The Leukotriene B4 and its Receptor BLT1 Act as Critical Drivers of Neutrophil Recruitment in Murine Bullous Pemphigoid-Like Epidermolysis Bullosa Acquisita  Tanya Sezin, Matthias Krajewski, Adam Wutkowski, Sadegh Mousavi, Lenche Chakievska, Katja Bieber, Ralf J. Ludwig, Markus Dahlke, Dirk Rades, Franziska S. Schulze, Enno Schmidt, Kathrin Kalies, Yask Gupta, Paul Schilf, Saleh M. Ibrahim, Peter König, Dominik Schwudke, Detlef Zillikens, Christian D. Sadik  Journal of Investigative Dermatology  Volume 137, Issue 5, Pages 1104-1113 (May 2017) DOI: 10.1016/j.jid.2016.12.021 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Alox5–/– and Ltb4r1–/– mice are resistant to skin inflammation in the antibody transfer EBA model. (a) Clinical course of antibody transfer EBA in WT, Alox5–/–, and Ltb4r1–/– mice. (b) Clinical presentation and histopathologies in wild-type, Alox5–/–, and Ltb4r1–/– mice, and (c) quantification of Ly-6G+ and Siglec-F+ cells in lesional skin of WT mice or corresponding skin sites of Alox5–/– and Ltb4r1–/– mice on day 9. Arrows indicate deposition of IgG or C3. (d) Effect of zileuton on antibody transfer EBA in WT mice. Stars indicate inflammatory infiltrates; arrows indicate subepidermal clefts. In a and d, n = 3–5 mice/group. In a, ∗∗∗P < 0.001 wild-type versus Alox5–/– or Ltbr4r1–/– mice; in d, ∗∗P < 0.01, ∗∗∗P < 0.001. Results in c were pooled from three independent experiments (n = –14 mice/group); ∗P < 0.05 compared with WT. Scale bar in H&E, IgG, and C3 stainings = 100 μm; scale bar in Ly-6G and Siglec-F stainings = 50 μm. ABSA, affected body surface area; EBA, epidermolysis bullous acquisita; H&E, hematoxylin and eosin; Pos., positive; WT, wild type. Journal of Investigative Dermatology 2017 137, 1104-1113DOI: (10.1016/j.jid.2016.12.021) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Neutrophils but not eosinophils are required for antibody transfer EBA. (a) Clinical course in anti-Ly-6G- or isotype control-treated WT mice (n = 3 mice/group), and (b) histopathologies of lesional skin of this experiment. (d) Clinical course of antibody transfer EBA in WT versus ΔdblGATA mice (n = 4 mice/group). (e) Comparison of WT and ΔdblGATA mice. All histopathologies were taken from lesional skin on day 12. Stars indicate inflammatory infiltrates; arrows indicate subepidermal clefts. In a and d, ∗∗P < 0.01; ∗∗∗P < 0.001. (c, f) Results in were pooled from two independent experiments (n = 5–7 mice/group). ∗P < 0.05; ∗∗P < 0.01. Scale bar in H&E stainings = 100 μm; scale bar in Ly-6G and Siglec-F stainings = 50 μm. ABSA, affected body surface area; H&E, hematoxylin and eosin; n.s., not significant; Pos., positive; WT, wild type. Journal of Investigative Dermatology 2017 137, 1104-1113DOI: (10.1016/j.jid.2016.12.021) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Lipid mediator profile in emerging pemphigoid disease-like skin inflammation. (a) Clinical presentation and histopathologies of anti-Col7-injected ears. The quantification of Ly-6G+ and Siglec-F+ cell infiltration was pooled from three independent experiments (n = 6–11 mice/group) and analyzed by Kruskal-Wallis test followed by Dunn multiple comparison test. ∗∗P < 0.01, ∗∗∗P < 0.001 compared with 0 hours. Stars indicate inflammatory infiltrates; arrows indicate subepidermal clefts. Scale bar in