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Nasal epithelial cell culture fluorescence recovery after photobleaching predicts cystic fibrosis therapeutic response

Timothy E. Corcoran, Carol A. Bertrand, Michael M. Myerburg, Daniel J. Weiner, Sheila A. Frizzell, Anna Li, Brittani Agostini, Robert S. Parker, Monica E. Shapiro, Ashok Muthukrishnan, Nicholas D. Hages, Brian P. Mulhern, Joseph M. Pilewski
ERJ Open Research 2022 8: 00382-2022; DOI: 10.1183/23120541.00382-2022
Timothy E. Corcoran
1Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA
2Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA
3Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
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  • ORCID record for Timothy E. Corcoran
  • For correspondence: corcorante@upmc.edu
Carol A. Bertrand
4Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA
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Michael M. Myerburg
1Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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Daniel J. Weiner
5Division of Pediatric Pulmonology, University of Pittsburgh, Pittsburgh, PA, USA
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Sheila A. Frizzell
1Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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Anna Li
6School of Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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Brittani Agostini
5Division of Pediatric Pulmonology, University of Pittsburgh, Pittsburgh, PA, USA
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Robert S. Parker
2Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA
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Monica E. Shapiro
2Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA
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Ashok Muthukrishnan
7Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
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Nicholas D. Hages
2Department of Chemical and Petroleum Engineering, University of Pittsburgh, Pittsburgh, PA, USA
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Brian P. Mulhern
1Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA
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Joseph M. Pilewski
1Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA
4Department of Pediatrics, University of Pittsburgh, Pittsburgh, PA, USA
8Department of Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA
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  • FIGURE 1
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    FIGURE 1

    In vivo mucociliary clearance (MMC) response to hypertonic saline (HS) inhalation correlates with in vitro measures of fluorescence recovery after photobleaching (FRAP) diffusion time measured in human nasal epithelial (HNE) cell cultures from the cystic fibrosis (CF) participants (R2=0.55, p=0.04). Only a portion of the CF group had FRAP measurements available (n=10).

  • FIGURE 2
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    FIGURE 2

    Relationship between fluorescence recovery after photobleaching (FRAP) diffusion time and airway surface liquid absorption rate in cystic fibrosis (CF) human nasal epithelial (HNE) cell cultures (R2=0.34, p=0.07). Only a portion of the CF group had FRAP measurements available (n=10).

  • FIGURE 3
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    FIGURE 3

    Mucociliary clearance (MCC) rate decreases with age in single cystic fibrosis transmembrane conductance regulator mutation carriers (CR group) (R2=0.44, p=0.002; n=16).

Tables

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  • Supplementary Materials
  • TABLE 1

    Demographics of study participants along with pulmonary function, Lung Clearance Index (LCI) and sweat chloride data

    CF (n=26)CR (n=16)HC (n=14)p-valuep-value
    (CF vs HC)
    p-value
    (CF vs CR)
    p-value
    (CR vs HC)
    Age (years)26.5 (19–39)48.0 (44–63.5)22.5 (20–23)0.00010.09<0.0001<0.0001
    Female/male (n)14/1210/66/80.560.510.290.43
    FEV1 % pred70 (50–93)97 (93–109)102 (95.5–113) (n=12)0.00030.00080.00140.30
    FVC % pred94.5 (76–104)104.0 (96–109)107.5 (100–116) (n=12)0.020.010.050.23
    FEF25–75% % pred40.5 (22–66)100.5 (74–119.5)83.0 (68–99.5) (n=12)0.00010.0040.00020.29
    LCI9.0 (7.6–13) (n=23)7.8 (7.1–8.3)7.2 (6.7–7.5) (n=11)0.00070.00050.010.10
    Sweat chloride (mmol·L−1)101 (91–110)36 (21–53) (n=14)22 (10–29) (n=12)0.0001<0.00010.00020.11

    Data are presented as median (interquartile range), unless otherwise stated. CF: cystic fibrosis; CR: single CF transmembrane conductance regulator mutation carrier; HC: healthy control; FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; FEF25–75%: forced expiratory flow at 25–75% of FVC; LCI: Lung Clearance Index (from multiple-breath washout testing). p-values comparing all groups by Kruskal–Wallis (nonparametric) except sex which is Chi-squared. Group comparisons by Dunn's test with Holm adjustment (nonparametric, multiple comparisons).

    • TABLE 2

      In vitro measures of human nasal epithelial (HNE) cell physiology across the groups

      CF (n=26)CR (n=16)HC (n=14)p-valuep-value
      (CF vs HC)
      p-value
      (CF vs CR)
      p-value
      (CR vs HC)
      Culture success/failed (n)23/39/714/00.0040.180.0040.001
      Cl− current (µA·cm−2)0.21 (−0.2–0.5) (n=16)4.0 (1.5–5.2) (n=7)5.4 (4.9–10.2) (n=13)0.0001<0.00010.0030.13
      Na+ current (µA·cm−2)22.1 (7.8–41.3) (n=16)9.3 (2.5–18.4) (n=7)37.2 (26.0–46.9) (n=13)0.0120.100.040.004
      i-ratio (Na+ current/
      Cl− current)
      29.4 (−3.9–112.3) (n=16)1.2 (0.7–3.9) (n=7)6.0 (4.2–8.0) (n=13)0.030.060.020.18
      TER (ohm·cm2)651 (453–807) (n=16)717 (205–1066) (n=7)556 (499–673) (n=13)0.630.500.370.66
      Cell ABS (% cleared per 24 h)49.7 (38.2–54.1) (n=23)42.4 (35.6–54.3) (n=9)36.8 (27.1–45.2) (n=14)0.030.010.190.22
      Normalised liquid absorption rate (% per 24 h)68.4 (61.2–80.2) (n=17)53.7 (39.5–72.5) (n=7)59.4 (37.2–66.5) (n=13)0.030.020.110.31
      HNE ASL FRAP diffusion time (τ/τsaline)3.5 (2.8–4.0) (n=10)2.7 (1.2–3.0) (n=5)2.1 (1.3–3.1) (n=13)0.030.020.080.40

      Data are presented as median (interquartile range), unless otherwise stated. CF: cystic fibrosis; CR: single CF transmembrane conductance regulator mutation carrier; HC: healthy control; TER: transepithelial resistance; ABS: technetium-99m-DTPA absorption rate; ASL: airway surface liquid; FRAP: fluorescence recovery after photobleaching. Cl− and Na+ currents and TER were measured using Ussing chamber assessments. Cell ABS is the absorption rate of technetium-99m-DTPA from the apical surface of the cultures after addition in a 10 µL volume. Liquid absorption is measured via an optical technique [6] based on changes in ASL volume after 10 µL volume addition. Not all sampled cultures were viable and available for all measurements. Data presented graphically in supplementary figure S3. p-values comparing all groups by Kruskal–Wallis (nonparametric) except for culture success/failure which is Chi-squared. Group comparisons by Dunn's test with Holm adjustment (nonparametric, multiple comparisons). The number of individual cell donors is indicated. A minimum of three cultures is included in each measurement.

      • TABLE 3

        Imaging-based measurements across the groups

        CF IS (n=26)CR (n=16)HC (n=12)CF HS (n=26)p-valuep-value
        (CF vs HC)
        p-value
        (CF vs CR)
        p-value
        (CR vs HC)
        p-value
        (CF HS vs CF IS)
        MCC
         Whole lung38 (26–49)36 (30–43)36 (26–47)55 (35–70)0.970.491.000.830.0001
         Peripheral lung36 (16–43)35 (31–39)35 (28–43)54 (35–62)0.810.840.630.44<0.0001
        ABS
         Whole lung21 (8–26)13 (7–24)6 (0–13)18 (6–25)0.030.010.140.140.21
         Peripheral lung20 (11–32)17 (5–22)7 (0–18)14 (8–25)0.050.030.150.160.03
        Cen%51 (47–57)52 (48–57)49 (46–51)52 (48–56)0.220.140.430.160.83

