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Specificity protein 1 is pivotal in the skin’s antiviral response

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Presentation on theme: "Specificity protein 1 is pivotal in the skin’s antiviral response"— Presentation transcript:

1 Specificity protein 1 is pivotal in the skin’s antiviral response
Lianghua Bin, MD, PhD, Michael D. Howell, PhD, Byung Eui Kim, MD, PhD, Joanne E. Streib, BA, Clifton F. Hall, MS, Donald Y.M. Leung, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 127, Issue 2, Pages e2 (February 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Inhibition of Sp1 enhances viral replication in NHK cells. A, Western blot detection of Sp1 and Sp3 protein expression in NHK cells after transfection with 3 independent siRNA duplexes targeting the Sp1 gene for 48 hours. TATA box binding protein (TBP) is used as a loading control for nuclear protein. B, Relative VV mRNA expression was evaluated by real-time quantitative PCR assay. NHK cells were transfected with 3 different Sp1 siRNA duplexes and scrambled siRNA for 48 hours and then inoculated with VV (MOI, 0.1) for 24 hours. C, Twenty-four–hour viral yield of VV was evaluated by a plaque assay. D, Relative HSV-1 mRNA expression was evaluated by real-time quantitative PCR assay. NHK cells were transfected with 3 different Sp1 siRNA duplexes and scrambled siRNA for 48 hours and then inoculated with HSV-1 (MOI, 0.05) for an additional 24 hours. All data are presented as mean ± SEM; ∗P <.05 and ∗∗P <.01 compared with scrambled siRNA. The data shown here are representative of 3 or 4 experiments. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Sp1 gene expression is decreased in skin biopsies from ADEH+ patients. A, Relative Sp1 mRNA expression in skin biopsies from nonatopic subjects (n = 31), ADEH− subjects (n = 55), and ADEH+ subjects (n = 20) evaluated by real-time quantitative PCR. B, Representative fluorescence images of Sp1 in skin biopsies from nonatopic, ADEH−, and ADEH+ subjects. Sp1 (red) presents as nuclear staining, and wheat germ agglutinin–conjugated fluorescein isothiocyanate (green) stains the cytoskeleton. Magnification ×630. C, MFI of Sp1 in skin biopsies from nonatopic, ADEH−;, and ADEH+ subjects. 18S, 18s ribosomal RNA. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Sp1 expression level is inversely correlated with VV replication. A, Upper panel, Western blot detection of Sp1 expression in NHK cells after transfection with different concentrations of Sp1 siRNA duplexes for 48 hours. The concentration of Sp1 siRNA duplexes is indicated above each lane. Lower panel, Relative VV mRNA expression was evaluated with different levels of Sp1 silencing in NHK cells with VV inoculation (MOI, 0.01) for 24 hours (corresponding to upper panel) by real-time quantitative PCR. All data are presented as mean ± SEM; ∗P <.05 and ∗∗P <.01 compared with scrambled siRNA control. B, Sp1 mRNA expression was inversely correlated with VV viral copies in skin explants (subject group adjusted; partial correlation r = –0.256; P = .009). 18S, 18s ribosomal RNA; TBP, TATA box binding protein. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 PKR/eIF2α pathway is impaired in Sp1-deficient NHK cells and skin of ADEH+ patients. A, Relative PKR mRNA expression in Sp1-silenced NHK cells evaluated by real-time quantitative PCR. Data are presented as mean ± SEM, representative 1 of 3 independent experiments. B, Western blot detection of phosphorylation of eIF2α (p-eIF2α), total eIF2α, Sp1, and β-actin in Sp1-silenced NHK cells and cells transfected with scrambled siRNA duplexes for 48 hours and then treated with VV (MOI, 0.1) and harvested at various time points. Time after treatment is indicated above each pair of lanes. C, VV mRNA expression in Sp1 and PKR–silenced NHK cells compared with cells transfected with scrambled siRNA as evaluated by real-time PCR. D, Relative PKR mRNA expression in skin explants from ADEH+ subjects (n = 20), ADEH− subjects (n = 55), and nonatopic subjects (n = 31). Data are presented as mean ± SEM. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 OAS2 is decreased in Sp1-silenced keratinocytes and ADEH+ patients. A-C, Real-time PCR results show that OAS2 is significantly downregulated, but OAS1 and OAS2 did not change significantly in Sp1-silenced NHK. Data are presented as mean ± SEM. D, OAS2 gene expression is significantly downregulated in skin biopsies from ADEH+ subjects compared with ADEH− and nonatopic subjects. ns, No significance. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 OAS2 reduction is part of the mechanism resulting in enhanced VV replication in Sp1-silenced keratinocytes. A, OAS2 gene expression in Sp1 and OAS2–silenced NHK cells compared with cells transfected with scrambled siRNA as evaluated by real-time PCR. B, VV mRNA expression in Sp1 and OAS2–silenced NHK cells compared with cells transfected with scrambled siRNA as evaluated by real-time PCR. All data are presented as mean ± SEM; ∗P < .05; ∗∗P < .01; ∗∗∗P < The data shown are representative of 3 experiments. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 IFN-γ treatment significantly augments antiviral responses in Sp1-deficient keratinocytes. A, OAS2 gene expression in Sp1-silenced NHK and control cells transfected with scrambled siRNA in the presence of different doses of IFN-γ as evaluated by real-time PCR. B, Protein phosphorylation of eIF2α in Sp1-silenced NHK and control cells transfected with scrambled siRNA in the absence and presense of IFN-γ (5 ng/mL) as evaluated by Western blot. C, VV mRNA expression in Sp1-silenced NHK and control cells transfected with scrambled siRNA in the presence of different doses of IFN-γ as evaluated by real-time PCR. All data are presented as mean ± SEM; ∗ P < .05; ∗∗P < .01. The data shown here are representative of 3 experiments. 18S, 18s ribosomal RNA. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Heatmap presentation of genes in GO group of that have greater than 1.5-fold change between Sp1 siRNA and scrambled siRNA. Journal of Allergy and Clinical Immunology  , e2DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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