Cigarette smoke extract induces thymic stromal lymphopoietin expression, leading to TH2-type immune responses and airway inflammation Yuki Nakamura,

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Cigarette smoke extract induces thymic stromal lymphopoietin expression, leading to TH2-type immune responses and airway inflammation Yuki Nakamura, Masanori Miyata, MD, Tetsuro Ohba, MD, Takashi Ando, MD, PhD, Kyosuke Hatsushika, MD, PhD, Fumiko Suenaga, Naomi Shimokawa, PhD, Yuko Ohnuma, Ryohei Katoh, MD, PhD, Hideoki Ogawa, MD, PhD, Atsuhito Nakao, MD, PhD Journal of Allergy and Clinical Immunology Volume 122, Issue 6, Pages 1208-1214 (December 2008) DOI: 10.1016/j.jaci.2008.09.022 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 CSE exposure induces TSLP expression in the mouse lung. A-C, BALB/c mice were intranasally challenged with CSE or PBS every day for a total of 7 days (days 0-6). The mouse lungs were removed on day 7 (3 hours after the final challenge), and real-time PCR (Fig 1, A), Western blot analysis (n = 3 in each group; Fig 1, B), and immunohistochemical staining (Fig 1, C) for TSLP were performed. Quantitative analysis of the TSLP immunoreactivity is shown in Fig 1, D. Representative results of 3 independent experiments are shown. ∗P < .05 in comparison with the corresponding control. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology 2008 122, 1208-1214DOI: (10.1016/j.jaci.2008.09.022) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 CSE-induced TSLP expression in the mouse lung depends on oxidative stress and TNF-α receptor I (TNFRI). A-C, BALB/c mice were intranasally challenged with CSE or PBS every day for a total of 7 days (days 0-6) with or without treatment with 150 mg/kg NAC. The mouse lungs were removed on day 7 (3 hours after the final challenge), and real-time PCR for TSLP (Fig 2, A), TNF-α (Fig 2, B), heme oxygenase 1 (HO-1; Fig 2, C), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed. D and E, BALB/c mice (Fig 2, D) or TNFRI-null mice and control C57BL/6 mice (Fig 2, E) were intratracheally challenged with CSE, and the mouse lungs were removed at the indicated times (Fig 2, D) or 3 hours after the challenge (Fig 2, E), and real-time RT-PCR was performed for TSLP and GAPDH. Representative results of 3 independent experiments are shown. ∗P < .05 in comparison with the corresponding control. Journal of Allergy and Clinical Immunology 2008 122, 1208-1214DOI: (10.1016/j.jaci.2008.09.022) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Intranasal exposure of CSE simultaneously with OVA induces airway inflammation in mice. BALB/c mice were intranasally challenged with CSE, OVA, or both (or PBS) 5 times per week for 8 weeks with or without treatment with 15 mg/kg per mouse anti-TSLP antibody or control antibody every week. The mouse lungs were removed at 2 (A), 4 (B), and 8 (C) weeks during the CSE challenge, the OVA challenge, or both, and then the tissue sections were stained with hematoxylin and eosin. Representative results of 3 independent experiments are shown. Journal of Allergy and Clinical Immunology 2008 122, 1208-1214DOI: (10.1016/j.jaci.2008.09.022) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Intranasal exposure of CSE simultaneously with OVA induces OVA-specific TH2-type immune responses in mice. BALB/c mice were intranasally challenged with CSE, OVA, or both (or PBS) 5 times per week for 8 weeks with or without treatment with 15 mg/kg per mouse anti-TSLP antibody or control antibody every week. The mice were killed at 3 hours after the final CSE challenge, OVA challenge, or both for further analysis. A-C, RNA was extracted from the mouse lungs, and real-time RT-PCR was performed for mEAR2 (Fig 4, A), GATA-3 (Fig 4, B), IL-13 (Fig 4, C), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). D, The mouse sera were collected, and the concentrations of OVA-specific IgE antibody were measured by means of ELISA. E and F, Spleen cells were collected and stimulated with 100 μg/mL OVA for 72 hours in vitro. The concentrations of IL-4 (Fig 4, E) and IL-13 (Fig 4, F) in the culture supernatants were measured by means of ELISA. Representative results of 3 independent experiments are shown. ∗P < .05 in comparison with the corresponding control. Journal of Allergy and Clinical Immunology 2008 122, 1208-1214DOI: (10.1016/j.jaci.2008.09.022) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions