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Umasundari Sivaprasad, PhD, David J. Askew, PhD, Mark B

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1 A nonredundant role for mouse Serpinb3a in the induction of mucus production in asthma 
Umasundari Sivaprasad, PhD, David J. Askew, PhD, Mark B. Ericksen, BS, Aaron M. Gibson, BS, Matthew T. Stier, Eric B. Brandt, PhD, Stacey A. Bass, Michael O. Daines, MD, Jamila Chakir, PhD, Keith F. Stringer, MD, Susan E. Wert, PhD, Jeffrey A. Whitsett, MD, Timothy D. Le Cras, PhD, Marsha Wills-Karp, PhD, Gary A. Silverman, MD, PhD, Gurjit K. Khurana Hershey, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 127, Issue 1, Pages e6 (January 2011) DOI: /j.jaci Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Generation and characterization of Serpinb3a-null mice. A, Schematic of the mouse and human SCCA locus (gray arrows indicate pseudogenes). B, Schematic of the targeting vector (top), WT (middle), and recombinant construct (bottom). B, BamH1; E, EcoR1 restriction sites. The probe used for Southern blotting is indicated in red. C, Genotyping by Southern blotting of BamH1-digested genomic DNA. The 2-kbp recombinant (red arrow) and 4.5-kbp WT alleles (black arrow) are indicated. The 6.5-kbp band is from WT Serpinb3b (green arrow). D, Expression of Serpinb3a (top), Serpinb3b (middle), and β-actin (bottom) in WT and Serpinb3a null (KO) mice. E, Percentage of B and T cells in the spleen. F, Percentage of B and T cells in the lymph node. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Serpinb3a-null mice show attenuated APTI after HDM challenge with no effect on sensitization APTI measurements (A), HDM-specific IgG1 levels (B), HDM-specific IgE levels in plasma (C), percentage of B cells (D), and percentage of CD3+ T cells (E) in the lymph node (LN). Experiments include 6 to 8 mice per group. P values are shown only for statistically significant differences. The horizontal bar for each group represents the mean. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Serpinb3a-null mice show no effect on inflammation after HDM challenge. A, Differential cell counts in BALF. B, H&E staining of airways, Experiments include 6 to 8 mice per group. P values are shown for statistically significant differences. Data shown are the means ± SDs. In B, the pictures were taken at ×200 magnification, and the inset panel was taken at ×1000 magnification. Eos, Eosinophils; KO, knock out; Lymph, lymphocytes; Macros, macrophages; Neut, neutrophils. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Decreased mucus production and goblet cell hyperplasia in Serpinb3a-null (KO) mice after HDM challenge. A, Representative images of Muc5AC staining (×200 magnification). B, Quantitation of Muc5AC staining. C, Representative images of CLCA3 staining. D, Quantitation of CLCA3 staining. B and D, Black bar, WT+HDM; white bar, KO+HDM. B, n = 10 for WT+HDM and n = 12 for KO+HDM. D, n = 9 for WT+HDM and n = 10 for KO+HDM. Frequency distribution across the groups was significantly different between the WT and KO mice for both Muc5AC (P = .005) and CLCA3 (P = .003). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Decreased SPDEF expression in Serpinb3a-null mice after HDM challenge. A, SPDEF staining in WT and Serpinb3a-null (KO) mice after PBS or HDM challenge. A representative image at ×200 magnification from each treatment is shown. B, Quantitation of SPDEF staining. Black bar, WT+HDM; white bar, KO+HDM. n = 13 for WT+HDM and KO+HDM. Frequency distribution across the groups was significantly different between the WT and KO mice (P < .001). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 The asthma phenotype is attenuated in the Serpinb3a-null mice (KO) after IL-13 treatment. A, APTI measurements (n = per group). B, Differential BALF cell counts (n = per group). P values are shown only for statistically significant differences. C, Representative images of Muc5AC staining (×200 magnification). Eos, Eosinophils; KO, knock out; Lymph, lymphocytes; Macros, macrophages; Neut, neutrophils; SAL, saline. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 SERPINB4 expression is induced by IL-13. A, Quantitative PCR for SERPINB4 in primary bronchial epithelial cells from controls and patients with asthma (6 subjects per group) treated with IL-13 for 24 hours. Data are presented as a fold increase in SERPINB4 expression in IL-13–treated cells compared with paired untreated controls. B, SERPINB4 expression after treatment with IL-4, IL-13, TNF-α, or IFN-γ. Data shown are representative of 3 independent experiments (each done in triplicate). The horizontal bar for each group represents the mean in A. B, Data shown are the means ± SDs. GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 There are no differences in levels of total IgG1 (A) and IgE (B) in the plasma of Serpinb3a-null (KO) mice compared with WT mice after allergen challenge. Statistically significant differences are indicated with the P value. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Decreased FOXA3 expression in Serpinb3a-null (KO) mice after HDM challenge. A, FOXA3 staining in WT and Serpinb3a-null mice after PBS or HDM challenge. A representative image at ×200 magnification from each treatment is shown. B, Quantitation of FoxA3 staining. Black bar, WT+HDM; white bar, KO+HDM. P values are shown only for statistically significant differences. n = 13 for WT+HDM and KO+HDM. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Representative airways for each grade used to quantify immunohistochemistry. A, Representative grade 0, 1, 2, and 3 airways for Muc5AC staining after HDM treatment. B, Representative grade 0, 1, 2, and 3 airways for CLCA3 staining after HDM treatment. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Representative airways for each grade used to quantify immunohistochemistry. A, Representative grade 0, 1, 2, and 3 airways for SPDEF staining after HDM treatment. B, Representative grade 0, 1, 2, and 3 airways for FOXA3 staining after HDM treatment. C, Representative grade 0, 1, 2, and 3 airways for Muc5AC staining after IL-13 treatment. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Quantitative PCR shows a modest but insignificant decrease in SPDEF mRNA in the lungs of SerpinB3a-null (KO) mice after HDM challenge. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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