Presentation is loading. Please wait.

Presentation is loading. Please wait.

Polyinosinic:polycytidylic acid induces protein kinase D–dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells 

Published bySilvester Andrews Modified over 5 years ago

Similar presentations

Presentation on theme: "Polyinosinic:polycytidylic acid induces protein kinase D–dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells "— Presentation transcript:

1 Polyinosinic:polycytidylic acid induces protein kinase D–dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells  Fariba Rezaee, MD, Nida Meednu, PhD, Jason A. Emo, MS, Bahman Saatian, MD, Timothy J. Chapman, PhD, Nayden G. Naydenov, PhD, Anna De Benedetto, MD, Lisa A. Beck, MD, Andrei I. Ivanov, PhD, Steve N. Georas, MD  Journal of Allergy and Clinical Immunology  Volume 128, Issue 6, Pages e11 (December 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 PolyI:C decreases the TEER of our model’s airway epithelial cell monolayers. 16HBE14o- cells were grown on Transwell inserts and stimulated with indicated concentrations of Pam3Cys (A), polyI:C (B), LPS (C), flagellin (D), and CpG oligonucleotides (E) for different time periods, followed by TEER measurements. Data are expressed as a percentage of control unstimulated cells and are means ± SEMs of 3 independent experiments per time point. ***P < .001, as determined by using ANOVA, followed by the Student t test with the Bonferonni correction for multiple comparisons. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 PolyI:C induces a dose-dependent increase in paracellular permeability. A, TEER was measured in 16HBE14o- cell monolayers stimulated for different times with different concentrations of polyI:C. B and C, Transepithelial flux of sodium fluorescein (Fig 2, B) or 3-kd fluorescein–conjugated dextran (Fig 2, C) was measured after 24 hours of incubation with polyI:C. Data are means ± SEMs of at least 6 independent experiments per group. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, as determined by using the Student t test. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 PolyI:C induces disassembly of AJs and TJs. Confluent 16HBE14o- cell monolayers were treated for 6 hours with either medium control or polyI:C (5 μg/mL). Localization of AJ proteins (E-cadherin and β-catenin) and TJ proteins (occludin and ZO-1) was determined by means of immunofluorescence labeling and confocal microscopy. Note the localization of junctional proteins at cell-cell contacts in control cells (arrows) and their translocation into the cytosolic compartment after polyI:C exposure (arrowheads). Images are a representative of at least 6 independent experiments. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Junctional disassembly in polyI:C-exposed epithelial cells is accompanied by remodeling of the apical actin cytoskeleton. Confluent 16HBE14o- cells were exposed for 24 hours to polyI:C (5 μg/mL), followed by fixation and dual-fluorescence labeling for F-actin and ZO-1. PolyI:C induced transformation of the perijunctional F-actin belt (arrows) into a disordered array of apical F-actin bundles (arrowheads). Images are a representative of at least 3 independent experiments. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Effect of PKC isoform inhibitors on polyI:C (pI:C)–induced junctional disassembly. 16HBE14o- cell monolayers were stimulated with polyI:C (5 μg/mL for 24 hours) with or without Gö6976 or Gö6983 (10 μmol/L). Localization of AJ and TJ proteins and architecture of the apical actin cytoskeleton were determined by means of fluorescence labeling and confocal microscopy. Note the normal localization of junctional proteins (arrows) and intact perijunctional F-actin belt (arrowhead) in polyI:C–exposed cells treated with the PKCμ (PKD) inhibitor Gö6976. Images are a representative of at least 3 independent experiments. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Pharmacologic inhibition of PKD attenuates polyI:C-induced epithelial permeability. Control and polyI:C-activated 16HBE14o- cell monolayers (5 μg/mL for 24 hours) were incubated with or without the inhibitor of classical PKC isoforms Gö6976 (10 μmol/L), and permeability was examined by measuring transmonolayer sodium fluorescein flux. Results are expressed as the fold change above control values (increasing values indicating greater permeability) and are presented as means ± SEMs of 3 independent experiments. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 PolyI:C-induced activation of PKD in epithelial cells. Confluent 16HBE14o- cell monolayers were treated for 0 to 120 minutes with either medium control or polyI:C (5 μg/mL). Expression of phospho-PKD and total PKD was examined by using Western blotting of whole-cell lysates. Images are representative of 3 independent experiments. PMA, Phorbol 12-myristate 13-acetate. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E1 Time course of TEER in 16HBE14o- cells. Although cells reached confluence about 3 days after seeding, TEER started to increase on day 4 and reached a plateau by day 7. All experiments were conducted at this time point. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E2 PolyI:C does not induce significant cell cytotoxicity. The cytotoxicity index of 16HBE14o- cells stimulated with various ligands for 24 hours compared with medium control or detergent lysed cells is shown. Results are means ± SEMs of 3 experiments. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E3 PolyI:C decreases TEER in primary NHBE cell monolayers. A, TEER was measured in NHBE cells grown at the air-liquid interface and exposed for the indicated times to polyI:C (5 μg/mL). B, NHBE cell cytotoxicity was measured at 48 hours, as in Fig E2. Data are expressed as the percentage of control unstimulated cells and are presented as means ± SEMs of 3 independent experiments. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E4 PolyI:C induces upregulation of dsRNA sensors. PolyI:C is a known ligand for TLR3 and can also be recognized by intracellular helicases, including RIG-I and MDA5. We examined the expression of these molecules using Western blotting of whole-cell lysates and observed that 16HBE14o- cells constitutively expressed TLR3 and MDA5 and that expression of TLR3, MDA5, and RIG-I was increased 24 hours after 5 μg/mL polyI:C treatment. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E5 PolyI:C-induced reduction of TEER is TLR3 dependent. 16HBE14o- cells were grown on Transwell inserts and transfected with nontarget (NT), TLR3, RIG-I, or MDA5 siRNA to investigate which receptors are involved in epithelial barrier disruption by polyI:C. Seventy-two hours after transfection, cells were treated with 5 μg/mL polyI:C for 24 hours, followed by analysis of TEER. Data are expressed as the percentage of resistance over untreated control and are means ± SEMs of 3 independent experiments. **P < .01, paired Student t test. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E6 PolyI:C does not significantly affect TJ or AJ protein expression. Total cell lysates of control and polyI:C-treated 16HBE14o- cells were subjected to SDS electrophoresis and immunoblotting analysis. Representative immunoblots and densitometric quantification or at least 3 independent experiments from the 6-hour time point are shown. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 Fig E7 Inhibition of NM II does not inhibit polyI:C-induced AJC disassembly. 16HBE14o- cells were stimulated with polyI:C (5 μg/mL for 24 hours), vehicle control (dimethyl sulfoxide), or inhibitors targeting NM II (blebbistatin), ROCK (Y-27632), or MLCK (ML-7), as indicated, and analyzed by means of immunofluorescence and confocal microscopy for expression of ZO-1. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

