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Exposure to airborne particulate <10 μm (PM 10 ) adversely affects the ocular surface. This study tested PM 10 on epithelial barrier integrity in immortalized human corneal epithelial cells (HCE-2) and mouse cornea, and whether antioxidant SKQ1 is restorative. HCE-2 were exposed to 100 μg/ml PM 10  ± SKQ1 for 24 h. An Electric Cell-Substrate Impedance Sensing (ECIS) system monitored the impact of PM 10 . RT-PCR, western blotting and immunofluorescence measured levels of barrier and associated proteins, stanniocalcin 2 (STC2), and a kit measured total calcium. In vivo, female C57BL/6 mice were exposed to either control air or PM 10 (±SKQ1) in a whole-body exposure chamber, and barrier associated proteins tested. Tight junction and mucins proteins in the cornea were tested. In HCE-2, PM 0 vs control significantly reduced mRNA and protein levels of tight junction and adherence proteins, and mucins. ECIS data demonstrated that PM 10 vs control cells exhibited a significant decrease in epithelial barrier strength at 4000 Hz indicated by reduced impedance and resistance. PM 10 also upregulated STC2 protein and total calcium levels. In vivo, PM 10 vs control reduced zonula occludens 1 and mucins. SKQ1 pre-treatment reversed PM 10 effects both in vitro and in vivo. In conclusion, PM 10 exposure reduced tight junction and mucin proteins, and compromised the seal between cells in the corneal epithelium leading to decreased epithelial barrier strength. This effect was reversed by SKQ1. Since the corneal epithelium forms the first line of defense against air pollutants, including PM 10 , preserving its integrity using antioxidants such as SKQ1 is crucial in reducing the occurrence of ocular surface disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Linda D. Hazlett reports financial support was provided by National Institutes of Health. Linda D Hazlett reports a relationship with National Institutes of Health that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

The effects of exposure to airborne particulate matter with a size of 10 μm or less (PM 10 ) 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 PM 10 for 2 weeks, infected, and the disease was documented by clinical score, PMN quantitation, bacterial plate count, RT-PCR and Western blot. PBS-treated, PM 10 -exposed corneas did not differ at 1 day postinfection (dpi), but exhibited earlier (3 dpi) corneal thinning compared to controls. By 3 dpi, PM 10 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. PM 10 resulted in higher viable bacterial plate counts at 1 and 3 dpi, but SKQ1 reduced them at 3 dpi. PM 10 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 PM 10 increased the susceptibility of C57BL/6 to PA infection; SKQ1 significantly reversed these effects, but was not effective as an adjunctive treatment.

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., (© 2024. The Author(s).)

We have previously shown that PM 10 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 PM 10 toxicity and SKQ1-mediated protection. HCE-2 were pre-treated with SKQ1 and then exposed to 100 μg/mL PM 10 . 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 PM 10 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, PM 10 exposure significantly increased DNA damage, phosphor-p53, p16 and p21 protein levels, and β -galactosidase ( β -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 PM 10 toxicity by preventing the restoration of cell viability and reduced oxidative stress. In conclusion, PM 10 induces inflammation, reduces Nrf2 transcriptional activity, and causes DNA damage, leading to a senescence-like phenotype, which is prevented by SKQ1.

Purpose: In vivo data indicate that mouse corneas exposed to PM 10 showed early perforation and thinning after infection with Pseudomonas aeruginosa . To understand the mechanisms underlying this finding, we tested the effects of PM 10 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 PM 10 or control air and assessed by clinical scores, slit lamp photography and western blot. HCET were exposed to 100μg/ml PM 10 for 24h before challenge with strain 19660 (MOI 20). A subset of cells were pre-treated with 50nM SKQ1 for 1h before PM 10 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 PM 10 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 , PM 10 induced a dose dependent reduction in cell viability of HCET and significantly increased mRNA levels of pro-inflammatory molecules compared to control. Exposure to PM 10 before bacterial challenge further amplified the reduction in cell viability and GSH levels. Furthermore, PM 10 exposure also exacerbated the increase in MDA and ROS levels and phase contrast microscopy revealed more rounded cells after strain 19660 challenge. PM 10 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: PM 10 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 PM 10 after bacterial challenge by reversing these effects. The findings provide insight into mechanisms underlying early perforation and thinning observed in infected corneas of PM 10 exposed mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Somayajulu, McClellan, Muhammed, Wright and Hazlett.)

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 (PM 10 ) in the mouse cornea and in vitro. C57BL/6 mice were exposed to control or 500 µg/m 3 PM 10 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 PM 10 ± SKQ1 and cell viability, MDA, mitochondrial ROS, ATP and Nrf2 protein were tested. In vivo, PM 10 vs. control exposure significantly reduced GSH, corneal thickness and increased MDA levels. PM 10 -exposed corneas showed significantly higher mRNA levels for downstream targets, pro-inflammatory molecules and reduced Nrf2 protein. In PM 10 -exposed corneas, SKQ1 restored GSH and Nrf2 levels and lowered MDA. In vitro, PM 10 reduced cell viability, Nrf2 protein, and ATP, and increased MDA, and mitochondrial ROS; while SKQ1 reversed these effects. Whole-body PM 10 exposure triggers oxidative stress, disrupting the Nrf2 pathway. SKQ1 reverses these deleterious effects in vivo and in vitro, suggesting applicability to humans.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Shamsa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Somayajulu, McClellan, Bessert, Pitchaikannu and Hazlett.)

