Tear fluid neutrophils and NETs: Implications in the pathogenesis of ocular graft vs host disease

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.