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.

The miR-183/96/182 cluster regulates sensory innervation, resident myeloid cells and functions of the cornea through cell type-specific target genes.

The conserved miR-183/96/182 cluster (miR-183C) is expressed in both corneal resident myeloid cells (CRMCs) and sensory nerves (CSN) and modulates corneal immune/inflammatory responses. To uncover cell type-specific roles of miR-183C in CRMC and CSN and their contributions to corneal physiology, myeloid-specific miR-183C conditional knockout (MS-CKO), and sensory nerve-specific CKO (SNS-CKO) mice were produced and characterized in comparison to the conventional miR-183C KO. Immunofluorescence and confocal microscopy of flatmount corneas, corneal sensitivity, and tear volume assays were performed in young adult naïve mice; 3' RNA sequencing (Seq) and proteomics in the trigeminal ganglion (TG), cornea and CRMCs. Our results showed that, similar to conventional KO mice, the numbers of CRMCs were increased in both MS-CKO and SNS-CKO vs age- and sex-matched WT control littermates, suggesting intrinsic and extrinsic regulations of miR-183C on CRMCs. The number of CRMCs was increased in male vs female MS-CKO mice, suggesting sex-dependent regulation of miR-183C on CRMCs. In the miR-183C KO and SNS-CKO, but not the MS-CKO mice, CSN density was decreased in the epithelial layer of the cornea, but not the stromal layer. Functionally, corneal sensitivity and basal tear volume were reduced in the KO and SNS-CKO, but not the MS-CKO mice. Tear volume in males is consistently higher than female WT mice. Bioinformatic analyses of the transcriptomes revealed a series of cell-type specific target genes of miR-183C in TG sensory neurons and CRMCs. Our data elucidate that miR-183C imposes intrinsic and extrinsic regulation on the establishment and function of CSN and CRMCs by cell-specific target genes. miR-183C modulates corneal sensitivity and tear production through its regulation of corneal sensory innervation.

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.

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.

The miR-183/96/182 cluster is a checkpoint for resident immune cells and shapes the cellular landscape of the cornea.

PURPOSE: The conserved miR-183/96/182 cluster (miR-183C) regulates both corneal sensory innervation and corneal resident immune cells (CRICs). This study is to uncover its role in CRICs and in shaping the corneal cellular landscape at a single-cell (sc) level. METHODS: Corneas of naïve, young adult [2 and 6 months old (mo)], female miR-183C knockout (KO) mice and wild-type (WT) littermates were harvested and dissociated into single cells. Dead cells were removed using a Dead Cell Removal kit. CD45+ CRICs were enriched by Magnetic Activated Cell Sorting (MACS). scRNA libraries were constructed and sequenced followed by comprehensive bioinformatic analyses. RESULTS: The composition of major cell types of the cornea stays relatively stable in WT mice from 2 to 6 mo, however the compositions of subtypes of corneal cells shift with age. Inactivation of miR-183C disrupts the stability of the major cell-type composition and age-related transcriptomic shifts of subtypes of corneal cells. The diversity of CRICs is enhanced with age. Naïve mouse cornea contains previously-unrecognized resident fibrocytes and neutrophils. Resident macrophages (ResMφ) adopt cornea-specific function by expressing abundant extracellular matrix (ECM) and ECM organization-related genes. Naïve cornea is endowed with partially-differentiated proliferative ResMφ and contains microglia-like Mφ. Resident lymphocytes, including innate lymphoid cells (ILCs), NKT and γδT cells, are the major source of innate IL-17a. miR-183C limits the diversity and polarity of ResMφ. CONCLUSION: miR-183C serves as a checkpoint for CRICs and imposes a global regulation of the cellular landscape of the cornea.

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.

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.

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.

The links of fine airborne particulate matter exposure to occurrence of cardiovascular and metabolic diseases in Michigan, USA.

Air pollutants, particularly airborne particulate matter with aerodynamic diameter < 2.5µm (PM2.5), have been linked to the increase in mortality and morbidity associated with cardiovascular and metabolic diseases. In this study, we investigated the dose-risk relationships between PM2.5 concentrations and occurrences of cardiovascular and metabolic diseases as well as the confounding socioeconomic factors in Michigan, USA, where PM2.5 levels are generally considered acceptable. Multivariate linear regression analyses were performed to investigate the relationship between health outcome and annual ground-level PM2.5 concentrations of 82 counties in Michigan. The analyses revelated significant linear dose-response associations between PM2.5 concentrations and cardiovascular disease (CVD) hospitalization. A 10 µg/m3 increase in PM2.5 exposure was found to be associated with a 3.0% increase in total CVD, 0.45% increase in Stroke, and a 0.3% increase in Hypertension hospitalization rates in Medicare beneficiaries. While the hospitalization rates of Total Stroke, Hemorrhagic Stroke, and Hypertension in urbanized counties were significantly higher than those of rural counties, the death rates of coronary heart disease and ischemic stroke in urbanized counties were significantly lower than those of rural counties. These results were correlated with the facts that PM2.5 levels in urbanized counties were significantly higher than that in rural counties and that the percentage of the population with health insurance and the median household income in rural counties were significantly lower. While obesity prevalence showed evidence of a weak positive correlation (ρ = 0.20, p-value = 0.078) with PM2.5 levels, there was no significant dose-response association between county diabetes prevalence rates and PM2.5 exposure in Michigan. In summary, this study revealed strong dose-response associations between PM2.5 concentrations and CVD incidence in Michigan, USA. The socioeconomic factors, such as access to healthcare resources and median household income, represent important confounding factors that could override the impact of PM2.5 exposure on CVD mortality.

