Ocular surface extracellular DNA and nuclease activity imbalance: a new paradigm for inflammation in dry eye disease

Abstract

Purpose: We determined whether nucleases are deficient in the tear fluid of dry eye disease (DED) patients, and whether this causes extracellular DNA (eDNA) and neutrophil extracellular trap (NET) accumulation in the precorneal tear film, thus causing ocular surface inflammation.

Methods: Exfoliated cells adhered to Schirmer test strips were collected on glass slides, and immunofluorescence confocal microscopy was used to evaluate neutrophils, eDNA, NETs, and their molecular components. Similar experiments were performed with mucoid films collected from the inferior conjunctival fornix or bulbar conjunctiva. We used quantitative PCR to evaluate eDNA signaling pathways and inflammatory cytokine expression. We also determined the amount of ocular surface eDNA and evaluated tear fluid nuclease activity.

Results: eDNA, NETs, and neutrophils were present on the ocular surface in DED patients and abundant in mucoid films. NETs consisted of eDNA, histones, cathelicidin, and neutrophil elastase. Tear fluid nuclease activity was decreased significantly in DED patients, whereas the amount of eDNA on the ocular surface was increased significantly. Expression of genes downstream of eDNA signaling, such as TLR9, MyD88, and type I interferon, as well as the inflammatory cytokines interleukin-6 and tumor necrosis factor-α, was significantly increased in DED patients.

Conclusions: 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.

Figure 1.

eDNA and neutrophil NETs in exfoliated material derived from Schirmer test strip impressions. (A1, A2) Impression cytology method using Schirmer test strips (A1, arrowhead) and silane coated glass slides (A2). (B) H&E staining showing exfoliated surface cells. (C) Wide-field fluorescent microscope image after DAPI stain of conjunctival impression material reveals short and sparse eDNA strands (arrowhead) in normal subjects (C1) and numerous long eDNA strands (C2) in DED patients (arrowhead). (D) Confocal immunofluorescence image after DAPI staining demonstrates numerous strands (arrowhead) and neutrophils with multilobed nucleus. (E) Confocal immunofluorescence staining image shows that histones (E1, green), neutrophil elastase (E2, red), and eDNA (E3, blue) are the molecular components of NETs (E4, overlay). Arrowhead points to the same area in the same section and indicates a NET strand. Inset in (E2) shows neutrophils with DAPI-stained multilobed nucleus. Scale bars: (B, C1, C2, D) 50 μm; (E1) 10 μm; (E2, inset) 10 μm. Scale bar in (E1) applies to (E1–E4).

Figure 2.

eDNA and NETs in mucoid films. (A) Clinical photographs of eyes of patients with severe tear-deficient DED. Arrowhead indicates a mucoid film over the cornea and bulbar conjunctiva (A1), and inferior fornix (A2). Inset shows magnified view of mucoid films. (B) cytological examination of the mucoid films. (B1) H&E staining shows the surface epithelial cells and numerous neutrophils. (B2) DAPI staining shows the presence of eDNA strands (arrowhead) and multilobed nucleus of neutrophils. (B3) Neutrophil elastase immunostaining (red) confirms the presence of neutrophils. (C) Confocal immunofluorescent staining image shows that neutrophil elastase (C1, red), histones (C2, green), and eDNA (C3, blue) are molecular components of NETs (C4, overlay). Arrowhead indicates a NET strand. (D) Laser capture microdissection (LCM) was performed to capture DAPI-stained strands to confirm the presence of DNA in them. (D1) Arrowhead indicates an eDNA strand. (D2) Asterisk occupies the area of the strand post LCM. (D3) GAPDH PCR product in eDNA strand lane confirms the presence of DNA material. Scale bars: (B1, B2, B3) 20 μm; (C, D1, D2) 50 μm. Scale bar in (C3) applies to (C1–C4).

Figure 3.

Confocal immunofluorescent staining image showing cathelicidin in NETs and neutrophils (A1-A4) Cathelicidin (green) is present in mucoid films (A1, arrow) and eDNA strands (A1, arrowhead). Cathelicidin colocalizes with neutrophil elastase (A2, red) and DAPI nuclear stain (A3, blue). (B1-B4) Cathelicidin (green) is present in neutrophils (B1, arrowhead) and colocalizes with neutrophil elastase (B2, red) and DAPI (B3, blue). (C1-C4) Cathelicidin (C1, arrowhead) and DAPI-stained nuclear material (C3, arrowhead) are extruded from a neutrophil to form NETs. Scale bars: 10 μm. Scale bar in (A1) applies to (A1–A4), in (B1) applies to (B1–B4), and in (C1) applies to (C1–C4).

Figure 4.

Amount of ocular surface eDNA and eDNA signaling pathway gene expression in DED patients and controls. (A) The average aqueous tear production, measured using Schirmer I test, was significantly lower in DED patients. (B) eDNA length was measured after impression of Schirmer strip on a glass slide. The eDNA length was significantly greater in DED patients. (C) eDNA amount on the Schirmer strips was measured using PicoGreen assay. DED patients have significantly greater eDNA amounts. (D) Genes in the eDNA signal transduction pathway were significantly overexpressed in exfoliated conjunctival cells from DED patients. (E) Inflammatory gene expression also was significantly increased in DED patients. *P < 0.05.

Figure 5.

Nucleases and DNAse I are present in tear fluid. (A1-A6) Confocal immunofluorescent microscope image showing DNAse1 in human lacrimal glands (A1–A3, red). The specificity of the staining was confirmed with peptide competition (A4–A6) and isotype control staining (not shown). (B) Nuclease activity in the tears of normal subjects. DNAse I ELISA showed that its concentration in normal tear fluid is 3.14 ng/ml. In the DNAse detection kit, normal tears completely degraded DNA (tears lane), Therefore, tear fluid nuclease activity is greater than 0.05 Kunitz units. (C1) Nuclease activity in tears was quantitated using a FRET assay. Plot of corrected fluorescence emission signal versus time for four normal (top four traces) and two DED patients (bottom two traces) is shown. The measured emission values were corrected by subtraction of the background signal from the probe substrate. The legend lists the intercepts in RFU and slopes in RFU/min for each trace; errors are given at the 95% confidence interval. (C2) Graph showing that the nuclease activity in DED patients was significantly lower compared to healthy individuals. *P < 0.05. Scale bar in (A1): 20 μm applies to (A1–A6).