Figure 1.
Infection of human ciliated cells by PIV in vitro and ex vivo.
(A) Scanning electron micrograph of HAE showing cilia and mucus on the luminal surface. (B) Histological cross-section of HAE showing pseudostratified, columnar airway epithelium with ciliated (cc) and mucin-secreting cells (mc) and basal epithelial cells (bc). (C) Representative confocal XZ sections of HAE or (D) histological sections of human tracheobronchial tissue inoculated with PIVGFP (106 PFU) and GFP expression assessed at 24 h pi. GFP was detected by indirect immunofluorescence with rabbit anti-GFP polyclonal antibodies (Ab) and goat anti-rabbit IgG-fluorescein (green). Ciliated cells were identified using mouse primary Ab against acetylated α-tubulin and detected with anti-mouse IgG-Texas Red (red). GFP colocalized to cells that were also positive for acetylated α-tubulin, confirming the targeting of ciliated cells in vitro and ex vivo by PIV. Bar represents 20 µm in (A); and 5 µm in (B, C, and D).
Figure 2.
Efficient targeting of CF ciliated cells by PIVCFTR.
(A–D) Representative immunodetection of human PIV fusion (F) glycoprotein (red) (A and B) en face or (C and D) in histological sections of CF HAE 48 h pi with PIVGFP (A and C) or PIVCFTR (B and D). F glycoprotein was detected by indirect immunofluorescence with a murine monoclonal anti-F Ab and anti-mouse IgG conjugated to AlexaFluor 594 (red). GFP-positive cells were identified in histological sections (C), using rabbit anti-GFP conjugated to fluorescein (green). Fluorescence was viewed using a Texas Red filter only (A and B) or a combined Texas Red-FITC-UV-filter (C and D). Bars represent 200 µm and 10 µm for (A and B, and C and D), respectively. (E and F) Quantitation of (E) numbers of PIV F-positive ciliated cells showing that PIVGFP and PIVCFTR infect similar numbers of ciliated cells at 48 h pi (n = 8); and (F) the efficiency of ciliated cell infection by PIVCFTR assessed at 24 h was similar with 5-min, 2-h, or 8-h inoculation times (n = 6). (G) CXCL8 protein levels secreted by HAE 48 h pi with mock, UV-inactivated PIV, PIVGFP, or PIVCFTR showing reduced levels for PIVCFTR versus PIVGFP. Responses positive/negative of the abscissa reflect CXCL8 secretion into apical/basolateral compartments, respectively (n = 5 for each). (H) Viral titers in apical compartment at 48 h pi for PIVGFP (green) and PIVCFTR (red) showing attenuated growth of PIVCFTR (n = 5 for each). An asterisk (*) denotes p<0.05; ns, not significantly different. Error bars indicate SEM .
Figure 3.
Expression of functional CFTR in CF ciliated cells targeted by PIV.