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-β-III tubulin antibody., Results: Anti-miR-183C downregulated miR-183C in the cornea. It resulted in an increase in IL-1β 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.

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.

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.

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-1β , 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-1β 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-1β 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.

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., (Copyright © 2020 The Authors.)

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.

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-1β, CXCL2, GPX1, GPX2, GR1, superoxide dismutase 2, and heme oxygenase 1) and HCET (high mobility group box 1, CXCL2, and IL-1β). 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 (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.

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.

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., (© 2019 The Author(s) Published by S. Karger AG, Basel.)

Purpose: High mobility group box 1 (HMGB1) contributes to adverse disease outcome in Pseudomonas aeruginosa keratitis. This study tests Box A, an HMGB1 antagonist, in a model of the disease., Methods: C57BL/6 mice (B6) were injected subconjunctivally (1 day before infection) with Box A or phosphate-buffered saline (PBS), infected with P. aeruginosa strain ATCC 19660, and injected intraperitoneally with Box A or PBS at 1 and 3 days postinfection (p.i.). Clinical scores, photographs with a slit lamp camera, real-time polymerase chain reaction (RT-PCR), western blot, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), myeloperoxidase (MPO), and bacterial plate count were used to assess disease outcome. In separate experiments, the therapeutic potential of Box A was tested as described above, but with treatment begun at 6 h p.i., Results: Box A versus PBS prophylactic treatment significantly reduced clinical scores, MPO activity, bacterial load, and expression of TLR4, RAGE, IL-1β, CXCL2, and TNF-α in the infected cornea. Box A blocked co-localization of HMGB1/TLR4 in infiltrated cells in the stroma at 3 and 5 days p.i., but only at 5 days p.i. for HMGB1/RAGE. Box A versus PBS therapeutic treatment significantly reduced clinical scores, MPO activity, bacterial load, and protein levels of IL-1β, CXCL2, and IL-6 in the infected cornea., Conclusion: Overall, Box A lessens the severity of Pseudomonas keratitis in mice by decreasing expression of TLR4, RAGE (their interaction with HMGB1), IL-1β, CXCL2 (decreasing neutrophil infiltrate), and bacterial plate count when given prophylactically. Therapeutic treatment was not as effective at reducing opacity (disease), but shared similar features with pretreatment of the mice.

Purpose: The purpose of this study is to establish the short tandem repeat (STR) profiles of several human cell lines commonly used in ocular surface research., Materials and Methods: Independently DNA was extracted from multiple passages of three human corneal epithelial cell lines, two human conjunctival epithelial cell lines and one meibomian gland cell line, from different laboratories actively involved in ocular surface research. The samples were then subjected to STR analysis on a fee-for-service basis in an academic setting and the data compared against that in available databases., Results: The STR profiles for the human corneal epithelial cells were different among the three cell lines studied and for each line the profiles were identical across the samples provided by three laboratories. Profiles for the human conjunctival epithelial cells were different among the two cell lines studied. Profiles for the meibomian gland cell line were identical across the samples provided by three laboratories. No samples were contaminated by elements of other cell lines such as HeLa., Conclusions: This comprehensive study provides verification of STR profiles for commonly used human ocular surface cell lines that can now be used as a reference by others in the field to authenticate the cell lines in use in their own laboratories.

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-1β., 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.

We selectively characterized three isolates from Pseudomonas aeruginosa keratitis patients and how glycyrrhizin (GLY) affected them. Type III toxins were determined using polymerase chain reaction (PCR). Minimum Inhibitory Concentration (MIC) of GLY and assays for its effects on: time kill, bacterial permeability, and biofilm/adhesion were done. In vivo, C57BL/6 (B6) mice were treated topically with GLY after G81007 infection. Clinical score, photography with a slit lamp and RT-PCR were used to assess treatment effects. Isolates expressed exoS and exoT , but not exoU . MIC for all isolates was 40 mg/mL GLY and bacteriostatic effects were seen for G81007 after treatment using time kill assays. From viability testing, GLY treatment significantly increased the number of permeabilized bacteria (live/dead assay). Isolates 070490 and G81007 formed more biofilms compared with R59733 and PAO1 (control). GLY-treated bacteria had diminished biofilm compared with controls for all isolates. GLY reduced adherence of the G81007 isolate to cultured cells and affected specific biofilm associated systems tested by reverse transcription PCR (RT-PCR). In vivo, after G81007 infection, GLY treatment reduced clinical score and messenger RNA (mRNA) expression of IL-1β, TNF-α, CXCL2 and HMGB1. This study provides evidence that GLY is bacteriostatic for G81007. It also affects biofilm production, adherence to cultured cells, and an improved keratitis outcome., Competing Interests: The authors declare no conflict of interest.

Psbetaeudomonas (P.) aeruginosa infection of the cornea in BALB/c mice does not result in perforation and the mice have been classified as resistant. However, regulation of this response via inflammasome activation remained untested. Therefore, BALB/c mice were infected with P. aeruginosa ATCC strain 19660 and NLRP3 and NLRC4 protein tested by ELISA. Since NLRC4 vs NLRP3 protein levels were significantly higher in the corneas of BALB/c at 1 and 5 days postinfection we used silencing to knockdown NLRC4. Silencing NLRC4 vs scrambled siRNA treatment exacerbated disease in BALB/c mice, reduced myeloperoxidase levels and elevated bacterial plate counts at 5 days postinfection. It also increased pro IL-1beta, but reduced total protein for IL-1beta and IL-18 at 5 days postinfection. Flow cytometry to identify cells affected by silencing, showed reduced caspase-1 levels in a CD11blowLy6Glow population of cells, (but not PMN or macrophages) from the infected cornea of siNLRC4 treated mice that produced less mature IL-1beta. These data provide evidence that the NLRC4 inflammasome contributes to resistance through regulation of caspase-1, IL-1beta and IL-18 in a CD11blowLy6Glow population of cells.

Purpose: High mobility group box 1 (HMGB1) contributes to poor disease outcome in Pseudomonas aeruginosa keratitis. This study tests the prophylactic effect of treatment with HMGB1 inhibitors, glycyrrhizin (GLY) and its derivative, carbenoxolone (CBX), for Pseudomonas keratitis., Methods: We treated C57BL/6 (B6) mice subconjunctivally with GLY or CBX, infected with a noncytotoxic clinical isolate (KEI 1025) or a cytotoxic strain (ATCC 19660) of P. aeruginosa, and injected intraperitoneally with either agent. Clinical score, photography with a slit lamp, real-time RT-PCR, ELISA, myeloperoxidase (MPO) assay, bacterial plate count, histopathology, and absorbance assays were used to assess treatment efficacy and bacteriostatic activity., Results: After KEI 1025 infection, GLY treatment reduced HMGB1 (mRNA and protein levels) and improved disease outcome with significant reduction in mRNA levels of IL-1β, TLR4, CXCL2, and IL-12; protein expression (IL-1β, CXCL2); neutrophil infiltrate; and bacterial load. Treatment with GLY enhanced antimicrobial proteins, including CRAMP and mBD2, but not mBD3. Glycyrrhizin also reduced clinical scores and improved disease outcome in corneas infected with strain 19660. However, neither HMGB1 mRNA or protein levels were reduced, but rather, CXCL2 expression (mRNA and protein), neutrophil infiltrate, and bacterial load were reduced statistically. Treatment with GLY initiated 6 hours after infection reduced plate count; GLY also was bacteriostatic for KEI 1025 and ATCC 19660., Conclusions: Glycyrrhizin reduces HMGB1 and is protective against P. aeruginosa-induced keratitis with a clinical isolate that is noncytotoxic. It was similar, but less effective when used after infection with a cytotoxic strain, which did not reduce HMGB1.

Macrophages are the predominant innate immune cells recruited to tissues following injury or infection. These early-responding, pro-inflammatory macrophages play an essential role in the amplification of inflammation. However, macrophage pro-inflammatory gene expression should be tightly regulated to avert host tissue damage. In this study, we identify the Kruppel-like transcription factor 6 (KLF6)-B cell leukemia/lymphoma 6 (BCL6) signaling axis as a novel regulator of macrophage inflammatory gene expression and function. Utilizing complementary gain- and loss-of-function studies, we observed that KLF6 is essential for macrophage motility under ex vivo and in vivo conditions. Concordant with these observations, myeloid-specific deficiency of KLF6 significantly attenuates macrophage pro-inflammatory gene expression, recruitment, and progression of inflammation. At the molecular level, KLF6 suppresses BCL6 mRNA and protein expression by elevating PR domain-containing 1 with ZNF domain (PRDM1) levels in macrophages. Interestingly, pharmacological or genetic inhibition of BCL6 in KLF6-deficient macrophages completely abrogated the attenuation of pro-inflammatory cytokine/chemokine expression and cellular motility. Collectively, our observations reveal that KLF6 repress BCL6 to enhance macrophage inflammatory gene expression and function., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Pseudomonas (P.) aeruginosa is a Gram negative bacterium widely dispersed in the environment which can cause acute and chronic infections in humans. According to the Centers for Disease Control and Prevention (CDC), the overall incidence of P. aeruginosa infections in USA hospitals averages about 0.4% (4/1000 discharges), and the bacterium is the fourth most commonly-isolated nosocomial pathogen accounting for 10.1% of all hospital-acquired infections. P. aeruginosa keratitis is a severe infection of the eye, progresses rapidly and remains a leading cause of corneal ulcers worldwide. Use of contact lenses is the major risk factor in the USA, while in less industrialized countries, trauma from agricultural accidents are of importance. Animal models of bacterial keratitis are of value in the study of this disease and suggest potential alternative therapeutic targets that are needed urgently due to increasing antibiotic resistance. Recently we have shown success and improved disease outcome after down-regulation of one promising target, high mobility group box1 (HMGB1) using small interfering RNA (siRNA). Testing more clinically relevant approaches are underway to reduce HMGB1 levels in P. aeruginosa keratitis which may hold promise for its treatment.

Purpose: Thrombomodulin (TM) is a multidomain, transmembrane protein with anti-inflammatory properties. Thrombomodulin domain (D) 1 is lectin-like, interacting with Lewis Y antigen on lipopolysaccharide, and with HMGB1, while TMD23 is associated with angiogenic and anti-inflammatory functions. Thus, we tested if TM is protective against Pseudomonas aeruginosa keratitis and whether it enhanced corneal vascularity., Methods: Eyes of C57BL/6 (B6) mice were injected with recombinant TM (rTM), rTMD1, or PBS subconjunctivally before and intraperitoneally after infection with P. aeruginosa. Clinical scores, photography with a slit lamp, RT-PCR, ELISA, myeloperoxidase (MPO) assay, viable bacterial plate counts, and India ink perfusion were used to assess the disease response and corneal vascularity (rTM only)., Results: Recombinant TM versus PBS treatment reduced clinical scores and corneal opacity. Corneal mRNA levels for HMGB1 were unchanged, but proinflammatory molecules IL-1β, CXCL2, NF-κB, TLR4, and RAGE were decreased; anti-inflammatory molecules SIGIRR and ST2 were increased. ELISA confirmed the mRNA data for HMGB1, IL-1β, and CXCL2 proteins. Both neutrophil influx and viable bacterial plate counts also were decreased after rTM treatment. Protein levels for angiogenic molecules VEGF, VEGFR-1, and VEGFR-2 were measured at 5 days post infection and were not different or reduced significantly after rTM treatment. Further, perfusion with India ink revealed similar vessel ingrowth between the two groups. Similar studies were performed with rTMD1, but disease severity, mRNA, proteins, MPO, and plate counts were not changed from controls., Conclusions: These data provide evidence that rTM treatment is protective against bacterial keratitis, does not reduce HMGB1, and is not angiogenic.

High-mobility group box 1 (HMGB1), a prototypic alarmin, mediates the systemic inflammatory response syndrome. Treatment with vasoactive intestinal peptide, an anti-inflammatory neuropeptide, downregulates proinflammatory cytokines and promotes healing in a susceptible (cornea perforates) model of Pseudomonas aeruginosa keratitis, and also significantly downregulates HMGB1 expression. Therefore, we examined targeting HMGB1 for the treatment of P. aeruginosa keratitis to avoid delivery and other issues associated with vasoactive intestinal peptide. For this, HMGB1 was silenced using small interfering RNA, whereas controls were treated with a nonspecific scrambled sequence small interfering RNA. Less disease was seen postinfection in siHMGB1 compared with control mice and was documented by clinical score and photographs with a slit lamp. Real-time RT-PCR and ELISA confirmed HMGB1 knockdown. RT-PCR analysis also revealed reduced mRNA levels of IL-1β, MIP-2, TNF-α, TLR4, and receptor for advanced glycation end products, whereas mRNA levels of anti-inflammatory TLRs single Ig IL-1-related receptor and ST2 were increased significantly. HMGB1 knockdown also decreased IL-1β and MIP-2 proteins, reducing polymorphonuclear cell number in the infected cornea. mRNA and protein levels of CXCL12 and CXCR4, as well as mononuclear cells, were reduced significantly after HMGB1 knockdown. Ab neutralization of HMGB1, infection with a clinical isolate, and recombinant HMGB1 treatment of resistant mice supported the silencing studies. These data provide evidence that silencing HMGB1 promotes better resolution of P. aeruginosa keratitis by decreasing levels of proinflammatory mediators (decreasing polymorphonuclear cell infiltration), increasing anti-inflammatory TLRs, reducing CXCL12 (preventing HMGB1/CXCL12 heterodimer formation), and signaling through CXCR4, reducing monocyte/macrophage infiltration., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Purpose: To determine if IL-17 regulates Mer tyrosine kinase-positive (MerTK+) cells in Pseudomonas aeruginosa keratitis., Methods: Interleukin 17 was tested in normal and infected cornea of susceptible C57BL/6 and resistant BALB/c mice. The latter were treated with recombinant mouse (rm) IL-17; both groups were treated with IL-17 neutralizing antibody. Mice were infected, and clinical score, PCR, ELISA, and myeloperoxidase (MPO) assays tested expression of proinflammatory and anti-inflammatory mediators and polymorphonuclear neutrophilic leukocyte (PMN) infiltrate. Fas and Fas ligand (FasL) protein levels were assessed in both mouse strains, while MerTK+ cells were examined by immunostaining and cell sorting before and after IL-17 neutralization., Results: The IL-17 mRNA and protein were higher in C57BL/6 versus BALB/c cornea after infection. The rmIL-17 treatment of BALB/c mice modified proinflammatory and anti-inflammatory mediators, but clinical score and MPO assay revealed no differences. However, only BALB/c mice treated with IL-17 neutralizing antibody showed increased disease, macrophage inflammatory protein (MIP) 2, and MPO levels. Fas and FasL protein levels, elevated earlier in BALB/c versus C57BL/6 mice, correlated with significantly more MerTK+ cells in BALB/c cornea at 3 days after infection. Neutralization of IL-17 in C57BL/6 mice elevated MerTK+ cells, while similar treatment of BALB/c mice significantly decreased them., Conclusions: These data provide evidence that IL-17 expression is higher in C57BL/6 versus BALB/c cornea after infection and that the latter group has more MerTK+ cells. Exogenous rmIL-17 failed to shift the disease response in resistant mice, but its neutralization resulted in worsened disease and reduced MerTK+ cells. Neutralization of IL-17 in C57BL/6 mice increased MerTK+ cells but did not dramatically shift the disease response., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The immune system protects the host from pathogenic microbes, but tight regulation of the evoked response is requisite to limit bystander damage. The interleukin (IL)-10 family of cytokines, composed of 9 members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and 3 distantly related members, IL-28A, IL-28B, and IL-29, plays a central role in this regulation. IL-10 family cytokines emerged before the adaptive immune response and elicit diverse host defense mechanisms, especially from epithelial cells during an infection. IL-10 family cytokines are also essential for maintenance and integrity of tissue epithelial layers. These cytokines promote innate immune responses from tissue epithelia that limit the damage caused by both viral and bacterial infections. They also facilitate tissue healing after infection/inflammation. In this regard, IL-10 suppresses pro-inflammatory responses, limiting tissue disruption resulting from an inflammatory response. Thus, a central functional theme of IL-10 family cytokines is their role in tissue protection. This review focuses on IL-10, the founding member of this family of cytokines, and integrates recent data on the function and regulation of IL-10 during bacterial infections. Emphasis is placed on the role of IL-10 in Pseudomonas aeruginosa keratitis and the subsequent infectious/inflammatory processes evoked.

Purpose: To determine whether rapamycin altered corneal growth factor levels to impact severity of Pseudomonas aeruginosa keratitis., Methods: BALB/c mice were injected intraperitoneally with rapamycin or PBS and infected with P. aeruginosa. Corneas were harvested and mRNA levels of growth factors (EGF, HGF, FGF-7/KGF), receptors (EGFR, c-met, FGFR-2), and signaling molecules (PI3K, Akt, S6K1, and IGF-1R) tested. ELISA determined HGF/c-met, IGF-1, and Substance P (SP) protein levels. Corneal application of recombinant (r)HGF was assessed by clinical score, photography with a slit lamp, real-time RT-PCR (mRNA for mT0R, IL-10, IL-12, IL-18, PI3KCα, Akt), and ELISA (total and phosphorylated [p]c-met); rIGF-1 effects also were tested by ELISA. In vitro, RAW cells and peritoneal macrophages were stimulated with LPS ± rHGF ± c-met inhibitor (CI) and mTOR mRNA levels tested., Results: Rapamycin disparately regulated infected corneal mRNA levels of EGF/EGFR and FGF-7/FGFR-2, but HGF/c-met mRNA levels both increased. ELISA confirmed elevated HGF protein. Rapamycin did not change PI3KCα or Akt signaling molecule expression, downregulated S6K1, but upregulated IGF-1R mRNA levels; IGF-1 and SP proteins also were upregulated. After infection, topical rHGF versus PBS increased mRNA levels of IL-12p40, IL-18, PI3KCα, and Akt; mTOR and IL-10 mRNA were downregulated; rIGF-1 increased HGF protein. In vitro, rHGF and LPS lowered RAW cell and macrophage mTOR levels; CI addition restored them., Conclusions: Collectively, these data provide evidence that enhanced corneal HGF levels increase signaling through the c-met receptor, decrease mTOR levels, and enhance proinflammatory cytokines, while decreasing anti-inflammatory cytokines, and that HGF signaling is central to disease outcome.

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, β, 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.

Purpose: To explore the role of triggering receptor expressed on myeloid cells 2 (TREM-2) in Pseudomonas aeruginosa (PA) keratitis., Methods: BALB/c mice were routinely infected with PA and evaluated at various postinfection time points for corneal expression of TREM-2, by real-time PCR, Western blot, and flow cytometry. Next, BALB/c and C57BL/6 mice were respectively treated with TREM-2 siRNA or agonistic anti-TREM-2 antibody, to determine the role of TREM-2 in PA keratitis. Bacterial load and neutrophil infiltration were tested by plate count and myeloperoxidase assay, respectively. Th1-/Th2-type and proinflammatory cytokine expression were tested by real-time PCR and ELISA after in vivo and in vitro silencing of TREM-2. Moreover, phosphorylated Akt levels were tested by Western blot in murine macrophages after treatment with agonistic anti-TREM-2 antibody. mRNA levels of proinflammatory cytokines were examined in murine macrophages after TREM-2 activation and lipopolysaccharide stimulation, following pretreatment with inhibitors for PI3K or Akt, to determine whether PI3K/Akt is required in TREM-2-mediated immune modulation. In addition, BALB/c mice were treated with wortmannin and analyzed for bacterial load and proinflammatory cytokine expression., Results: TREM-2 expression was elevated in the infected BALB/c corneas at 3 or 5 days postinfection. Silencing of TREM-2 accelerated disease progression by enhancing bacterial load and corneal inflammation, whereas activation of TREM-2 promoted host resistance to PA keratitis. PI3K/Akt signaling is required in the TREM-2-mediated immune modulation, and inhibition of PI3K resulted in worsened disease after PA corneal infection., Conclusions: TREM-2 promoted host resistance to PA infection by suppressing corneal inflammation via activation of the PI3K/Akt pathway.

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.

Purpose: Exogenous vasoactive intestinal peptide (VIP) down-regulates pro-inflammatory but up-regulates anti-inflammatory cytokines, growth factors (GFs) and Toll-like receptors promoting healing in experimental Pseudomonas aeruginosa (P. aeruginosa) keratitis. Whether VIP is required for GF or GF receptor (R) expression in normal and infected corneas is unknown and is the purpose of this study., Methods: VIP knockout ((-/-)) and wild-type (WT) C57BL/6 (B6) mice were infected and tested using PCR array, real-time RT-PCR, ELISA, and immunostaining. VIP antagonist treatment studies also were done using B6 and BALB/c mice., Results: Infected corneas of VIP(-/-) versus WT B6 mice perforated earlier (2 vs. 5 days postinfection [p.i.]), and array data showed that GFs were differentially changed between groups. RT-PCR revealed that the infected cornea of VIP(-/-) versus WT mice expressed higher mRNA levels of epidermal growth factor (EGF) and hepatocyte growth factor (HGF), reduced FGF, EGFR, and HGFR, with no difference in FGFR; differences between groups were not seen in normal cornea. Immunostaining for GF and GFR in the normal cornea of VIP(-/-) versus WT mice was similar. However, at 1 day p.i., VIP(-/-) versus WT mice had more intense EGF and HGF, similar FGFR, and reduced FGF, EGFR, and HGFR staining. VIP antagonist treatment decreased protein levels for GFR at 5 days p.i. in both B6 and BALB/c mice, with no significant changes in normal cornea., Conclusions: The data showed that endogenous VIP is not requisite for GF or GFR expression in the normal cornea but, after infection, its absence or reduction is critical for their regulation.

Purpose: Studies from our laboratory have demonstrated that vasoactive intestinal peptide (VIP) directly converts the normally susceptible C57BL/6J (B6) mouse to resistant after ocular infection through modulation of the inflammatory response. This study examines mechanisms by which VIP influences the healing phase following infection--specifically reconstitution of the extracellular matrix (ECM)., Methods: B6 mice received daily intraperitoneal (IP) injections of VIP, while control mice were similarly injected with sterile phosphate buffered saline (PBS). Real-time RT-PCR, ELISA, and immunofluorescent staining were used to assess the effects of VIP treatment on ECM molecule expression after Pseudomonas aeruginosa-induced keratitis. We also compared the effect of VIP treatment on lipopolysaccharide (LPS)-stimulated B6- and BALB/c-derived fibroblasts., Results: In vivo analyses revealed that VIP treatment of P. aeruginosa-infected B6 corneas led to a significant increase in ECM molecules associated with healing/homeostasis, while those associated with ECM degradation were significantly down-regulated when compared to wild-type (WT) controls. In vitro studies revealed that VIP treatment of lipopolysaccharide-stimulated fibroblasts derived from susceptible B6 and resistant BALB/c mice expressed distinct differences in ECM molecule expression, whereby the latter expressed higher levels of ECM molecules aimed at reconstitution. Furthermore, differential expression of VIP receptor-1/VIP receptor-2 (VIPR1/VIPR2) was observed between B6 and BALB/c after VIP treatment of LPS-stimulated fibroblasts., Conclusions: VIP treatment functions to enhance ECM reconstitution, which appears to be carried out in large part by fibroblasts via VIPR2. Overall, the data from this study suggest that VIP not only regulates disease pathogenesis, but also functions to restore integrity of the corneal stroma.

Purpose: This study analyzed the influence of substance P (SP) on growth factors related to wound healing in mice in the presence of infectious keratitis., Methods: Naturally resistant mice were injected intraperitoneally with SP or phosphate-buffered saline and infected with Pseudomonas aeruginosa, and corneal messenger RNA (mRNA) levels of growth factors and apoptosis genes were tested. Enzyme-linked immunosorbent assay determined the protein levels, whereas immunohistochemistry tested the distribution, macrophage phenotype, and cell quantitation. In vitro, macrophages were stimulated with lipopolysaccharide (LPS; with or without SP) and mRNA levels of proinflammatory and antiinflammatory cytokines and apoptosis genes were tested., Results: After SP, epidermal growth factor mRNA and protein levels were disparately regulated early, with no differences later in the disease. Hepatocyte growth factor and fibroblast growth factor-7 mRNA and protein levels were increased after SP treatment. Enumerating dual-labeled stromal cells revealed no difference between SP-treated versus phosphate-buffered saline-treated groups in the percentage of epidermal growth factor-labeled fibroblasts or macrophages, but there were significant increases in both hepatocyte growth factor- and fibroblast growth factor-7-labeled cells. Type 2 (M2) macrophages and caspase-3 mRNA levels were decreased, whereas B-cell lymphoma-2 mRNA expression was increased after SP treatment. In vitro, mRNA levels of several proinflammatory cytokines and B-cell lymphoma-2 were elevated, whereas transforming growth factor β was decreased after macrophage stimulation with SP (with LPS) over LPS alone. (Mice: n = 105 control; 105 experimental.), Conclusions: These data show that treatment with SP in infectious keratitis elevates growth factors but also adversely affects the disease by enhancing the inflammatory response and its sequelae.

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.

Purpose: Vasoactive intestinal peptide (VIP) is an anti-inflammatory neuropeptide that downregulates proinflammatory cytokines and promotes healing in a susceptible model of P. aeruginosa keratitis. Growth factors also play a role in corneal healing and restoration of tissue homeostasis after wounding. However, whether VIP treatment modulates growth factors to promote healing in the infected cornea remains untested and is the purpose of this study., Methods: C57BL/6 (B6) mice were injected with VIP and mRNA and protein levels, and immunostaining for EGF, FGF, HGF, and VEGF-A were done. Exogenous treatment with a mixture of the growth factors also was tested and levels of cytokines, defensins, and bacterial counts were determined., Results: Real-time RT-PCR, immunostaining, and ELISA data demonstrated that treatment with VIP enhanced levels of EGF, FGF, and HGF during disease, and that VEGF-A, and associated angiogenic molecules also were increased by VIP. Moreover, immunohistochemical studies confirmed that both epithelial and stromal cells participated in growth factor production. Most notably, treatment with a mixture of EGF, FGF, and HGF after disease onset, prevented corneal perforation when compared with controls. This outcome was associated with downregulation of proinflammatory cytokines such as macrophage inflammatory protein-2 (MIP-2), upregulation of anti-inflammatory cytokines such as TGF-β, and antimicrobials β-defensins 2 and 3, as well as decreased plate counts at 1 day postinfection (p.i.) (P = 0.0001)., Conclusions: Collectively, the data provide evidence that VIP treatment modulates growth factors, angiogenic molecules, and defensins in the infected cornea and that this in turn promotes healing and restoration of tissue homeostasis.

Purpose: Testican-1 (or SPOCK) is a highly conserved chimeric proteoglycan encoded by the SPOCK1 gene. Protease regulatory activity has recently been demonstrated by this molecule and its family members testican-2 and -3. The present study tested the hypothesis that testican-1 regulates corneal matrix metalloproteinase (MMP)-2 expression, thus improving disease outcome after Pseudomonas aeruginosa-induced keratitis., Methods: C57BL/6 (B6) and BALB/c mice were routinely infected with P. aeruginosa and were evaluated at various postinfection (pi) times for corneal expression of testican-1 and MMP-2, by PCR array, real-time RT-PCR, ELISA, activity assays, zymography, and immunohistochemistry. Next, B6 mice were treated with recombinant human (rh) testican-1, and expression was knocked down in BALB/c ice by siTestican-1 treatment, to determine the relationship between the two molecules., Results: BALB/c versus B6 mice expressed significantly higher mRNA and protein levels of testican-1 after P. aeruginosa-induced ocular infection. MMP-2 expression and activation was also disparate between the two mouse strains. After rhTestican-1 treatment in B6 mice, overall disease response was significantly improved, whereas siRNA treatment of BALB/c mice converted the normally resistant response to susceptible. Testican-1 was shown to influence MMP-2 expression, activation, and regulation, as well., Conclusions: This study demonstrates corneal expression of testican-1 and its temporal regulation of MMP-2 expression and activation after induction of bacterial keratitis. Furthermore, the data collectively indicate that testican-1 is a novel target for disease treatment to promote better disease outcome regarding chronic inflammation and infection and diseases involving pathologic tissue destruction.

As a novel family of cell surface receptors, triggering receptors expressed on myeloid cells (TREMs) play an important role in inflammatory responses. However, the role of TREMs in the ocular immune system remains unknown. In this study, we examined the expression and function of TREM-1 in Pseudomonas aeruginosa keratitis, one of the most common sight-threatening ocular diseases. TREM-1 was significantly increased in human corneas after P. aeruginosa infection. Consistent with TREM-1 expression at the human ocular surface, TREM-1 levels (mRNA and protein) were also elevated in the infected corneas of C57BL/6 (B6) mice at 1, 3, and 5 days postinfection. To determine whether TREM-1 dictates the outcome of P. aeruginosa keratitis in susceptible mice, TREM-1 signaling in B6 mice was blocked with a soluble mTREM-1/Fc fusion protein. The results indicated that blockade of TREM-1 reduced the severity of corneal disease, polymorphonuclear neutrophil infiltration, Th1/proinflammatory cytokine expression and Toll-like receptor (TLR) activation but enhanced the production of Th2 cytokines, murine β-defensin 2 (mBD2), single Ig interleukin-1R-related molecule (SIGIRR), and ST2. Furthermore, we also used agonistic anti-mTREM-1 antibody to activate TREM-1 signaling in B6 mice and found that TREM-1 activation resulted in worsened disease and earlier corneal perforation in infected B6 mouse corneas and elevated production of proinflammatory cytokines and TLR signaling molecules but reduced expression of mBD2, SIGIRR, and ST2. To the best of our knowledge, this study provides the first evidence that TREM-1 functions as an inflammatory amplifier in P. aeruginosa keratitis by modulating TLR signaling and Th1/Th2 responses.

Purpose: This study tested the hypothesis that the neuropeptide vasoactive intestinal peptide (VIP) regulates adhesion molecule expression, reduces inflammatory cell migration and infiltration into the Pseudomonas aeruginosa-infected cornea of susceptible B6 mice, and promotes corneal healing and resistance., Methods: B6 mice received daily intraperitoneal (IP) injections of VIP from -1 through 5 days after infection. Control mice were similarly injected with sterile phosphate-buffered saline (PBS). Transcript levels of adhesion molecules were determined by PCR array, then select molecules were tested individually by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and confirmed at the protein level by enzyme-linked immunosorbent assay (ELISA) or immunofluorescent staining with confocal laser scanning microscopy at various time points after infection to assess the effects of VIP treatment in the regulation of adhesion molecule expression., Results: Injection of B6 mice with VIP compared with PBS resulted in significant downregulation of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, platelet-endothelial cell adhesion molecule-1, and P-selectin and L-selectin mRNA expression. Protein levels for ICAM-1 and VCAM-1, detected by ELISA, supported the mRNA data at similar time points. Immunofluorescence staining further confirmed the effects of VIP treatment, showing reduced corneal expression of ICAM-1/leukocyte function-associated antigen (LFA-1) and VCAM-1/very late antigen-4 (VLA-4) at select time points compared with PBS-treated animals., Conclusions: VIP treatment downregulates the production of adhesion molecules integral to the transmigration process of host inflammatory cells (polymorphonuclear neutrophils, macrophages) into the infected cornea. This results directly in reduced cellular infiltration, less stromal destruction, and better disease outcome.

