1. Serum amyloid P attenuates M2 macrophage activation and protects against fungal spore–induced allergic airway disease 
Ana Paula Moreira, PhD, Karen A. Cavassani, PhD, Rikki Hullinger, Rogério S. Rosada, PhD, Daniel J. Fong, BS, Lynne Murray, PhD, Dave P. Hesson, PhD, Cory M. Hogaboam, PhD 
Journal of Allergy and Clinical Immunology 
Volume 126, Issue 4, Pages e7 (October 2010)
DOI: /j.jaci
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2. Fig 1
Short pentraxins inhibited M2 differentiation both in vitro and in vivo. A, Binding of FITC-labeled SAP or CRP on CD11b cells. B, Ym-1 and arginase transcript expression in BMDMs under M2 conditioning medium and SAP, murine SAP, or CRP. C, Transcript expression in interstitial lung macrophages cultured with M2 conditioning medium and SAP. D, A schematic representation of the A fumigatus allergic model. E, FIZZ1 transcripts in alveolar macrophages from mice with A fumigatus allergy treated with PBS, CRP, or SAP from days 15 to 30. F, FIZZ1+ cells (stained brown) evaluated by using immunohistochemistry (×200 and ×400 magnification). G, Morphometric analysis of FIZZ1+ cells. Data are presented as means ± SEMs of 2 independent experiments (n = 4-5 mice per group; Fig 1, E-G). ∗P <.05, †P < .01, and ‡P < .001 compared with WT M2 conditioning medium alone. ∗P < .05 compared with PBS in panel G.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3. Fig 2
SAP inhibited STAT6 phosphorylation in M2 macrophages. A, Immunoblotting for p-STAT6 (Tyr641) or total STAT6 in CD11b+ cells cultured with M2 conditioning medium with or without SAP and CRP (20 μg/mL). B, BMDMs from allergic mice were cultured with M2 conditioning medium with or without SAP and CRP. CD11bhigh cells were analyzed for the activation of STAT6. The percentage of cells is indicated in each histogram, and the percentage of STAT6-activated CD11b+ cells is represented in C. Data are presented as means ± SEMs of 2 independent experiments (n = 4 mice per group). ∗P < .05 compared with medium alone.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4. Fig 3
Exogenous SAP therapy prevented or reversed established airway resistance in a fungal allergic airway disease model. A and B, Airway resistance at days 15 (Fig 3, A) or 30 (Fig 3, B) after conidia challenge in mice with A fumigatus allergy treated with PBS, CRP, or SAP. C, Cytokine levels in whole-lung samples from allergic mice at day 30 after conidia challenge. Data are presented as means ± SEMs of 1 to 3 independent experiments (n = 5 mice per group). †P < .05 compared with baseline airway resistance. ∗∗P < .001 compared with PBS-treated mice after methacholine. ∗P < .05 compared with PBS-treated mice. MIP, Macrophage inflammatory protein.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5. Fig 4
SAP reduced histologic evidence of airway inflammation and remodeling. A, Lung sections of allergic mice stained with hematoxylin and eosin (HE), Masson trichrome, or periodic acid–Schiff (PAS; ×400 and ×200 magnification). B, Morphometric analysis of the percentage of fibrosis in the airway. C, Transcript expression in whole lung. D, Fibrinogen levels in lungs of allergic mice at 30 days after conidia challenge. E, Lung cell differentials in allergic mice. F, Number of CD11b+CD11c+ cells with activated STAT6 in lungs of allergic mice. Data are presented as means ± SEMs of 1 to 3 independent experiments (n = 5 mice per group). ∗P < .05 compared with the PBS (control)–treated group.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6. Fig 5
SAP treatment of adoptively transferred M2 macrophages restored the antiallergic properties of these cells during chronic fungal allergic disease. A, Lung tissues stained with hematoxylin and eosin (HE), Masson trichrome, or periodic acid–Schiff (PAS; ×400 and ×200 magnification) or immunofluorescence for p-STAT6 antibody (red) in allergic mice transferred with M2-differentiated macrophages activated with or without SAP. Nuclei were stained with 4′-6-diamidino-2-phenylindole dihydrochloride. B, Morphometric analysis of the percentage of fibrosis in the airway. C and D, Airway resistance (Fig 5, C) and BIOPLEX results (Fig 5, D) for p-STAT6 in lungs of transferred allergic mice. E, Fold increase in IL-10 and IL-13 levels in whole-lung samples from transferred allergic mice. Data are presented as means ± SEMs of 2 experiments (n = 10 mice per group). ∗P < .05 compared with M2 macrophage-transferred mice.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
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7. SAP decreased lung FIZZ1 levels
SAP decreased lung FIZZ1 levels. FIZZ1 levels in bronchoalveolar lavage fluid from allergic PBS-, CRP-, or SAP-treated mice are shown. Data are presented as means ± SEMs (n = 5 mice per group). ∗P < .05 compared with CRP-treated mice.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8. The protective effect of SAP was not dependent on the endogenous production of murine SAP or CRP in the lungs. Murine CRP (A) and murine SAP (B) levels in whole-lung samples from allergic PBS-, CRP-, or SAP- treated mice are shown. Data are presented as means ± SEMs (n = 5 mice per group). ∗P < .05 compared with naive mice and †P < .05 compared with PBS (control)–treated mice.
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
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9. A fumigatus conidia in the lungs, serum nitrite, and whole-lung inducible nitric oxide synthase levels in PBS-, SAP-, and CRP-treated allergic mice. A and B, Gomori methenamine silver (GMS) staining (Fig E3, A) and morphometric analysis (Fig E3, B) of lung sections at 30 days after conidia challenge are shown. Original magnification was ×100 (n = 5). Photomicrographs are representative of all sections (n = 5 per treatment group). C, Nitrite/nitrate levels measured in serum with a Griess reaction. D, Transcript expression of inducible nitric oxide synthase in whole-lung samples from PBS-, CRP-, or SAP- treated mice. Data are means ± SEMs (n = 5 mice per group).
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10. Inflammatory effect and lung injury observed after treatment of allergic mice with a higher dose of CRP. Lung sections were stained with hematoxylin and eosin. Photomicrographs are representative of all sections. Original magnification was ×100 and ×400 (n = 5 per treatment group).
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
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11. Activation of STAT6 in different populations of myeloid cells in the lungs of allergic mice. Dot plots gated in viable cells (forward scatter [FSC] × side scatter [SSC]) obtained from lungs of PBS-, CRP-, and SAP-treated mice are shown. CD11b+CD11c+, CD11b+CD11c−, and CD11b−CD11c+ gated cells were analyzed for intracellular activation of STAT6 (phosphorylation at Tyr641; n = 5 mice per group).
Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci )
Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions