Neutrophil Extracellular Traps accumulate on ocular surface of Dry Eye Disease patients: Potential for a new therapeutic strategy using DNase I eye drops

Abstract

Purpose: We hypothesize that Neutrophil Extracellular Traps (NETs) and Extracellular DNA (eDNA) production and clearance mechanisms are dysregulated in Dry Eye Disease (DED). We investigated the abundance of NETs and eDNA as well as the clinical benefit of clearing them with DNase I eye drops.

Methods: Mucoid films (MF) from the ocular surface and exfoliated cells that adhered to Schirmer test strips were collected and mounted on glass slides. Immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. eDNA abundance was determined in the tear fluid of patients with DED and of healthy control subjects using PicoGreen dye assay. Two patients with DED were treated with DNase I eye drops.

Results: MFs from DED patients showed presence of numerous Neutrophils and NETs admixed with epithelial cells. High infiltration of Neutrophils (>15 per 20x field) was observed in nine (64%) patients with graft versus host disease (GVHD). Larger than normal neutrophil diameter and abnormal nuclear morphology (indistinct lobes or diffuse) pointed towards activated neutrophils. PicoGreen dye assay from DED patients’ tear fluid showed two important findings. First, the fluorescence signal measurements at short incubation times (2 to 5 minutes) measured eDNA levels in. Second, the increase in fluorescence signal on kinetic assay over 20 minutes occurred due to PicoGreen dye assay buffer-induced degeneration of admixed cells and consequent release of intracellular DNA into the medium. The eDNA abundance in healthy control subjects’ tear fluid was 1.4±0.2 μg/ml. eDNA abundance in tear fluid of patients with non-autoimmune DED, autoimmune DED, and GVHD was significantly higher (2.9±0.6, 5.2±1.2, and 9.1±2.3 μg/ml, respectively; p<0.05). Treatment of DED patients with DNase I eye drops reduced eDNA abundance, abrogated signal increase, and improved comfort.

Conclusions: Our findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface. An approach for managing DED can be to measure eDNA abundance in tear fluid with the PicoGreen dye assay and reduce excessive amounts with DNase I eye drops.

Keywords: 480 cornea: basic science