Neutrophil extracellular traps (NETs) contribute to pathological changes of ocular graft-vs.-host disease (oGVHD) dry eye: Implications for novel biomarkers and therapeutic strategies

Abstract

Purpose: To investigate the role of neutrophil extracellular traps (NETs) and NET-associated proteins in the pathogenesis of oGVHD and whether dismantling of NETs with heparin reduces those changes.

Methods: Ocular surface washings from oGVHD patients and healthy subjects were analyzed. Isolated peripheral blood human neutrophils were stimulated to generate NETs and heparinized NETs. We performed in vitro experiments using cell lines (corneal epithelial, conjunctival fibroblast, meibomian gland (MG) epithelial and T cells), and in vivo experiments using murine models, and compared the effects of NETs, heparinized NETs, NET-associated proteins and neutralizing antibodies to NET-associated proteins.

Results: Neutrophils, exfoliated epithelial cells, NETs and NET-associated proteins (extracellular DNA, Neutrophil Elastase, Myeloperoxidase, Oncostatin M (OSM), Neutrophil gelatinase-associated lipocalin (NGAL) and LIGHT/TNFSF14) are present in ocular surface washings (OSW) and mucocellular aggregates (MCA). Eyes with high number of neutrophils in OSW have more severe signs and symptoms of oGVHD. NETs (and OSM) cause epitheliopathy in murine corneas. NETs (and LIGHT/TNFSF14) increase proliferation of T cells. NETs (and NGAL) inhibit proliferation and differentiation of MG epithelial cells. NETs enhance proliferation and myofibroblast transformation of conjunctival fibroblasts. Sub-anticoagulant dose Heparin (100 IU/mL) dismantles NETs and reduces epithelial, fibroblast, T cell and MG cell changes induced by NETs.

Conclusion: NETs and NET-associated proteins contribute to the pathological changes of oGVHD (corneal epitheliopathy, conjunctival cicatrization, ocular surface inflammation and meibomian gland disease). Our data points to the potential of NET-associated proteins (OSM or LIGHT/TNFSF14) to serve as biomarkers and NET-dismantling biologics (heparin eye drops) as treatment for oGVHD.

Keywords: Biomarkers; Dry eye; Heparin; NETs; Ocular GVHD.

Conflict of interest statement

Conflict of Interest:

Sandeep Jain, MD: Consultant, Ocugen, Inc; Stock Ownership, Advaite, LLC; Patent application

Copyright © 2019 Elsevier Inc. All rights reserved.

Figures

Figure 1.. Immunofluorescent staining of mucocellular aggregates (MCA) in oGVHD patients to demonstrate the presence of NETs.

(A1 & A2) Clinical photographs of oGVHD patients showing translucent and whitish MCAs on the ocular surface (arrows). (A3) Hematoxylin and Eosin (H&E) staining of MCA shows numerous neutrophils, surface epithelial cells and extracellular DNA strands. (B1-B4) Confocal immunofluorescent staining of MCA showing co-localization of neutrophil elastase (NE) (B1, red), Citrulline H3 (B2, citH3, green) and DAPI nuclear staining (B3, blue) in extracellular strands (B4, arrows) confirming that these extracellular strands are NETs. Confocal immunofluorescent staining of MCA showing co-localization of NE (C1, red), myeloperoxidase (C2, green) and DAPI nuclear stain (C3, blue) in extracellular strands (C4, white arrows) further confirming that these extracellular strands are NETs. (D1-E4): Analysis of a MCA adhered to the cornea of an oGVHD patient. (D1) Clinical photograph of an oGVHD patient showing MCAs adhered to the cornea. (D2) Clinical photograph of the same patient after application of a filter paper to the cornea to lift the MCA. (D3) H&E staining of the filter paper shows that MCA comprises of neutrophils, surface epithelial cells and extracellular DNA strands. (E1-E4) Confocal immunofluorescent staining of the MCA shows co-localization of citH3 (E1, red), NE (E2, green) and DAPI nuclear staining (E3, blue) in extracellular strands (E4, white arrows) confirming that these extracellular strands within the MCA are NETs.