(A) CFTR mRNA levels in CF HAE 48 h after inoculation with PIVGFP or PIVCFTR and relative to mRNA levels in CF HAE mock-inoculated (n = 12). (B) Representative western blot of CFTR protein in lysates of CF HAE 48 h pi with mock (lane 1), PIVGFP (lane 2), and PIVCFTR (lanes 3–5), with lanes 4 and 5 representing serial 10-fold dilution of cell lysates. For comparison, non-CF HAE lysates also were included to detect endogenous CFTR levels (lane 6). Markers indicate the fully glycosylated mature form of CFTR (Band C) and immature nonglycosylated CFTR (Band B). Data are representative of experiments with cells derived from two separate patients. (C) Representative confocal images of CFTR immunoreactivity in CF HAE (i and ii), and non-CF HAE (iii and iv), 48 h after inoculation with PIVGFPCFTR (i and iii) or PIVGFP (ii and iv). CFTR was detected with CFTR monoclonal Ab (clone 596) and secondary Abs conjugated to AlexaFluor 594 (red). CF HAE inoculated with PIVGFPCFTR (i) showed immunolocalization of CFTR at apical domains of ciliated cells that were also GFP-positive, but not in GFP-negative ciliated cells or in CF HAE infected with PIVGFP (ii). Robust apical domain CFTR and GFP were detected in non-CF HAE ciliated cells inoculated with PIVGFPCFTR and endogenous CFTR levels detected in GFP-negative ciliated cells (iii, arrowheads). Endogenous CFTR was detected at the apical membranes of GFP-positive and -negative ciliated cells after inoculation with PIVGFP (iv, arrowheads). Bar represents 5 µm. (D) Representative traces of short-circuit current measurements (Isc) from CF HAE 48 h pi by PIVCFTR, PIVGFP, or mock showing post amiloride responses to sequentially added forskolin (Fsk), and CFTR172. A representative Fsk-induced Isc response by a non-CF HAE is shown for comparison. No Fsk responses were seen in CF HAE inoculated with PIVGFP or mock, consistent with the absence of CFTR. (E) Summary data for Fsk-activated changes in Isc (ΔIsc) in CF HAE 48 h pi with mock, PIVGFP, PIVΔF508CFTR, or PIVCFTR. Fsk responses for non-CF HAE are shown as comparison. CFTR delivery to ciliated cells resulted in restoration of functional Cl− channel activity in CF HAE to levels exhibited in non-CF HAE. Each bar represents at least 11 cultures from four different patients. ns denotes not significantly different. Error bars indicate SEM.
Figure 4.
CFTR activity in ciliated cells is regulated by factors other than CFTR levels.
(A) Percentage inhibition of Fsk-activated CFTR by CFTR172 applied to either the apical (AP) or basolateral (BL) surfaces of PIVCFTR-corrected CF HAE. CFTR activity was inhibited by apical, but not basolateral, addition of CFTR172 for at least 15 min (n = 4). (B) Driving force for Cl− secretion does not dictate magnitude of the Fsk response determined by measurement of Fsk-mediated ΔIsc under physiological Cl− concentration (KBR) or Cl−-free solutions (HKLC) in CF HAE 48 h after inoculation by PIVGFP (green bars) or PIVCFTR (red bars) and compared to non-CF HAE (white bars) (n = 8). (C) UTP-mediated Cl− secretion in KBR (solid bars) and HKLC (hatched bars) bathing solutions after inoculation of CF HAE with vehicle alone (black bars), PIVGFP (green bars), or PIVCFTR (red bars) (n = 6). Note that under conditions of increased driving force for Cl− secretion, responses far exceed the maximal responses achieved with Fsk-mediated CFTR activation in the presence of overexpressed CFTR. (D) Fsk-induced ΔISC in non-CF HAE inoculated with mock (black bars), PIVGFP (green bars), or PIVCFTR (red bars), showing that overexpression of CFTR on top of endogenous CFTR does not significantly enhance the Fsk-mediated secretory response under physiological conditions (n = 5 for each). ns denotes not significantly different. Error bars indicate SEM .
Figure 5.
Expression of CFTR in CF ciliated cells restores normal ASL homeostasis and MCT to CF HAE.
