Our results indicate that while tear-film neutrophils are alive, they do not respond to inflammatory stimuli in the same manner as blood-isolated neutrophils. This refractory phenotype may be due to exposure to anti-inflammatory factors present in the tear film.

PurposeThis 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.MethodsFourteen 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.ResultsFollowing 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.ConclusionsNeutrophils 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.

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.

PurposeMidday 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.MethodsSubjects 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.ResultsThirty-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).ConclusionsLeukocytes, 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.

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.