Figure 2.. Presence of NETs and their molecular components in ocular surface washings of oGVHD patients and comparison with experimentally induced NETs.

(A1): Clinical photograph showing ocular surface of an oGVHD patient. (A2): H&E staining shows numerous enlarged neutrophils and surface epithelial cells from ocular surface washings of an oGVHD patient. (A3): Sytox Green staining shows extracellular strands in ocular surface washings of a definite oGVHD patient. Confocal immunofluorescent staining of ocular surface washings of an oGVHD patient shows co-localization of NE (A4, red), MPO (A5, green) and DAPI nuclear stain (A6, blue) in extracellular strands (A7, merged image) confirming presence of NETs. (A8): Boxplot shows level of eDNA from healthy subjects (n=40 eyes), none oGVHD (n=35 eyes) and definite oGVHD patients (n=55 eyes). (A9 & A10): Graphs showing the level of MPO-DNA complex (A9) and NE-DNA complex (A10) measured by ELISA from ocular surface washings of healthy subjects (n=10 eyes), none oGVHD (n=10 eyes) and definite oGVHD (n=10 eyes) patients. (A11): Representative FEMTO pulse capillary electrophoresis data showing the size of eDNA from ocular surface washings of healthy subjects, none oGVHD and definite oGVHD patients. A12-A17: Graphs showing the amount of NE (A12), MPO (A13), IL-8 (A14), OSM (A15), LIGHT (A16) and TNFa (A17) measured by Luminex from ocular surface washings of healthy subjects, none oGVHD, and definite oGVHD patients. (B1): H&E staining shows multi-lobed nucleus of intact isolated human neutrophils from peripheral venous blood. (B2): H&E staining shows enlarged neutrophils from human peripheral blood after stimulation with 1 nM PMA. (B3): Sytox Green staining shows extracellular DNA strands from human peripheral neutrophils stimulated with 1 nM PMA. (B4-B7): Confocal immunofluorescent staining of PMA activated human neutrophils from peripheral venous blood shows co-localization of NE (B4, red), MPO (B5, green) and DAPI (B6, blue) in extracellular strands (B7, merged image) confirming them to be NETs. (B8): Boxplot shows level of eDNA in supernatant from RPMI culture media alone (n=9), unstimulated neutrophils (no NETs, n=10) and neutrophils stimulated with PMA to induce NETosis (NETs, n=11). (B9 & B10): Graphs showing the level of MPO-DNA complex (B9) and NE-DNA complex (B10) measured by ELISA from RPMI medium (n=3) alone and supernatants of unstimulated neutrophils (no NETs, n=15) and PMA stimulated neutrophils (NETs, n=15). (B11): FEMTO pulse capillary electrophoresis data showing the size of eDNA from PMA stimulated human neutrophils (NETs). (B12-B17): Graph showing the amount of NE (B12), MPO (B13), IL-8 (B14), OSM (B15), LIGHT (B16) and TNFa (B17) measured by Luminex from RPMI medium alone and supernatants of unstimulated neutrophils (no NETs) and PMA stimulated neutrophils. (C1): Boxplot shows the number of neutrophils in ocular surface washings of healthy subjects (n=40 eyes), none oGVHD (n=35 eyes) and definite oGVHD (n=55 eyes) patients. (C2): Scatter plot showing correlation of eDNA (n=53 eyes) and number of neutrophils (n=53 eyes) for definite oGVHD patients. (C3): Representative H&E staining of ocular surface wash in definite oGVHD patients shows numerous epithelial cells and few neutrophils (N<E group). (C4): Representative H&E staining of ocular surface wash in definite oGVHD patients shows numerous neutrophils and few epithelial cells (N>E group). (C5-C8): Graphs comparing signs and symptoms between N<E and N>E groups in definite oGVHD patients. (C5): OSDI score between N<E group (n=19 eyes) and N>E group (n=36 eyes). (C6) Bulbar redness score between N<E group (n=18 eyes) and N>E group (n=32 eyes). (C7) Composite score for definite oGVHD patients between N<E group (n=18 eyes) and N>E group (n=36 eyes). (C8) eDNA amount in ocular surface wash between N<E group (n=19 eyes) and N>E group (n=30 eyes).

Figure 3.. Effect of heparin on NETs in oGVHD patients.

(A1-A3): Representative images of Sytox Green staining showing dismantling of NETs with Heparin after experimental NETosis. (A1): naïve neutrophils; (A2): PMA stimulated human neutrophils showing abundant NETs; (A3): Sub-anticoagulant dose of heparin (100 IU/mL) for 1 h dismantles the NETs. (A4-A7): Graphs showing protein-associated DNA fragments in supernatants to confirm dismantling of NETs by Heparin. (A4): eDNA amount from supernatants of naïve adherent NETs (n=4) and Heparin dismantled NETs (n=4); (A5): Histone-associated DNA fragments measured in supernatants of naïve adherent NETs (n=15) and Heparin dismantled NETs (n=13). (A6): MPO-associated DNA fragment measured in supernatants of naïve adherent NETs (n=15) and Heparin dismantled NETs (n=15). (A7): NE-associated DNA fragment measured in supernatants of naïve adherent NETs (n=15) and Heparin dismantled NETs (n=15). (B1-B8): Dismantling of NETs in MCAs by Heparin. (B1, B2, B5, B6): Naïve MCAs are collected from the ocular surface and stained with H&E (B1, B5) and Sytox Green (B2, B6) to show the presence of NETs. The physical appearance of isolated MCA is shown as an inset in B5. (B3, B4, B7, B8): Naïve MCAs are incubated with Heparin 100 IU/mL (B3, B4) or RPMI as control (B7, B8). H&E staining (B3, B7) and Sytox Green staining (B4, B8) show dismantling of NETs in MCA with Heparin (B3, B4) but not with RPMI control (B7, B8). (B9): Graph showing amount of Histone-associated DNA fragments in the supernatant of MCAs incubated in the presence (n=5) or absence of Heparin (n=6). (B10 & B11): Representative clinical images of an oGVHD patient who had adherent MCA over a keratoprosthesis (B10) and treated with heparin (100 IU/mL) eye drops three times a day for 4 weeks (B11). Adherent MCA were much reduced after heparin eye drop use. (C1-C8): Representative images showing scratch wound assay in immortalized human corneal epithelial cells that are incubated with various doses of Heparin. Representative kinetic curve over 30 hours (C9) and graph at 30 hour time point (C10) showing relative wound density with various doses of Heparin after epithelial wound scratch. (C11): Graph showing the cell proliferation (MTS assay) of various doses of heparin after epithelial scratch wound. (C12): Graph showing cytotoxicity for various doses of heparin in epithelial scratch wounds as measured by LDH assay (n=6/group, 3 separate experiments). (C13): Representative kinetic assay showing effect of Heparin on NETosis. Sytox Green fluorescence intensity in supernatants from neutrophils stimulated with 1 nM PMA (naïve NETs), 1 nM PMA with heparin (dismantled NETs) and heparin alone (no NETs, control).

Figure 4.. Pathological effects of NETs on corneal epithelial cells.

