Effect of low-level light therapy in individuals with dry eye disease.

INTRODUCTION: Low-level light therapy (LLLT) or photobiomodulation, the application of red light to the eye, is used for the treatment of dry eye. Limited studies have investigated the efficacy of LLLT as a stand-alone treatment. The investigation aimed to evaluate the effect of LLLT on signs and symptoms of dry eye. METHODS: Participants with mild to moderate dry eye were recruited for this three-visit study. Visits were 7 (±3) days apart and all participants received 633 nm LLLT (eye-light®) for 15 min at each visit. Clinical measures including first and average non-invasive keratograph tear break-up time (NIKBUT), tear meniscus height (TMH), meibomian gland (MG) loss for upper and lower eyelids, ocular surface disease index (OSDI) score, tear film lipid layer thickness, meibum quality score, Schirmer's test, corneal fluorescein staining and eyelid temperature for external upper (EUL) and external lower (ELL) eyelids were measured from the right eye of participants before and after treatment. RESULTS: Thirty participants (mean [SD] age: 31.1 [9.5] years) completed the study. Treatment with LLLT resulted in significant differences in first and average NIKBUT, TMH, tear film lipid layer thickness, OSDI score, Schirmer's test, meibum quality score and eyelid temperature over time (all p < 0.05). Compared to baseline, TMH, tear film lipid layer thickness and eyelid temperature significantly increased by 0.06 mm (95% CI: 0.01-0.11), 12.9 nm (95% CI: 1.18-24.55), and 7.0°C, respectively, for both EUL (95% CI: 6.17-7.84) and ELL (95% CI: 6.17-7.73). The respective decrease in the OSDI score and Schirmer's test was 10.2 (95% CI: -15.15 to -5.26) and 4.4 mm (95% CI: -7.31 to -1.42; all p < 0.05). There was no significant difference in corneal fluorescein staining and MG loss after LLLT. CONCLUSION: Low-level light therapy treatment significantly improved signs and symptoms of dry eye in the early phases of treatment, suggesting its efficacy for dry eye management.

Potential pro-inflammatory impact of scleral lens midday fogging on human corneal epithelial cells: An in vitro study.

PURPOSE: Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown. This study examines the in vitro pro-inflammatory effect of the FR on human corneal epithelial cells in varying degrees of MDF. METHODS: Normal SL neophytes were recruited to wear SL 8 h daily for 4 days. Following 8 h on days 1 and 4, optical coherence tomography (OCT) images were acquired for MDF quantification using ImageJ, and the FR was collected. FR samples from the same eye were later pooled, diluted 2-fold and applied on human telomerase-immortalized corneal epithelial (hTCEpi) cells cultured on Terasaki microwell plates. Tumor necrosis factor (TNF)-α and culture media were used as positive and negative controls, respectively. After a 30-minute treatment, the nuclear factor-kappa B (NF-κB) pathway was measured by NF-κB-p65 immunofluorescence and images were analyzed with ImageJ. Pearson's correlation was conducted to determine the association between median nuclear fluorescence and MDF. RESULTS: Fourteen FR samples with a mean volume of 22 ± 16 µl were tested. Mean MDF severity following 8 h of SL wear was 25 ± 17 units (range 7 - 64). The median nuclear fluorescence (NF-κB-p65 translocation) in cultured hTCEpi cells ranged from 31.43 to 45.16 while the negative and positive controls were 44.71 ± 1.72 and 108.77 ± 68.38, respectively. Although a potential positive trend between MDF and median nuclear fluorescence was observed, Pearson's correlation analysis revealed no significant association (r = +0.48, P = 0.09). CONCLUSIONS: The results suggest that the FR can trigger NF-κB-p65 translocation in hTCEpi cells, which may be associated with MDF severity. This study introduces the use of Terasaki microwell plates for immunofluorescence studies of the FR. The technique is simple, minimizes sample usage, and does not require expensive instrumentation.

Nonpolar Lipids Contribute to Midday Fogging During Scleral Lens Wear.

Purpose: To determine correlations between lipids in the fluid reservoir (FR) and the severity of midday fogging (MDF) in scleral lens (SL) wear. Methods: SL neophytes were recruited to wear custom SL for 4 days, examined after 8 hours on days 1 and 4. Lens vault and MDF were quantified from anterior segment optical coherence tomography (AS-OCT), and the FR was collected and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Relative abundance of lipids was compared to MDF scores using nonparametric correlation testing (Spearman rank). Ocular surface and SL fitting characteristics (lens vault, fitting curves) were likewise compared to MDF. Results: Thirteen participants (26 eyes, 69% female, 28 ± 9 years old) were included in this study. MDF severity after 8 hours of SL wear was 33 ± 29 units on day 1 and 28 ± 24 units on day 4 (r = .94; P < 0.01). Twelve samples were analyzed using LC-MS/MS, and a total of 170 distinct lipid species were detected. The lipid classes with greatest correlation to MDF were the wax esters (r = .73, P = 0.01), cholesteryl esters (r = .59; P = 0.049), and triacylglycerols (r = .64, P = 0.03). Polar lipids were observed abundantly in all samples. None of the measured ocular surface or fitting outcomes were correlated to MDF. Conclusions: Nonpolar lipids were the greatest contributors to MDF among these normal participants. Polar lipids may be due to cellular debris, although they do not appear contributory to MDF.

Changes in tear glucose and insulin concentrations following an oral glucose tolerance test.

CLINICAL RELEVANCE: Tear glucose and insulin are responsible for the health of the ocular surface; thus, it is important for clinicians to detect the tear glucose and insulin using point-of-care methods. AIM: To determine if changes in blood glucose and insulin levels following an oral glucose tolerance test are reflected in the tears and to test the association between gene expression and tear insulin and glucose. METHODS: Twenty healthy young adults were enrolled. Basal tears and peripheral blood samples were collected to assess glucose and insulin using a point-of-care glucometer and ELISA assays in fasted subjects, and 1.5 and 3 h after an oral glucose challenge. Conjunctival impression cytology was collected to determine gene expression of insulin receptor (INSR) and glucose transporters (GLUT1 and GLUT4). Changes were examined using non-parametric one-way ANOVA. Spearman tests were conducted to examine associations between variables. RESULTS: Glucose and insulin levels increased 1.5 h after oral glucose in both blood (P < 0.001) and tears (P < 0.049) and returned to near baseline values after 3 h. There was a positive correlation between glucose levels in the blood and tears (rho = 0.57, P < 0.001), but not between blood and tear insulin levels (P = 0.18). Glucose and insulin levels in tears were correlated (rho = 0.32, P = 0.048). Tear glucose concentration at 1.5 h after oral glucose was associated with INSR expression (rho = 0.49, P = 0.03), and there was a trend with GLUT1 (P = 0.06) but not GLUT4. CONCLUSION: Tear glucose reflected blood glucose levels but this correspondence was not observed for insulin. Further studies are required to determine the role of glucose and insulin on the ocular surface in both health and diabetes.

Anti-inflammatory and wound healing potential of medicinal maggot excretions/secretions at the ocular surface.

PURPOSE: In the skin, Lucilia sericata maggot excretions/secretions (ES) accelerate wound healing and limit inflammation. This study aimed to determine whether ES have similar beneficial effects at the ocular surface. METHODS: Human corneal epithelial cells (HCEC) were cultured with ES and cell viability was determined by the MTT assay. Additionally, mRNA expression of growth factors, antimicrobial peptides (AMPs) and cytokines was assessed by qPCR. ES ability to modulate TLR-induced IL-6 and IL-8 expression was determined by qPCR and ELISA. ES potential to promote corneal healing was evaluated in vitro by a migration assay in HCEC, and in vivo using a mouse model. RESULTS: ES did not impair HCEC viability up to 25 µg/ml. Among the factors evaluated, only hBD-2 was upregulated (2.5-fold) by 1.5 µg/ml ES after 6 hrs (P = 0.04). In HCEC, ES reduced Poly I:C-induced IL-6 and IL-8 mRNA (P ≤ 0.001) and protein (P ≤ 0.0001) expression. A similar effect was observed with Flagellin (TLR5 agonist) but it was less robust for FSL-1 (TLR2/6 agonist) and Pam3CSK4 (TLR1/2 agonist). The greatest in vitro migration effect was observed with 6.2 µg/ml ES after 44 hrs where gap area compared to vehicle was 53.3 ± 3.7% vs. 72.6 ± 5.4% (P = 0.001). In the mouse model, the maximum healing effect was present with 1.5 µg/ml ES after 12 hrs with a wound area of 19.0 ± 2.7% vs. 60.1 ± 21.6% (P = 0.003) or 77% reduction of the wound area compared to the negative control. CONCLUSIONS: ES significantly reduce in vitro TLR-induced production of inflammatory cytokines and promote corneal wound healing.

Impact of Low Humidity on Damage-associated Molecular Patterns at the Ocular Surface during Dry Eye Disease.

SIGNIFICANCE: Dry eye is one of the leading causes for individuals to seek eye care, whereas the pathogenesis is poorly understood. One mechanism in which dry eye inflammation may ensue is by the release of damage-associated molecular patterns (DAMPs) by damaged cells to stimulate the production of cytokines and matrix metalloproteinases. Examining DAMP levels on the ocular surface during dry eye disease (DED) will increase our understanding of their potential involvement in the pathogenesis of DED. PURPOSE: This study aimed to quantitate DAMPs, high-mobility group box 1 (HMGB1), and heat shock proteins on the ocular surface of normal and dry eye subjects and to examine the impact of low-humidity environment (LHE) on DAMPs and inflammation in dry eye subjects. METHODS: Basal tears (10 to 20 µL) and conjunctival impression cytology samples were analyzed for HMGB1, HSP-27, HSP-60, HSP-70, and HSP-90α by ELISA or Luminex assays in normal (n = 15) and DED (n = 15) subjects. In addition, a subset of DED subjects were exposed to LHE for 2 hours. The level of DAMPs in the tear film was evaluated by ELISA or Luminex assay. Interleukin 6, interleukin 8, or metalloproteinase (MMP) 9 mRNA were quantitated by real-time polymerase chain reaction from conjunctival impression cytology samples. RESULTS: Compared with age-matched normal subjects, HMGB1 was significantly elevated in the tear film of DED subjects (P = .03), whereas there was no significant difference in heat shock proteins. Conjunctival impression cytology samples revealed no significant difference in intracellular DAMP levels between both groups. After exposure to an LHE, there was an increase in corneal staining (P = .005), HSP-60 levels in the tear film (P = .01), and MMP-9 mRNA in the conjunctiva (P = .001). CONCLUSIONS: Dry eye subjects had higher levels of HMGB1 in their tear film. Exposure to an LHE worsened corneal staining, increased conjunctival MMP-9 mRNA expression, and increased tear film HSP-60 levels. Larger studies are needed to understand the involvement of DAMPs in stimulating dry eye inflammation.

Proteoglycan 4 (PRG4) expression and function in dry eye associated inflammation.

Dry eye disease (DED) affects hundreds of millions of people worldwide. It is characterized by the production of inflammatory cytokines and chemokines as well as damaging matrix metalloproteinases (MMPs) at the ocular surface. While proteoglycan 4 (PRG4), a mucin-like glycoprotein present at the ocular surface, is most well known as a boundary lubricant that contributes to ocular surface integrity, it has been shown to blunt inflammation in various cell types, suggesting a dual mechanism of action. Recently, full-length recombinant human PRG4 (rhPRG4) has been shown to improve signs and symptoms of DED in humans. However, there remains a significant need for basic science research on rhPRG4's biological properties and its potential therapeutic mechanisms of action in treating DED. Therefore, the objectives of this study were to characterize endogenous PRG4 expression by telomerase-immortalized human corneal epithelial (hTCEpi) cells, examine whether exogenous rhPRG4 modulates cytokine and chemokine secretion in response to dry eye associated inflammation (TNFα and IL-1ß), explore interactions between rhPRG4 and MMP-9, and understand how experimental dry eye (EDE) in mice affects PRG4 expression. PRG4 secretion from hTCEpi cells was quantified by Western blot and expression visualized by immunocytochemistry. Cytokine/chemokine production was measured by ELISA and Luminex, while rhPRG4's effect on MMP-9 activity, binding, and expression was quantified using an MMP-9 inhibitor kit, surface plasmon resonance, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Finally, EDE was induced in mice, and PRG4 was visualized by immunohistochemistry in the cornea and by Western blot in lacrimal gland lysate. In vitro results demonstrate that hTCEpi cells synthesize and secrete PRG4, and PRG4 secretion is inhibited by TNFα and IL-1ß. In response to these pro-inflammatory stresses, exogenous rhPRG4 significantly reduced the stimulated production of IP-10, RANTES, ENA-78, GROα, MIP-3α, and MIG, and trended towards a reduction of MIP-1α and MIP-1ß. The hTCEpi cells were also able to internalize fluorescently-labelled rhPRG4, consistent with a mechanism of action that includes downstream biological signaling pathways. rhPRG4 was not digested by MMP-9, and it did not modulate MMP-9 gene expression in hTCEpi cells, but it was able to bind to MMP-9 and inhibited in vitro activity of exogenous MMP-9 in the presence of human tears. Finally, in vivo results demonstrate that EDE significantly decreased immunolocalization of PRG4 on the corneal epithelium and trended towards a reduction of PRG4 in lacrimal gland lysate. Collectively these results demonstrate rhPRG4 has anti-inflammatory properties on corneal epithelial cells, particularly as it relates to mitigating chemokine production, and is an inhibitor of MMP-9 activity, as well as that in vivo expression of PRG4 can be altered in preclinical models of DED. In conclusion, these findings contribute to our understanding of PRG4's immunomodulatory properties in the context of DED inflammation and provide the foundation and motivation for further mechanistic research of PRG4's properties on the ocular surface as well as expanding clinical evaluation of its ability as a multifunctional therapeutic agent to effectively provide relief to those who suffer from DED.

The Role of Endogenous Antimicrobial Peptides in Modulating Innate Immunity of the Ocular Surface in Dry Eye Diseases.

The ocular surface has the challenging responsibility of maintaining a clear moist refractive surface while protecting the eye from exogenous pathogens and the environment. Homeostasis of the ocular surface, including its innate immune components, is altered in ocular surface disease states. In this review, we focus on antimicrobial peptides and the role they play in the immune response of the ocular surface during healthy states and dry eye diseases. Antimicrobial peptides are of special interest to the study of the ocular surface because of their various roles that include microbial threat neutralization, wound healing, and immune modulation. This review explores current literature on antimicrobial peptides in ocular surface diseases and discusses their therapeutic potential in ocular surface diseases and dry eye.

Obese Mice with Dyslipidemia Exhibit Meibomian Gland Hypertrophy and Alterations in Meibum Composition and Aqueous Tear Production.

BACKGROUND: Dyslipidemia may be linked to meibomian gland dysfunction (MGD) and altered meibum lipid composition. The purpose was to determine if plasma and meibum cholesteryl esters (CE), triglycerides (TG), ceramides (Cer) and sphingomyelins (SM) change in a mouse model of diet-induced obesity where mice develop dyslipidemia. METHODS: Male C57/BL6 mice (8/group, age = 6 wks) were fed a normal (ND; 15% kcal fat) or an obesogenic high-fat diet (HFD; 42% kcal fat) for 10 wks. Tear production was measured and meibography was performed. Body and epididymal adipose tissue (eAT) weights were determined. Nano-ESI-MS/MS and LC-ESI-MS/MS were used to detect CE, TG, Cer and SM species. Data were analyzed by principal component analysis, Pearson's correlation and unpaired t-tests adjusted for multiple comparisons; significance set at p ≤ 0.05. RESULTS: Compared to ND mice, HFD mice gained more weight and showed heavier eAT and dyslipidemia with higher levels of plasma CE, TG, Cer and SM. HFD mice had hypertrophic meibomian glands, increased levels of lipid species acylated by saturated fatty acids in plasma and meibum and excessive tear production. CONCLUSIONS: The majority of meibum lipid species with saturated fatty acids increased with HFD feeding with evidence of meibomian gland hypertrophy and excessive tearing. The dyslipidemia is associated with altered meibum composition, a key feature of MGD.

IOP and Optic Nerve Head Morphology during Scleral Lens Wear.

SIGNIFICANCE: Scleral lenses (SLs) are increasing in scope, and understanding their ocular health impact is imperative. The unique fit of an SL raises concern that the landing zone causes compression of conjunctival tissue that can lead to resistance of aqueous humor outflow and increased intraocular pressure (IOP). PURPOSE: This study aimed to assess changes in optic nerve head morphology as an indirect assessment of IOP and evaluate other IOP assessment methods during SL wear. METHODS: Twenty-six healthy adults wore SL on one randomly selected eye for 6 hours, whereas the fellow eye served as a control. Global minimum rim width (optical coherence tomography) and IOP (Icare, Diaton) were measured at baseline, 2 and 6 hours after SL application, and again after SL removal. Central corneal thickness, anterior chamber depth, and fluid reservoir depth were monitored. RESULTS: Minimum rim width thinning was observed in the test (-8 µm; 95% confidence interval [CI], -11 to -6 µm) and control (-6 µm; 95% CI, -9 to -3 µm) eyes after 6 hours of SL wear (P < .01), although the magnitude of thinning was not significantly greater in the lens-wearing eyes (P = .09). Mean IOP (Icare) significantly increased +2 mmHg (95% CI, +1 to +3 mmHg) in the test eyes (P = .002), with no change in the control eyes. Mean IOP changes with Diaton were +0.3 mmHg (95% CI, -0.9 to +3.2 mmHg) in the test eyes and +0.4 mmHg (95% CI, -0.8 to +1.7 mmHg) in the control eyes. However, Diaton tonometry showed poor within-subject variation and poor correlation with Icare. No clinically significant changes were observed in central corneal thickness or anterior chamber depth. CONCLUSIONS: This study suggests that SLs have a minimal effect on IOP homeostasis in the normal eye during SL wear and an insignificant impact on the optic nerve head morphology in healthy adult eyes.

Lipid Analysis on Block Copolymer-containing Packaging Solution and Lens Care Regimens: A Randomized Clinical Trial.

SIGNIFICANCE: Lotrafilcon B lenses packaged in and cared for with block copolymer-containing (polyoxyethylene-polyoxybutylene; EOBO) lens care solutions resulted in lower cholesterol extraction than each of the habitual silicone hydrogel lens/multipurpose solution (MPS) regimens tested. PURPOSE: This study aimed to compare the extracted cholesterol of lotrafilcon B lenses packaged in and cared for with EOBO-containing lens care solutions with the extracted cholesterol of habitual silicone hydrogel lenses cared for with MPS not containing EOBO. METHODS: In this prospective, randomized, observer-masked parallel study, habitual wearers of senofilcon C, senofilcon A, comfilcon A, and samfilcon A contact lenses using a non-EOBO MPS were randomized 1:1 to lotrafilcon B lenses packaged in and cared for with EOBO-containing solutions or to their habitual lenses and MPS. Subjects randomized to lotrafilcon B were further randomized to one of two EOBO-containing lens care solutions, OPTI-FREE PUREMOIST or CLEAR CARE PLUS with HydraGlyde (Alcon Laboratories, Inc., Fort Worth, TX). A subset of right eye lenses was collected after wear, and total cholesterol was extracted and measured using a fluorometric enzymatic assay. RESULTS: Of 143 lenses analyzed, 95 were from subjects randomized to their habitual lenses/MPS and 48 to lotrafilcon B + EOBO lenses plus CLEAR CARE PLUS with HydraGlyde or OPTI-FREE PUREMOIST. The mean amounts of cholesterol extracted from lotrafilcon B + EOBO lenses cared for with CLEAR CARE PLUS with HydraGlyde (0.28 ± 0.18 µg/lens) and OPTI-FREE PUREMOIST (0.28 ± 0.48 µg/lens) were significantly lower than those extracted from senofilcon C (4.18 ± 3.25 µg/lens), senofilcon A (2.19 ± 2.69 µg/lens), comfilcon A (2.17 ± 1.47 µg/lens), and samfilcon A (2.07 ± 1.48 µg/lens) lenses used with MPS (P < .0001 each). CONCLUSIONS: Cholesterol sorption was significantly lower in wearers of lotrafilcon B lenses cared for with polyoxyethylene-polyoxybutylene-containing lens care solutions than in users of habitual silicone hydrogel lenses cared for with non-polyoxyethylene-polyoxybutylene MPS.

Scleral lens wear: Measuring inflammation in the fluid reservoir.

PURPOSE: To measure inflammatory mediators in the scleral lens fluid reservoir (FR) in healthy eyes and to compare them to basal tear samples after 8-hs (8h) and 4-days (4d) of scleral lens (SL) wear. METHODS: Fifteen normal, habitual soft contact lens wearers were fitted with 14.8- or 15.4-mm SLs (Zenlens, Alden Optical, USA). Basal ocular surface tears and FR samples were collected after 8h and 4d of daily SL wear. Levels of interleukin (IL) -4 and -8, matrix metalloproteinase (MMP)-7, -9, and -10, and tissue inhibitor of MMPs (TIMPs) 1-4 were measured in all samples using Luminex assays. Visual acuity, corneal and conjunctival staining, and comfort assessments were completed at the baseline, 8h and 4d time points. RESULTS: MMP-9 and MMP-10 were greater in FR than basal ocular surface tears. After 8h of SL wear, the median concentration of MMP-9 in the FR and basal tears were 62.7 and 15.2 ng/mL, respectively (p = 0.047). Likewise, MMP-10 was significantly greater in FR compared to basal tears, after 8h (25.8 ng/mL vs 2.8 ng/mL, p < 0.001) and 4d (2.1 ng/mL vs17.2 ng/mL, p = 0.047). IL-4 and IL-8 levels were greater in FR but not significantly at 8h (2.2 vs 3.1 ng/mL; and 0.1 vs 0.4 ng/mL, respectively) or 4d (0.9 vs 3.5 ng/mL; 0.0 vs 0.2 ng/mL). MMP-7 was not affected by SL wear after 8h (46.0 basal vs 54.4 ng/mL FR) or 4d (34.2 vs 87.5 ng/mL). Visual acuity, corneal and conjunctival staining did not change; comfort was reduced in SL compared to soft contact lens wear. CONCLUSIONS: This is the first study to compare the FR with the basal ocular surface tears. MMP-9 and MMP-10 were elevated in the FR after several hours of SL wear, suggesting potential clinical implications of SL wear and deserves further investigation.

Dyslipidemia and Meibomian Gland Dysfunction: Utility of Lipidomics and Experimental Prospects with a Diet-Induced Obesity Mouse Model.

Meibomian gland dysfunction (MGD) is the leading cause of dry eye disease and loss of ocular surface homeostasis. Increasingly, several observational clinical studies suggest that dyslipidemia (elevated blood cholesterol, triglyceride or lipoprotein levels) can initiate the development of MGD. However, conclusive evidence is lacking, and an experimental approach using a suitable model is necessary to interrogate the relationship between dyslipidemia and MGD. This systematic review discusses current knowledge on the associations between dyslipidemia and MGD. We briefly introduce a diet-induced obesity model where mice develop dyslipidemia, which can serve as a potential tool for investigating the effects of dyslipidemia on the meibomian gland. Finally, the utility of lipidomics to examine the link between dyslipidemia and MGD is considered.

MyD88 Deficiency Protects Against Dry Eye-Induced Damage.

Purpose: Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation. Toll-like receptors (TLRs) are integral in the initiation of inflammatory signaling. Therefore, we evaluated the effect of TLR-deficiency on dry eye-related ocular surface damage and inflammation using a mouse model of experimental dry eye (EDE). Methods: C57BL/6 wild-type (WT), MyD88-/-, and IL-1R-/- mice were exposed to EDE conditions for 5 days. Tear production was measured by phenol red thread test and ocular surface damage assessed with fluorescein staining. Corneal homogenates were obtained for matrix metalloproteinase (MMP) and cytokine expression analysis by Luminex assay and quantitative PCR. In addition, whole eyes and eyelids were dissected and goblet cells and Meibomian glands were imaged, respectively. Results: Following 5 days of EDE, WT mice had extensive ocular surface staining, while MyD88-/- mice had no increased staining above non-EDE conditions. Similarly, MyD88-/- mice did not have increased corneal MMP-2, 3, or 8 concentrations, as seen with WT mice. MyD88-deficiency also resulted in decreased corneal cytokine levels. In addition, MyD88-/- mice had significantly lower conjunctival goblet cell counts compared with both WT (EDE) and IL-1R-/- (non-EDE) mice. However, there was no difference in Meibomian gland morphology between WT, IL-1R-/-, and MyD88-/- mice. Conclusions: These studies demonstrate the importance of TLR signaling in dry eye development. Mice lacking TLR signaling, MyD88-/-, were protected from EDE-induced ocular surface damage and inflammatory mediator expression, warranting further investigation into TLR inhibition as a potential therapeutic for DED.

Neutrophils cause obstruction of eyelid sebaceous glands in inflammatory eye disease in mice.

Meibomian glands (MGs) are sebaceous glands of the eyelid margin that secrete lipids needed to avert tear evaporation and to help maintain ocular surface homeostasis. Obstruction of MGs or other forms of MG dysfunction can promote chronic diseases of the ocular surface. Although chronic eyelid inflammation, such as allergic eye disease, is an associated risk factor for obstructive MG dysfunction, it is not clear whether inflammatory processes contribute to the pathophysiology of MG obstruction. We show that polymorphonuclear neutrophils (PMNs) promoted MG obstruction in a chronic inflammatory model of allergic eye disease in mice. Analysis of leukocytes in tears of patients with MG dysfunction showed an increase in PMN numbers compared to healthy subjects. Moreover, PMN numbers in tears positively correlated with clinical severity of MG dysfunction. Our findings point to a role for PMNs in the pathogenesis and progression of MG dysfunction.

High-Mobility Group Box 1 in Dry Eye Inflammation.

Purpose: To determine high-mobility group box 1 (HMGB1) expression during experimental dry eye (EDE) and dry eye-like culture conditions and elucidate its role in corneal dry eye-related inflammation. Methods: EDE was induced in 8- to 12-week-old C57BL/6 mice. Corneal tissue sections and lysates from EDE and untreated mice were evaluated for HMGB1 expression by immunostaining and quantitative real-time PCR (qPCR). For in vitro studies, human corneal epithelial cells (HCEC) were treated with hyperosmolar media, toll-like receptor (TLR) agonists, or proinflammatory cytokines to determine HMGB1 expression. HCEC were also treated with human recombinant HMGB1 (hrHMGB1) alone or in combination with inflammatory stimuli, and TNFα, IL-6, and IL-8 expression evaluated by qPCR and ELISA. Nuclear factor-κB (NF-κB) p65 nuclear translocation was determined by immunostaining. Results: EDE mice had higher corneal HMGB1 RNA and protein expression compared to untreated animals. In HCEC, hyperosmolar stress and TNFα treatment stimulated HMGB1 production and secretion into culture supernatants. However, in vitro stimulation with hrHMGB1 did not induce secretion of TNFα, IL-6, or IL-8 or NF-κB p65 nuclear translocation. In addition, the inflammatory response elicited by TLR agonists fibroblast-stimulating lipopeptide-1 and lipopolysaccharide was not enhanced by hrHMGB1 treatment. Conclusions: HMGB1 expression was enhanced by dry eye conditions in vivo as well as in vitro, during hyperosmolar stress and cytokine exposure, suggesting an important role for HMGB1 in dry eye disease. However, no direct inflammatory effect was observed with HMGB1 treatment. Therefore, under these conditions, HMGB1 does not contribute directly to dry eye-induced inflammation and its function at the ocular surface needs to be explored further.

MyD88 contribution to ocular surface homeostasis.

The cornea must maintain homeostasis, enabling rapid response to injury and microbial insult, to protect the eye from insult and infection. Toll-like receptors (TLRs) are critical to this innate immune response through the recognition and response to pathogens. Myeloid differentiation primary response (MyD88) is a key signaling molecule necessary for Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R)-mediated immune defense and has been shown to be necessary for corneal defense during infection. Here, we examined the intrinsic role of TLR signaling in ocular surface tissues by determining baseline levels of inflammatory mediators, the response to mechanical stimuli, and corneal infection in MyD88-deficient mice (MyD88-/-). In addition, cytokine, chemokine, and matrix metalloproteinase (MMP) expression was determined in ocular surface cells exposed to a panel of TLR agonists. Compared to wild-type (WT) animals, MyD88-/- mice expressed lower MMP-9 levels in the cornea and conjunctiva. Corneal IL-1α, TNFα, and conjunctival IL-1α, IL-2, IL-6, and IL-9 levels were also significantly reduced. Additionally, CXCL1 and RANTES expression was lower in both MyD88-/- tissues compared to WT and IL-1R-/- mice. Interestingly, MyD88-/- mice had lower corneal sensitivities (1.01±0.31 gm/mm2) than both WT (0.59±0.16 gm/mm2) and IL-1R-/- (0.52±0.08 gm/mm2). Following Pseudomonas aeruginosa challenge, MyD88-/- mice had better clinical scores (0.5±0.0) compared to IL-1R-/- (1.5±0.6) and WT (2.3±0.3) animals, but had significantly more corneal bacterial isolates. However, no signs of infection were detected in inoculated uninjured corneas from either MyD88 or IL-1R-deficient mice. This work furthers our understanding of the importance of TLR signaling in corneal defense and immune homeostasis, showing that a lack of MyD88 may compromise the baseline innate response to insult.

Analysis of tear inflammatory mediators: A comparison between the microarray and Luminex methods.

PURPOSE: Inflammatory mediators have been shown to modulate dry eye (DE) disease and may correlate with disease severity, yet the methods used and the associated findings vary significantly in the literature. The goal of this research was to compare two methods, the quantitative microarray and the magnetic bead assay, for detecting cytokine levels in extracted tear samples across three subject groups. METHODS: Tears were collected from Schirmer strips of the right and left eyes of 20 soft contact lens wearers (CL), 20 normal non-contact lens wearers (NOR), and 20 DE subjects and stored at -80 °C. Tear proteins were eluted and precipitated using ammonium bicarbonate and acetone. The right and left eye samples were combined for each subject. Following the Bradford protein quantitation method, 10 µg of total protein was used for each of the two analyses, Quantibody® Human Inflammation Array 3 (RayBiotech) and High Sensitivity Human Cytokine Magnetic Bead Kit (Millipore). The assays were run using the GenePix® 4000B Scanner (Molecular Devices) or the Luminex MagPix® plate reader (Luminex), respectively. The data were then compared between the two instruments and the three subject groups. RESULTS: Of the 40 proteins on the Quantibody® microarray, seven had average expression levels above the lower limit of detection: ICAM-1, MCP-1, MIG, MCSF, TIMP-1, TIMP-2, and TNF-RI. Significant differences in expression levels (p<0.05) were detected between the CL and DE groups for MCSF, TIMP-1, and TNF R1, between the NOR and DE groups for ICAM-1, and between the CL and NOR groups for ICAM-1, MCP-1, MCSF, TIMP-1, TIMP-2, and TNF-R1 when using the Student t test. Of the 13 proteins tested with Luminex, IL-1ß, IL-4, IL-6, IL-7, and IL-8 had expression levels above the minimum detectable level, and these were most often detected using the Luminex assay compared to the Quantibody® microarray. Contrarily, IL-2, IL-12, IL-13, INF-g, and GM-CSF were detected more frequently using the Quantibody® microarray than the Luminex assay. Significant differences in expression levels (p<0.05) were only detected between the CL and DE groups for IL-7 and IL-8 and between the CL and NOR subjects for IL-8. CONCLUSIONS: In addition to detecting more significant differences between the subject groups, the Quantibody® microarray detected more inflammatory cytokines in total within the range of detection than the Luminex assay. Differences were also noted in the types of cytokines each assay could detect from the limited protein samples. Both methods offer advantages and disadvantages; therefore, these factors should be considered when determining the appropriate assay for analyzing tear protein samples.

