Brushed nasal epithelial cells are a surrogate for bronchial epithelial CFTR studies

Abstract

Recent advances in the management of cystic fibrosis (CF) target underlying defects in the CF transmembrane conductance regulator (CFTR) protein, but efficacy analyses remain limited to specific genotype-based subgroups. Patient-derived model systems may therefore aid in expanding access to these drugs. Brushed human nasal epithelial cells (HNEs) are an attractive tissue source, but it remains unclear how faithfully they recapitulate human bronchial epithelial cell (HBE) CFTR activity. We examined this gap using paired, brushed HNE/HBE samples from pediatric CF subjects with a wide variety of CFTR mutations cultured at the air-liquid interface. Growth and structural characteristics for the two cell types were similar, including differentiation into mature respiratory epithelia. In electrophysiologic analysis, no correlation was identified between nasal and bronchial cultures in baseline resistance or epithelial sodium channel (ENaC) activity. Conversely, robust correlation was demonstrated between nasal and bronchial cultures in both stimulated and inhibited CFTR activity. There was close correlation in modulator-induced change in CFTR activity, and CFTR activity in both cell types correlated with in vivo sweat chloride measurements. These data confirm that brushed HNE cell cultures recapitulate the functional CFTR characteristics of HBEs with fidelity and are therefore an appropriate noninvasive HBE surrogate for individualized CFTR analysis.

Keywords: Epithelial transport of ions and water; Ion channels; Monogenic diseases; Pulmonology.

Figure 1. Center-specific growth characteristics of brushed human nasal and bronchial epithelial cells (bHNEs and bHBEs).

(A) Initial sample cell count for bHNEs (white circles, n = 102) and bHBEs (gray circles, n = 22). **P < 0.01 by 2-tailed t test; bars represent mean ± SEM. (B) Cumulative cell growth. Note that the passage 0 (P0) time point reflects the initial cell counts shown in A; error bars indicate SEM. (C) Time between each passage, with no significant difference between bHNE (white bars) and bHBE (gray bars) cultures at any passage. Boxes represent median (center line), 25th and 75th quartiles (box limits), and minimum/maximum (whiskers) values. Note that in B and C, the number of ongoing cultures in each tissue type dwindles with advancing passage (P1: 87/22; P2: 32/15; P3: 13/3, P4: 3/3 for bHNE/bHBE, respectively) due both to cell-specific (e.g., contamination) and protocol-specific (e.g., systematic banking at P2) factors. Using multiple t tests (with Holm-Šidák correction), no significant difference was noted between bHNE and bHBE total cell count (B) or time to passage (C) at any passage beyond P0.

Figure 2. Brushed human nasal and bronchial epithelial cells (bHNEs and bHBEs) share similar characteristics of a mature respiratory epithelium.

(A) Detection of adherens junctions (E-cadherin, green), mucus-producing cells (muc5AC, red), and ciliated cells (acetylated α-tubulin, purple) in both bHNE (left) and bHBE (right) samples. (B) Similar CFTR (green) detection between paired bHNE (left) and bHBE (right) samples. All samples were taken from subject J (Table 1) and are representative of other subjects (3 paired samples; see Supplemental Figure 2 for additional subject images), though CFTR staining varied with mutation. Scale bars: 25 μm

Figure 3. There is poor correlation between brushed human nasal epithelial cells (bHNEs and bHBEs) for baseline resistance and epithelial sodium channel (ENaC) activity.

Baseline resistance values for bHNE (white bars) and bHBE (gray bars) samples for each subject (A) and aggregate data for all donors (B). ENaC activity (as measured by amiloride-inhibited short-circuit current [I_sc]) in bHNE (white bars) and bHBE (gray bars) samples for each subject (C) and aggregate data for all donors (D). (E) Baseline resistance in bHNE (x axis) and bHBE (y axis) is plotted for each subject; linear regression was nonsignificant. (F) ENaC activity is similarly plotted, with no significant correlation. n = 3–6 inserts per sample and condition; individual replicates are noted by circles in A and C to demonstrate distribution. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-way ANOVA with Tukey’s correction for multiple comparisons was used for A and C, while unpaired, 2-sided t tests were used for B and D. Individual sample labels correlate with donor codes shown in Table 1. Error bars in A, C, E, and F represent SEM. Boxes in B and D represent median (center line), 25th and 75th quartiles (box limits), and minimum/maximum (whiskers) values.

Figure 4. Stimulated and inhibited CFTR activity correlate well between brushed human nasal and bronchial epithelial cells (bHNEs and bHBEs).

(A) Both stimulated (forskolin/IBMX, white; VX-770, light gray) and inhibited (Inh172, dark gray) CFTR-dependent short-circuit current (Isc) for paired bHNE (solid bars) and bHBE (hatched bars) samples for all subjects. (Note the different y-axis scales used for subjects A–F to the left and subjects G–J to the right.) (B) Aggregate data for all donors. (C) Total stimulated CFTR current (forskolin/IBMX+VX-770) in bHNEs (x axis) and bHBEs (y axis) are plotted for each subject; linear regression with 95% CI (dotted lines) is shown. (D) Inhibited CFTR currents (Inh172) are similarly plotted; linear regression with 95% CI (dotted lines) is shown. n = 3–6 inserts per sample and condition; individual replicates are noted by circles in A (stimulated: forskolin/IBMX+VX-770) to demonstrate distribution. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two-way ANOVA with Tukey’s correction for multiple comparisons was used to compare stimulated (forskolin/IBMX+VX-770) and inhibited (Inh172) CFTR currents in A, while unpaired, 2-sided t test was used for the same comparison in B. Individual sample labels correlate with donor codes shown in Table 1. Error bars in A, C, and D represent SEM. Boxes in B represent median (center line), 25th and 75th quartiles (box limits), and minimum/maximum (whiskers) values.

Figure 5. Absolute VX-809–induced changes in CFTR currents correlate well for same-subject brushed human nasal and bronchial epithelial (bHNE and bHBE) samples.

(A) Absolute change in amiloride-inhibited currents in VX-809–pretreated bHNE (x axis) and bHBE (y axis) paired samples; linear regression was nonsignificant. Absolute VX-809–induced change in stimulated (forskolin/IBMX+VX-770) (B) and inhibited (Inh172) (C) CFTR currents for paired bHNE (x axis) and bHBE (y axis) samples, along with linear regression characteristics with 95% CI (dotted lines). n = 3-6 inserts per sample and condition. Individual sample labels correlate with donor codes shown in Table 1. Error bars represent SEM.

Figure 6. Similar, nonlinear inverse correlation between CFTR activity and sweat chloride (SC) in both brushed human nasal and bronchial epithelial cells (bHNEs and bHBEs).

Stimulated CFTR short-circuit current (Isc; forskolin/IBMX+VX-770, y axis) is plotted against SC values (x axis) for both bHNE (white circles) and bHBE (gray circles) samples. Similar nonlinear (log-log) regression lines are plotted for bHNE (solid line) and bHBE (dotted line) samples; regression values are as indicated. For subject J (2 paired samples), Isc values from repeated samples were averaged. n = 1 SC measurement for each subject, 3–6 inserts per tissue sample per subject. Individual sample labels correlate with donor codes shown in Table 1. Error bars represent SEM.