Prophylactic Knockdown of the miR-183/96/182 Cluster Ameliorates Pseudomonas aeruginosa-Induced Keratitis.

Purpose: Previously, we demonstrated that miR-183/96/182 cluster (miR-183C) knockout mice exhibit decreased severity of Pseudomonas aeruginosa (PA)-induced keratitis. This study tests the hypothesis that prophylactic knockdown of miR-183C ameliorates PA keratitis indicative of a therapeutic potential. Methods: Eight-week-old miR-183C wild-type and C57BL/6J inbred mice were used. Locked nucleic acid-modified anti-miR-183C or negative control oligoribonucleotides with scrambled sequences (NC ORNs) were injected subconjunctivally 1 day before and then topically applied once daily for 5 days post-infection (dpi) (strain 19660). Corneal disease was graded at 1, 3, and 5 dpi. Corneas were harvested for RT-PCR, ELISA, immunofluorescence (IF), myeloperoxidase and plate count assays, and flow cytometry. Corneal nerve density was evaluated in flatmounted corneas by IF staining with anti-ß-III tubulin antibody. Results: Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1ß at 1 dpi, which was decreased at 5 dpi; fewer polymorphonuclear leukocytes (PMNs) at 5 dpi; lower viable bacterial plate count at both 1 and 5 dpi; increased percentages of MHCII+ macrophages (Mϕ) and dendritic cells (DCs), consistent with enhanced activation/maturation; and decreased severity of PA keratitis. Anti-miR-183C treatment in the cornea of naïve mice resulted in a transient reduction of corneal nerve density, which was fully recovered one week after the last anti-miR application. miR-183C targets repulsive axon-guidance receptor molecule Neuropilin 1, which may mediate the effect of anti-miR-183C on corneal nerve regression. Conclusions: Prophylactic miR-183C knockdown is protective against PA keratitis through its regulation of innate immunity, corneal innervation, and neuroimmune interactions.

Targeting Inflammation Driven by HMGB1 in Bacterial Keratitis-A Review.

Pseudomonas (P.) aeruginosa is a Gram-negative bacteria that causes human infectionsinfections. It can cause keratitis, a severe eye infection, that develops quickly and is a major cause of ulceration of the cornea and ocular complications globally. Contact lens wear is the greatest causative reason in developed countries, but in other countries, trauma and predominates. Use of non-human models of the disease are critical and may provide promising alternative argets for therapy to bolster a lack of new antibiotics and increasing antibiotic resistance. In this regard, we have shown promising data after inhibiting high mobility group box 1 (HMGB1), using small interfering RNA (siRNA). Success has also been obtained after other means to inhinit HMGB1 and include: use of HMGB1 Box A (one of three HMGB1 domains), anti-HMGB1 antibody blockage of HMGB1 and/or its receptors, Toll like receptor (TLR) 4, treatment with thrombomodulin (TM) or vasoactive intestinal peptide (VIP) and glycyrrhizin (GLY, a triterpenoid saponin) that directly binds to HMGB1. ReducingHMGB1 levels in P. aeruginosa keratitis appears a viable treatment alternative.

Nano-Biomaterials for Retinal Regeneration.

Nanoscience and nanotechnology have revolutionized key areas of environmental sciences, including biological and physical sciences. Nanoscience is useful in interconnecting these sciences to find new hybrid avenues targeted at improving daily life. Pharmaceuticals, regenerative medicine, and stem cell research are among the prominent segments of biological sciences that will be improved by nanostructure innovations. The present review was written to present a comprehensive insight into various emerging nanomaterials, such as nanoparticles, nanowires, hybrid nanostructures, and nanoscaffolds, that have been useful in mice for ocular tissue engineering and regeneration. Furthermore, the current status, future perspectives, and challenges of nanotechnology in tracking cells or nanostructures in the eye and their use in modified regenerative ophthalmology mechanisms have also been proposed and discussed in detail. In the present review, various research findings on the use of nano-biomaterials in retinal regeneration and retinal remediation are presented, and these findings might be useful for future clinical applications.

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.

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.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

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.

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.

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.

MicroRNAs in Ocular Infection.

MicroRNAs (miRNAs) are small, non-coding, regulatory RNA molecules and constitute a newly recognized, important layer of gene-expression regulation at post-transcriptional levels. miRNAs quantitatively fine tune the expression of their downstream genes in a cell type- and developmental stage-specific fashion. miRNAs have been proven to play important roles in the normal development and function as well as in the pathogenesis of diseases in all tissues and organ systems. miRNAs have emerged as new therapeutic targets and biomarkers for treatment and diagnosis of various diseases. Although miRNA research in ocular infection remains in its early stages, a handful of pioneering studies have provided insight into the roles of miRNAs in the pathogenesis of parasitic, fungal, bacterial, and viral ocular infections. Here, we review the current status of research in miRNAs in several major ocular infectious diseases. We predict that the field of miRNAs in ocular infection will greatly expand with the discovery of novel miRNA-involved molecular mechanisms that will inform development of new therapies and identify novel diagnostic biomarkers.