Anionic surfactants and commercial detergents decrease tight junction barrier integrity in human keratinocytes Mu Xian, MD, PhD, Paulina Wawrzyniak,

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Presentation transcript:

Anionic surfactants and commercial detergents decrease tight junction barrier integrity in human keratinocytes Mu Xian, MD, PhD, Paulina Wawrzyniak, MSc, Beate Rückert, Sci Tec, Su Duan, MD, PhD, Yifan Meng, MD, Milena Sokolowska, MD, PhD, Anna Globinska, MSc, Luo Zhang, MD, PhD, Mübeccel Akdis, MD, PhD, Cezmi A. Akdis, MD Journal of Allergy and Clinical Immunology Volume 138, Issue 3, Pages 890-893.e9 (September 2016) DOI: 10.1016/j.jaci.2016.07.003 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Anionic surfactants and detergents decreased TER and increased paracellular flux in ALI-cultured NHEKs. A, Decreased TER over time in NHEKs in response to 72 hours of stimulation with 3 μg/mL SDS, 1 μg/mL SDBS, 1 μg/mL BZC, and 30 μg/mL Tween 20 (TW20), respectively. B, Increase in FITC-dextran paracellular permeability across NHEKs treated with the same surfactants as in Fig 1, A. C and D, TER kinetics (Fig 1, C) and paracellular permeability of FITC-dextran (Fig 1, D) in NHEKs stimulated with 10−6 (vol/vol) detergent A or 10−6 (vol/vol) detergent B. Data are expressed as means ± SEMs (n = 5 donors per group in duplicates). *P < .05, **P < .01, and ***P < .001. u.s., Unstimulated. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Anionic surfactants and detergents reduce expression of TJ proteins. A, Decreased immunofluorescence detection of occludin (green) in ALI-cultured NHEKs treated with surfactants or detergents. Representative pictures from 3 donors are shown. Cells were fixed after 72 hours of stimulation with 3 μg/mL SDS, 1 μg/mL SDBS, 10−6 (vol/vol) detergent A (Det. A), or 10−6 (vol/vol) detergent B (Det. B). 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei. Bar = 20 μm. B, Claudin 1 and occludin protein expression in ALI-cultured NHEKs measured by means of Western blotting. C, Quantified protein expression levels of claudin-1 and occludin in NHEK lysates measured by means of Western blotting (n = 3). Each protein was normalized to the β-actin loading control. Data are expressed as percentages of each treatment to unstimulated. *P < .05. u.s., Unstimulated. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Cytotoxicity of surfactants and commercial detergents to skin keratinocytes. Surfactants (A) and commercial detergents (B) were applied at concentrations ranging from 0.1 to 30 μg/mL and 10−8 (vol/vol) to 10−4 (vol/vol) into medium of NHEK cultures. Culture supernatants were collected at 24 hours after addition of the surfactants or detergents, and LDH release was measured. Data are expressed as means ± SEMs (n = 3 donors per group in triplicates). TW20, Tween 20. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Surfactants decreased the TER of ALI-cultured NHEKs in a dose-dependent manner. Barrier integrity determined by TER in response to SDS (A), SDBS (B), BZC (C), and Tween 20 (TW20; D) stimulation is shown. All stimulations were done at the start of experiments. In comparison with medium alone, SDS of 1 and 3 μg/mL and SDBS of 1 μg/mL decreased barrier integrity significantly, whereas the effects of BZC and Tween 20 were not significant. Data are expressed as means ± SEMs (n = 3 donors per group in duplicates). *P < .05 and **P < .01. u.s., Unstimulated. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Dose-dependent increase in FITC-dextran paracellular permeability across NHEKs treated with different types of surfactants. Barrier integrity determined based on paracellular flux in response to SDS (A), SDBS (B), BZC (C), and Tween 20 (TW20; D) stimulation is shown. Measurements were done at 72 hours of stimulation. In comparison with medium alone, SDS of 3 μg/mL and SDBS of 1 μg/mL decreased barrier integrity significantly, whereas BZC and Tween 20 did not show any effect. Data are expressed as means ± SEMs (n = 4 donors per group in duplicates). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Commercial detergents decreased the TER of NHEKs in a dose-dependent manner. Barrier integrity determined by using TER in response to detergent A and detergent B stimulation is shown. Both stimulations were started at t = 0 and maintained until the end of the experiments. In comparison with medium alone, both detergents of 10−6 (vol/vol) decreased barrier integrity significantly after 72 hours of stimulation. Data are expressed as means ± SEMs (n = 3 donors per group in duplicates). **P < .01. u.s., Unstimulated. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Commercial detergents increased the FITC-dextran paracellular permeability across NHEKs in a dose-dependent manner. Barrier integrity determined based on paracellular flux in response to detergent A and detergent B stimulation is shown. Measurements were done at 72 hours of stimulation. In comparison with medium alone, both detergents of 10−6 (vol/vol) decreased barrier integrity significantly. Data are expressed as means ± SEMs (n = 3 donors per group in duplicates). *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Barrier integrity returns to initial levels after removal of surfactants and detergents. A, Decreased TER over time in NHEKs in response to 72 hours of stimulation with 3 μg/mL SDS, 1 μg/mL SDBS, 10−6 (vol/vol) detergent A (Det. A), or 10−6 (vol/vol) detergent B (Det. B), respectively. After the surfactant or detergent was removed, TER increased gradually within 72 hours. B, FITC-dextran paracellular permeability across NHEKs treated with the same surfactant or detergent as in Fig E6, A, increased for 72 hours and decreased after the stimulants were removed. Data are expressed as means ± SEMs (n = 4 donors per group in duplicates). u.s., unstimulated. *P < .05. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 mRNA expression of TJ-related molecules was not altered in response to surfactants or detergents. Cultured cell samples were harvested after 72 hours of stimulation with 3 μg/mL SDS, 1 μg/mL SDBS, 1 μg/mL BZC, 30 μg/mL Tween 20 (TW20), 10−6 (vol/vol) detergent A (deter A), or 10−6 (vol/vol) detergent B (deter B), respectively. Primer sequences are shown in Table E1. Data are expressed as means ± SEMs (n = 3-5 donors per group in duplicates). u.s., Unstimulated. Journal of Allergy and Clinical Immunology 2016 138, 890-893.e9DOI: (10.1016/j.jaci.2016.07.003) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions