June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Tear fluid neutrophils and NETs: Implications in the pathogenesis of ocular graft vs host disease
Author Affiliations & Notes
  • ILANGOVAN RAJU
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • SEUNGWON AN
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Disha Varma
    Orthopedics, Rush University, Chicago, Illinois, United States
  • Anubhav Pradeep
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Bayasgalan Surenkhuu
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Azucena Lopez
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Anisha D'costa
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Christine Mun
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Anna Ahn
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Satyabrata Sinha
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Shilpa Gulati
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Sandeep Jain
    Ophthalmology and Visual Sciences, University of Illinois-Chicago, LIERI, Chicago, Illinois, United States
  • Footnotes
    Commercial Relationships   ILANGOVAN RAJU, None; SEUNGWON AN, None; Disha Varma, None; Anubhav Pradeep, None; Bayasgalan Surenkhuu, None; Azucena Lopez, None; Anisha D'costa, None; Christine Mun, None; Anna Ahn, None; Satyabrata Sinha, None; Shilpa Gulati, None; Sandeep Jain, None
  • Footnotes
    Support  NIH Grant EY024966
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3934. doi:
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      ILANGOVAN RAJU, SEUNGWON AN, Disha Varma, Anubhav Pradeep, Bayasgalan Surenkhuu, Azucena Lopez, Anisha D'costa, Christine Mun, Anna Ahn, Satyabrata Sinha, Shilpa Gulati, Sandeep Jain; Tear fluid neutrophils and NETs: Implications in the pathogenesis of ocular graft vs host disease. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3934.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : The contribution of neutrophil extracellular traps in the pathogenesis of oGVHD is not fully understood. The purpose of the present investigation is to determine whether the severity of ocular disease in oGVHD is related to the presence of neutrophils and their products (NETs) in tear fluid, and whether neutrophils in oGVHD patients are abnormally responsive to stimuli that cause NETosis.

Methods : Conjunctival washings from the ocular surface of healthy subjects (n=10) and pre-transplant (n=20) and definite oGVHD patients (n=33)) were collected and analyzed for abundance of extracellular DNA (eDNA), neutrophils, and epithelial cells. The tear fluid eDNA was measured by PicoGreen kinetic assay. Neutrophils were isolated from peripheral blood and stimulated with PMA or hyperosmolar solutions, and the amount of NETosis was determined by Sytox Green plate reader assay.

Results : Patients with excess neutrophils in the tear fluid had more severe disease as evidenced by significantly higher composite score (8.53 ± 0.52, p < 0.05) as compared to patients with epithelial cells excess (6.6 ± 0.48). The clinical signs that were significantly worse in patients with neutrophil excess include: (i) ocular redness score, (ii) corneal staining, and (iii) MMP-9 test. Patients with oGVHD who had an excess of neutrophils in their tear fluid had significantly more severe ocular surface disease (composite score) and a higher amount of tear fluid eDNA. The amount of eDNA was significantly higher in oGVHD patients (7.25 ± 1.86 μg/mL, p < 0.05) compared to pre-transplant patients (1.85 ± 0.37) and healthy subjects (1.47 ± 0.16). The concentration of tear fluid eDNA showed a significant moderate positive correlation with OSDI (r = 0.45), ocular redness score (r = 0.45), corneal staining (r = 0.35), and the composite score (r = 0.42). Peripheral blood neutrophils in oGVHD patients were hyperresponsive to NETosing stimuli.

Conclusions : Our results suggest that neutrophils and their extracellular products (NETs) may contribute towards ocular GVHD pathology, and make a case for investigating the clinical value of manipulating them for treating oGVHD. Potential therapeutic strategies may include: (i) enhancing the clearance of NETs from the ocular surface using nucleases; (ii) reducing the responsiveness of neutrophils to NETosing stimuli; and (iii) reducing the egress of neutrophils onto the ocular surface.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

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