H&E stainings = 100 μm; scale bar in Ly-6G and Siglec-F stainings = 50 μm. (b) Heat map of lipid mediator expression in anti-Col7– versus PBS-treated skin. (c) Time kinetics of LTB4 and 5-HETE levels in anti-Col7– versus PBS-treated skin. Data in b and c were pooled from three independent experiments (n = 5–10 mice/group/time point). In b, groups were compared by two-way analysis of variance with Benjamini-Hochberg correction; (∗P < 0.05, ∗∗P < 0.01) and in c, by two-way analysis of variance with Bonferroni post hoc test (∗P < 0.05, ∗∗P < 0.01). H&E, hematoxylin and eosin; hrs, hours; LTB4, leukotriene B4; PBS, phosphate buffered saline; PQN, probablistic quotient normalization. Journal of Investigative Dermatology 2017 137, 1104-1113DOI: (10.1016/j.jid.2016.12.021) Copyright © 2017 The Authors Terms and Conditions

Figure 4 5-LO and BLT1 expression solely on hematopoietic cells drive antibody transfer EBA. (a) Course of disease, (b) clinical presentation and histopathologies, and (c) quantification of Ly-6G+ cell infiltration in lesional skin on day 9 in bone marrow chimera between WT and Alox5–/– mice in the EBA transfer model. (d) Course of disease, (e) clinical presentation and histopathologies, and (f) quantification of Ly-6G+ cell infiltration in lesional skin in bone marrow chimera between WT and Ltb4r1–/– mice in the EBA transfer model on day 9. In a, ##P < 0.01 for WT→WT versus Alox5–/–→Alox5–/– and Alox5–/– →WT mice; ∗∗P < 0.01 for WT→Alox5–/– versus Alox5–/–→Alox5–/– and Alox5–/–→WT mice. In d, ##P < 0.01 and ###P < 0.001 for WT→WT versus Ltb4r1–/–→Ltb4r1–/– and Ltb4r1–/–→WT mice; ∗∗P < 0.01 and ∗∗∗P < 0.01 for WT→Ltb4r1–/– versus Ltb4r1–/–→Ltb4r1–/– and Ltb4r1–/–→WT mice; n = 6 mice/group. Stars indicate inflammatory infiltrates; arrows indicate subepidermal clefts. Scale bar in H&E stainings = 100 μm; scale bar in Ly-6G stainings = 50 μm. 5-LO, 5-lipoxygenase; ABSA, affected body surface area; EBA, epidermolysis bullous acquisita; H&E, hematoxylin and eosin; Pos., positive; WT, wild type. Journal of Investigative Dermatology 2017 137, 1104-1113DOI: (10.1016/j.jid.2016.12.021) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Adoptive transfer of neutrophils into the skin precipitates pemphigoid disease-like skin lesions in anti-Col7–treated Ltb4r1–/– mice. (a) Number of mice per group displaying skin lesions by day 9 after adoptive transfer of WT or Ltb4r1–/– neutrophils (PMNs) subcutaneously. Analysis by F test did not find statistically significant differences between WT and Ltb4r1–/– neutrophils in their general capability to precipitate disease. Results were pooled from three independent experiments. (b) Clinical presentation and histopathology on day 9 of the experiment. Arrows indicate inflammatory skin lesions. (c) Direct immunofluorescence for IgG (indicated by arrows), Ly-6G, and Siglec-F and quantification of the latter two pooled from three independent experiments (n = 10–11 mice/group), statistically analyzed by Mann-Whitney U test. Scale bar in H&E and IgG stainings = 100 μm; scale bar in Ly-6G and Siglec-F stainings = 50 μm. H&E, hematoxylin and eosin; PMN, Ltb4r1–/– neutrophil; Pos., positive; WT, wild type. Journal of Investigative Dermatology 2017 137, 1104-1113DOI: (10.1016/j.jid.2016.12.021) Copyright © 2017 The Authors Terms and Conditions