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Chronic rhinosinusitis with nasal polyps is characterized by B-cell inflammation and EBV-induced protein 2 expression  Kathryn E. Hulse, PhD, James E.

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Presentation on theme: "Chronic rhinosinusitis with nasal polyps is characterized by B-cell inflammation and EBV-induced protein 2 expression  Kathryn E. Hulse, PhD, James E."— Presentation transcript:

1 Chronic rhinosinusitis with nasal polyps is characterized by B-cell inflammation and EBV-induced protein 2 expression  Kathryn E. Hulse, PhD, James E. Norton, MS, Lydia Suh, BS, Qiu Zhong, MD, Mahboobeh Mahdavinia, MD, PhD, Patrick Simon, MD, Robert C. Kern, MD, David B. Conley, MD, Rakesh K. Chandra, MD, Bruce K. Tan, MD, Anju T. Peters, MD, Leslie C. Grammer, MD, Kathleen E. Harris, BS, Roderick G. Carter, BS, Atsushi Kato, PhD, Robert P. Schleimer, PhD  Journal of Allergy and Clinical Immunology  Volume 131, Issue 4, Pages e7 (April 2013) DOI: /j.jaci Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 NPs contain increased numbers of inflammatory cells. Cells were isolated from sinus tissue biopsy specimens after 4 days and analyzed by using flow cytometry. A, Cell viability was assessed by using DAPI exclusion. B, Total cell yield was determined by dividing the number of cells recovered after 4 days by the original weight of the biopsy specimen. C, CD45+ cell numbers were calculated based on the total number of cells recovered and the frequency of CD45+ cells within the DAPI− gate and divided by the original weight of the biopsy specimen. Data represent means ± SEMs. *P < .05 and **P < .01, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 B cells and plasma cells were more abundant in NPs. Cells were isolated from sinus tissue biopsy specimens after 4 days and analyzed by means of flow cytometry. A, Representative flow cytometric plots. APC, Allophycocyanin; Pe-Cy7, phycoerythrin-cyanin 7. B, Numbers of B cells (CD19+) and plasma cells (PC; CD138+) were calculated as above from within the DAPI−CD45+ population. C, Mean side scatter A (SSC-A) fluorescence intensity of CD19+ B cells. D, Representative ×20 immunofluorescence image of CD19+ (red) and CD138+ (green) cells in NP tissue. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Antibody levels were increased in NPs. A, Antibody isotype levels in tissue extracts were normalized to total protein. B, Antibody isotype levels measured in nasal lavage fluid. C, Gene expression of pIgR in tissue extracts. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Differential expression of IgA1 and IgA2 in NPs. Total IgA1 and IgA2 levels in tissue extracts (A) and nasal lavage fluid (B) are shown. sIgA1 and sIgA2 levels in tissue extracts (C) and nasal lavage fluid (D) are shown. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 EBI2 was highly expressed in NPs. A, Gene expression analysis of EBI2 from sinus tissue biopsy specimens was normalized to β-glucuronidase. B, Representative Western blot showing EBI2 and β-actin. C, Average of EBI2 expression normalized to β-actin in sinus tissue. D, Correlations between normalized EBI2 gene expression and CH25H, CD138, and B lymphocyte–induced maturation protein (BLIMP-1; n = 60 in each plot). Data in Fig 5, A and C, represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 Representative gating strategy used for analysis of flow cytometric data. Debris was gated out based on forward scatter A (FSC-A) versus side scatter A (SSC-A), DAPI+ dead cells were excluded, and we analyzed B lineage subsets within the CD45+ gate. All gates are based on fluorescence minus one controls. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 Sinus tissue antibody levels. A, Tissue levels of IgG2, IgG3, and IgM. B, Nasal lavage fluid levels of IgG2, IgG3, and IgM. Data represent means ± SEMs. *P < .05, **P < .01, and ***P < .001, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 Serum antibody levels. Data represent means ± SEMs. *P < .05 and ***P < .001, Kruskal-Wallis test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 Antibody levels in NPs and sera normalized to albumin. Similar results were found for all UT compared with sera (data not shown). Data represent means ± SEMs. ***P < .0001, Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E5 EBI2 expression correlates with markers of plasma cells. Gene expression was assessed by using microarray analysis (see the Methods section in this article's Online Repository for details) and confirms gene expression data from an independent sample set assessed by using quantitative RT-PCR (n = 24 in each plot). For further information, see Fig 5. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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