16 Fig E8 The effect of polyI:C on junctional disassembly is not due to autocrine signaling. We tested this hypothesis by exposing 16HBE cells to supernatants of polyI:C-treated cells. The entire supernatant was collected from medium control and polyI:C-treated cells after 24 hours and was placed on untreated confluent epithelial monolayers. Although polyI:C induced a significant decrease in TEER, the supernatant collected from polyI:C-treated cells did not affect TEER. The results are representative of 2 independent studies and expressed as percentage resistance versus untreated control. **P < .01, ANOVA. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

17 Fig E9 A neutralizing type I interferon receptor antibody does not inhibit the effects of polyI:C on barrier dysfunction. 16HBE14o- cells were stimulated with or without 5 μg/mL polyI:C, as indicated in the absence or presence of a neutralizing IFN-α/β receptor antibody (αIFNAR) used at 2 and 10 μg/mL, followed by analysis of TEER at the indicated time points. Results are expressed relative to control untreated cells and are means ± SEMs of 3 experiments per condition. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Download ppt "Polyinosinic:polycytidylic acid induces protein kinase D–dependent disassembly of apical junctions and barrier dysfunction in airway epithelial cells "

Similar presentations

Volume 63, Issue 4, Pages (April 2003)

Volume 63, Issue 4, Pages (April 2003)
Immune deficiency caused by impaired expression of nuclear factor-κB essential modifier (NEMO) because of a mutation in the 5′ untranslated region of.

Immune deficiency caused by impaired expression of nuclear factor-κB essential modifier (NEMO) because of a mutation in the 5′ untranslated region of.
Dorothea Dijkstra, MSc, Rob Leurs, PhD, Paul Chazot, PhD, Fiona C

Dorothea Dijkstra, MSc, Rob Leurs, PhD, Paul Chazot, PhD, Fiona C
Volume 127, Issue 3, Pages (September 2004)

Volume 127, Issue 3, Pages (September 2004)
Rhinovirus infection causes steroid resistance in airway epithelium through nuclear factor κB and c-Jun N-terminal kinase activation  Alberto Papi, MD,

Rhinovirus infection causes steroid resistance in airway epithelium through nuclear factor κB and c-Jun N-terminal kinase activation  Alberto Papi, MD,
Steve N. Georas, MD, Fariba Rezaee, MD 

Steve N. Georas, MD, Fariba Rezaee, MD 
Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo  Jin-Ah Park, PhD, Asma S.

Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo  Jin-Ah Park, PhD, Asma S.
Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic.

Cell-specific activation profile of extracellular signal-regulated kinase 1/2, Jun N-terminal kinase, and p38 mitogen-activated protein kinases in asthmatic.
IL-13 and TH2 cytokine exposure triggers matrix metalloproteinase 7–mediated Fas ligand cleavage from bronchial epithelial cells  Samuel J. Wadsworth,

IL-13 and TH2 cytokine exposure triggers matrix metalloproteinase 7–mediated Fas ligand cleavage from bronchial epithelial cells  Samuel J. Wadsworth,
TNF-α–mediated bronchial barrier disruption and regulation by src-family kinase activation  Michelle A. Hardyman, PhD, Emily Wilkinson, BSc, Emma Martin,

TNF-α–mediated bronchial barrier disruption and regulation by src-family kinase activation  Michelle A. Hardyman, PhD, Emily Wilkinson, BSc, Emma Martin,
IgE cross-linking impairs monocyte antiviral responses and inhibits influenza-driven TH1 differentiation  Regina K. Rowe, MD, PhD, David M. Pyle, MD,

IgE cross-linking impairs monocyte antiviral responses and inhibits influenza-driven TH1 differentiation  Regina K. Rowe, MD, PhD, David M. Pyle, MD,
IL-10 disrupts the Brd4-docking sites to inhibit LPS-induced CXCL8 and TNF-α expression in monocytes: Implications for chronic obstructive pulmonary disease 

IL-10 disrupts the Brd4-docking sites to inhibit LPS-induced CXCL8 and TNF-α expression in monocytes: Implications for chronic obstructive pulmonary disease 
Defective nuclear entry of hydrolases prevents neutrophil extracellular trap formation in patients with chronic granulomatous disease  Susana Romao, PhD,

Defective nuclear entry of hydrolases prevents neutrophil extracellular trap formation in patients with chronic granulomatous disease  Susana Romao, PhD,
Ting-ting Zhang, MSc, Klaus Okkenhaug, PhD, Baher F

Ting-ting Zhang, MSc, Klaus Okkenhaug, PhD, Baher F
Activation of protease-activated receptor 2 leads to impairment of keratinocyte tight junction integrity  Peter Nadeau, BS, Mason Henehan, BS, Anna De.

Activation of protease-activated receptor 2 leads to impairment of keratinocyte tight junction integrity  Peter Nadeau, BS, Mason Henehan, BS, Anna De.
Allergen-dependent solubilization of IL-13 receptor α2 reveals a novel mechanism to regulate allergy  Michael O. Daines, MD, Weiguo Chen, PhD, Yasuhiro.

Allergen-dependent solubilization of IL-13 receptor α2 reveals a novel mechanism to regulate allergy  Michael O. Daines, MD, Weiguo Chen, PhD, Yasuhiro.
Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells  Rommy Koetzler, MD, MSc, Raza S. Zaheer,

Nitric oxide inhibits human rhinovirus-induced transcriptional activation of CXCL10 in airway epithelial cells  Rommy Koetzler, MD, MSc, Raza S. Zaheer,
Julie M. Caldwell, PhD, Carine Blanchard, PhD, Margaret H

Julie M. Caldwell, PhD, Carine Blanchard, PhD, Margaret H
FcεRI-mediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells  Shigeru Okumura, DDS, PhD, Hironori Sagara,

FcεRI-mediated amphiregulin production by human mast cells increases mucin gene expression in epithelial cells  Shigeru Okumura, DDS, PhD, Hironori Sagara,
Activation of Epidermal Toll-Like Receptor 2 Enhances Tight Junction Function: Implications for Atopic Dermatitis and Skin Barrier Repair  I-Hsin Kuo,

Activation of Epidermal Toll-Like Receptor 2 Enhances Tight Junction Function: Implications for Atopic Dermatitis and Skin Barrier Repair  I-Hsin Kuo,

Similar presentations

Ads by Google