Fig 1: Genetic deficiency in Clec4d, Clec4e, and Clec4n in experimental EBA. (A) Affected Body Surface Area (BSA; clinical score) in antibody transfer-induced EBA in neutrophil-specific Clec4n (Clec4nΔPMN ) mutant mice). (B) Area under the curve analysis (AUC) of BSA over time. (C) Representative histology of CLEC4MUT and CLEC4WT following 2 weeks of antibody transfer reveals comparable inflammatory infiltrate. Similar findings are noted in the (D) BSA, (E) AUC, and (F) histology of Clec4d−/− vs. Clec4e+/+ WT, as well as (G) BSA (H) AUC, and (I) histology of Clec4e−/− vs. Clec4e+/+ WT. All results are presented as mean ± SEM. Data were merged from two to three independent experiments (n = 10–11 mice/group). (A, D, G) were analyzed by two-way ANOVA; (B, E, H) were analyzed by Student’s t-test. EBA, epidermolysis bullosa acquisita; BSA, body surface area; IgG, immunoglobulin G; AUC, area under the curve; n.s., not significant.
Fig 2: Expression of CLEC4 markers in human and experimental EBA neutrophils. (A) Flow cytometry of blood demonstrates an increased percentage of Ly6G+CD45+ cells in EBA relative to control mice. (B) Immunofluorescent staining of FFPE from mice with EBA reveals co-expression of Ly6G (green) and CLEC4 markers (red) overlaid with DAPI. Dectin-2, Dectin-3, and Mincle are expressed on a majority of neutrophils in experimental EBA. (C) Flow cytometry of blood neutrophils demonstrates that CLEC4N and CLEC4D are expressed on both control and EBA neutrophils, while CLEC4E is expressed only on a small number of neutrophils in both EBA and control mice (n = 3–4). (D) Immunofluorescent staining of FFPE from patients with inflammatory EBA demonstrates expression of CLEC4N/D/E (green) on MPO+ cells (red). Notes: Epidermis and dermis are demarcated by a white line. The inset demonstrates characteristic nuclear morphology of neutrophils on dually stained cells. Images from mouse tissue are representative of n = 10 from three independent experiments. Flow cytometry density plots are shown of a single biologic replicate and are representative of n = 3–4 mice. Images shown from patients with inflammatory EBA are representative of n = 5. Scale bars, 300 μm (B); 300 μm (D). EBA, epidermolysis bullosa acquisita; IgG, immunoglobulin G; FMO, fluorescent minus one.
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