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Sandeep Jain, SEUNGWON AN, ILANGOVAN RAJU, Bayasgalan Surenkhuu, Satyabrata Sinha, Damiano Rondelli, Christine Mun; Neutrophil Extracellular Traps (NETs) in Patients with Dry Eye Disease Secondary to ocular Graft-vs-Host-Disease (oGVHD): Pathological Consequences and Therapeutic Implications. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4873.
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© ARVO (1962-2015); The Authors (2016-present)
To establish the presence of NETs on the ocular surface of oGVHD patients and to determine whether NETs can induce pathological changes that underlie oGVHD and whether dismantling of NETs abrogates those pathological changes.
Ocular surface washings from age matched patients with ‘definite’ oGVHD, ‘none’ oGVHD and healthy subjects were analyzed. Isolated peripheral blood human neutrophils were stimulated to generate NETs and heparinized NETs. We performed in vitro experiments using cell lines (corneal epithelial and conjunctival fibroblast) in scratch wound models, and in vivo experiments using murine corneal epitheliopathy models, and compared the effects of naïve NETs with heparinized NETs.
NETs and their molecular components (extracellular DNA, neutrophil elastase, myeloperoxidase and oncostatin M) were present in ocular surface washings and mucocellular aggregates. Neutrophil numbers and amount of eDNA in ocular surface washings of oGVHD patients showed a strong correlation. Incubation with peripheral blood neutrophils generated NETs caused significant delay in closure of scratch wounds in cultured corneal epithelial cells. Application of NETs to murine corneas caused significantly greater epitheliopathy and significantly reduced healing of epithelial defects. Corneas exposed to NETs also had significantly higher abundance of inflammatory cytokines (IP-10 and IL-1β). Incubation with NETs enhanced proliferation and myofibroblast transformation of primary human conjunctival fibroblasts. Incubation with NETs significantly increased proliferation of T-cells in allogeneic mixed lymphocytic reaction (MLR). Incubation of NETs with sub-anticoagulant dose of Heparin (100 IU/mL) dismantled NETs as evidenced by reduced levels of histone-associated DNA fragments (which represent intact NETs). NETs incubated with heparin abrogates epithelial, fibroblast and T-cell changes induced by NETs.
NETs are present over ocular surface of oGHVD patients. NETs delay epithelial healing, promote fibroblast proliferation and myofibroblast transformation and enhance allogeneic T-cell proliferation. Dismantling with sub-anticoagulant dose heparin abrogates NET-induced epithelial, fibroblast and T-cell affects, pointing to its therapeutic potential.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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