(A1-A3): Representative clinical images of an ocular GVHD patient showing corneal disease. (A1) corneal fluorescein staining showing superficial punctate keratopathy; (A2) conjunctival lissamine green staining showing punctate epitheliopathy; (A3) fluorescein staining showing corneal epithelial defect. (A4, A5): Boxplot showing lissamine green staining scores in healthy subjects (n=40 eyes), none oGVHD (n=35 eyes) and definite oGVHD patients (n=48 eyes); (A4) corneal staining score; (A5) conjunctival staining score. (B1-B8): Representative image showing scratch wound assay in immortalized human corneal epithelial cells incubated with RPMI culture medium, unstimulated neutrophils (no NETs), PMA-stimulated neutrophils (naive NETs) and heparinized NETs. (B9): Kinetic curve showing the relative wound density at different time points. (B10): Higher magnification image showing scratch wound assay in immortalized human corneal epithelial cells incubated with naive NETs for 30 hours. The inset shows elongated spindle shaped cells resembling fibroblasts. (C1-C48): Representative confocal immunofluorescent staining images of scratch wounds in corneal epithelial cells to show evidence for epithelial mesenchymal transition (EMT). Epithelial cells were incubated with RPMI culture medium (C1-C12), unstimulated neutrophils (no NETs, C13-C24), PMA-stimulated neutrophils (naive NETs, C25–36) and heparinized NETs (C37–48). (D1): Representative Western blots probed with antibodies for mesenchymal markers. (D2-D5): Quantitative data from Western blots showing the fold change in EMT markers (three independent experiments); (D2) alpha-smooth muscle actin; (D3) CCN2; (D4) vimentin; (D5) E-cadherin.

Figure 5.. Pathological effects of NETs on murine ocular surface.

(A1-A6): Representative images of murine corneas (n=5/group) showing fluorescein staining after topical application of unstimulated murine neutrophils supernatant (no NETs), PMA stimulated murine neutrophils (naive NETs), and heparinized murine NETs (dismantled NETs). (A7): Graph comparing fluorescein staining data between groups at Day 0 and Day 7. (A8): Graph showing amount of IP-10 in corneal lysates prepared after 7 days of topical application of RPMI (n=12 corneas), no NETs (n=12 corneas), NETs (n=12 corneas), and heparinized NETs (n=27 corneas). (A9): Graph showing amount of IL-1β in corneal lysates prepared after 7 days of topical application of RPMI (n=12 corneas), no NETs (n=12 corneas), NETs (n=24 corneas) and heparinized NETs (n=15 corneas). (B1-B16): Representative images of murine corneas showing fluorescein staining after epithelial scratch and application of RPMI culture medium, unstimulated murine neutrophils supernatant (no NETs), PMA-stimulated murine neutrophils (naïve NETs), and heparinized murine NETs (dismantled NETs). (B17): Graph comparing the epithelial defect area between groups at Day 1 to Day 3 (n=5/group).

Figure 6.. Pathological effect of oncostatin M (OSM) on ocular surface.

(A1-A5): H&E and confocal immunofluorescent images of mucocellular aggregates (MCA) collected from ocular surface of a definite oGVHD patient; (A1) H&E staining; (A2) neutrophil elastase (NE, red); (A3) OSM (green); (A4) DAPI nuclear stain (blue); (A5) Merged image showing co-localization of NE and OSM. (A6-A10): H&E and cytospin preparations of ocular surface washings from a definite oGVHD patient; (A6) H&E staining; (A7) neutrophil elastase (NE, red); (A8) OSM (green); (A9) DAPI nuclear stain (blue); (A10) Merged image showing co-localization of NE and OSM. (B1): Graph showing data demonstrating OSM (10 ng/mL) and heparin (100 IU/mL) binding. (B2): Graph showing data demonstrating OSM presence in murine NETs. (C1-C6): Representative fluorescein stained images of murine corneas showing pathological effect of naïve NETs, heparinized NETs and NETs with OSM neutralizing antibodies that were topically applied to murine corneas for five days. (C1-C3): At Day 0, corneas in all groups do not show fluorescein staining. (C4-C6): At day 5, significant corneal fluorescein staining is seen with naïve NETs (C4) but not with heparinized NETs (C5) or NETs incubated with OSM neutralizing antibody (C6). (C7): Graph showing data comparing corneal fluorescein staining between groups. (C8-C10): Graph showing amount of IL-1β (C8), IL-6 (C9) and IP-10 (C10) in corneal lysates (n=5/group) prepared after 5 days of topical application. (D1-D6): Representative fluorescein stained images of murine corneas showing pathological effect of recombinant mouse OSM protein, recombinant mouse OSM protein + heparin, recombinant mouse OSM protein +OSM neutralizing antibody that were topically applied to murine corneas for five days. (D1-D3): At Day 0, corneas in all groups do not show fluorescein staining. (D4-D6): At day 5, significant corneal fluorescein staining is seen with recombinant OSM protein (D4) but not with recombinant OSM protein + heparin (D5) or recombinant OSM protein + OSM neutralizing antibody (D6). (D7): Graph showing data comparing corneal fluorescein staining between groups. (D8-D10): Graph showing amount of IL-1β (D8), IL-6 (D9) and IP-10 (D10) in corneal lysates (n=5/group) prepared after 5 days of topical application. (E1-E4): Treatment of an oGVHD patient with severe ocular surface disease with Heparin 100IU/mL eye drops twice a day for 4 weeks. Lissamine green staining showed severe corneal (E1) and conjunctival (E2) epitheliopathy prior to treatment with Heparin eye drops. After 4 weeks of treatment, corneal (E3) and conjunctival (E4) staining was significantly reduced.

