Research ArticleCystic Fibrosis

Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis

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Science Translational Medicine  03 Apr 2019:
Vol. 11, Issue 486, eaav3488
DOI: 10.1126/scitranslmed.aav3488
  • Fig. 1 Mucins in CF and non-CF disease controls.

    (A) BALF total mucin concentrations from subjects with CF (n = 121) and non-CF controls (n = 27). (B) Total concentrations of mucins in BALF after stratification of patients by the absence (n = 14 non-CF and 82 CF; left) or presence (n = 13 non-CF and 39 CF; right) of pathogenic infection (infxn) on culture of BALF. (C) Representative SEM of a flake from a non-CF subject. (D) Representative SEM of a flake from a CF subject. (E and F) Representative IHC of MUC5B (green) and MUC5AC (red) in a cytospin from (E) a non-CF and (F) a CF BALF. (G) Mucin staining intensities, an index of flake number, of MUC5B and MUC5AC in CF (n = 109) and non-CF (n = 21) BALF. (H) Flake granularity, measured via image analysis of RMS roughness in CF (n = 17) and non-CF (n = 59) BALF. *P < 0.05; **P < 0.01 after multivariate analysis.

  • Fig. 2 Mucins and markers of airway disease in early CF.

    (A) Correlation between mucins and neutrophil counts in BALF from subjects with CF (blue; r2 = 0.38, P < 0.001, n = 62) and non-CF controls (orange; n = 16, not significant). (B and C) Correlations between mucins and other markers associated with neutrophilic inflammation including (B) IL-8 (r2 = 0.29, P < 0.001, n = 62) and (C) DNA (r2 = 0.43, P < 0.001, n = 62). (D to F) Correlations between mucins and BALF metabolite concentrations measured by metabolomics in 60 CF BALF samples, including (D) hypoxanthine (r2 = 0.47, P < 0.001) as a marker of inflammation, (E) lactate (r2 = 0.48, P < 0.001) as a marker of hypoxia, and (F) ratio of oxidized to reduced glutathione (GSSG/GSH) as a marker of oxidative stress (r2 = 0.33, P < 0.001).

  • Fig. 3 Bacterial cultures and the microbiome in early CF.

    (A) Pathogen recovery frequency (left) and burden of pathogens in samples with positive cultures (right) in BALF from subjects with CF and from non-CF controls (n = 124 CF and 32 non-CF). (B) Total bacterial burden, as measured by 16S qPCR, in BALF from subjects with CF and non-CF controls (n = 46 CF and 15 non-CF). (C) qPCR and distribution of taxa from microbiome analyses for all CF (left) and non-CF samples (right). Bar height represents total qPCR signal, with detected taxa represented proportionally within the bar. For clarity, taxa with average prevalence <1% were grouped (light gray bars). The pattern observed from washes of sterile bronchoscopes (w) is shown for reference (n = 6). Taxa associated with environmental contamination are shown in green; taxa associated with the oral cavity, including Moraxella, Haemophilus, and Streptococcus as common upper airway commensals (72), are shown in blue; and known pathogens are shown in red. Taxa were grouped as previously described (39). (D) Concentrations of MUC5B and MUC5AC as measured by IHC in mucin flakes recovered by BALF from subjects with CF with no structural lung disease (CF-NSD, n = 37) and non-CF controls (n = 21) (significant by parameter; P = 0.054 postmultivariate analyses). (E) Neutrophil (PMN) counts in CF-NSD versus non-CF. (F) qPCR in CF-NSD and non-CF samples. (G) Microbiome analyses of CF-NSD. Triangles represent samples from subjects who received antibiotics ≤3 months before bronchoscopy. See (C) for taxa legend. *P < 0.05, P < 0.05 by nonparametric analysis.

  • Fig. 4 Treatment of mucin flakes in early CF.

    (A) IHC of MUC5B (green), MUC5AC (red), and DNA (blue) on a cytospin of unprocessed CF BALF treated with (PBS), DNase (300 U/ml), or the reducing agent DTT (10 mM) for 60 min at 37°C. (B) Staining intensities of MUC5B and MUC5AC in DTT-treated samples relative to those with vehicle (PBS) or DNase (n = 5 per group). Lack of effect of DNase on DNA staining intensities reflected the intracellular location of most DNA. All intensities were normalized to the average values from the PBS group (n = 10 per group). (C) Flake density in PBS and DTT-treated samples analyzed by SEM. (D) Flake density and structure by SEM in samples treated with NAC (10 mM, 1 hour at 37°C) or the novel therapeutic P2062 (10 mM, 1 hour at 37°C). (E) Imaging analysis of flake density in CF BALF samples treated with NAC or P2062 (n = 7 per group). (F) TMV, a measure of mucociliary clearance, in sheep treated with nebulized P2062 (solid purple line; 2.5 ml, 50 mM concentration) or saline (dashed blue line) (n = 3 per group). (G) TMV in sheep exposed to inhaled neutrophil elastase followed by treatment with nebulized P2062 (solid purple line) or saline (dashed blue line) did not (n = 3 per group).

  • Table 1 Demographics of AREST CF and non-CF disease control populations.

    See the Supplementary Materials for definitions of infection and pathogens. P values are calculated by Student’s t test for parametric variables, Mann-Whitney for nonparametric variables, and by χ2 for discrete variables.

    CFNon-CFP
    Subjects, n4616
    Age, years3.3 ± 1.73.2 ± 2.00.837
    Gender, male (%)19 (41%)11 (69%)0.083
    CFTR mutations, F508del
    homozygous
    25 (54%)
    CFTR mutations, residual function2 (4.3%)
    Study visits, n (range per subject)62 (1–3)16 (1)
    BALF samples, n12432
    BALF TCC, ×106 cells/ml (RML only)6.7 ± 7.21.3 ± 1.0<0.001
    BALF % neutrophils (RML only)18.6 ± 22.34.4 ± 5.1<0.001
    Antibiotic treatment*43 (69%)7 (44%)0.080

    *Antibiotics within 3 months of bronchoscopy.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/11/486/eaav3488/DC1

      Fig. S1. Mucin flakes in CF.

      Fig. S2. Molecular microbiology in CF samples.

      Fig. S3. CF microbiology and antibiotic use.

      Fig. S4. Microrheologic effects of treatment.

      Fig. S5. P2062.

      Fig. S6. Disease pathogenesis in normal and early CF lung.

      Table S1. Diagnoses in non-CF disease control populations.

      Table S2. Bacterial infection by conventional culture in AREST CF and non-CF disease control populations as a function of bacterial density.

      Table S3. Multivariate analyses for BALF mucin parameters.

      Table S4. Correlation of mucins, MUC5B and MUC5AC to markers of inflammation and metabolomic markers of airway environment in CF BALF.

      Table S5. Multivariate analyses of mucins versus BALF metabolomic and inflammatory markers.

      Table S6. BALF markers in CF-NSD versus non-CF controls.

      Data file S1. Raw data (provided as separate Excel file).

    • The PDF file includes:

      • Fig. S1. Mucin flakes in CF.
      • Fig. S2. Molecular microbiology in CF samples.
      • Fig. S3. CF microbiology and antibiotic use.
      • Fig. S4. Microrheologic effects of treatment.
      • Fig. S5. P2062.
      • Fig. S6. Disease pathogenesis in normal and early CF lung.
      • Table S1. Diagnoses in non-CF disease control populations.
      • Table S2. Bacterial infection by conventional culture in AREST CF and non-CF disease control populations as a function of bacterial density.
      • Table S3. Multivariate analyses for BALF mucin parameters.
      • Table S4. Correlation of mucins, MUC5B and MUC5AC to markers of inflammation and metabolomic markers of airway environment in CF BALF.
      • Table S5. Multivariate analyses of mucins versus BALF metabolomic and inflammatory markers.
      • Table S6. BALF markers in CF-NSD versus non-CF controls.
      • Legend for data file S1

      [Download PDF]

      Other Supplementary Material for this manuscript includes the following:

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