Staphylococcus aureus enhances the tight junction barrier integrity in healthy nasal tissue, but not in nasal polyps Can Altunbulakli, MSc, Rita Costa,

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Staphylococcus aureus enhances the tight junction barrier integrity in healthy nasal tissue, but not in nasal polyps Can Altunbulakli, MSc, Rita Costa, MSc, Feng Lan, MD, PhD, Nan Zhang, MD, PhD, Mubeccel Akdis, MD, PhD, Claus Bachert, MD, PhD, Cezmi A. Akdis, MD Journal of Allergy and Clinical Immunology Volume 142, Issue 2, Pages 665-668.e8 (August 2018) DOI: 10.1016/j.jaci.2018.01.046 Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 S aureus upregulates TJs and improves barrier integrity in ITs, but not in NPs. A, mRNA expression of TJ and junction-associated molecules in IT (n = 11) tissue infected with S aureus (2 × 107 CFU/mL) (n = 8) or S epidermidis (2 × 107 CFU/mL) (n = 3). B, mRNA expression of TJ and junction-associated molecules in NP (n = 10) tissue infected with S aureus (2 × 107 CFU/mL) (n = 7) or S epidermidis (2 × 107 CFU/mL) (n = 3). TJ gene expression measured by quantitative RT-PCR. Expression values calculated as arbitrary units (2(−ΔCt) × 1000), percentage of glyceraldehyde 3-phosphate dehydrogenase expression. *P < .05. **P < .01. Journal of Allergy and Clinical Immunology 2018 142, 665-668.e8DOI: (10.1016/j.jaci.2018.01.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A, Immunofluorescence stainings of human claudin-4 and -7 molecules in IT (n = 3) tissue infected with S aureus (2 × 107 CFU/mL) or S epidermidis (2 × 107 CFU/mL) (n = 3). B, Immunofluorescence stainings of human claudin-4 and -7 molecules nasal tissue with NP (n = 3) infected with S aureus (2 × 107 CFU/mL) or S epidermidis (2 × 107 CFU/mL) (n = 3). C, Mean fluorescence intensity values of claudin-4 and -7 in ITs and NPs (n = 3). Measurements were taken from 3 separate epithelial regions of equal area from each donor. *P < .05. **P < .01. ***P < .001. Journal of Allergy and Clinical Immunology 2018 142, 665-668.e8DOI: (10.1016/j.jaci.2018.01.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Protein measurement of (A) IL-1β and (B) IL-6 on IT nasal tissue (n = 3) supernatants stimulated with increasing doses of S aureus. C, mRNA expression of IL-1β and IL-6 in IT (n = 11) tissue infected with S aureus (2 × 107 CFU/mL) or S epidermidis (2 × 107 CFU/mL). Gene expression measured by quantitative RT-PCR. Expression values calculated as arbitrary units (2(−ΔCt) × 1000), percentage of glyceraldehyde 3-phosphate dehydrogenase expression. Journal of Allergy and Clinical Immunology 2018 142, 665-668.e8DOI: (10.1016/j.jaci.2018.01.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Quantification of S aureus abundance on IT and NP tissue after initial stimulation dose of 2 × 107 CFU/mL. *P < .05. Journal of Allergy and Clinical Immunology 2018 142, 665-668.e8DOI: (10.1016/j.jaci.2018.01.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 A and B, mRNA expression of other TJ and related molecules in IT (n = 11) and nasal NP (n = 11) tissue infected with S aureus (2 × 107 CFU/mL) or S epidermidis (2 × 107 CFU/mL). Tight junction gene expression measured by quantitative RT-PCR. Expression values calculated as arbitrary units (2(−ΔCt) × 1000), percentage of glyceraldehyde 3-phosphate dehydrogenase expression. *P < .05. **P < .01. Journal of Allergy and Clinical Immunology 2018 142, 665-668.e8DOI: (10.1016/j.jaci.2018.01.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Immunofluorescence staining of TUNEL in IT tissue and NP tissue infected with S aureus (2 × 107 CFU/mL) or S epidermidis (2 × 107 CFU/mL). Journal of Allergy and Clinical Immunology 2018 142, 665-668.e8DOI: (10.1016/j.jaci.2018.01.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions