Journal: bioRxiv

Article Title: Mechanical Skin Stress-Induced Lesion Development via ATP-Amplified Neutrophil Extracellular Trap Formation

doi: 10.64898/2026.03.11.710999

Figure Lengend Snippet: (A) Representative clinical images and H&E-stained ear sections from EBA mice treated with vehicle (EBA+vehicle) or DNase1 (EBA+DNase1) on day 12. White arrows indicate lesional skin. Dotted lines demarcate major dermal-epidermal separations. Scale bar = 100 µm. (B) Affected surface area defined as ((total affected body surface area)/(total body surface area) × 100) was quantified every 2 days for 12 days. Dot plots indicate all individual scores and lines indicate group means. N = 7 for each group. *P<0.05 (Two-way ANOVA (time and group as two variables)). (C) Histological blister scores defined as ((combined total length of all blistered regions)/(combined total length of all dermal-epidermal junction examined) × 100) were quantified from H&E staining images at day 12. Data shown as dot plots with mean ± standard deviation. N = 7 for each group. *P<0.05 (Mann-Whitney U test). (D) Representative immunohistochemistry of ear sections from EBA+vehicle and EBA+DNase1 mice stained for myeloperoxidase (MPO), citrullinated Histone H3 (CitH), and 4, 6-diamidino-2-phenylindole (DAPI). Co-distributed area of MPO and CitH indicates NETosis. Dotted lines indicate the dermal-epidermal junction. Scale bar = 40 µm. Quantification of NETosis area (E: (NETosis area of upper dermis)/(total area of upper dermis) × 100), infiltrating neutrophils (F: (neutrophil number in upper dermis)/(total area of upper dermis)), and NET index (G: (NETosis area of upper dermis)/(neutrophil number in upper dermis)). Data shown as dot plots with mean ± standard deviation. N = 7 for each group. *P<0.05, **P<0.01 (Mann-Whitney U test). IL-8 secretion by HaCaT cells (H) or fibroblasts (I) following 12-hour stimulation with 0.02, 0.2, or 0.5 mL of supernatant with or without NET components. IL-8 levels measured by ELISA. Data shown as dot plots with mean ± standard deviation. N = 4 for each group. *P<0.05, **P<0.01, ***P<0.001 (Two-way ANOVA (dose and group as two variables) with multiple comparison test). Large asterisks in the center indicate ANOVA significance, and small asterisks show pairwise comparisons.

Article Snippet: Human adult low calcium temperature keratinocytes (HaCaT) (300493F; Cell Lines Service GmbH., Eppelheim, Germany) and human fibroblast cells (PCS-201-030; American Type Culture Collection., Manassas, Virginia, U.S.) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (041-29775, 044-29765; FUJIFILM Wako Pure Chemical Corporation., Osaka, Japan) supplemented with 10% fetal bovine serum (10270-106; Thermo Fisher Scientific, Inc.), 100 U/mL penicillin and 100 μg/mL streptomycin (168-23191; FUJIFILM Wako Pure Chemical Corporation.).

Techniques: Staining, Standard Deviation, MANN-WHITNEY, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: Mechanical Skin Stress-Induced Lesion Development via ATP-Amplified Neutrophil Extracellular Trap Formation

doi: 10.64898/2026.03.11.710999

Figure Lengend Snippet: (A) Representative immunohistochemistry of human skin sections from healthy control (HC), Behçet disease (BD), Sweet syndrome (SS), pyoderma gangrenosum (PG), and epidermolysis bullosa acquisita (EBA) for myeloperoxidase (MPO), citrullinated Histone H3 (CitH), and 4ʹ,6-diamidino-2-phenylindole (DAPI). Dotted lines indicate the dermal-epidermal junction. Scale bar = 40 µm. (B) Quantification of NET index (NETosis area of upper or lower dermis)/(Neutrophil number in upper or lower dermis). The dot plots indicate all individual scores and error bars indicate mean ± standard deviation. N = 3 (HC), 3 (BD), 5 (SS), 5 (PG), and 6 (EBA). *P<0.05, **P<0.01 (Mann-Whitney U test). (C) Proposed model of the mechanical stimulation-ATP-NETs axis in EBA. Mechanical stimulation triggers ATP release from keratinocytes. In combination with C5a, extracellular ATP promotes NETosis. NET components induce IL-8 release from keratinocytes and fibroblasts, resulting in further neutrophil recruitment.

Article Snippet: Human adult low calcium temperature keratinocytes (HaCaT) (300493F; Cell Lines Service GmbH., Eppelheim, Germany) and human fibroblast cells (PCS-201-030; American Type Culture Collection., Manassas, Virginia, U.S.) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (041-29775, 044-29765; FUJIFILM Wako Pure Chemical Corporation., Osaka, Japan) supplemented with 10% fetal bovine serum (10270-106; Thermo Fisher Scientific, Inc.), 100 U/mL penicillin and 100 μg/mL streptomycin (168-23191; FUJIFILM Wako Pure Chemical Corporation.).

Techniques: Immunohistochemistry, Control, Standard Deviation, MANN-WHITNEY