title: Hyperosmolar stress induces neutrophil extracellular trap formation: implications for dry eye disease.
abstract: OBJECTIVE
  To determine if hyperosmolar stress can stimulate human neutrophils to form neutrophil extracellular traps (NETs) and to investigate potential strategies to reduce formation of NETs (NETosis) in a hyperosmolar environment.
  
  
  METHODS
  Neutrophils were isolated from peripheral venous blood of healthy subjects and incubated in iso-osmolar (280 mOsM) or hyperosmolar (420 mOsM) media for 4 hours. Neutrophil extracellular traps were quantified using a PicoGreen dye assay to measure extracellular DNA. Two known inhibitors of NETosis, staurosporine and anti-β2 integrin blocking antibody, and two proresolution formyl peptide receptor 2 (FPR2) agonists, annexin/lipocortin-1 mimetic peptide and 15-epi-lipoxin A4, were evaluated as possible strategies to reduce hyperosmolarity-induced NETosis.
  
  
  RESULTS
  The amount of NETs induced by hyperosmolar medium (420 mOsM) increased linearly over time to 3.2 ± 0.3 times that induced by iso-osmolar medium at 4 hours (P < 0.05). NETosis increased exponentially with increasing osmolarity and was independent of the stimulus used to increase osmolarity. Upon neutrophil exposure to hyperosmolar stress, restoration of iso-osmolar conditions decreased NET formation by 52.7% ± 5% (P < 0.05) but did not completely abrogate it. Among the strategies tested to reduce NETosis in a hyperosmolar environment, annexin-1 peptide was the most efficacious.
  
  
  CONCLUSIONS
  Hyperosmolarity induces formation of NETs by neutrophils. This NETosis mechanism may explain the presence of excessive NETs on the ocular surface of patients with dry eye disease. Because they reduce hyperosmolarity-induced NETosis, FPR2 agonists may have therapeutic potential in these patients.
authors.affiliation: Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, United States.
authors.forename: Sapna
authors.initials: S
authors.lastname: Tibrewal
authors.affiliation: Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, United States.
authors.forename: Yair
authors.initials: Y
authors.lastname: Ivanir
authors.affiliation: Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, United States.
authors.forename: Joy
authors.initials: J
authors.lastname: Sarkar
authors.affiliation: Department of Ophthalmology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, United States.
authors.forename: Neema
authors.initials: N
authors.lastname: Nayeb-Hashemi
authors.affiliation: Department of Ophthalmology, Loyola University Chicago Stritch School of Medicine, Maywood, Illinois, United States.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Bouchard
authors.affiliation: Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, United States.
authors.forename: Eunjae
authors.initials: E
authors.lastname: Kim
authors.affiliation: Corneal Neurobiology Laboratory, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, College of Medicine, Chicago, Illinois, United States.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Jain
mesh_terms: D015352:Dry Eye Syndromes / Q000503:physiopathology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D065206:Extracellular Traps / Q000378:metabolism*; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D019272:Leukocyte Elastase / Q000378:metabolism; D009504:Neutrophils / Q000502:physiology*; D009994:Osmolar Concentration; D044042:Receptors, Formyl Peptide / Q000819:agonists; D044067:Receptors, Lipoxin / Q000819:agonists; D013312:Stress, Physiological / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; C105101:FPR2 protein, human; D006982:Hypertonic Solutions; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; D019272:Leukocyte Elastase
keywords: dry eye; extracellular DNA; hyperosmolar stress; neutrophil extracellular traps