        Data are presented as median (interquartile range), unless otherwise stated. CF: cystic fibrosis; IS: isotonic saline; CR: single CF transmembrane conductance regulator mutation carrier; HC: healthy control; HS: hypertonic saline; MCC: mucociliary clearance rate; ABS: technetium-99m-DTPA absorption rate; Cen%: percentage of radioactive counts deposited in the central lung zone (see supplementary material). All groups inhaled IS during the MCC/ABS scan. CF subjects performed an additional study day where they inhaled 7% HS during the scan. Data are presented graphically in supplementary figures S4 and S5 (CF HS versus CF IS). p-values comparing all groups by Kruskal–Wallis (nonparametric). Group comparisons by Dunn's test with Holm adjustment (nonparametric, multiple comparisons). HS versus IS comparison for CF group by Wilcoxon matched-pairs signed-ranks test (nonparametric, paired).

        • TABLE 4

          Variables that correlated with therapeutic response to hypertonic saline (HS) in the cystic fibrosis group

          β0β1R2p-value
          Baseline MCCadjusted31.62−0.440.200.01
          HNE ASL FRAP diffusion time−23.5211.230.550.04

          MCCadjusted: mucociliary clearance adjusted based on aerosol distribution; HNE: human nasal epithelial; ASL: airway surface liquid; FRAP: fluorescence recovery after photobleaching. Therapeutic response (=β1 (listed variable)+β0) is the improvement in MCCadjusted after inhaling HS compared with a baseline measurement made after isotonic saline inhalation.

          • TABLE 5

            Multivariable regression models of forced expiratory volume in 1 s (FEV1) % pred in the cystic fibrosis (CF) group

            ModelGroupModel ofAge (years)Chronic Pseudomonas aeruginosa#Sex (female)Sweat chloride (mmol·L−1)HNE ASL FRAPModel
            β1p-valueβ2p-valueβ3p-valueβ4p-valueβ5p-valueR2p-valueβ0
            1CFFEV1 % pred (n=25)−1.10.001−22.70.007−10.90.12−0.260.050.66<0.0001145
            2CFFEV1 % pred (n=10)¶−0.850.11−30.00.09−27.80.03−0.210.45−13.80.020.910.01187
            3CFFEV1 % pred (n=10)¶−1.10.09−7.950.70−24.80.07−0.250.570.760.03142

            MCC: mucociliary clearance; P. aeruginosa: Pseudomonas aeruginosa; HNE: human nasal epithelial; ASL: airway surface liquid; FRAP: fluorescence recovery after photobleaching. FEV1 % pred=β1(age)+β2(P. aeruginosa)+β3(sex)+β4(sweat chloride)+β5(FRAP)+β0. #: chronic P. aeruginosa is defined as two or more positive throat or sputum cultures in the previous year; ¶: only a portion of the CF group had FRAP measurements available (n=10).

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              Supplementary material 00382-2022.SUPPLEMENT

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            Nasal epithelial cell culture fluorescence recovery after photobleaching predicts cystic fibrosis therapeutic response
            Timothy E. Corcoran, Carol A. Bertrand, Michael M. Myerburg, Daniel J. Weiner, Sheila A. Frizzell, Anna Li, Brittani Agostini, Robert S. Parker, Monica E. Shapiro, Ashok Muthukrishnan, Nicholas D. Hages, Brian P. Mulhern, Joseph M. Pilewski
            ERJ Open Research Oct 2022, 8 (4) 00382-2022; DOI: 10.1183/23120541.00382-2022

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            Nasal epithelial cell culture fluorescence recovery after photobleaching predicts cystic fibrosis therapeutic response
            Timothy E. Corcoran, Carol A. Bertrand, Michael M. Myerburg, Daniel J. Weiner, Sheila A. Frizzell, Anna Li, Brittani Agostini, Robert S. Parker, Monica E. Shapiro, Ashok Muthukrishnan, Nicholas D. Hages, Brian P. Mulhern, Joseph M. Pilewski
            ERJ Open Research Oct 2022, 8 (4) 00382-2022; DOI: 10.1183/23120541.00382-2022
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