Jump to:

Peer-reviewed articles


Jin,Y., Minten,C., Jenkins,M., Jones,L., Gorbet,M. Investigation of the rhythmic recruitment of tear neutrophils to the ocular surface and their phenotypes Scientific Reports 2024;147061 [ Show Abstract ]

Hundreds of thousands of polymorphonuclear neutrophils (PMNs) are collected from the ocular surface upon waking, while few are harvested during daytime. This study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs, including changes in IL-8 and C5a in tears, and their phenotypes across different time points in a 24-h cycle. Tear PMNs were collected using a gentle eyewash after 2-h and 7-h of sleep (eye closure, EC) at night, after 2-h EC during the day, and towards the end of the afternoon. Significantly fewer cells were collected after 2-h EC during the day compared to 2-h EC at night. A positive correlation between IL-8 and PMN numbers existed, but not with C5a. Tear PMNs collected after 2-h EC at night were less degranulated and possessed a larger activation potential compared to 7-h EC. Tear PMNs from 7-h EC at night exhibited hyper-segmented nuclei and more NETosis compared to 2 h EC night, indicating an aged and activated phenotype. The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by increased IL-8 in nighttime tears. Higher degranulation and NETs point to the significant activation of tear PMNs on the ocular surface during prolonged eye closure at night.


Jin,Y., Dixon,B., Jones,L., Gorbet,M. The Differential Reactive Oxygen Species Production of Tear Neutrophils in Response to Various Stimuli In Vitro International Journal of Molecular Sciences 2021;22(23):12899 [ Show Abstract ]

A large number of polymorphonuclear neutrophils (PMNs) invade the ocular surface during prolonged eye closure (sleep); these leukocytes are commonly referred as tear PMNs. PMNs contribute to homeostasis and possess an arsenal of inflammatory mediators to protect against pathogens and foreign materials. This study examined the ability of tear PMNs to generate reactive oxygen species (ROS), an essential killing mechanism for PMNs which can lead to oxidative stress and imbalance. Cells were collected after sleep from healthy participants using a gentle eye wash. ROS production in stimulated (phorbol-12-myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucyl-phenylalanine (fMLP)) and unstimulated tear PMNs was measured using luminol-enhanced chemiluminescence for 60 min. A high level of constitutive/spontaneous ROS production was observed in tear PMNs in the absence of any stimulus. While tear PMNs were able to produce ROS in response to PMA, they failed to appropriately respond to LPS and fMLP, although fMLP-stimulated tear PMNs generated ROS extracellularly in the first three minutes. Higher ROS generation was observed in isolated tear PMNs which may be due to priming from the magnetic bead cell separation system. The differential responses of tear PMNs in ROS generation provide further evidence of their potential inflammatory roles in ocular complications involving oxidative stress.


Jin,Y., Jones,L., Gorbet,M. Investigation of the response of tear-film neutrophils to interleukin 8 and their sensitivity to centrifugation, fixation, and incubation Scientific report 2020;10(1):19690 [ Show Abstract ]

During eye closure, a large number of neutrophils (polymorphonuclear neutrophils, PMNs) invade the ocular surface and are often referred to as tear-film PMNs. While immunophenotyping experiments have been performed on tear-film PMNs, the impact of commonly used experimental procedures on their phenotype as well as their response to interleukin-8 (IL-8), a physiological inflammatory mediator, have not yet been investigated. A gentle eye wash method was used to collect cells at home. In the morning upon awaking, participants washed their eyes with sterile phosphate buffer saline (PBS) and collected the runoff into a sterile polypropylene tube. The cell collection was then delivered to the lab within two hours. The effects of centrifugation, incubation and fixation with paraformaldehyde (PFA) before (pre-fixed staining) or after (post-fixed staining) incubation with antibodies were characterized. Tear-film PMNs as well as blood PMNs (used for comparison) were also stimulated with IL-8. To assess the reproducibility of cell collection and variability in receptor expression over time, participants were also asked to collect cells three times over a period of a month. The change in expression of surface receptors, CD11b, CD16, CD55, CD66b, important inflammatory and activation markers, and CD45 (PAN leukocyte marker) was assessed by flow cytometry. Fixing tear-film PMNs prior to the staining with antibodies resulted in a significant (fivefold or more) reduction in the expression of CD11b, CD16 and CD45 when compared to unfixed samples, while CD16 was the only receptor to undergo significant downregulation upon post-staining fixation. Furthermore, additional centrifugation step prior to antibody incubation as well as long (4 h) incubation at 37 °C resulted in significant reductions in expression of CD11b, CD16 and CD55 when compared to control samples. As opposed to blood PMNs, stimulating tear-film PMNs with IL-8 did not induce any significant changes in expression of CD11b, CD16, CD55 and CD66b. When working with collected tear-film PMNs, our results suggest that any additional centrifugation and incubation step should be avoided, or at least limited, and post fixation staining is recommended in order to preserve cell phenotype and cell integrity of tear film PMNs. Our study also adds further information on the reproducibility of the gentle eye wash as well as the inability of tear-film PMNs to modulate their surface receptors upon stimulation with IL-8. The latter may be due to prior exposure to IL-8, activation in the closed-eye environment, or a reduced ability to respond to inflammatory stimulus. Further mechanistic studies will be needed to gain a better understanding of the tear-film neutrophil phenotype.

Scientific Presentations


Jin Y, Jones L, Gorbet M. Investigation of the Circadian Recruitment of Tear Neutrophils to the Ocular Surface and Their Phenotypes  The Association for Research in Vision and Ophthalmology, New Orleans, LA, USA, April, 2023 [ Show Abstract ]

Purpose: Hundreds of thousands of leukocytes (with approximately 60% of the population being polymorphonuclear neutrophils (PMNs)) can be collected from the ocular surface immediately on waking, while only a few are harvested during the daytime. To better understand their role in ocular surface homeostasis and inflammation, this study aimed to investigate potential factors contributing to the circadian infiltration of tear PMNs and changes in phenotype across different time points in a 24-hour cycle.

Methods: Tear leukocytes were collected from 30 participants using a gentle eyewash after 2-hr and 7-hr of sleep at night, after 2-hr sleep during the day, and towards the end of the day (around 5 pm). Cell count and morphology were determined using a Moxi Z cell counter and May-Grunwald stain, respectively. Cells were stimulated by fMLP. Changes in the degranulation (lactoferrin, CD66b, CD63) and cell aging state (CD184) of PMNs were measured via flow cytometry. Neutrophil extracellular traps (NETs) were also identified by flow cytometry and microscopy following staining with myeloperoxidase, citrullinated histones, and CD15.

Results: Significantly more cells were collected from the nighttime compared to the daytime (p<0.001). There was a positive correlation between IL-8 concentrations and PMN numbers, but not with C5a, suggesting that the recruitment of tear PMNs to the ocular surface may be driven mainly by IL-8. 2hr-sleep-derived tear PMNs were less degranulated than 7hr-sleep-derived tear PMNs (p<0.03) and possessed a larger functional activation potential in response to stimulus (p<0.03). Furthermore, 7hr-sleep-derived tear PMNs exhibited hyper-segmented nuclei and were prone to aggregation, when compared to 2hr-night-derived tear PMNs, suggesting an aged and activated phenotype. A significantly increased number of NETs were present in 7hr-night-derived tear samples (p<0.05).

Conclusions: The diurnal-nocturnal recruitment pattern of tear PMNs may be driven by the increase in IL-8 in nighttime tears. Higher levels of degranulation and NETs indicate that tear PMNs become more activated on the ocular surface during prolonged eye closure at night. This PMN inflammatory response must then be balanced by other anti-inflammatory processes to prevent ocular surface damage.