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Tilda Barliya, Dardik Rima, Yael Nisgav, Mor Dachbash, Dan Gaton, Gili Kenet, Dov Weinberger, Tami Livnat; Involvement of NETosis in LPS-induced ocular inflammation in a mouse model.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3297.
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© ARVO (1962-2015); The Authors (2016-present)
NETosis is a process of microbial killing where neutrophils form Neutrophil Extracellular Traps (NETs). NETs are net-like structures comprised of DNA, histones, and antimicrobial proteins which create a net that captures and eliminates the pathogen. The occurrence of NETosis in ocular inflammatory conditions has not been explored yet. We therefore aimed to evaluate the involvement of NETosis in LPS -induced ocular inflammation in a mouse model.
Inflammation was induced in the right eye of 20 C57BL/6J male mice by intravitreal injections of 100 ng/1μl/mice lipopolysaccharide (LPS) from E.coli. Control eyes were injected with saline. Eyes were clinically evaluated for inflammation progression at 0, 3, 6, 24 and 48 hrs post injection using slit lamp and indirect ophthalmoscope. At each time point eyes were enucleated, fixed and stained with H&E for histopathology. Presence of neutrophils and NETs was evaluated using immunofluorescence staining using the following markers; CD11b - a marker for neutrophils, neutrophil elastase (NE) – a serine protease with high affinity to DNA which helps destroy bacteria, myeloperoxidase (MPO) – expressed in neutrophils and helps to carry out the antimicrobial activity and Citrullinated Histone 3 (CitH3) –citrullinated chromatin, a common marker for proper NET existence
Clinical and histological signs of inflammation were observed as early as 3 hrs and peaked at 24 hrs post immunization, which was associated with massive influx of inflammatory cells to the vitreous. The inflammatory cells were positive for CD11b+ thereby indicating the presence of activated neutrophils. A subpopulation of these cells excreted to their surroundings an extracellular net-like structure positive for NE, MPO and CitH3, components that are positive markers for NETosis and powerful anti-microbial agents.
Our study shows for the first time elevation of NETosis markers associated with inflammation induced in murine eyes. Our results suggest that neutrophil recruitment together with NETosis may play a role in inflammatory processes in the eye.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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