Targeting integrin α5β1 ameliorates severe airway hyperresponsiveness in experimental asthma - PubMed (original) (raw)

Targeting integrin α5β1 ameliorates severe airway hyperresponsiveness in experimental asthma

Aparna Sundaram et al. J Clin Invest. 2017.

Abstract

Treatment options are limited for severe asthma, and the need for additional therapies remains great. Previously, we demonstrated that integrin αvβ6-deficient mice are protected from airway hyperresponsiveness, due in part to increased expression of the murine ortholog of human chymase. Here, we determined that chymase protects against cytokine-enhanced bronchoconstriction by cleaving fibronectin to impair tension transmission in airway smooth muscle (ASM). Additionally, we identified a pathway that can be therapeutically targeted to mitigate the effects of airway hyperresponsiveness. Administration of chymase to human bronchial rings abrogated IL-13-enhanced contraction, and this effect was not due to alterations in calcium homeostasis or myosin light chain phosphorylation. Rather, chymase cleaved fibronectin, inhibited ASM adhesion, and attenuated focal adhesion phosphorylation. Disruption of integrin ligation with an RGD-containing peptide abrogated IL-13-enhanced contraction, with no further effect from chymase. We identified α5β1 as the primary fibronectin-binding integrin in ASM, and α5β1-specific blockade inhibited focal adhesion phosphorylation and IL-13-enhanced contraction, with no additional effect from chymase. Delivery of an α5β1 inhibitor into murine airways abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. Finally, α5β1 blockade enhanced the effect of the bronchodilator isoproterenol on airway relaxation. Our data identify the α5β1 integrin as a potential therapeutic target to mitigate the severity of airway contraction in asthma.

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Conflict of interest statement

The authors have declared that no conflict of interest exists.

Figures

Figure 1

Figure 1. Chymase abrogates cytokine-enhanced airway contraction.

(A) Force exerted on human bronchial rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline (control), then for 20 minutes with rhChy (30 nM) or vehicle, in response to a range of concentrations agonist Mch. n = 3–9 rings per group. **P < 0.01 and ***P < 0.001, for IL-13 versus IL-13/rhChy; repeated measures of variance. (B) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-17A (100 ng/ml) or saline (control), then for 20 minutes with rhChy (30 nM) or vehicle in response to a range of concentrations of the contractile agonist Mch. n = 4–5 rings per group. ***P < 0.001, for IL-17A versus IL-17A/rhChy; repeated measures of variance. All data represent the mean ± SEM.

Figure 2

Figure 2. Chymase impairs smooth muscle adhesion to fibronectin and focal adhesion complex phosphorylation.

(A) Schematic of alternate pathways important in the transmission of tension. Vh, vinculin head domain; Vt, vinculin tail domain. (B) Adhesion of human ASM cells to fibronectin measured by absorbance of crystal violet at 595 nm. Fibronectin (0.1 μg/ml) or cells were treated with the indicated doses of rhChy for 20 minutes and then chymostatin (10 μg/ml) prior to assessment of adhesion. (C) Adhesion of human ASM cells to collagen I (0.1 μg/ml), vitronectin (0.3 μg/ml), or laminin I (10 μg/ml), as measured by absorbance of crystal violet at 595 nm. Ligands were treated with the indicated doses of rhChy for 20 minutes and then chymostatin (10 μg/ml) prior to assessment of adhesion. (B and C) Data represent the mean ± SEM from triplicate experiments. (D) Representative Western blots and quantitative densitometry for phosphorylated and total vinculin and FAK in human ASM cells plated on poly-

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-lysine (300 μg/ml) alone or poly-

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-lysine with either fibronectin (1 μg/ml) or collagen I (1 μg/ml), with the addition of rhChy (30 nM) or vehicle for 20 minutes, followed by chymostatin (10 μg/ml). GAPDH was used as a loading control. Poly, poly-

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-lysine; FN, fibronectin; C, collagen I. *P < 0.05 and **P < 0.01 versus Poly; #P < 0.05 and ##P < 0.01 versus Poly/FN by 2-way ANOVA. n = 3 distinct experiments.

Figure 3

Figure 3. Chymase cleaves fibronectin and disrupts integrin binding to abrogate IL-13–enhanced contraction.

(A) Coomassie staining (5 μg, left) and representative Western blot (50 μg, right) for fibronectin cleavage products in human plasma fibronectin after treatment with rhChy (30 nM) or vehicle for 20 minutes. (B) Representative Western blot for fibronectin cleavage products in mouse posterior tracheal smooth muscle strips after treatment with rhChy (30 nM) or vehicle for 20 minutes. Ctrl, control. (C) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline (control), then for 1 hour with GRGDSP peptide (RGD, 10 μg/ml) or vehicle in response to a range of concentrations of the contractile agonist Mch. *P < 0.05, **P < 0.01, and ***P < 0.001, for IL-13 versus IL-13/RGD; repeated measures of variance. (D) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline (control), then for 1 hour with GRGDSP (10 μg/ml), 20 minutes with rhChy (30 nM), or both, in response to a range of concentrations of Mch. ***P < 0.001, for IL-13 versus IL-13/RGD plus rhChy. NS, for IL-13/RGD versus IL-13/rhChy versus IL-13/RGD plus rhChy; repeated measures of variance. All data represent the mean ± SEM. n = 4–5 rings per group.

