Claudin-1 expression in airway smooth muscle exacerbates airway remodeling in asthmatic subjects Hiroyuki Fujita, MD, PhD, Maciej Chalubinski, MD, PhD,

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Claudin-1 expression in airway smooth muscle exacerbates airway remodeling in asthmatic subjects Hiroyuki Fujita, MD, PhD, Maciej Chalubinski, MD, PhD, Claudio Rhyner, PhD, Philippe Indermitte, Dipl Ing FH, Norbert Meyer, MD, Ruth Ferstl, PhD, Angela Treis, MSc, Enrique Gomez, PhD, Ahmet Akkaya, MD, Liam O’Mahony, PhD, Mübeccel Akdis, MD, PhD, Cezmi A. Akdis, MD Journal of Allergy and Clinical Immunology Volume 127, Issue 6, Pages 1612-1621.e8 (June 2011) DOI: 10.1016/j.jaci.2011.03.039 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Claudin-1 is upregulated by IL-1β and TNF-α and downregulated by IL-4 and IL-13 in human primary ASM cells. Human primary ASM cells were incubated with 50 ng/mL (A), different concentrations (B), or 10 ng/mL (C and D) of cytokines. Relative claudin-1 mRNA expression (Fig 1, A and B) and claudin-1 protein levels were detected by means of immunofluorescence staining at 24 hours (Fig 1, C), Western blotting (Fig 1, D), and multispectral imaging flow cytometry (E). Results are representative of 2 (Fig 1, D and E) or 3 independent experiments with 2 different ASM cell lines. Fig 1, A and B, shows means ± SEMs. FSC, Forward scatter; ic, isotype control; us, unstimulated. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Claudin-1 overexpression stimulates ASM cell proliferation. An empty vector (pORF) or a vector containing claudin-1 (claudin-1–pORF) was transfected into primary ASM cells. Relative mRNA expression of TJ molecules was analyzed (A), and claudin-1 protein levels were analyzed by means of immunofluorescence (B) and Western blotting (C). Cell proliferation was determined by using a CFSE proliferation assay (D), a tritiated thymidine incorporation assay (E), and a wound-healing assay (F). Fig 2, A, B, D, and F are representative of at least 3 independent experiments. Fig 2, E, is from 4 independent experiments. Fig 2, A and E, show means ± SEMs. *P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 siRNA knockdown of claudin-1 decreased ASM cell proliferation. Scrambled siRNA or claudin-1 siRNA was transfected. Claudin-1 expression (A and B) and cell proliferation (C-E) were analyzed as in Fig 2. siRNA-transfected ASM cells were stimulated with 10 ng/mL TNF-α 24 hours after transfection, and claudin-1 protein (F) and cell proliferation (G and H) were analyzed under TNF-α–stimulated conditions. Fig 3, A, B, C, E, F, and H, are representative of at least 2 independent experiments. Fig 3, D and G, are from at least 4 independent experiments. Fig 3, A, D, and G, show means ± SEMs. ∗P < .05 and ∗∗P < .01. ic, Isotype control. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Claudin-1 expression is suppressed by dexamethasone (Dex). ASM cells were incubated with and without 50 ng/mL of cytokines in the presence or absence of each compound, and relative claudin-1 mRNA expression (A) and protein levels (B) were analyzed after 24 hours. Data are representative of 3 independent experiments. Fig 4, A, shows means ± SEMs. ∗P < .05 and ∗∗P < .01. ic, Isotype control; Rapa, rapamycin; us, unstimulated. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Claudin-1 overexpression upregulates VEGF and downregulates IL-6, IL-8, and IP-10 in ASM cells. Twenty-four hours after the transfection, ASM cells were left unstimulated (A) or stimulated with PMA and ionomycin (B) for 24 hours. Cytokine and chemokine concentrations in supernatants were determined by using the luminometric bead array, and relative percentage change against the negative control was shown. The ranges of secreted cytokines and chemokines were as follows: IL-6, 296 to 4,448 pg/mL; IL-8, 257 to 3,435 pg/mL; IP-10, 662 to 81,462 pg/mL; and VEGF, 263 to 2,500 pg/mL. Data are from 6 independent experiments in triplicate of 18 samples. ∗∗P < .01. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Claudin-1 expression is increased in asthmatic lungs. A, Human bronchial biopsy specimens from patients with severe persistent asthma and nonasthmatic subjects were analyzed. B, An ovalbumin (OVA)–induced murine model of airway inflammation was analyzed. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), claudin-1 was stained with Alexa Fluor 488 (green), and α-smooth muscle actin (α-SMA) was stained with Cy3 (red). Data shown are from 1 representative staining. H&E, Hematoxylin and eosin; ic, isotype control. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Expression of claudin-4, occludin, ZO-1, and ZO-2 in human primary ASM cells. Human primary ASM cells were incubated with 50 ng/mL of each cytokine, and relative claudin-4, occludin, ZO-1, and ZO-2 mRNA expressions were analyzed at 6 and 24 hours. Results are representative of 3 independent experiments. Means ± SEMs are shown. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Effect of TH2 cytokines on claudin-1 expression. Human primary ASM cells were stimulated with 10 ng/mL of each cytokine, and the percentage of claudin-1 mRNA suppression at 24 hours was shown. Results are representative of 3 independent experiments. Means ± SEMs are shown. ∗∗P < .01. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Kinetic change of claudin-1 and ZO-2 expression. Human primary ASM cells were stimulated with 10 ng/mL of each cytokine, and relative claudin-1 and ZO-2 mRNA expressions were analyzed at each time point. Results are representative of 3 independent experiments. Means ± SEMs are shown. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Effect of cytokines on ASM cell proliferation. ASM cells were incubated and stimulated with 10 ng/mL of each cytokine in a 96-well flat-bottom plate for a certain period, and tritiated thymidine was added to each well in the last 24 hours. The proliferation of stimulated ASM cells was compared with that of unstimulated (us) ASM cells, and the ratio was shown. Results are means ± SEMs from 3 independent experiments. ∗P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Quantification of the wound-healing model. The wound-healing models shown in Figs 2, F; 3, E; and 3, H, were quantified as A, B, and C, respectively. D, Comparison of the role of claudin-1 in wound healing in the presence or absence of TNF-α. The intensity of cell growth inside the scratch fields was calculated, and that of each time point was compared with that of the starting point. ∗P < .05 and ∗∗P < .01. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Time course of claudin-1 expression by salbutamol. Human primary ASM cells were stimulated with 50 ng/mL of each cytokine in the presence or absence of 10 μmol/L salbutamol, and claudin-1 mRNA expression was analyzed at each time point. Results are representative of 2 independent experiments. Means ± SEMs are shown. us, Unstimulated. Journal of Allergy and Clinical Immunology 2011 127, 1612-1621.e8DOI: (10.1016/j.jaci.2011.03.039) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions