Ocular Surface Extracellular DNA and Nuclease Activity Imbalance: A New Paradigm for Inflammation in Dry Eye Disease

Abstract

Extracellular DNA production and clearance mechanisms are dysregulated in DED. Nuclease deficiency in tear fluid allows eDNA and NETs to accumulate in precorneal tear film, and results in ocular surface inflammation. These findings point to novel therapeutic interventions in severe DED based on clearance of eDNA, NETs, and other molecular components from the ocular surface.

EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples

“…Furthermore, our group conceived independently the endogenous DNase activity assay used in this study; however, previous similar protocols were identified after literature research [25,26]. In conclusion, these evidences showed that the blood DNases are active in serum and the widely used anticoagulant EDTA also works as an indirect blood's DNase inhibitor that avoids ex vivo degradation of the ccfDNA.…”

Hyperosmolar Stress Induces Neutrophil Extracellular Trap Formation: Implications for Dry Eye Disease

“…3,[6][7][8] We have recently reported the role of NETs and eDNA as possible sources of inflammation in dry eye disease (DED), a chronic inflammatory disorder of the ocular surface. [9][10][11] We reported the presence of excessive amounts of eDNA and molecular components of NETs on the ocular surface of patients with DED, along with a deficiency of tear fluid nucleases, and we showed that eDNA abundance was highest in patients with severe DED. Also, we showed that treatment with recombinant human DNase I eye drops (rhDNase I; 0.1% four times a day) in patients with severe refractory DED and 5 excessive tear fluid eDNA resulted in symptomatic improvement, resolution of corneal rose bengal staining, and a reduction in tear fluid eDNA abundance.…”

“…9 We also performed experiments to evaluate whether in neutrophils that have been exposed to hyperosmolar stress, NETosis decreases when iso-osmolarity is restored. For these experiments, supernatant hyperosmolar culture medium was gently removed at 2 hours of incubation, new culture medium (iso-osmolar or hyperosmolar) was added, and neutrophils cultured for another 2 hours.…”

“…9 Slides were analyzed using an inverted Zeiss microscope to visualize neutrophil morphology and identify NETs. NETs were identified as extracellular strands positive for both neutrophil elastase and 4',6-diamidino-2-phenylindole (DAPI).…”

Neutrophil extracellular traps as the main source of eDNA

“…Moreover, it was presented that limitation of NET formation using PAD inhibitors considerably affect lupus and atherosclerosis phenotype in murine models [18,19]. An imbalance between NETsderived eDNA production and nuclease efficiency is also observed in patients with dry eye disease, which results in ocular surface inflammation [20]. Additionally, increased NETosis also impairs wound healing in diabetes and treatment with plasma DNase 1 because the agent cleaving NETs was demonstrated as the approach to considerably improve re-epithelialisation in diabetic mice [21].…”