Neutrophil and T-Cell Homeostasis in the Closed Eye

Abstract

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.

Frontline Science: Aggregated neutrophil extracellular traps prevent inflammation on the neutrophil-rich ocular surface

“…Postnikoff et al. recently reported that after 7 h of sleep, closed eyes were infiltrated by neutrophils, T cells, and monocytes . The presence of viable neutrophils in tear films was also observed by trypan blue and PI exclusion in another study .…”

“…The neutrophilrelated proteins like MPO, NE, and CG and protein S100 were iden- monocytes. 24 The presence of viable neutrophils in tear films was also observed by trypan blue and PI exclusion in another study. 25 In the evening TF, C5a showed higher values (P < 0.01, Fig.…”

The closed eye harbours a unique microbiome in dry eye disease

“…In this study, human subjects collected samples of sterile saline rinse applied to the ocular surface upon awakening in the morning. Using saline eye wash to collect microbiome samples is novel and less invasive than swabs of the ocular surface that has previously been used for the collection of tear leukocytes 18,20 . In a series of pooled samples from both eyes, the closed eye tear microbiome in individuals with dry eye disease was distinct from that in healthy controls.…”

“…The evidence may also suggest the immune system of the closed eye cannot regulate the ocular surface microbiome during sleep. In the closed eye, preliminary evidence suggests higher numbers of neutrophils with an enhanced degranulation response in dry eye disease 18,20 , indicating a dysregulated immune response. This dysregulation may permit increased microbial diversity.…”

The closed eye harbors a unique microbiome in dry eye disease

“…In this study, human subjects collected samples of sterile saline rinse applied to the ocular surface upon awakening in the morning. Using saline eye wash to collect microbiome samples is novel and less invasive than swabs of the ocular surface, which has been routinely used for the collection of tear leukocytes 18,20 . In a series of pooled samples from both eyes, the closed eye tear microbiome in individuals with dry eye disease was distinct from that in healthy controls.…”