including TLR2, TLR4, TLR9, and MyD88; and the transcription factor NF-kappaB. Additionally, in vivo studies indicated that mBD2 silencing enhanced corneal nitrite levels and NF-kappaB activation. Collectively, the data provide evidence that mBD2, but not mBD1, is required for host resistance against P. aeruginosa-induced corneal infection.

Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

Defensins play an important role in both innate and adaptive immunity due to their antimicrobial, regulatory, and chemotactic effects. Nonetheless, the role of murine beta-defensins (mBD) 3 and 4, the murine homologs of human beta-defensins (hBD) 2 and 3, remains unknown in Pseudomonas aeruginosa keratitis. This study explored their role in corneal infection and potential synergy with mBD2, a defensin associated with better outcome in this disease. Immunostaining and real-time RT-PCR data demonstrated that mBD3 and mBD4 expression was inducible and differentially regulated in the infected cornea of resistant BALB/c vs susceptible C57BL/6 (B6) mice. Knockdown studies using small interfering RNA treatment indicated that mBD3, but not mBD4, is required in ocular defense. Moreover, in vivo studies demonstrated individual and combined effects of mBD2 and mBD3 that modulate bacterial load, polymorphonuclear neutrophil (PMN) infiltration, and production of IFN-gamma, MIP-2, IL-1beta, TNF-alpha, inducible NO synthase (iNOS), TLR2, TLR4, MyD88, and NF-kappaB. Most notably, bacterial load was increased at 5 days postinfection by silencing either mBD2 or mBD3, but it was elevated at both 1 and 5 days postinfection when silencing both defensins. PMN infiltration was increased at 1 day postinfection by silencing both defensins or mBD3, but not mBD2 alone. iNOS expression was elevated by silencing mBD2, but it was reduced after silencing mBD3 or both defensins. Additionally, cell sources of mBD2 (macrophages, PMN and fibroblasts) and mBD3 (PMN) in corneal stroma were identified by dual label immunostaining after infection. Collectively, the data provide evidence that mBD2 and mBD3 together promote resistance against corneal infection.

Ocular Effects of Glycyrrhizin at Acidic and Neutral pH.

PURPOSE: To test the effects of acidic vs. neutral pH glycyrrhizin (GLY) on the unwounded and wounded normal mouse cornea and after infection with Pseudomonas aeruginosa isolates KEI 1025 and multidrug-resistant MDR9. METHODS: Acidic or neutral GLY vs. phosphate-buffered saline (PBS) was topically applied to normal or wounded corneas of C57BL/6 mice. In unwounded corneas, goblet cells and corneal nerves were stained and quantitated. After wounding, corneas were fluorescein stained and photographed using a slit lamp. Mice also were infected with KEI 1025 or MDR9 and the protective effects of GLY pH evaluated comparatively. RESULTS: In the unwounded cornea, application of acidic or neutral GLY vs. PBS reduced the number of bulbar conjunctival goblet cells but did not alter corneal nerve density. Similar application of GLY to scarified corneas delayed wound closure. After KEI 1025 infection, none of the GLY vs. PBS-treated corneas perforated; GLY treatment also decreased plate count (neutral pH more effective) and reduced MPO and several cytokines. Similarly, for MDR9, GLY at either pH was protective and also enhanced the effects of moxifloxacin to which MDR9 is resistant. CONCLUSION: Acidic or neutral pH GLY decreased goblet cell number but had no effect on nerve density. After corneal wounding, GLY at either pH (1) delayed wound closure and, (2) after infection, decreased keratitis when used alone or in combination with moxifloxacin. Neutral pH did not alter the therapeutic effect of GLY and would be preferred if used clinically.

Effects of Glycyrrhizin Treatment on Diabetic Cornea.