Figure 7.. Pathological effects of NETs on conjunctival fibroblasts.

(A1-A3): Representative clinical images of an ocular GVHD patient showing conjunctival cicatricial disease; (A1) Conjunctival subepithelial fibrosis (CSEF) under upper lid palpebral conjunctiva; (A2) conjunctival fornix foreshortening; (A3) symblepheron formation. (A4): Graph showing data comparing presence of CSEF (% eyes) between healthy subjects (n=15 eyes), none oGVHD (n=39 eyes) and definite oGVHD (n=87 eyes) patients. (B1-B8): Representative image showing scratch wound assay in primary human conjunctival fibroblast cells incubated with RPMI culture medium, PMA-stimulated neutrophils (naive NETs), heparinized NETs, and heparin alone. (B9): Kinetic curve showing the relative wound density at different time points. (B10): Graph showing data comparing collagen concentration measured from the supernatants of conjunctival fibroblast scratch wound assays (RPMI n=12; NETs n=15; heparinized NETs n=15; heparin alone n=5). (C1-C12): Representative confocal immunofluorescent staining images of scratch wounds in conjunctival fibroblasts to show the evidence of myofibroblast transformation. Conjunctival fibroblasts were incubated with RPMI culture medium (C1-C3), PMA-stimulated neutrophils (naive NETs, C4-C6), heparinized NETs (C7-C9) and heparin alone (C10-C12). (C13): Graph showing data comparing myofibroblast transformation (fluorescent intensity in immunofluorescent staining images) between conjunctival fibroblasts incubated with RPMI (n=6); NETs (n=4); heparinized NETs (n=5) or heparin alone (n=5). (C14, C15): Representative Western blot image (C14) and graph (C15) to compare α-SMA abundance between conjunctival fibroblasts incubated with RPMI; NETs; heparinized NETs or heparin alone. Tubulin was shown as the loading control. (D1-D4): Representative confocal immunofluorescent staining images of scratch wounds in conjunctival fibroblasts to show the evidence of fibroblast proliferation using α-Ki67 antibody. Conjunctival fibroblasts were incubated with RPMI (D1); NETs (D2); heparinized NETs (D3) or heparin alone (D4). (D5): Graph showing data comparing fibroblast proliferation (fluorescent intensity of α-Ki67 positive cells in immunofluorescent staining images) between conjunctival fibroblasts incubated with RPMI (n=6); NETs (n=4); heparinized NETs (n=9) or heparin alone (n=7). (E1-E8): Representative images from a collagen gel contraction assay. Conjunctival fibroblasts were seeded in collagen matrices and incubated with RPMI (E1, E5; n=9); NETs (E2, E6; n=9); heparinized NETs (E3, E7; n=9) and heparin alone (E4, E8; n=9). Collagen gel images were obtained at baseline (E1-E4) and after 24 hours incubation (E5-E8). (E9): Graph showing data comparing collagen gel contraction (%) between collagen matrices incubated with RPMI (n=9); NETs (n=9); heparinized NETs (n=9); heparin alone (n=9). (E10, E11): Representative Western blot image (E10) and graph (E15) to compare α-SMA abundance between collagen matrices incubated with RPMI; NETs; heparinized NETs or heparin alone. Tubulin blot is shown as the loading control.

