Influence of Th2 Cytokines on the Cornified Envelope, Tight Junction Proteins, and β- Defensins in Filaggrin-Deficient Skin Equivalents  Stefan Hönzke, Leonie Wallmeyer, Anja Ostrowski, Moritz Radbruch, Lars Mundhenk, Monika Schäfer-Korting, Sarah Hedtrich  Journal of Investigative Dermatology  Volume 136, Issue 3, Pages 631-639 (March 2016) DOI: 10.1016/j.jid.2015.11.007 Copyright © 2015 The Authors Terms and Conditions

Figure 1 Impact of IL-4 and IL-13 on histology, epidermal thickness, and skin surface pH of normal and filaggrin knockdown skin equivalents. (a) Representative histologic staining of untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Bar = 100 μm. (b) Epidermal thickness of untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Values are given as mean ± SEM. n = 3–4. (c) Skin equivalent surface pH values. Values are given as mean ± SEM. n = 3. Plus sign indicates statistical significance over FLG– untreated: +++P ≤ 0.001. Journal of Investigative Dermatology 2016 136, 631-639DOI: (10.1016/j.jid.2015.11.007) Copyright © 2015 The Authors Terms and Conditions

Figure 2 Impact of IL-4 and IL-13 on protein expression of filaggrin, involucrin, and loricrin. (a–c) Western blots and relative protein expression semiquantified via densitometry of untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Values are given as mean ± SEM. n = 3. Asterisk indicates statistical significance from FLG+ untreated: *P ≤ 0.05, **P ≤ 0.01. Plus sign indicates statistical significance from FLG– untreated: +P ≤ 0.05, ++P ≤ 0.01. (d, e) Representative immunostaining against filaggrin (FLG, red), involucrin (IVL, green), and loricrin (LOR, green) in untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Bar = 100 μm. Exposure times: red channel 1/10 second; green channel 1/13 second; blue channel 1/55 second. Counterstaining was performed with 4′,6-diamidin-2-phenylindol (DAPI; blue). Journal of Investigative Dermatology 2016 136, 631-639DOI: (10.1016/j.jid.2015.11.007) Copyright © 2015 The Authors Terms and Conditions

Figure 3 Tight junction protein expression in normal and filaggrin knockdown skin equivalents. Western blots and relative protein expression of (a) occludin (OCLN), (b) claudin-1 (CLDN-1), and (c) claudin-23 (CLDN-23) semiquantified via densitometry of untreated and IL-4, IL-13, and IL-4/IL-13-supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Values are given as mean ± SEM. n = 3. Asterisk indicates statistical significance over FLG+ untreated: *P ≤ 0.05. Plus sign indicates statistical significance from FLG– untreated: +P ≤ 0.05, ++P ≤ 0.01. Journal of Investigative Dermatology 2016 136, 631-639DOI: (10.1016/j.jid.2015.11.007) Copyright © 2015 The Authors Terms and Conditions

Figure 4 Impact of IL-4 and IL-13 on thymic stromal lymphopoietin (TSLP) expression in normal and filaggrin knockdown skin equivalents. (a) Western blots and relative protein expression of TSLP semiquantified via densitometry of untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Values are given as mean ± SEM. n = 3. Asterisk indicates statistical significance over FLG+ untreated: *P ≤ 0.05. (b) Representative immunostaining against TSLP (green) in untreated and IL-4, IL-13, and IL-4/IL-13 supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Bar = 100 μm. Exposure times: green channel 1/13 second; blue channel 1/55 second. The skin equivalent sections were counterstained with 4′,6-diamidin-2-phenylindol (DAPI; blue). Journal of Investigative Dermatology 2016 136, 631-639DOI: (10.1016/j.jid.2015.11.007) Copyright © 2015 The Authors Terms and Conditions

Figure 5 β-Defensin 2 (hBD-2) expression in normal and filaggrin knockdown skin equivalents. (a) hBD-2 levels in untreated and IL-4, IL-13, IL-4/IL-13, or tumor necrosis factor-α (TNF-α) supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents, quantified via ELISA (calculated per [g] protein). Values are given as mean ± SEM. n = 3. Asterisk indicates statistical significance over FLG+ untreated: *P ≤ 0.05. Plus sign indicates statistical significance from FLG–: +P ≤ 0.05. (b) Representative immunostaining against hBD-2 (green) in untreated and IL-4, IL-13, IL-4/IL-13, or TNF-α supplemented normal (FLG+) and filaggrin knockdown (FLG–) skin equivalents. Bar = 100 μm. Exposure time: green channel 1/8 second; blue channel 1/55 second. The skin equivalent sections were counterstained with 4′,6-diamidin-2-phenylindol (DAPI; blue). Journal of Investigative Dermatology 2016 136, 631-639DOI: (10.1016/j.jid.2015.11.007) Copyright © 2015 The Authors Terms and Conditions