Purpose: To test how glycyrrhizin (GLY) affects mouse corneal epithelial cells (MCEC) and the diabetic murine cornea. Methods: Viability of MCEC grown under normal or high glucose (HG) with/without GLY was tested by an MTT assay. In addition, C57BL/6 mice were injected with streptozotocin and a subset of control and diabetic mice received GLY in their drinking water. mRNA and protein levels of proinflammatory and oxidative stress molecules were tested by reverse transcription-polymerase chain reaction (RT-PCR) in both models. Ex vivo studies using human diabetic versus control corneas analyzed proinflammatory and oxidative stress markers using RT-PCR and enzyme-linked immunosorbent assay. Results: GLY protected against loss of cell viability induced by HG and significantly reduced HMGB1, IL-1ß, TLR2, TLR4, NLRP3, COX2, SOD2, HO-1, GPX2, and GR1. In vivo, corneas of GLY-treated diabetic mice showed significantly decreased mRNA expression for CXCL2, iNOS, and all molecules listed above; GLY also lowered HMGB1 and IL-1ß proteins (in vitro and in vivo). Ex vivo studies using diabetic human corneas revealed elevated mRNA levels of inflammatory and oxidative stress molecules (as listed above for in vivo) versus normal age-matched controls. Protein levels for HMGB1 and IL-1ß also were elevated in diabetic human versus control corneas. Conclusions: The data provide evidence that GLY treatment attenuates inflammation and oxidative stress in vitro in MCEC and in vivo in the cornea of diabetic mice. Ex vivo data support the similarities of proinflammatory and oxidative stress data in mouse compared to human, suggesting that GLY treatment would have relevancy to patient care.

The miR-183/96/182 Cluster Regulates the Functions of Corneal Resident Macrophages.

Tissue-resident macrophages (ResMϕ) play important roles in the normal development and physiological functions as well as tissue repair and immune/inflammatory response to both internal and external insults. In cornea, ResMϕ are critical to the homeostasis and maintenance, wound healing, ocular immune privilege, and immune/inflammatory response to injury and microbial infection. However, the roles of microRNAs in corneal ResMϕ are utterly unknown. Previously, we demonstrated that the conserved miR-183/96/182 cluster (miR-183/96/182) plays important roles in sensory neurons and subgroups of both innate and adaptive immune cells and modulates corneal response to bacterial infection. In this study, we provide direct evidence that the mouse corneal ResMϕ constitutively produce both IL-17f and IL-10. This function is regulated by miR-183/96/182 through targeting Runx1 and Maf, key transcriptional regulators for IL-17f and IL-10 expression, respectively. In addition, we show that miR-183/96/182 has a negative feedback regulation on the TLR4 pathway in mouse corneal ResMϕ. Furthermore, miR-183/96/182 regulates the number of corneal ResMϕ. Inactivation of miR-183/96/182 in mouse results in more steady-state corneal resident immune cells, including ResMϕ, and leads to a simultaneous early upregulation of innate IL-17f and IL-10 production in the cornea after Pseudomonas aeruginosa infection. Its multiplex regulations on the simultaneous production of IL-17f and IL-10, TLR4 signaling pathway and the number of corneal ResMϕ place miR-183/96/182 in the center of corneal innate immunity, which is key to the homeostasis of the cornea, ocular immune privilege, and the corneal response to microbial infections.

Airborne Particulates Affect Corneal Homeostasis and Immunity.

Purpose: To determine the effects of airborne particulate matter (PM) <2.5 µm in vitro and on the normal and Pseudomonas aeruginosa (PA)-infected cornea. Methods: An MTT viability assay tested the effects of PM2.5 on mouse corneal epithelial cells (MCEC) and human corneal epithelial cells (HCET). MCEC were tested for reactive oxygen species using a 2',7'-dichlorodihydrofluorescein assay; RT-PCR determined mRNA levels of inflammatory and oxidative stress markers in MCEC (HMGB1, toll-like receptor 2, IL-1ß, CXCL2, GPX1, GPX2, GR1, superoxide dismutase 2, and heme oxygenase 1) and HCET (high mobility group box 1, CXCL2, and IL-1ß). C57BL/6 mice also were infected and after 6 hours, the PM2.5 was topically applied. Disease was graded by clinical score and evaluated by histology, plate count, myeloperoxidase assay, RT-PCR, ELISA, and Western blot. Results: After PM2.5 (25-200 µg/mL), 80% to 90% of MCEC and HCET were viable and PM exposure increased reactive oxygen species in MCEC and mRNA expression levels for inflammatory and oxidative stress markers in mouse and human cells. In vivo, the cornea of PA+PM2.5 exposed mice exhibited earlier perforation over PA alone (confirmed histologically). In cornea, plate counts were increased after PA+PM2.5, whereas myeloperoxidase activity was significantly increased after PA+PM2.5 over other groups. The mRNA levels for several proinflammatory and oxidative stress markers were increased in the cornea in the PA+PM2.5 over other groups; protein levels were elevated for high mobility group box 1, but not toll-like receptor 4 or glutathione reductase 1. Uninfected corneas treated with PM2.5 did not differ from normal. Conclusions: PM2.5 triggers reactive oxygen species, upregulates mRNA levels of oxidative stress, inflammatory markers, and high mobility group box 1 protein, contributing to perforation in PA-infected corneas.

Effects of Glycyrrhizin on Multi-Drug Resistant Pseudomonas aeruginosa.

The effects of glycyrrhizin (GLY) on multi-drug resistant (MDR) systemic (MDR9) vs. ocular (B1045) Pseudomonas aeruginosa clinical isolates were determined. Proteomes of each isolate with/without GLY treatment were profiled using liquid chromatography mass spectrometry (LC-MS/MS). The effect of GLY on adherence of MDR isolates to immortalized human (HCET) and mouse (MCEC) corneal epithelial cells, and biofilm and dispersal was tested. Both isolates were treated with GLY (0.25 minimum inhibitory concentration (MIC), 10 mg/mL for MDR9 and 3.75 mg/mL for B1045) and subjected to proteomic analysis. MDR9 had a greater response to GLY (51% of identified proteins affected vs. <1% in B1045). In MDR9 vs. controls, GLY decreased the abundance of proteins for: antibiotic resistance, biofilm formation, and type III secretion. Further, antibiotic resistance and type III secretion proteins had higher control abundances in MDR9 vs. B1045. GLY (5 and 10 mg/mL) significantly reduced binding of both isolates to MCEC, and B1045 to HCET. MDR9 binding to HCET was only reduced at 10 mg/mL GLY. GLY (5 and 10 mg/mL) enhanced dispersal for both isolates, at early (6.5 h) but not later times (24-72 h). This study provides evidence that GLY has a greater effect on the proteome of MDR9 vs. B1045, yet it was equally effective at disrupting adherence and early biofilm dispersal.

Glycyrrhizin Use for Multi-Drug Resistant Pseudomonas aeruginosa: In Vitro and In Vivo Studies.

Purpose: Our purpose was to test glycyrrhizin (GLY) effects and ciprofloxacin interactions on multidrug resistant (MDR) isolates of Pseudomonas aeruginosa in vitro and in vivo in a mouse model of keratitis. Methods: A Hardy-disk tested antibiotic sensitivity of isolates MDR9 (nonocular) and B1045 (ocular). GLY MIC (both isolates) and ciprofloxacin was determined spectrophotometrically. A live/dead assay using confocal microscopy and plate count, tested GLY effects on bacterial permeabilization/viability. Proteomics profiled bacterial efflux pumps (MDR9 vs. PAO1); RT-PCR comparatively tested GLY effects on their mRNA expression levels. The activity of efflux pumps was tested using ethidium bromide (EB); and scanning electron microscopy (SEM) visualized the effects of GLY treatment of bacteria. A combination of GLY and ciprofloxacin was tested in C57BL/6 mice (begun 18 hours after infection) and disease scored, photographed and MPO and plate counts done. Results: MDR9 was resistant to 6/12 and B1045 to 7/12 antibiotics (both to ciprofloxacin). MIC GLY for MDR9 was 40 mg/mL and 15 mg/mL for B1045. Ciprofloxacin MIC (32 µg/mL) was reduced 2-fold to 16 µg/mL when ciprofloxacin and GLY were combined. GLY altered bacterial membrane permeability and reduced viability. Proteomics revealed increased efflux pumps in MDR9 versus PAO1; GLY reduced their mRNA expression levels and EB suggested decreased activity. In C57BL/6 mice, treatment with GLY and ciprofloxacin versus ciprofloxacin, significantly reduced clinical scores, plate count, and MPO. Conclusions: GLY decreases MDR by: altering bacterial parameters, including viability and efflux pump activity. In vivo, it increases the effectiveness of ciprofloxacin, reducing ocular disease, plate count, and MPO activity.

The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa.

Macrophages (Mϕ) are an important component of the innate immune system; they play critical roles in the first line of defense to pathogen invasion and modulate adaptive immunity. MicroRNAs (miRNAs) are a newly recognized, important level of gene expression regulation. However, their roles in the regulation of Mϕ and the immune system are still not fully understood. In this report, we provide evidence that the conserved miR-183/96/182 cluster (miR-183/96/182) modulates Mϕ function in their production of reactive nitrogen (RNS) and oxygen species (ROS) and their inflammatory response to Pseudomonas aeruginosa (PA) infection and/or lipopolysaccharide (LPS) treatment. We show that knockdown of miR-183/96/182 results in decreased production of multiple proinflammatory cytokines in response to PA or LPS treatment in Mϕ-like Raw264.7 cells. Consistently, peritoneal Mϕ from miR-183/96/182-knockout versus wild-type mice are less responsive to PA or LPS, although their basal levels of proinflammatory cytokines are increased. In addition, overexpression of miR-183/96/182 results in decreased production of nitrite and ROS in Raw264.7 cells. We also provide evidence that DAP12 and Nox2 are downstream target genes of miR-183/96/182. These data suggest that miR-183/96/182 imposes global regulation on various aspects of Mϕ function through different downstream target genes.

