Tear Film miRNAs and Their Association With Human Dry Eye Disease.

PURPOSE: miRNAs can regulate inflammatory pathways. The purpose of this work was to determine if inflammatory-related tear film miRNAs are associated with extracellular vesicles (EVs) in human non-Sjögren's Syndrome dry eye disease (DED) participants. METHODS: Five DED and 5 non-DED human participants were recruited. Tears samples were collected by washing the ocular surface of both eyes with phosphate buffered saline, pooling samples from the right and left eyes, and purifying EVs from the samples with a polyethylene glycol (PEG) 8000 precipitation procedure. Samples were directly analyzed via ELISA or transmission electron microscopy (TEM), or RNA was isolated first from the EVs and evaluated with RNA-Seq. RESULTS: EVs were identified in the tear film of both groups using TEM and ELISA. Following EV purification and RNA isolation, RNA-Seq determined that there were 126 EV miRNAs differentially expressed between the two groups when comparing their RNA cargoes. Ingenuity Pathways Analysis found 9 upregulated miRNAs that were associated with inflammation (miR-127-5p, miR-1273h-3p, miR-1288-5p, miR-130b-5p, miR-139-3p, miR-1910-5p, miR-203b-5p, miR-22-5p, and miR-4632-3p; all p < 0.049; fold regulation range = 1.43-1.67). CONCLUSION: This study determined that EVs are present in the tear film and that tear EVs contain miRNAs that may be associated with DED inflammatory pathways.

Enhanced closed eye neutrophil degranulation in dry eye disease.

PURPOSE: Closed eye neutrophils have demonstrated increased prevalence in dry eye disease, but the phenotype and extent of activation of these cells has yet to be described. METHODS: 12 normal subjects and 12 subjects with dry eye disease were recruited and trained for self-collection of closed eye leukocytes, immediately upon awakening. Tear leukocytes were isolated and peripheral blood was collected, and stained with a panel of fluorescently-labeled antibodies to determine the activation phenotype of neutrophils. Extracellular matrix metalloproteinase 9 (MMP9) and neutrophil elastase (NE) was quantified by an enzyme-linked immunosorbent assay. RESULTS: Total numbers of tear leukocytes recovered, at awakening, from normal and dry eye subjects were similar. Tear neutrophils from dry eye subjects had increased expression of membrane receptor CD66b, a marker associated with secondary granule degranulation. There was also a higher proportion of monocytes in the dry eye cohort, as compared to the normal cohort. Extracellular MMP9 was significantly higher in subjects with dry eye disease, and while NE was also elevated, it did not achieve statistical significance. CONCLUSIONS: Increased inflammation can be observed in the closed eye tears of subjects with dry eye disease, and neutrophils may be a potential source of pathogenic species in dry eye disease. Further research is required to determine the diagnostic potential of closed eye tears.

The closed eye harbours a unique microbiome in dry eye disease.

Dry eye affects millions of individuals. In experimental models, dry eye disease is associated with T helper cell 17-mediated inflammation of the ocular surface that may cause persistent damage to the corneal epithelium. However, the initiating and perpetuating factors associated with chronic inflammation of the ocular surface remain unclear. The ocular microbiota alters ocular surface inflammation and may influence dry eye disease development and progression. Here, we collected serial samples of tears on awakening from sleep, closed eye tears, during a randomized clinical trial of a non-pharmaceutical dry eye therapy and used 16S rRNA metabarcoding to characterize the microbiome. We show the closed dry eye microbiome is distinct from the healthy closed eye microbiome, and that the microbiome remains distinct despite daily saline eye wash upon awakening. The ocular microbiome was described only recently, and this report implicates a distinct microbiome in ocular disease development. Our findings suggest an interplay between microbial commensals and inflammation on the ocular surface. This information may inform future studies of the pathophysiological mechanisms of dry eye disease.

Identification of Leukocytes Associated With Midday Fogging in the Post-Lens Tear Film of Scleral Contact Lens Wearers.

Purpose: Midday fogging is a frequent complaint among scleral contact lens (ScCL) wearers, and the mechanism and cause of this is unknown. The purpose of this investigation was to understand the relation between midday fogging, ocular surface leukocytes, and ScCL fitting characteristics. Methods: Subjects arrived at a clinical exam having worn ScCLs for at least 4 hours. ScCL were removed, and 150 µL of phosphate-buffered saline (PBS) was used to wash the bowl of the ScCL. Eyes were washed post-ScCL removal with 5 mL PBS per eye. Wash solutions were collected and leukocytes were then isolated and counted, followed by assessment with flow cytometry. Samples from the post-lens tear fluid were stained with fluorescently labeled antibodies to detect leukocyte distributions. Results: Thirty-nine eyes from 19 adapted, full-time, ScCL wearers were included, and 46% presented with midday fogging. ScCL corneal clearance was 246 ± 61 µm for nonfoggers, while it was 308 ± 98 µm for those with fogging (P < 0.05). On average, the number of leukocytes collected from the ScCL bowl (9551 ± 18,926) was greater than the number of leukocytes recovered from the eye wash (2195 ± 4384, P < 0.02). ScCL corneal clearance was associated with the presence of fogging, with an odds ratio of 2.24 (95% confidence interval = 1.48-3.38, P < 0.001). Conclusions: Leukocytes, predominated by neutrophils, are present in the post-lens tear film of ScCL wearers, and in particular wearers with greater ScCL corneal clearance have greater odds of having midday fogging.

Leukocyte Distribution in the Open Eye Tears of Normal and Dry Eye Subjects.

PURPOSE: Leukocytes accumulate in the eye with sleep, but little is known about the presence or absence of leukocytes in awake, open eye tears. This study sought to compare normal and dry eye subjects for daily variation in open eye leukocyte composition. MATERIALS AND METHODS: Ten normal subjects and nine dry eye subjects were enrolled. Subjects were trained for self-collection of tear samples using an ocular surface wash with 5 mL of phosphate buffered saline per eye. Subjects performed washes at awakening, between 8 and 9 am, between 11 am and 12 pm, and between 4 pm and 5 pm on four separate days. Leukocytes were isolated from the wash and were counted with a cell counter before staining with an anti-CD45 antibody and viability stain. Stained leukocytes were then analyzed via flow cytometry. Side scatter characteristics were used to distinguish granulocytes from lymphocytes. Results were interpreted both by time of wash as well as time from awakening. RESULTS: At awakening, dry eye subjects had approximately twice as many recovered leukocytes and had a statistically significantly higher granulocyte-to-lymphocyte ratio as compared to normals. Leukocytes were rapidly cleared from the eye with a significant decrease in leukocyte counts at the 8 am time point as compared to awakening. Leukocyte counts across all open eye time points appeared to be consistent, with no differences between normal and dry eye subjects. CONCLUSIONS: There is a low level, constitutively expressed population of leukocytes in the open eye tears of normal and dry eye subjects. Higher levels of granulocytes in dry eye disease subjects warrants further investigation into this population of cells, and their role in homeostasis and dysregulation.

Neutrophil and T-Cell Homeostasis in the Closed Eye.

Purpose: This study sought to examine the changes and phenotype of the tear neutrophil and T-cell populations between early eyelid closure and after a full night of sleep. Methods: Fourteen healthy participants were recruited and trained to wash the ocular surface with PBS for at-home self-collection of ocular surface and tear leukocytes following up to 1 hour of sleep and a full night of sleep (average 7 hours), on separate days. Cells were isolated, counted, and incubated with fluorescently labeled antibodies to identify neutrophils, monocytes, and T cells. For neutrophil analysis, samples were stimulated with lipopolysaccharide (LPS) or calcium ionophore (CaI) before antibody incubation. Flow cytometry was performed. Results: Following up to 1 hour of sleep, numerous leukocytes were collected (2.6 × 105 ± 3.0 × 105 cells), although significantly (P < 0.005) more accumulated with 7 hours of sleep (9.9 × 105 ± 1.2× 106 cells). Neutrophils (65%), T cells (3%), and monocytes (1%) were identified as part of the closed eye leukocyte infiltration following 7 hours of sleep. Th17 cells represented 22% of the total CD4+ population at the 7-hour time point. Neutrophil phenotype changed with increasing sleep, with a downregulation of membrane receptors CD16, CD11b, CD14, and CD15, indicating a loss in the phagocytic capability of neutrophils. Conclusions: Neutrophils begin accumulating in the closed eye conjunctival sac much earlier than previously demonstrated. The closed eye tears are also populated with T cells, including a subset of Th17 cells. The closed eye environment is more inflammatory than previously thought and is relevant to understanding ocular homeostasis.

Development of a curved, stratified, in vitro model to assess ocular biocompatibility.

PURPOSE: To further improve in vitro models of the cornea, this study focused on the creation of a three-dimensional, stratified, curved epithelium; and the subsequent characterization and evaluation of its suitability as a model for biocompatibility testing. METHODS: Immortalized human corneal epithelial cells were grown to confluency on curved cellulose filters for seven days, and were then differentiated and stratified using an air-liquid interface for seven days before testing. Varying concentrations of a commercial ophthalmic solution containing benzalkonium chloride (BAK), a known cytotoxic agent, and two relevant ocular surfactants were tested on the model. A whole balafilcon A lens soaked in phosphate buffered saline (BA PBS) was also used to assess biocompatibility and verify the validity of the model. Viability assays as well as flow cytometry were performed on the cells to investigate changes in cell death and integrin expression. RESULTS: The reconstructed curved corneal epithelium was composed of 3-5 layers of cells. Increasing concentrations of BAK showed dose-dependent decreased cell viability and increased integrin expression and cell death. No significant change in viability was observed in the presence of the surfactants. As expected, the BA PBS combination appeared to be very biocompatible with no adverse change in cell viability or integrin expression. CONCLUSIONS: The stratified, curved, epithelial model proved to be sensitive to distinct changes in cytotoxicity and is suitable for continued assessment for biocompatibility testing of contact lenses. Our results showed that flow cytometry can provide a quantitative measure of the cell response to biomaterials or cytotoxic compounds for both the supernatant and adherent cell populations. As a specifically designed in vitro model of the corneal epithelium, this quantitative model for biocompatibility at the ocular surface may help improve our understanding of cell-material interactions and reduce the use of animal testing.