In Vivo Induction of Cutaneous Inflammation Results in the Accumulation of Extracellular Trap-Forming Neutrophils Expressing RORγt and IL-17 Romy R.M.C.

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In Vivo Induction of Cutaneous Inflammation Results in the Accumulation of Extracellular Trap-Forming Neutrophils Expressing RORγt and IL-17 Romy R.M.C Keijsers, Anke G.M. Hendriks, Piet E.J. van Erp, Bram van Cranenbroek, Peter C.M. van de Kerkhof, Hans J.P.M. Koenen, Irma Joosten Journal of Investigative Dermatology Volume 134, Issue 5, Pages 1276-1284 (May 2014) DOI: 10.1038/jid.2013.526 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Application of LTB4 causes thickening of the epidermis, influx of immune cells, and small abscesses. (a) Hematoxylin and eosin (H&E) stainings of the skin 0, 8, 24, 32, 48, and 72hours after the application of leukotriene B4 (LTB4). LTB4 causes a gradual increase of epidermal thickness (indicated by arrow), influx of immune cells (indicated by #), and small abscesses in the subcorneal zone of the epidermis (indicated by *). Bar=200μm. (b) Mean thickening of the epidermis in μm at all time points after application of LTB4 (n=10). (c) Means±SEM number, as analyzed by immunohistochemistry of CD3+, CD4+, CD8+, Foxp3+, IL-17+, RORγ+, elastase+, and tryptase+ cells mm-2, present in the dermis at six consecutive time points after the application of LTB4 (n=10). *P<0.01 to the previous measurement in time. Journal of Investigative Dermatology 2014 134, 1276-1284DOI: (10.1038/jid.2013.526) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dual-color immunofluorescent stainings of inflamed skin after the application of LTB4 and after tape-stripping.Immunofluorescence of (a) RORγ (red) and elastase (green), (b) IL-17 (red) and RORγ (green), (c) IL-17 (red) and tryptase (green), (d) RORγ (red) and tryptase (green), (e) CD3 (red) and IL-17 (green), and (f) CD3 (red) and RORγ (green). 4',6-Diamidino-2-phenylindole (DAPI) was used as a counterstain. Arrowheads point toward double positively stained cells. Bar=100μm. Journal of Investigative Dermatology 2014 134, 1276-1284DOI: (10.1038/jid.2013.526) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Extracellular trap formation of neutrophils after the application of LTB4. Confocal laser scanning microscopy of (a) neutrophils showing neutrophil extracellular traps formed of nuclear material and (b) neutrophils with intact nuclei. Both images show neutrophils that display both elastase (green) and IL-17 (red). Bar=10μm. Journal of Investigative Dermatology 2014 134, 1276-1284DOI: (10.1038/jid.2013.526) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Accumulated neutrophils in the subcorneal zone of the LTB4-challenged skin contain IL-17 mRNA. (a) Immunohistochemical stainings of microabscesses in the subcorneal zone of the epidermis, caused by the application of LTB4. The microabscesses stain positively for elastase and IL-17 (arrows). Bar=100μm. (b) Representative immunofluorescent mRNA stainings (from n=4 experiments) for IL-17 (red), RPLP0 (housekeeping gene) (green), and myeloperoxidase (MPO) (green) on LTB4-challenged skin and normal skin. As a negative control, anti-rat probes were used instead of anti-human probes. DAPI was used as a nuclear stain. Microabscesses are marked by the white dotted line. Journal of Investigative Dermatology 2014 134, 1276-1284DOI: (10.1038/jid.2013.526) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Tape-stripping results in the thickening of the epidermis with hyperkeratosis, parakeratosis, and influx of immune cells. (a) Hematoxylin and eosin stainings of the skin 0, 16, 24, 48, and 72hours after tape-stripping. Tape-stripping leads to (partial) removal of the stratum corneum at t=16hours. This results in a gradual thickening of the epidermis (indicated by an arrow) with hyperkeratosis, parakeratosis (indicated by *), and influx of immune cells (indicated by #). Bar=200μm. (b) Mean thickening of the epidermis in μm at all time points after tape-stripping (n=10). (c) Means±SEM number, as analyzed by immunohistochemistry, of CD3+, CD4+, CD8+, Foxp3+, IL-17+, RORγ+, elastase+, and tryptase+ cells mm-2, present in the dermis at five consecutive time points after tape-stripping (n=10). *P<0.01 to the previous measurement in time. Journal of Investigative Dermatology 2014 134, 1276-1284DOI: (10.1038/jid.2013.526) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions