Eosinophils increase airway sensory nerve density in mice and in human asthma

Abstract

In asthma, airway nerve dysfunction leads to excessive bronchoconstriction and cough. It is well established that eosinophils alter nerve function and that airway eosinophilia is present in 50 to 60% of asthmatics. However, the effects of eosinophils on airway nerve structure have not been established. We tested whether eosinophils alter airway nerve structure and measured the physiological consequences of those changes. Our results in humans with and without eosinophilic asthma showed that airway innervation and substance P expression were increased in moderate persistent asthmatics compared to mild intermittent asthmatics and healthy subjects. Increased innervation was associated with a lack of bronchodilator responsiveness and increased irritant sensitivity. In a mouse model of eosinophilic airway inflammation, the increase in nerve density and airway hyperresponsiveness were mediated by eosinophils. Our results implicate airway nerve remodeling as a key mechanism for increased irritant sensitivity and exaggerated airway responsiveness in eosinophilic asthma.

Conflict of interest statement

Competing interests: D.B.J. formerly consulted for GlaxoSmithKline on the development of mepolizumab. A.D.F. is a consultant in lung toxicology for the fragrance industry. R.W.C. is funded by GlaxoSmithKline and is a consultant for GlaxoSmithKline, TEVA, Genentech, and Novartis on technologies related to medication adherence. All other authors declare that they have no competing interests.

Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1.. Airway sensory innervation and substance P expression are increased in moderate persistent asthma.

(A) Representative 3D nerve models generated from bronchoscopic human airway biopsies immunolabeled with antibody to the pan-neuronal protein PGP9.5. (B and C) Bar graphs showing nerve length (B) and branch points (C) in samples derived from healthy subjects (control) and patients with mild intermittent asthma (intermittent) and moderate persistent asthma (persistent). (D) Correlation between nerve length and branch points in control and asthmatic patients (r2 = 0.87, P < 0.0001). (E) Representative 3D nerve models generated from bronchoscopic human airway biopsies obtained from healthy subjects (control) and patients with mild intermittent asthma (intermittent) and moderate persistent asthma (persistent), immunolabeled with antibody to neuronal substance P, and the pan-neuronal protein PGP9.5. Movies of nerve modeling are available in the online supplement (movies S1 to S3). Data are presented as means ± SEM. Asterisk (*) indicates P < 0.05 compared to all other groups. Statistical significance was determined using one-way analysis of variance (ANOVA) with a Bonferroni post hoc test (B, C, and E) and linear regression (D). In total, 63 subjects were included in the final analysis. Three biopsy specimens were analyzed per subject with 10 randomized z-stack images obtained per specimen.

Fig. 2.. Airway and peripheral blood eosinophils are associated with increased airway innervation in asthma.

(A) Eosinophil peroxidase (EPX) in human airway biopsies from healthy subjects (control, white bars) and from patients with mild intermittent (blue bars) and moderate persistent asthma (red bars). EPX was quantified above and below the epithelial basement membrane (BM). Data points represent the average of three biopsies per subject. A total of 57 subjects were evaluated. (B) Correlation between blood eosinophils and airway EPX for each subject. r2 = 0.01, P = 0.8. Colored dots correspond with mild intermittent asthma (blue), moderate persistent asthma (red), or control (white). (C and D) Nerve length and branch points in patients stratified into terciles by peripheral blood eosinophil count. n = 62 subjects. (E and F) Nerve length and branch points in subgroups stratified by type 2 asthma phenotype and airway EPX. Type 2-low versus type 2-high asthma was defined by blood eosinophils less than or greater than 300 cells/μl. High EPX was defined as greater than 5500 positive voxels. n = 57 subjects. (G and H) Correlation of nerve length (G) and branch points (H) with airway EPX in patients with moderate persistent asthma. Linear regression for length r2 = 0.1 and P = 0.09 and for branch points r2 = 0.14 and P < 0.05. n = 28. Bar graphs represent means ± SEM. Asterisk (*) indicates P < 0.05. Statistical significance was determined using one-way ANOVA with a Bonferroni post hoc test (A and C to F) and linear regression.

Fig. 3.. Increased airway nerve density is associated with lack of bronchodilator responsiveness and increased sensitivity to environmental stimuli.

(A and B) Nerve length and branch points in patients with asthma stratified by the presence or absence of airflow obstruction on pulmonary function testing (defined as a ratio of FEV1/FVC less than 0.7). n = 43. (C and D) Nerve length and branch points in patients with asthma stratified by the presence or absence of bronchodilator responsiveness to inhaled albuterol. Bronchodilator responsiveness was defined as an increase in postbronchodilator FEV1 and/or FVC > 200 ml and >12%. n = 22. (E and F) Nerve length and branch points in patients with asthma stratified by their sensitivity to environmental stimuli, determined by the Asthma Quality of Life Questionnaire. Moderate or worse sensitivity to environmental triggers was defined as an environmental stimuli domain score of ≤5, whereas scores >5 indicated minimal or no sensitivity. n = 26. Colored dots indicate mild intermittent (blue) and moderate persistent (red) asthma. Data are presented as means ± SEM. Asterisk (*) indicates P < 0.05. Statistical significance was determined by t test (unpaired, two-tailed).

Fig. 4.. Airway eosinophils increase sensory innervation and cause neuronally mediated airway hyperresponsiveness in mice.

(A) Airway responsiveness to aerosolized serotonin in WT mice, transgenic mice with airway eosinophilia driven by airway-specific Il5 overproduction (Il5tg), eosinophil-deficient mice (PHIL) and eosinophil-deficient mice over-expressing airway Il5 (Il5tg/PHIL). (B) Airway responsiveness to aerosolized serotonin after vagotomy. n = 6 to 9 per group. (C and D) Nerve length and branch points in the proximal trachea of WT, Il5tg, PHIL, and Il5tg/PHIL mice. n = 4 to 5 per group. (E and F) Nerve length and branch points at the carina in WT, Il5tg, PHIL, and Il5tg/PHIL mice. n = 4 to 8 per group. (G) Bronchoalveolar lavage (BAL) eosinophils (Eos). n = 4 to 5 per group. Data are presented as means ± SEM. Asterisk (*) indicates P < 0.05 compared to all other groups. Statistical significance was determined using two-way repeated measures ANOVA (A and B) and one-way ANOVA with a Bonferroni post hoc test (C to G).