(A) The contribution of Cl− and Na+ to transepithelial electrical potential difference (Vt) under thin-film conditions in CF HAE mock-inoculated (black bars), PIVGFP (green bars), or PIVCFTR (red bars). Vt changes for non-CF HAE are shown for comparison (white bars). Bar graphs depict percentage change in Vt in response to Cl− channel inhibitor bumetanide (Bum) and Na+ channel inhibitor benzamil (Bum/Benz). Each bar represents eight cultures derived from three different patients, and an asterisk (*) indicates p<0.05, and ns indicates not significant. (B) ASL height measurements 24 h after apical addition of 25 µl of PBS containing Texas Red dextran to CF HAE 48 h pi with mock (black), PIVGFP (green), or PIVCFTR (red) and compared to ASL height in non-CF HAE (white). Each bar represents nine cultures from three patients. (C) Cilia beat frequency measurements (CBF in beats per second [bps]) from CF HAE 24 h after the addition of 25 µl of PBS to CF HAE 48 h pi with mock (black), PIVGFP (green), or PIVCFTR (red) and compared to non-CF HAE (white). Each bar represents six cultures derived from two patients. (D) MCT rates measured 24 h after bead addition to CF HAE inoculated with mock (black), PIVGFP (green), or PIVCFTR (red). MCT for non-CF HAE are shown for comparison (white). Each bar represents at least nine cultures derived from three patients. (E) Representative photomicrographs showing time-lapse (3-s exposures) movement 24 h after addition of green fluorescent microspheres (as an index of MCT) on CF HAE 48 h after inoculation with mock (i), PIVGFP (ii), or PIVCFTR (iii). Note: GFP-positive cells are also observed below beads in PIVGFP-inoculated CF HAE. Bar represents 60 µm, and asterisk (*) denotes p<0.05, and ns not significantly different compared to non-CF HAE (iv). Error bars indicate SEM.
Figure 6.
Duration of CFTR functional correction is limited by shedding of PIV-infected ciliated cells.
(A) Duration of CFTR functional activity in PIV corrected CF HAE. CF HAE were inoculated with PIVCFTR (red bars) or PIVGFP (green bars) at day 0, and ion transport studies were performed at days specified (n = 8 cultures derived from 3 different patients). Significant CFTR function could be measured for up to 8 d but waned by 21 d pi in CF HAE inoculated with PIVCFTR. No significant CFTR function was detected in CF HAE inoculated with PIVGFP. (B) Histological Cytospin assessment of cells shed into apical surface secretions at day 6 pi showing few shed ciliated cells in mock-inoculated CF HAE (i and iii), whereas significant numbers of cells were shed after PIVGFP (ii and iv). Cytospinned apical washes were counterstained with Giemsa (i and ii) or probed with Abs to GFP (green) and acetylated alpha-tubulin (red) to show ciliated cells. Bar represents 20 µm. Images are representative of Cytospins from two individual experiments. (C) Loss of PIV F-positive cells over time after inoculation with PIVCFTR (red bars) or PIVGFP (green bars), showing that PIVCFTR and PIVGFP infected equal numbers of ciliated cells at day 2, but that ciliated cell shedding was delayed for PIVCFTR versus PIVGFP by day 8. Data derived from same dataset in (A). An asterisk (*) denotes p<0.05. Error bars indicate SEM.
Figure 7.
How many CF airway epithelial cells require CFTR to restore ASL homeostasis and MCT to non-CF HAE levels?
(A) Quantitation of the percentage of PIV F-positive cells 24 h after inoculation with different concentrations of PIVCFTR (103–106 PFU) and correlation of percentage of PIVCFTR-positive cells to: (B) increased CFTR mRNA expression levels; (C) Fsk-activated changes in Isc; (D) amil-induced changes in Isc; (E) ASL height; and (F) MCT rates. All measurements were made 24 h after inoculation with PIVCFTR (closed red circles) or controls (PIVGFP or PIVΔF508CFTR, closed green triangles) as described in Materials and Methods. n>11 for each data point representing cultures from 2 or 3 different donors. Dashed lines and grey shaded regions represent mean and standard deviations of ASL heights and MCT rates measured in parallel experiments with non-CF HAE. For these experiments, approximately 80% of surface cells were ciliated cells. (G) Schematic representation depicting the role of CFTR and ENaC in ASL homeostasis in non-CF airway epithelium; the presence of CFTR modulates ENaC activity and combined regulation of these ion channels dictates ASL depth regulation at a level sufficient for effective MCT (black arrow). In CF airway epithelium, the absence of CFTR reduces fluid secretion and leads to dysregulation of ENaC activity overall resulting in hyperabsorption of surface fluid, dehydration of ASL, and mucostasis with accumulation of mucus plugs. Delivery of CFTR to ciliated cells of CF HAE by PIV restores CFTR function, ENaC regulation, ASL homeostasis, and MCT. Error bars indicate SEM.