Advances in understanding host innate/adaptive immunity and abrogation of immune privilege in ocular viral and bacterial infections have been accomplished using animal models. In Pseudomonas aeruginosa keratitis, mouse models have shown that IL-12-driven IFN-gamma production in Th1 responder strains such as C57BL/6 contributes to corneal perforation, while IL-18-driven IFN-gamma production is associated with bacterial killing and less disease in Th2 responders (BALB/c). The role of neuropeptides, macrophages, and regulation of neutrophil apoptosis is discussed. The potentially blinding Th1 CD4 T-cell-mediated immunopathology referred to as herpes stromal keratitis (HSK) is characterized by breakdown of the normal barrier to blood and lymph angiogenesis in the cornea, a dramatic increase in mature professional antigen-presenting cells, and a heavy leukocytic infiltrate composed primarily of neutrophils. HSK is more frequent and severe in BALB/c than C57BL/6 mice, and varies in severity with the strain and dose of HSV-1 used to infect the cornea.

Purpose: The role of the Fas pathway was tested in Pseudomonas aeruginosa-infected mouse cornea by contrasting the responses of FasL(-/-) and wild-type (WT) mice., Methods: TUNEL staining, real-time RT-PCR, immunostaining, and ELISA assay were used., Results: Compared with WT (resistant) mice, BALB/c FasL(-/-) exhibited significantly elevated bacterial counts and polymorphonuclear leukocyte numbers at 1 and 3 days postinfection (p.i.) and worse outcomes from disease. Similar bacterial challenges in C57BL/6 FasL(-/-) compared with WT mice also led to worsened disease as evidenced by earlier corneal perforation in the susceptible mouse strain. Intense TUNEL staining of apoptotic cells was seen earlier (1 day vs. 3 days) p.i. in BALB/c WT than in knockout mice, This earlier apoptotic pattern correlated with increased expression of caspases 3, 8, and 9 and BAX and with decreased expression of the antiapoptotic molecule Bcl-2. Furthermore, expression levels of the proinflammatory molecule TNF-alpha and its receptor, MIP-2, inducible nitric oxide synthase (iNOS), and nitrite also were significantly elevated in the infected cornea of BALB/c FasL(-/-) compared with WT mice. In vitro, LPS-stimulated Mphi from BALB/c FasL(-/-) mice expressed significantly less caspase 3 and 9, BAX, and IL-10 and more TNF-alpha, MIP-2, and IL-1beta than did cells from WT mice., Conclusions: Fas-FasL interaction in the cornea balances the host innate immune response to improve disease outcome by promoting earlier apoptosis and regulating proinflammatory cytokines/chemokines and nitric oxide (nitrite) production. Dysregulation of this interaction contributes to bystander tissue damage, enhancing nutrients for bacterial growth and worsened disease outcome after P. aeruginosa infection.

Purpose: To determine the role of IL-33 in resistance to Pseudomonas aeruginosa keratitis., Methods: Corneal IL-33 mRNA and protein levels were tested in susceptible C57BL/6 (B6) and resistant BALB/c mice. B6 mice were injected with recombinant mouse IL-33 (rmIL-33) and disease severity, bacterial load, polymorphonuclear neutrophils (PMN) infiltrate, gene expression of inflammatory, and T-helper (Th)1/Th2 cytokines were tested by RT-PCR. IL-33 signaling and macrophage (Mvarphi) polarization also were examined., Results: IL-33 mRNA and protein were expressed constitutively in the normal corneas of both groups and were significantly elevated at 1 to 5 days after infection in BALB/c over B6 mice. rmIL-33-treated B6 mice showed less severe disease than did PBS controls and exhibited decreased bacterial load, PMN infiltrate, and corneal mRNA levels for IL-1beta, MIP-2, and TNF-alpha. Th2-type cytokines (IL-4, -5, -10) also were significantly upregulated, and protein levels for TNF-alpha and IL-10 confirmed the mRNA data. To further investigate IL-33 in corneal inflammation, it was overexpressed in Mvarphi (RAW264.7 cells). This significantly increased IL-5 and IL-10, while it decreased IFN-gamma and other pro-inflammatory cytokines. The role of the Mvarphi was further tested in infected rmIL-33 compared with PBS-injected mice. Immunostaining showed that rmIL-33 injection shifted Mvarphi polarization from NO synthase 2 to arginase production. Furthermore, peritoneally elicited cells (B6 mice) treated with lipopolysaccharide and rmIL-33 exhibited elevated ST2 levels and a shift from IL-12 to IL-10 mRNA production., Conclusions: These data provide evidence that IL-33 promotes a Th2-type immune response and reduces inflammation by polarizing the Mvarphi production of anti-inflammatory mediators in the cornea.