Figure 4

Figure 4. Integrin α5β1 is critical for smooth muscle cell adhesion to fibronectin.

(A) Human ASM cells in suspension were labeled with primary antibodies specific for cell-surface integrins β1, αv, β6, β3, β5, α8, α5 and a secondary antibody conjugated to APC. The cells were analyzed by flow cytometry and gated for live cells. The resultant population was analyzed for APC expression (solid line). Human ASM cells labeled with a secondary antibody alone served as a control (dashed line). Representative histograms of APC expression versus cell counts are shown. The x axis represents APC expression (mean fluorescence intensity); the y axis represents the cell count (percentage of maximum). (B) Representative Western blot for expression of integrin αvβ1 was determined by IP. Lysates from human ASM cells underwent pulldown with mouse IgG or anti-αv antibody, followed by immunoblotting (IB) for β1. Immunoblotting for αv was performed to confirm enrichment of αv. n = 3 distinct experiments. (C) Adhesion (measured by absorbance of crystal violet at 595 nm) of human ASM cells to fibronectin (0.1 μg/ml) after treatment with the indicated specific integrin-blocking antibodies. Data represent the mean ± SEM from triplicate experiments. (D) Schematic of fibronectin, with arrows marking chymase cleavage sites identified during N-terminal sequencing of the 3 cleaved fibronectin bands seen on the Coomassie staining in Figure 3A. The underlined amino acids correspond to aligned sequences detected during N-terminal sequencing.

Figure 5

Figure 5. Blockade of α5β1 inhibits focal adhesion phosphorylation and impairs IL-13–enhanced tracheal ring contraction.

(A) Representative Western blot and quantitative densitometry for phosphorylated and total vinculin and FAK in human ASM cells treated for 20 minutes with integrin α5–blocking antibody (P1D6, 10 μg/ml) or mouse IgG control, then plated on poly-

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-lysine (300 μg/ml) or poly-

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-lysine with either fibronectin (1 μg/ml) or collagen I (1 μg/ml). GAPDH was used as a loading control. *P < 0.05 and **P < 0.01 versus Poly; #P < 0.05 versus Poly/FN. n = 3 distinct experiments. GAPDH was used as a loading control. *P < 0.05 and **P <0.01 versus Poly; #P < 0.05 versus Poly/FN, by 2-way ANOVA n = 3 distinct experiments. Data represent the mean ± SEM. (B) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline and for 12 hours with α5-blocking antibody (300 μg/ml) or rat IgG in response to a range of concentrations of the contractile agonist Mch. ***P < 0.001, for IL-13/IgG versus IL-13/anti-α5. (C) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline, then for 1 hour with a small-molecule inhibitor directed against the α5β1 synergy site of fibronectin (ATN-161, 100 μg/ml) or vehicle in response to a range of concentrations of Mch. **P < 0.01 and ***P < 0.001, for IL-13/vehicle versus IL-13/ATN-161. (D) Contractile force of mouse tracheal rings measured after incubation for 12 hours in DMEM with IL-13 (100 ng/ml) or saline (control), then for 1 hour with ATN-161 (100 μg/ml), 20 minutes with rhChy (30 nM), or both, in response to a range of concentrations of Mch. ***P < 0.001, for IL-13/vehicle versus IL-13/ATN-161 plus rhChy. NS, for IL-13/ATN-161 versus IL-13/rhChy versus IL-13/ATN-161 plus rhChy. (B–D) Data represent the mean ± SEM; n = 3–5 rings per group; significance was determined by repeated measures of variance.

Figure 6

Figure 6. Inhibition of α5β1 protects against airway hyperresponsiveness and enhances the effect of bronchodilators.

(A) Pulmonary resistance measurements in WT C57Bl/6 mice following immunization and i.n. challenge with OVA, with i.n. administration of ATN-161 (12.5 mg/kg) or vehicle (5% DMSO, 0.9% saline) 1 hour prior to measurements. n = 8 animals per group. (B) Tension of mouse tracheal rings measured after 12 hours in DMEM with IL-13 (100 ng/ml) and incubation for 15 minutes with Mch (10–6 M) in response to a range of concentrations of the β-adrenergic agonist isoproterenol in the presence of ATN-161 (100 μg/ml) or vehicle. n = 5–8 rings per group. Data represent the mean ± SEM. **P < 0.01 and ***P < 0.001; repeated measures of variance.

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