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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Ocul Surf. 2019 Apr 6;17(3):589–614. doi: 10.1016/j.jtos.2019.03.010

Figure 7. Pathological effects of NETs on conjunctival fibroblasts.

Figure 7.

(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.