Figure 8.. Pathological effect of NETs on immune cell proliferation.

(A1): H&E staining of mucocellular aggregates (MCA) showed numerous neutrophils, surface epithelial cells and mononuclear cells. (A2-A5): Confocal immunofluorescent staining images of MCA showing the presence of neutrophil elastase (NE) (A2, red), CD3 positive T cells (A3, green), DAPI nuclear staining (A4, blue) and merged image (A5). (B): Graph shows NET-induced T cell proliferation in mixed lymphocyte reaction (MLR). NETs promote proliferation of MLR. Heparin inhibits MLR proliferation. (C1): H&E staining of mucocellular aggregates (MCA) showed numerous neutrophils, surface epithelial cells and mononuclear cells. (C2-C5): Confocal immunofluorescent staining images of MCA showing the presence of neutrophil elastase (NE) (C2, red), LIGHT protein (C3, green), DAPI nuclear staining (C4, blue) and merged image (C5). (D): Graph shows effect of human recombinant LIGHT/TNFSF14 protein induced on T cell proliferation and in MLR. LIGHT/TNFSF14 induces T cell proliferation. LIGHT/TNFSF14 neutralizing antibodies inhibit NET-induced MLR proliferation.

Figure 9.. Pathological effect of NETs on Meibomian glands.

Representative infrared images of the lower lid showing the presence of normal meibomian glands in healthy subjects (A1) and severe Meibomian gland atrophy in a patient with definite oGVHD (A2). (A3): Graph showing data comparing meiboscale (score 0–4 based on severity of meibomian gland atrophy) between healthy subjects (n=40 eyes), none oGVHD (n=35 eyes) and definite oGVHD (n=54 eyes) patients. (B1): Graph showing data comparing amount of NGAL in conjunctival washings between healthy subjects, none oGVHD and definite oGVHD. (B2): Graph showing data comparing amount of NGAL in RPMI, unstimulated human neutrophils (no NETS) and PMA stimulated neutrophils (NETs). (C1): H&E staining of mucocellular aggregates (MCA) showed numerous neutrophils, surface epithelial cells and mononuclear cells. (C2-C5): Confocal immunofluorescent staining images of MCA showing the presence of neutrophil elastase (NE) (C2, red), NGAL protein (C3, green), DAPI nuclear staining (C4, blue) and merged image (C5). (D1-D8): Effect of NETs and NGAL on immortalized Meibomian gland (MG) epithelial cell differentiation. Lipid accumulation was detected using LipidTOX staining (green). (D1): Keratinocyte serum free medium (KSFM) only; (D2): differentiation medium (DFM) only; (D3): DFM + NETs; (D4) DFM + NETs + NGAL neutralizing antibody (NAb); (D5) DFM + NETs + heparin 100 IU/mL; (D6) DFM + human recombinant NGAL protein (rNGAL); (D7): DFM + rNGAL + NGAL Nab; (D8) DFM + rNGAL + heparin. (D9): Graph showing data comparing LipidTox staining (fluorescence intensity) under various differentiation culture conditions. (D10): Graph showing data comparing MG cell proliferation that was determined by measuring the cellular DNA content (fluorescence intensity) using a commercially available dye binding kit under various culture conditions.