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Maud Gorbet, Mark D P Willcox; Interactions of tear-film neutrophils with clinical bacteria.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4048.
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During sleep, in the closed-eye environment, a shift in tear film composition leads to the recruitment of leukocytes to the ocular surface. Neutrophils (also known as polymorphonuclear leukocytes; PMN) are considered our first line of defense and play an essential role in preventing infection. Following chemical stimulus, a lack of upregulation of cell activation markers has previously been observed on tear film neutrophils (TF-PMN). This pilot study was conducted to investigate the response of TF-PMN to clinical bacteria and assess whether overnight lens wear effected on their response to bacteria.
Acuvue Oasys lens wearers and non-lens wearers were recruited to collect their cells upon awakening using an eye wash “at-home collection kit”. Collected cells were counted and resuspended in tubes containing either ATS-2% NHS (artificial tear solution supplemented with 2% of normal human serum) or PBS-10% Heat Inactivated (HI) FBS. Collected cells were incubated with Pseudomona aeruginosa 6294 (an invasive clinical strain) and P. aeruginosa 6206 (a cytotoxic clinical strain) at bacteria to PMN ratios of 10:1 and 1:10. Following incubation, samples were serially diluted in PBS, plated on agar and incubated overnight at 35oC. The next day, bacteria colonies were counted. To further characterize the response to bacteria, TF-PMN were analysed by flow cytometry for receptor upregulation and oxidative burst.
With a bacteria:TF-PMN ratio of 10:1 in ATS-2% NHS, no reduction in bacteria growth was observed for 6206 and 6294. Fewer 6294 CFU were counted when interactions with TF-PMN occurred in PBS-HIFBS. Furthermore, regardless of incubation medium, bacteria:TF-PMN interactions at a ratio of 1:10 resulted in a significant increase in the number of 6206 cells (p<0.05). For both 6206 and 6294, interactions at the 1:10 ratio with TF-PMN collected following overnight lens wear led to a significant increase in CFU when compared to the 10:1 ratio (p<0.04). Flow cytometry results confirmed the differential TF-PMN response to 6206 and 6294.
Our results suggest that at low bacteria to PMN ratio, a condition that may closely mimic the closed-eye environment, TF-PMN are unable to phagocytose clinical strains of Pseudomona aeruginosa and may be releasing factors that promote bacterial survival/growth. This may have an important impact during overnight lens wear and may contribute to the higher risks of microbial keratitis.
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