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.