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Role of IL-17A and neutrophils in fibrosis in experimental hypersensitivity pneumonitis  Simon A. Hasan, BSc, Bertus Eksteen, MBChB, FRCP (UK), PhD, Danielle.

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Presentation on theme: "Role of IL-17A and neutrophils in fibrosis in experimental hypersensitivity pneumonitis  Simon A. Hasan, BSc, Bertus Eksteen, MBChB, FRCP (UK), PhD, Danielle."— Presentation transcript:

1 Role of IL-17A and neutrophils in fibrosis in experimental hypersensitivity pneumonitis 
Simon A. Hasan, BSc, Bertus Eksteen, MBChB, FRCP (UK), PhD, Danielle Reid, MSc, Heather V. Paine, BSc, Abrar Alansary, BSc, Kerri Johannson, MD, FRCPC, Carol Gwozd, MLT, Kimberly-Ann R. Goring, MSc, Tina Vo, BSc, David Proud, PhD, FRCS, Margaret M. Kelly, MBChB, FRCPC, PhD  Journal of Allergy and Clinical Immunology  Volume 131, Issue 6, Pages e5 (June 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Lung histology after exposure to S rectivirgula antigen. A, Bronchial associated lymphoid tissue (magnification, ×100), lower insert magnification ×400, upper insert goblet cell metaplasia (magnification, ×100; alcian blue/PAS). B, Band of neutrophils (short arrows) and multinucleated giant cells (long arrows) in subpleural region (magnification, ×400; H&E). C, Subpleural fibrosis with infiltrating neutrophils (arrows; H&E). D, CD3+ lymphocytes (arrowheads; diaminobenzidine, brown stain). Area stained with picrosirius red (Fig 1, C) and viewed under brightfield (E) or polarized light (F) to show collagen deposition. G, Myofibroblasts, (α-smooth muscle actin stain, brown) in fibrotic areas close to neutrophils (arrows). H, Tissue neutrophils (arrows) still present after 6 weeks of S rectivirgula antigen (H&E). Exposure was 2 weeks (Fig 1, A and B), 3 weeks (Fig 1, D-G), and 6 weeks (Fig 1, H). All magnification was ×200, unless stated otherwise. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Exposure to S rectivirgula antigen for 1 to 3, 5, or 6 weeks. BAL fluid and lung tissue were collected 4 days after the last exposure in each group. A, Total inflammatory cells. B, Soluble collagen measured from homogenized right lung (μg/RL). C, KC, IL-17A, and active TGF-β1 (D; pg/mL). *P < .05 compared with weeks 3, 5, and 6. #P < 0.05 compared with weeks 2, 3, 5, and 6. **P < .005 compared with all time points (n = 6 to 8 mice per group). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Effect of inhibiting active TGF-β on inflammation and fibrosis in EHP. C57BL/6 mice were exposed to 3 weeks of intratracheal PBS or S rectivirgula antigen (SR Ag) and intraperitoneal (IP) anti–TGF-β antibody (Ab) or isotype control. BAL fluid and lung tissue were collected 4 days after last treatment in each group and total inflammatory cells (A), BAL fluid IL-17A (B; pg/mL), and soluble collagen (C; μg/right lung [RL]) were quantified. *P < .05 and **P < .002. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Effect of IL-17A and neutrophil depletion on total BAL fluid inflammatory cells and fibrosis after chronic exposure to S rectivirgula antigen (SR Ag). Anti–IL-17A (A and B) or Gr-1 (C and D) neutralizing antibody (Ab) were administered to C57BL/6 mice during the last 2 weeks of 3 weeks of exposure to S rectivirgula antigen. A and C, Total BAL fluid inflammatory cells (×103) and (B and D) soluble collagen (μg/right lung [RL]) were measured 4 days after the last exposure to S rectivirgula antigen. Inflammation and fibrosis were compared with saline and appropriate isotype control. *P < .05 compared with saline control (n = 6 to 8 mice per group). IP, Intraperitoneal. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Loss of IL-17A signaling on structural and/or bone marrow–derived cells during chronic exposure to S rectivirgula antigen. Wild-type (C57BL/6) mice, IL-17RA–deficient (Il17ra−/−) and their bone marrow chimeric mice were exposed to S rectivirgula antigen for 3 weeks. Four days after the last SR exposure, total BAL fluid inflammatory cells (A), BAL fluid KC (B; pg/mL) and soluble collagen (C; μg/right lung [RL]) were measured. *P < .05 compared with Il17ra−/− and strIl17ra−/−, **P < .005 compared with all other groups. #P < .05 compared with Il17ra−/−. Absence of IL17RA on structural cells (strIl17ra−/−) or on bone marrow–derived cells (bmIl17ra−/−); n = 5 to 8 mice per group. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Neutrophils and monocytes/macrophages are the main IL-17A–secreting cells in EHP. Inflammatory cells were isolated from lung homogenates of mice exposed to 3 weeks of S rectivirgula antigen. IL-17A secreting and nonsecreting cell populations were then isolated. A, FACS-assisted characterization of inflammatory cell populations before and after IL-17A enrichment. ***P < .001. B, Diff-quik and (C) H&E stained cytospins prepared from IL-17A–positive cell fraction show neutrophils (white arrows), macrophages (black arrows) as the main cell types. Representative of 3 independent experiments (n = 2 per experiment). Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Neutrophils and monocytes/macrophages are the main cells that express IL-17A mRNA and protein in EHP in wild-type (WT) and RAG-2–deficient (RAG-2−/−) mice. C57BL/6 or RAG-2–deficient mice were exposed to S rectivirgula antigen for 3 weeks. Leucocytes were isolated and purified, treated with brefeldin A and ionomycin, and stained to assess IL-17A expression by Gr-1–expressing cells A, Representative flow cytometric histograms gated on Gr-1 expression (white peaks, isotype control; gray peaks, IL-17A staining). B, Graphical summary representation of data. C, Gr-1 high and intermediate cells underwent FACS to >99% purity. Representative before and after sort histograms. D, Quantitative reverse-transcription PCR on cell populations that underwent FACS: lymphocyte (CD3), monocyte (Gr-1int), and neutrophil (Gr-1hi). *P < .05 compared with C57BL/6 Gr-1int. **P < .01 compared with C57BL/6 Gr-1int. ***P < .001 compared with C57BL/6 CD3. #P < .001 compared with C57BL/6 Gr-1int. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E1 Protocol for administration of Saccharopolyspora rectivirgula antigen (SR Ag). S rectivirgula antigen is administered by intratracheal aerosol on 3 consecutive days each week (black arrows) for up to 6 weeks. The mice were sacrificed 4 days after the last aerosol (white arrows), and BAL fluid, blood, and lung tissue were collected. Because inflammation and fibrosis were established after 3 weeks of exposure to S rectivirgula antigen, most outcomes were assessed at this time point. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E2 TGF-β activity in the lung in EHP. A, Immunohistochemistry for β6 integrin, a marker of αvβ6 integrin, which is an activator of TGF-β. Strongly positive epithelial cells (arrowheads) are seen in areas of subpleural fibrosis. B, Active TGF-β (pg/mL) in BAL fluid from mice exposed to 3 weeks of intratracheal PBS only or Saccharopolyspora rectivirgula antigen (SR Ag) with intraperitoneal (IP) anti-TGF-β antibody (Ab) or isotype control; n = 5 to 8 mice per group. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E3 Effect of Gr-1 antibody (Ab) on blood counts after 3 weeks of chronic exposure to Saccharopolyspora rectivirgula antigen (SR Ag) compared with PBS. Gr-1 Ab or isotype control was administered intraperitoneally (IP) twice weekly to wild-type mice exposed to SR Ag or PBS during the last 2 weeks of 3 weeks of pulmonary exposure. A, Blood neutrophils and (B) mononuclear cells (×106/mL) were quantified 4 days after the last exposure to assess efficacy of Gr-1 Ab on systemic neutrophil depletion; n = 6 to 8 mice per group. NS, Not signficant. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E4 Identification of cell populations that produce IL-17A in EHP with the use of flow cytometry. Inflammatory cells were isolated from whole lung homogenates of wild-type mice after chronic Saccharopolyspora rectivirgula antigen (SR Ag) exposure. Flow cytometry was performed before or after IL-17A capture, using markers for T lymphocytes (CD4/CD3; A), B lymphocytes (CD19/CD3; B), and myeloid-derived cells (C and D). Gr-1high, neutrophils; Gr-1int, CD11c, and MHC II, monocytes/antigen-presenting cells. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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