TNF-α–mediated bronchial barrier disruption and regulation by src-family kinase activation Michelle A. Hardyman, PhD, Emily Wilkinson, BSc, Emma Martin,

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Presentation transcript:

TNF-α–mediated bronchial barrier disruption and regulation by src-family kinase activation Michelle A. Hardyman, PhD, Emily Wilkinson, BSc, Emma Martin, BMedSci, Nivenka P. Jayasekera, MD, Cornelia Blume, PhD, Emily J. Swindle, PhD, Neil Gozzard, PhD, Stephen T. Holgate, MD, DSc, Peter H. Howarth, MD, DM, Donna E. Davies, PhD, Jane E. Collins, PhD Journal of Allergy and Clinical Immunology Volume 132, Issue 3, Pages 665-675.e8 (September 2013) DOI: 10.1016/j.jaci.2013.03.005 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Effect of TNF-α on HBEC barrier function and TJ staining. A, Day 4 TER (n = 9). B, Day 4 FITC-dextran permeability (n = 8). Representative immunofluorescent photomicrographs (n = 8) are controls (C, E, G, and I) or TNF-α (D, F, H, and J) for occludin (C and D), claudin-3 (E and F), claudin-4 (G and H), and claudin-8 (I and J) with TJ staining green and nucleii stained red. XZ stacks are labeled apical and basolateral. Scale bar, 50 μm. Ap, Apical; Baso, basolateral; cl3, claudin-3; cl4, claudin-4; cl8, claudin-8; con, control; occl, occludin; TNF, TNF-α. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 TNF-α caused an increase in innate immune cytokine release from primary differentiated HBECs. Basolateral medium taken every 24 hours from cells treated with TNF-α was assayed for cytokines and TNF mRNA. TNF-α caused significant increases in TNF-α mRNA (D) (n = 3) and release of endogenous TNF-α (A), IL-1β (B), IL-6 (C), IL-8 (E) (all n = 7), and TSLP (F) (n = 5). Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 SU6656 inhibited TNF-α–mediated changes in macromolecular barrier function and TJ staining. Effect of TNF-α without (light gray bars) or with (dark gray bars) SU6656 on TER (A) (n = 8) or the FITC-dextran permeability (B) (n = 8); data are expressed relative to controls without or with SU6656. Representative immunofluorescent photomicrographs (n = 8) are control (C and G), TNF-α (D and H), SU6656 alone (E and I), or SU6656/TNF-α (F and J) for claudin-4 (C-F) or occludin (G-J). XZ stacks are labeled apical and basolateral. Scale bar, 50 μm. Ap, Apical; Baso, basolateral; cl4, claudin-4; con, control; occl, occludin; SU, SU6656; TNF, TNF-α. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 The SFK inhibitor SU6656 inhibited TNF-α–mediated changes in E-cadherin and p120-catenin staining and reduced endogenous TNF-α release. Representative immunofluorescent photomicrographs (n = 3) are control (A and E), TNF-α (B and F), SU6656 alone (C and G), or SU6656/TNF-α (D and H) for E-cadherin (A-D) or p120 catenin (E-H). XZ stacks are labeled apical and basolateral. Bar 50 μm. I, shows basolateral concentration of endogenous TNF-α without or with SU6656 (n = 5). Ap, Apical; Baso, basolateral; con, control; E-cad, E-cadherin; p120, p120 catenin; SU, SU6656 alone; TNFSU, SU6656/TNF-α; TNF, TNF-α. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 The effect of TNF-α and SU6656 on p120-catenin phosphorylation at residues Y228 and Y280. A, Representative Western blot analysis of p120-PY228, p120-PY280, total p120-catenin, and β-actin control. Shown are lysates from control cells and cells treated with TNF-α, SU6656, or SU6656/TNF-α. B, Ratio of signal intensities of p120-PY228 and total p120-catenin after normalization to β-actin. TNF-α–mediated increase in p120-PY228 was significantly reduced with SU6656 (n = 3). C, Control; T, TNF-α; S, SU6656; ST, SU6656/TNF-α. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effect of SU6656 on barrier function and TJ staining in HBECs from severe asthma. A, Day 4 TER (n = 4). B, Day 4 FITC-dextran permeability (n = 4). Representative immunofluorescent photomicrographs (n = 4) are controls (C, E, G, and I) or SU6656 (D, F, H, and J) for claudin-4 (C and D), occludin (E and F), E-cadherin (G and H), and p120 (I and J) with TJ staining green and nucleii stained red. XZ stacks are labeled apical and basolateral. Scale bar, 50 μm. Ap, Apical; Baso, basolateral; C, control; cl4, claudin-4; occl, occludin; SU, SU6656. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 7 Localization of E-cadherin, p120, and neutrophil elastase. Representative photomicrographs of healthy (n = 4) (A-C) and severe asthmatic epithelium (n = 4) (D-F) showing staining of glycolmethacrylate serial sections of bronchial biopsies with E-cadherin (A and D), p120 catenin (B and E) and neutrophil elastase (C and F). In patients with asthma, neutrophil elastase–positive cells were intraepithelial, and junctional staining was often lost (insets and arrows indicate positive staining). Scale bar, 50 μm. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Effect of TNF-α on HBECs showing dose (A-C) and time (A and D) responses for changes in TER (A and B), and FITC dextran permeability (C and D). E, Shown is LDH release over time in response to 10 ng/mL TNF-α compared with controls. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Western blot analysis in upper panel shows HBECs treated for 4 days with TNF-α, compared with untreated controls and stained for claudin-3, -4, -8, and occludin. No consistent or significant differences in expression levels were observed, as seen from the quantitation of band density shown in lower panel (n = 3). Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 TNF-α did not promote the release of IL-10 or IL-33 from differentiated HBECs. Basolateral medium, taken every 24 hours from cells treated with 10 ng/mL TNF-α, was assayed by ELISA for IL-10 (A) or IL-33 (B) and showed no significant change in either cytokine (n = 4). Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 TNF-α (shaded bars) caused an increased release of proMMP-9 from primary airway epithelial cells, whereas TIMP-1 expression was unaffected. Basolateral medium was assayed every 24 hours from cells treated with 10 ng/mL TNF-α. A, Zymography (n = 4) showed the presence of proMMP-9; no other type of MMP was detected on the gels. B, MSD MMP-9 array (n = 4) showed that TNF-α caused a significant increase in proMMP-9 release after 24 hours compared with controls. MMP-9 release increased further for 3 days, although these later time points were not statistically different from controls because of the range of responses. C, Low proteolytic activity was detected in the basolateral media from control HBECs, but this was not significantly different from TNF-α treatment, confirming that most proMMP-9 was not activated (n = 4). D, The levels of TIMP-1 in the basolateral media of control and TNF-α–treated HBECs was determined by ELISA and was not significantly different from controls over time (n = 4). Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 The effect of SU6656 on the TNF-α–mediated TER and cytokine effects in primary HBECs. A, Shown is the TER over time with no significant effect at day 4 (n = 8) (see Fig 3, A for P values). Concentration of innate immune cytokines into the basolateral media was assayed every 24 hours and subjected to MSD assay for IL-1β (B), IL-6 (C), and IL-8 (D). The SU6656 inhibitor had no significant effect on the release of cytokines in the presence of 10 ng/mL exogenous TNF-α. Controls, light shaded bars; TNF-alpha, dark shaded bars. Journal of Allergy and Clinical Immunology 2013 132, 665-675.e8DOI: (10.1016/j.jaci.2013.03.005) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions