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Innate IL-13–producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity  Jillian L. Barlow, PhD, Agustin Bellosi,

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Presentation on theme: "Innate IL-13–producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity  Jillian L. Barlow, PhD, Agustin Bellosi,"— Presentation transcript:

1 Innate IL-13–producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity  Jillian L. Barlow, PhD, Agustin Bellosi, PhD, Clare S. Hardman, BSc, Lesley F. Drynan, MSc, See Heng Wong, PhD, James P. Cruickshank, Andrew N.J. McKenzie, PhD  Journal of Allergy and Clinical Immunology  Volume 129, Issue 1, Pages e4 (January 2012) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Nuocytes infiltrate during OVA-induced lung allergy. A-C, Nuocytes in the BAL fluid were gated as side scatter–low (SSClow) lineage− (Fig 1, A) and then further defined as ICOS+T1/ST2variable in a 12-day (Fig 1, B) or 25-day (Fig 1, C) model. D, Number of BAL nuocytes. E, Percentage of lung nuocytes. There were 3 to 4 mice per group. The results are representative of 2 independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 T cells and nuocytes make IL-13 but rarely IL-4 during allergic lung inflammation. Detection of IL-4eGFP and IL-13Tom production in BAL lineage−ICOS+T1/ST2variable nuocytes on days 12 (A) and 25 (B). Detection of cytokine reporter expression in total BAL cells on days 12 (C) and 25 (D). Cytokine reporter expression in T cells and non–T cells (E) and in lineage− cells or nuocytes (F) in BAL fluid from the day 12 model. Cytokine reporter expression in T cells and non–T cells (G) and in lineage− cells or nuocytes (H) in BAL fluid from the day 25 model. There were 3 to 4 mice per group, and the results are representative of 2 similar experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 IL-25 and IL-33 induce lung nuocyte infiltration. A, Side scatter–low (SSClow) lineage− nuocyte infiltration in the BAL fluid after cytokine administration. B and C, Number of BAL nuocytes (Fig 3, B) and number of T1/ST2+ and T1/ST2– nuocytes expressing IL-4eGFP+, IL-13Tom+, and IL-4eGFP/IL-13Tom+ (Fig 3, C) after intranasal administration of IL-25 or IL-33. D and E, Cytokine reporter expression in total BAL cells after IL-25 (Fig 3, D) or IL-33 (Fig 3, E) stimulation. There were 3 to 4 mice per group. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Nuocytes can be purified from the BAL fluid. A, Giemsa-stained cytospin preparations of purified nuocytes isolated directly from the BAL fluid of mice treated intranasally with IL-25 (×100 magnification). B, Nuocyte number in cultures containing IL-7 and IL-33. C, Flow cytometric analysis of in vitro cultured nuocytes after 6 days. Cultured nuocytes are from 30 wild-type mice, and data are representative of more than 3 independent experiments. D, Airway resistance in wild-type (WT) and Il13−/− mice receiving PBS, IL-25, or 80,000 nuocytes per mouse. i.v., Intravenous; ns, not significant. There were 6 mice per group. The experiment is representative of 2 independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Nuocytes restore IL-25–induced AHR in Il13−/− mice. A, Nuocyte (lineage−ICOS+T1/ST2variable) migration in BAL fluid. B and C, Airway resistance (Fig 5, B) and numbers of nuocytes, eosinophils, and neutrophils in the BAL fluid in Il13−/− mice receiving 500,000 wild-type or Il13−/− nuocytes in combination with PBS or IL-25 (Fig 5, C). There were 6 mice per group. *P = .05 and **P = .005 between Il13−/− IL-25 intranasal (i.n.)/wild-type (WT) nuocytes intravenous (i.v.) and Il13−/− IL-25 i.n./Il13−/− nuocytes i.v. groups. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Generation of Il13+/Tom reporter mice. A, Targeting construct. B and C, Verification of homologous recombination (Fig E1, B) and removal of neomycin cassette (Fig E1, C) by means of Southern analysis. D, PCR genotyping of IL-13Tom mice. E and F, Verification of Il4+/eGFPIl13+/Tom dual-reporter mice after OVA lung challenge on days 12 (Fig E1, E) and 25 (Fig E1, F) by using wild-type mice and Il4+/eGFP and Il13+/Tomato single-reporter mice. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 T cells and nuocytes make IL-13 but rarely IL-4 during allergic lung inflammation. A and B, Detection of IL-4eGFP and IL-13Tom production in lung lineage−ICOS+T1/ST2variable nuocytes on days 12 (Fig E2, A) and 25 (Fig E2, B). C and D, Detection of IL-4eGFP+, IL-13Tom+, and IL-4eGFP/IL-13Tom+ cells in total lung cells on days 12 (Fig E2, C) and 25 (Fig E2, D). E and F, Cytokine reporter expression in T cells and non–T cells (Fig E2, E) and in lineage− cells or nuocytes (Fig E2, F) in lung tissue of the day 12 model. G and H, Cytokine reporter expression in T cells and non–T cells (Fig E2, G) and in lineage− cells or nuocytes (Fig E2, H) in lung tissue of the day 25 model. All cell numbers are based on flow cytometric data in Il4+/eGFPIl13+/Tom mice. There were 3 to 4 mice per group, and the experiment was representative of 2 similar experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 Cytokine production by T cells in the mediastinal lymph nodes. A, Number of cells expressing IL-4eGFP+, IL-13Tom+, and IL-4eGFP/IL-13Tom+ in the day 12 and day 25 models. B and C, Cytokine reporter expression in CD4 T cells and non-CD4+ cells from mediastinal lymph node cells in the day 12 (Fig E3, B) and day 25 (Fig E3, C) models. All cell numbers are based on flow cytometric data in Il4+/eGFPIl13+/Tom mice. There were 3 to 4 mice per group. The experiment is representative of 2 independent experiments. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 Wild-type, but not Il13−/−, nuocytes restore IL-25–induced eosinophilia in Il13−/− mice. A and B, Flow cytometric analysis of nuocytes (lineage−ICOS+T1/ST1var; Fig E4, A) and eosinophils (CD11c-SiglecF+Gr1int) and neutrophils (CD11c-SiglecF-Gr1high; Fig E4, B) after wild-type or Il13−/− nuocytes into IL-25 intranasally treated wild-type and Il13−/− mice. C, Differential counts for eosinophils and polymorphonuclear leukocytes on Giemsa-stained cytospin preparations. i.n., Intranasal; i.v., intravenous; WT, wild-type. Journal of Allergy and Clinical Immunology  , e4DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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