Neutrophil Extracellular Traps (NETs) delay Epithelial Wound Healing in-vitro: Implications in chronic Ocular GVHD

Abstract

Purpose : Previously we have observed numerous neutrophils admixed with epithelial cells in tear fluid and mucoid debris in patients with ocular GVHD (oGVHD). Through these experiments we tried to determine whether epithelial cells contribute to formation of NETs and whether NETs effect epithelial healing or expression of cytokines/chemokines.

Methods : Immortalized Human Corneal Epithelial cells (HCETs) were cultured in DMEM growth medium with 10% FBS. Human neutrophils were isolated from peripheral blood. Cells were cultured in 96-well plates seeded with 30,000 cells per well. Upon 100% confluency, a scratch-wound was generated in each well using the IncuCyte Wound maker kit and images were taken using the IncuCyte Zoom system. The following experimental conditions were employed. (i) Epithelial cells only; (ii) Epithelial cells + 1nM PMA; (iii) Epithelial cells + 10nM PMA (iv) Epithelial cells + Neutrophils; (v) Epithelial cells + Neutrophils + 1nM PMA and lastly; (vi) Epithelial cells + Neutrophils + 10nM PMA. Neutrophils were added in serum free RPMI medium and incubated. Images were captured for every 8 h and quantitated the wound healing by IncuCyte software.

Results : At 0 h time point the relative density of cells in the scratch wound region were at baseline (0). On addition of neutrophils to the epithelial cells there was a decline in the rate of closure of the scratch wound, with relative cell density in the wound being -2.83 ± 3.4 compared to 75.34 ± 11.38 when no neutrophils were added. Furthermore, this reduction was significantly higher in PMA pre-incubated epithelial cells [-5.9 ± 6.98 for 1nM PMA treated group (p≤ 0.0001) and -29.38 ± 3.67 for 10nM PMA treatment (p≤0.0001). Our results also showed elevated levels of the cytokine FGF2 in the supernatant of epithelial cells in presence of neutrophils (9.15 ± 0.85). However, the levels of FGF2 were significantly higher in the PMA treated epithelial cells [13.5 ± 0.5 for 1nM PMA treatment (p ≤ 0.04) & 20.05 ± 1.05 (p ≤0.01) for 10nM PMA treatment] as compared to untreated control.

Conclusions : NETs retard epithelial wound closure in-vitro therefore, may contribute to non-healing epithelial defects in chronic oGVHD. Expression of FGF2 during NETs-epithelial interaction may contribute to sub-epithelial fibrosis in chronic oGVHD.

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