Merritt L. Fajt, MD, Stacy L

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Prostaglandin D2 pathway upregulation: Relation to asthma severity, control, and TH2 inflammation Merritt L. Fajt, MD, Stacy L. Gelhaus, PhD, Bruce Freeman, PhD, Crystal E. Uvalle, BS, John B. Trudeau, BA, Fernando Holguin, MD, MPH, Sally E. Wenzel, MD Journal of Allergy and Clinical Immunology Volume 131, Issue 6, Pages 1504-1512.e12 (June 2013) DOI: 10.1016/j.jaci.2013.01.035 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 PGD2 levels measured by using ELISA in BAL fluid samples. Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 A, qRT-PCR of HPGDS mRNA transcripts in epithelial cell brushings relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *The difference between HCs and the Mild-Mod/ICS group tended to be different (P = .01) but did not reach significance. B, Correlation between HPGDS and tryptase epithelial cell brushing mRNA levels. Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Epithelial brushing cell cytospin preparations. A, Comparison of the percentage of total HPGDS+ cells, as determined by means of IHC. B, Cytospin preparations (fixed in paraformaldehyde) stained with HPGDS antibody. Red arrows, HPGDS+ cells from an SA. Panels are shown at ×40 magnification. Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 qRT-PCR of DP1 and CRTH2 mRNA transcripts in BAL cells relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *The difference between the Mild-Mod/ICS group versus SAs tended to be different (P = .01) but did not reach significance. Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 BAL cell cytospin preparations. A and C, Comparison of the percentage of total BAL cells positive for DP1 (Fig 5, A) and CRTH2 (Fig 5, C), as determined by means of IHC, is shown. B, Cytospin preparations stained with DP1 antibody. Red arrows, DP1+ cells. Upper left panel, HC; lower left panel, Mild-Mod/ICS subject; right panel, SA. Panels are shown at ×40 magnification. D, Cytospin preparations from an SA double stained with CRTH2 and CD3 antibodies. The upper panel is shown at ×40 magnification. The lower panel is shown at ×100 magnification. Red arrow, CRTH2+/CD3+ lymphocyte; blue arrow, CRTH2+ eosinophil; black arrow, CD3+ lymphocyte. Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Final selection of the study population. In total, 88 (79%) had data from each of the 3 sample types (BAL fluid, BAL cells, and epithelial brushings). Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 PGD2 levels measured by means of ELISA in BAL fluid samples in nonoverlapping subjects (n = 79). Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Correlation between PGD2 levels measured by using ELISA and LCMS. Relative levels for BAL fluid PGD2 (in picograms per milliliter) by using ELISA and LCMS are shown on a log10 scale. In this subset (n = 10) the values were compared by using Spearman correlation and strongly correlated (rs = 0.80, P = .006), confirming the ELISA results. Journal of Allergy and Clinical Immunology 2013 131, 1504-1512.e12DOI: (10.1016/j.jaci.2013.01.035) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions