Research ArticleAsthma

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

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Science Translational Medicine  05 Sep 2018:
Vol. 10, Issue 457, eaar8477
DOI: 10.1126/scitranslmed.aar8477
  • 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).

  • Table 1 Clinical characteristics of study subjects.

    Values are means ± SD unless otherwise stated. NA, not applicable.

    Asthma severity
    ControlIntermittentPersistent
    Characteristicn = 19n = 13n = 31
    Age (range)56.1 (21–76)58.9 (40–75)55.9 (28–78)
    Female sex—no. (%)14 (73)8 (62)17 (55)
    Body mass index*26.9 ± 4.927.3 ± 4.528.4 ± 5.5
    Former smoker—no. (%)1 (5)1 (7)7 (23)
    FEV1 before bronchodilation
      Mean (liters)2.78 ± 0.72.43 ± 0.82.11 ± 0.9
      Percent predicted value106.4 ± 15.187.5 ± 11.172.9 ± 22.5
    FEV1/FVC ratio before bronchodilation77.5 ± 4.067.7 ± 6.561.1 ± 13.9±
    FEV1 after bronchodilation
      Mean (liters)NA2.45 ± 0.62.41 ± 1.0
      Percent predicted valueNA88.9 ± 11.280.9 ± 22.7
    FEV1/FVC ratio after bronchodilationNA67.7 ± 6.570.3 ± 15.6
    Blood eosinophil count (cells/μl)182 ± 93277 ± 289301 ± 225
    Use of inhaled corticosteroid—no. (%)0 (0)0 (0)31 (100)
    Use of long-acting β2 agonist (%)0 (0)0 (0)28 (90)
    Use of >2 controller medications (%)0 (0)0 (0)21 (68)
    Use of daily systemic steroids—no. (%)0 (0)0 (0)4 (11)
    AQLQ score§NA5.4 ± 1.14.1 ± 1.4
    Symptoms§NA4.1 ± 1.73.1 ± 1.6
      Emotional function§NA4.0 ± 1.82.9 ± 1.9
      Environmental stimuli§NA4.5 ± 2.13.4 ± 1.9
      Activity limitation§NA4.4 ± 1.43.8 ± 2.4

    *The body mass index is the weight in kilograms divided by the height squared.

    P < 0.05 compared to control.

    ±P < 0.05 compared to all other groups.

    §Minimally important differences are reflected by differences in scores >0.5 points.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/10/457/eaar8477/DC1

      Fig. S1. Airway innervation was not associated with sex, body mass index, or age.

      Table S1. Raw data of airway nerve modeling and eosinophilia in mice.

      Movie S1. Quantitative 3D model of airway epithelial sensory nerves from a healthy nonasthmatic subject.

      Movie S2. Quantitative 3D model of airway epithelial sensory nerves from a subject with moderate persistent asthma.

      Movie S3. Quantitative 3D model of airway epithelial sensory nerves costained for substance P from a subject with moderate persistent asthma.

    • The PDF file includes:

      • Fig. S1. Airway innervation was not associated with sex, body mass index, or age.
      • Table S1. Raw data of airway nerve modeling and eosinophilia in mice.
      • Legends for movies S1 to S3

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

      • Movie S1 (.mp4 format). Quantitative 3D model of airway epithelial sensory nerves from a healthy nonasthmatic subject.
      • Movie S2 (.mp4 format). Quantitative 3D model of airway epithelial sensory nerves from a subject with moderate persistent asthma.
      • Movie S3 (.mp4 format). Quantitative 3D model of airway epithelial sensory nerves costained for substance P from a subject with moderate persistent asthma.

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