Functions of Peptidoglycan Recognition Proteins (Pglyrps) at the Ocular Surface: Bacterial Keratitis in Gene-Targeted Mice Deficient in Pglyrp-2, -3 and -4.

PURPOSE: Functions of antimicrobial peptidoglycan recognition proteins (Pglyrp1-4) at the ocular surface are poorly understood. Earlier, we reported an antibacterial role for Pglyrp-1 in Pseudomonas aeruginosa keratitis. Here we investigated functions of three other related genes Pglyrp-2, -3 and -4 in a mouse model of P. aeruginosa keratitis. METHODS: Wild type (WT) and each of the Pglyrp-null genotypes were challenged with P. aeruginosa keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-α, IFN-γ, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of Pglyrp-1-4, mouse beta defensins (mBD)-2,-3, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the Pglyrp-null mouse types. RESULTS: The Pglyrp-2-/- mice showed reduced disease and lower induction of pro-inflammatory TNF-α (p = 0.02) than WT or the other Pglyrp null mice. Viable bacterial yield was significantly lower in the Pglyrp-2-/- (p = 0.0007) and the Pglyrp-4-/- (p = 0.098) mice. With regards to expression of these antimicrobial genes, Pglyrp-2 expression was induced after infection in WT mice. Pglyrp-3 expression was low before and after infection in WT mice, while Pglyrp-4 expression was slightly elevated after infection in WT, Pglyrp-2 and -3 null mice. Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance. CONCLUSIONS: Efficient resolution of keratitis in the Pglyrp-2-/- mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

Dry eye modulates the expression of toll-like receptors on the ocular surface.

We aimed to determine if toll-like receptor (TLR) expression is modulated in response to dry eye-associated conditions and in dry eye syndrome (DES). Primary human corneal epithelial cells (HCEC), an SV40 HCEC cell line or a normal human conjunctival epithelial cell line (IOBA-NHC) were cultured under hyperosmolar stress (HOS) (400-500 mOsm/kg) or with DES associated cytokines (IL-1α/ß, TNFα or TGFß) at concentrations ranging from 1 to 1000 ng/ml for up to 24 h. Epithelial cells were harvested from a human cornea organ culture model following 24 h of desiccation. Conjunctival impression cytology samples were harvested from subjects with DES and age and gender-matched normal subjects. TLR4, TLR5 or TLR9 mRNA or protein was examined by quantitative RT-PCR, western blotting or flow cytometry. TLR functionality was evaluated in terms of addition of TLR agonists and quantitation of secreted inflammatory cytokines by the use of ELISA and Luminex assays. In SV40 HCEC, HOS significantly increased TLR4 by 8.18 fold, decreased TLR9 by 0.58 fold, but had no effect on TLR5 mRNA expression. TLR4 and TLR9 protein were decreased by 67.7% and 72% respectively. TLR4 mRNA was also significantly up-regulated by up to 9.70 and 3.36 fold in primary HCEC and IOBA-NHC respectively. DES associated cytokines had no effect on TLR4, TLR5 and TLR9 expression. In response to desiccation, TLR4 and TLR5 mRNA were significantly up-regulated by 4.81 and 2.51 fold respectively, while TLR9 mRNA was down-regulated by 0.86 fold in HCEC. A similar trend for TLR4 and TLR9 protein was observed. TLR9 mRNA was significantly down-regulated by almost 59.5% in DES subjects. In conclusion, changes in TLR expression occur in dry eye and could have an important role in ocular surface susceptibility to inflammation and infection.