The Phenotype of Tear Neutrophils and Their Role in Ocular Homeostasis and Inflammation

Abstract

Introduction: Neutrophils (polymorphonuclear neutrophils, PMNs) are part of the innate immune system with potent killing mechanisms against pathogens. Although the anterior segment of the eye has a unique characteristic, immune privilege, a significant influx of PMNs has been detected after a prolonged eye closure at night, such as sleep. However, the functions and roles of tear PMNs in ocular homeostasis and complications remain largely unknown. The overall objectives of this thesis were to examine and compare the phenotype and functions of tear PMNs from healthy participants and participants with ocular allergy.

Methods: Participants used a gentle eye wash method to collect tear PMNs from the ocular surface by washing their eyes with sterile phosphate buffer saline (PBS).

• Study 1: Tear PMNs were either isolated from the eyewash by using the MACS-Column or EasySep cell separation system or reconcentrated by centrifugation. Stimulated (phorbol-12myristate-13-acetate (PMA), lipopolysaccharides (LPS) or N-Formylmethionyl-leucylphenylalanine (fMLP)) and unstimulated ROS production by both isolated and non-isolated tear PMNs were measured using luminol-enhanced chemiluminescence for 60 min.

• Study 2: Tear PMNs were incubated with different medium conditions, Hank’s Balanced Salt Solution (HBSS) only, HBSS with lactoferrin, lysozyme, mucin, albumin, and IgG (ATS), and ATS without lactoferrin and lysozyme (MAI). Unstimulated or PMA-stimulated ROS production by tear PMNs was measured using luminol-enhanced chemiluminescence and flow cytometry with DCFH-DA.

• Study 3: Tear leukocytes were collected at four different time points, 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). After stimulation with fMLP, changes in the degranulation (lactoferrin, CD66b, CD63) and cell aging state (CD184) of tear PMNs were measured via flow cytometry. Neutrophil extracellular traps (NETs) were quantified by flow cytometry and visualized by microscopy following staining with myeloperoxidase, citrullinated histones, and CD15.

• Study 4: Tear PMNs were collected from participants suffering from ocular allergy on two consecutive days, after a full night of sleep in the morning on Day 1 and at the end of the day, i.e., around 4:30 pm, on Day 2. Tear PMNs were either activated with fMLP or left unstimulated, followed by staining with antibodies against degranulation markers (CD66b and CD63), adhesion markers (CD11b and CD54), eosinophil marker (CD193), aging markers (CD184 and CD62L), as well as with the fluorescent probe DCFH-DA. The intensity of fluorescence was measured via flow cytometry.

Results:

• Study 1: Tear PMNs have a high level of constitutive/spontaneous ROS production even in the absence of any stimulus. However, tear PMNs failed to appropriately respond to LPS and fMLP, although they were able to produce ROS in response to PMA. Higher ROS generation was observed in isolated tear PMNs which may be caused by priming from the magnetic bead cell separation system.

• Study 2: The presence of tear proteins significantly reduced the unstimulated and PMAstimulated ROS production by tear PMNs in HBSS and ATS. However, the findings on ROS production by PMA-stimulated PMNs incubated in MAI were different between the flow cytometry and chemiluminescence, suggesting that lactoferrin and lysozyme may have a greater impact on extracellular ROS production.

• Study 3: Significantly more cells were collected from the nighttime compared to the daytime. 2hr EC night tear PMNs were less degranulated than 7hr EC night tear PMNs and possessed a higher activation ratio in response to fMLP. Furthermore, 7hr EC night tear PMNs exhibited hypersegmented nuclei and were prone to aggregation, when compared to 2hr EC night tear PMNs, suggesting an aged and activated phenotype. A significantly increased number of NETs were present in 7hr nocturnal closed-eye tear samples.

• Study 4: There were significantly more tear PMNs collected from individuals with ocular allergy compared to healthy participants upon awakening. Tear PMNs from ocular allergy participants exhibited a less activated phenotype but a higher activation potential in response to fMLP compared to healthy participants, which was correlated with their younger maturation state. However, no significant difference in the production of ROS by tear PMNs between these two groups was observed.

Conclusion: Tear PMNs become more aged and activated with increasing eye closure time at night, which can potentially aid in ocular surface surveillance. The presence of tear proteins may limit the ROS release by tear PMNs, thereby protecting the ocular environment from potential damage. However, in individuals suffering from ocular allergy, tear PMNs may be constantly recruited to the ocular surface, contributing to symptoms of ocular allergy and development of ocular complications associated with inflammation. This thesis identified some of the functionalities and potential roles of tear PMNs in maintaining ocular homeostasis and regulating inflammation. While further investigation is needed to comprehensively characterize the underlying mechanisms of tear PMNs involved in ocular inflammation, the current results may support the development of new therapeutical strategies to reduce ocular surface inflammation.