Tear fluid extracellular DNA: diagnostic and therapeutic implications in dry eye disease

Abstract

Purpose: To determine the abundance of extracellular DNA (eDNA) in tear fluid of patients with dry eye disease (DED) and to report clinical outcomes after DNase I eyedrops use to reduce excessive tear fluid eDNA.

Methods: Tear fluid was collected from healthy control subjects and patients with DED. The eDNA abundance was determined with the PicoGreen dye assay. The DED symptoms and clinical signs were recorded and correlated with eDNA abundance. Two patients with DED having excessive eDNA in tear fluid were treated with DNase I eyedrops.

Results: The PicoGreen dye assay measures tear fluid eDNA abundance after a 2-minute incubation time. With longer incubations, admixed cells also contribute to eDNA measurements. The mean (SE) eDNA abundance in healthy control subjects' tear fluid was 1.4 (0.2) μg/mL. The mean (SE) eDNA abundance in tear fluid of patients with nonautoimmune DED, autoimmune DED, and graft versus host disease was significantly higher: the values were 2.9 (0.6), 5.2 (1.2), and 9.1 (2.3) μg/mL, respectively (P < 0.05). In most of these patients, the PicoGreen dye kinetic assay of tear fluid showed an increase in fluorescence signal due to the presence of viable cells in tear fluid. Tear fluid eDNA had the best correlation with corneal Rose Bengal staining (r = 0.55). Treatment of patients having DED with DNase I eyedrops reduced eDNA abundance, abrogated signal increase, and improved comfort.

Conclusions: Excessive eDNA is present in tear fluid of patients with dry eyes. A novel therapeutic approach for managing DED may be to measure eDNA abundance in tear fluid with the PicoGreen dye assay and reduce excessive amounts with DNase I eyedrops.

Keywords: DNase I; PicoGreen dye assay; dry eye; extracellular DNA; tear fluid.

Figures

Figure 1

The PicoGreen dye assay to measure DNA abundance. (A) Fluorescence signal intensity measurement of λDNA with the PicoGreen dye (1:200 dilution) shows a linear relationship (r = 0.996). (B) The PicoGreen dye kinetic assay of λDNA and tear fluid from a patient having DED with and without DNase I addition. Stable emission of fluorescence is observed from the λDNA-PicoGreen dye complex. The addition of exogenous DNase I to the λDNA-PicoGreen dye complex leads to an exponential decay in fluorescence signal due to DNA degradation. Similarly, on the addition of DNase I to tear fluid eDNA-PicoGreen dye complex, fluorescence signal intensity decreases exponentially over time, indicating that the eDNA present in tear fluid is degraded by DNase I. RFU, relative fluorescence units.

Figure 2

Fluorescence signal intensity change of tear fluid eDNA or λDNA measured with the PicoGreen dye kinetic assay. (A1) Representative curves of tear fluid from a healthy control subject showing decreasing signal and two patients with GVHD showing increasing signal intensity. The λDNA (100 ng/mL) is shown for comparison. (A2) Percentage change in fluorescence signal in healthy control subjects and patients with nonautoimmune DED, autoimmune DED, or GVHD over 20 minutes. (B) Staining of tear fluid cells with AO/PI dye shows viable cells (green). (C) Kinetic assays of mixtures of λDNA (50 ng/mL) and varying numbers of HCLE cells (0, 100, 500, or 1000) show changes in fluorescence signal dependent on the number of HCLE cells. Increased signal is seen with the addition of 500 and 1000 cells. AO/PI, acridine orange/propidium iodide; DED, nonautoimmune DED; N, normal healthy control subjects; RFU, relative fluorescence units. Scale bar: 50 μm.

Figure 3

The DNA abundance measurement (eDNA or iDNA) in the PicoGreen dye assay. (A) The DNA abundance measured from a mixture of λDNA (50 ng/mL) and HCLE cells (0, 100, 500, or 1000) at different incubation durations (2, 5, or 25 minutes). At 2 minutes, the change in DNA abundance with the addition of HCLE cells is minimal. Thus, the measured DNA abundance at 2 minutes is due to eDNA. A significant increase in DNA abundance is seen with the addition of 500 or more cells at 25-minute incubation. Thus, the measured DNA abundance at 25 minutes is due to both eDNA and iDNA. (B) The HCLE cells stained with the PicoGreen dye in assay buffer (1× TE) at different incubation durations (2 and 25 minutes). (B1) Overlay of brightfield and fluorescent images at 2 minutes shows healthy HCLE cells with bright nuclear staining. Arrowheads indicate stained nuclei. (B2) Overlay of brightfield and fluorescent images at 25 minutes shows evidence of cell degeneration with an absence of nuclear staining. Arrowheads indicate swollen cells with vacuoles. The arrow points to a nucleus showing fragmentation. *P < 0.05. Scale bars: 50 μm.

Figure 4

Case 1: Ocular surface findings of a patient with severe DED and excessive eDNA in tear fluid treated with rhDNase (Pulmozyme) 0.1% eyedrops. (A) Clinical photograph showing mucoid debris (arrow) and corneal filaments (arrowheads) on the ocular surface. (B1–4) Confocal immunofluorescence staining of the mucoid films was performed as described previously. (B1) The eDNA strands (arrow) stain with DAPI (blue). Arrowheads point to multilobed neutrophil nuclei. (B2) Cathelicidin staining within neutrophils (green, arrowheads) with goat polyclonal anti-cathelicidin (clone C-14; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). (B3) Neutrophil elastase staining within neutrophils (red, arrowheads) with mouse monoclonal anti-human neutrophil elastase (clone NP57; Dako Denmark A/S, Glostrup, Denmark). (B4) Overlay. The secondary antibodies were Dylight 488 anti-goat IgG for cathelicidin (1:1000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and Dylight 594 conjugated anti-mouse IgG for neutrophil elastase. (C1) Clinical photograph taken before initiating rhDNase treatment shows extensive ocular surface staining with Rose Bengal dye. (C2) Clinical photograph taken after rhDNase treatment (0.1% four times a day) for 2 months shows resolution of corneal filaments and surface staining. (D1) The PicoGreen dye kinetic assay of the patient's tear fluid before initiating rhDNase treatment shows increasing signal over 20 minutes. (D2) Kinetic assay after rhDNase treatment for 2 months shows signal decay. (D3) Kinetic assay performed 1 month after discontinuation of rhDNase treatment shows an increase in signal. (E) Staining of tear fluid cells with AO/PI dye shows viable cells (green) after discontinuation of rhDNase treatment for 1 month. AO/PI, acridine orange/propidium iodide; RFU, relative fluorescence units. Scale bars: 50 μm.

Figure 5

Case 2: Ocular surface findings of a patient with severe DED and excessive eDNA in tear fluid treated with rhDNase 0.1% eyedrops. Widefield fluorescence image after DAPI staining of Schirmer I test impressions (A) and mucoid film (B) processed as described previously. The eDNA strands (arrows) and abundant exfoliated cells (arrowheads) are seen. (C1) The PicoGreen dye kinetic assay of the patient's tear fluid before initiating rhDNase treatment shows an increase in signal over 20 minutes. (C2) Kinetic assay after rhDNase treatment for 1 month shows signal decay. RFU, relative fluorescence units. Scale bars: 50 μm.