Substance P promotes susceptibility to Pseudomonas aeruginosa keratitis in resistant mice: anti-inflammatory mediators downregulated.

PURPOSE: Studies have shown that blocking substance P (SP) binding to neurokinin 1 receptor with spantide I prevents Pseudomonas aeruginosa-induced corneal perforation in susceptible C57BL/6 mice. This study tested the effect of SP injection on the resistance response (cornea heals) of BALB/c mice. METHODS: The day before infection, mice were injected intraperitoneally with SP or PBS. Disease was graded by clinical score, slit lamp, plate count, real-time RT-PCR, and ELISA assays, and polymorphonuclear neutrophils (PMNs) were quantitated using a myeloperoxidase assay. In additional experiments, BALB/c mice were injected intraperitoneally with vasoactive intestinal peptide (VIP) antagonist and similarly analyzed. RESULTS: Mice injected with SP exhibited worsened disease on days 1 to 7 after infection compared with controls. SP injection resulted in elevated PMN levels and viable bacterial counts in the cornea 3 and 5 days after infection. mRNA expression for NFkappaB and type 1 cytokines (e.g., IFN-gamma), as well as for TNF-alpha, MIP-2, IL-18, IL-6, and IL-1beta, were significantly elevated, whereas VIP and cytokines TGF-beta and IL-10 were significantly reduced. Differences in mRNA expression were selectively confirmed at the protein level by ELISA for NFkappaB, IL-1beta, and IL-10. VIP antagonist treatment also resulted in exacerbated disease scores, elevated proinflammatory mediators, and reduced anti-inflammatory mediators. CONCLUSIONS: These data provide evidence that the neuropeptide SP, among its broad systemic effects, is a potent neuroimmunoregulator that promotes susceptibility in the resistant BALB/c mouse by overcoming the anti-inflammatory effects of VIP and IL-10 and that a balance between SP and VIP levels may be critical in disease resolution.

Topical Glycyrrhizin Is Therapeutic for Pseudomonas aeruginosa Keratitis.

PURPOSE: Glycyrrhizin (GLY), an inhibitor of high-mobility group box 1 (HMGB1) protects prophylactically against Pseudomonas aeruginosa keratitis. However, the therapeutic potential of GLY to enhance an antibiotic has not been tested and is our purpose. METHODS: C57BL/6 mice (B6) were infected with a clinical isolate (KEI 1025) of P. aeruginosa and treated topically at 6 h postinfection (p.i.) with GLY or phosphate-buffered saline (PBS). Clinical scores, photography with a slit lamp, enzyme-linked immunosorbent assay, myeloperoxidase assay, bacterial plate counts, histopathology, reactive oxygen/nitrogen species (ROS/RNS) assays, and in vitro macrophage (Mφ) stimulation assays were used to assess effects of GLY treatment. In separate similar experiments, the ability of GLY to bioenhance the antibiotic, tobramycin (TOB), was assessed. RESULTS: In vivo, GLY versus PBS topical treatment began at 6 h p.i., improved disease outcome by significantly reducing clinical scores, proinflammatory proteins (HMGB1, RAGE, TLR4, TNF-α, and CXCL2), neutrophil infiltrate, bacterial load, ROS/RNS, and nitric oxide. In vitro, GLY downregulated iNOS and COX-2 expression (mRNA) in both mouse and human (THP-1) Mφ. At 6 and 24 h p.i., treatment with GLY enhanced the effects of TOB compared with TOB alone by significantly reducing corneal bacterial load and/or protein levels of cytokines CXCL2 and IL-1ß. CONCLUSIONS: Data provide evidence that GLY is not only therapeutic for Pseudomonas keratitis through its ability to reduce HMGB1, bacterial load, and oxidative damage but also through its bioenhancement of an antibiotic, even when treatment is initiated at 24 h after infection.