AE (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.doi:10.1371/journal.pbio.1000155.g005
The epithelial Na+ channel (ENaC) is rate-limiting for Na+ absorption and is negatively regulated by CFTR expression [24]–[26]. Since ENaC activity is regulated by mediators present in the ASL (e.g., nucleotides and proteases [27],[28]), and Ussing chamber (“thick-film”) studies result in washing away of these critical ENaC regulatory factors, we determined how CFTR delivery to CF ciliated cells affected the regulated activity of ENaC under thin-film conditions using microelectrodes. The change in Vt, in response to the Na+ channel blocker, benzamil (10−5 M), indicated that Na+ transport accounted for approximately 40% of the total Vt in non-CF HAE (Figure 5A, white bars) and approximately 80% of transport in CF HAE (black bars), consistent with a Na+ hyperabsorptive phenotype for CF airway epithelia. Expression of CFTR, but not GFP, in CF ciliated cells significantly reduced the contribution of CF HAE Na+ transport to levels measured in non-CF HAE (Figure 5A, red bars). These data show that delivery of CFTR to CF ciliated cells restored both Cl− secretion and the regulation of Na+ absorption by CF airway epithelia to normal, non-CF HAE levels. The simplest conclusion drawn from these data is that ENaC and CFTR both reside in ciliated cells in the human airway epithelium.
CFTR Expression in CF Ciliated Cells Restores Regulation of Apical Surface Hydration
During these experiments, it was noted that the lumenal surfaces of PIVCFTR-corrected CF HAE appeared hydrated compared to the dehydrated surfaces of CF HAE, suggesting that the rebalancing of Na+ absorption and Cl− secretion consequent to delivery of CFTR to ciliated cells restored hydration to the lumenal surfaces of CF HAE. Therefore, we initiated experiments to measure ASL height regulation in CF HAE in the absence or presence of transduced CFTR in ciliated CF cells.
ASL volume regulation was assessed by measuring ASL height with XZ-plane confocal microscopy 48 h after addition of 25 µl of PBS to the apical surfaces of CF HAE. In control CF HAE (inoculated with mock or PIVGFP), CF epithelia absorbed almost all fluid from their surfaces, resulting in an ASL height of 3 µm, i.e., the minimal space of compacted folded-over cilia and consistent with mucostasis (Figure 5B) [29]. However, in PIVCFTR-corrected CF HAE, ASL height stabilized at approximately 8 µm, a height similar to that of non-CF HAE (Figure 5B). These data show that delivery of CFTR to CF ciliated cells fully restored the regulation of ASL height to levels maintained by non-CF HAE, thus establishing the critical role of CFTR and ciliated cells in ASL height homeostasis.
We next investigated whether the depletion of ASL over time impaired cilia beat by measuring ciliary beat frequency (CBF) of CF HAE under thin-film conditions. Immediately after addition of 25 µl of PBS to the apical surface of CF HAE, CBF was approximately 8 Hz, a value not different than exhibited by non-CF HAE (Figure 5C). However, consistent with the decreased ASL height in control CF HAE, effective CBF was reduced 48 h later in CF HAE compared to non-CF HAE (Figure 5C). In contrast, effective CBF was maintained in PIVCFTR-inoculated, but not PIVGFP-inoculated, CF HAE at levels similar to those measured in non-CF HAE (Figure 5C). These data strongly argue that ineffective cilia beat observed in CF HAE reflects a defect in ASL height regulation, not ciliary function, and restoration of ASL height regulation with PIVCFTR is sufficient to restore effective cilia beat in CF HAE.
CFTR Expression in CF Ciliated Cells Restores Effective MCT
A novel feature of the HAE model is the recapitulation of MCT, reflecting coordinated cilia beat that produces rotational flow of mucus over hydrated epithelial apical surfaces [6]. In CF HAE, the rotational flow is abolished due to ASL depletion, mimicking mucostasis described for CF airways in vivo [6]. To determine whether expression of CFTR in CF ciliated cells could prevent mucostasis, CF HAE were inoculated with PIVCFTR, PIVGFP, or mock and, 24 h later, a small bolus of 1-µm fluorescent beads added to the apical surfaces and cultures maintained at >95% humidity for 24 h. For control CF HAE, rotational flow of beads was rarely observed at 48 h pi (Figure 5D, 5Ei, and 5Eii), i.e., mucostasis occurred. In contrast, PIVCFTR-corrected CF HAE exhibited significant rotational flow of beads, indicating that MCT had been restored (Figure 5D and 5Eiii). Under these conditions at 48 h pi, PIVCFTR restored approximately 50% of the MCT measured in parallel non-CF HAE (Figure 5D and 5Eiv). These data are the first demonstration, to our knowledge, that MCT can be restored to CF airway epithelia by delivering CFTR to ciliated cells, indicating that the cumulative effects of CFTR deficiency on mechanical innate defense can be reversed by this strategy.
Why MCT was not completely restored to normal levels, especially when ion transport processes and ASL volume regulation were fully corrected, remains to be determined. A possible explanation may relate to potentially subtle cytopathic effects of PIV infection at 48 h pi. To address this possibility, we inoculated non-CF HAE with PIVGFP or PIVCFTR, and assessed MCT 48 h later. PIVGFP decreased MCT to 40±3% (n = 5) of normal levels, whereas PIVCFTR decreased MCT to approximately 74±11% (n = 5) of normal levels. These data strongly suggest that after 48 h of PIV infection, virus-induced cytopathic events, likely linked to virus replication capacity, limit complete restoration of MCT to non-CF levels.
Duration of CFTR correction is limited by shedding of PIV-infected ciliated cells. Experiments to determine the duration of PIVCFTR-mediated bioelectric correction showed that significant functional correction was maintained for at least 1 wk with detectable, but decreased, levels of function remaining at 21 d pi (Figure 6A). Previously, we have shown that PIV-infected ciliated cells are shed from HAE 3–7 d pi by a poorly understood process of extruding infected ciliated cells from the epithelium onto the lumenal surfaces of HAE [17]. This process likely represents an innate defense function of the epithelium to rid itself of PIV-infected ciliated cells. To confirm that PIV-infected ciliated cells were being shed from the epithelium, we assessed the cellular composition of apical secretions 6 d after PIVGFP inoculation. By morphologic and immunodetection of GFP-positive cells and ciliated cells, we determined that PIV-infected ciliated cells were shed into apical secretions at a rate far exceeding that of natural ciliated cell shedding (Figure 6B). Therefore, the temporally related loss of CFTR functional activity in PIVCFTR-corrected CF HAE likely reflects shedding of infected ciliated cells and suggests that the extent of correction is directly related to the numbers of ciliated cells expressing CFTR. To further explore this relationship, we counted the numbers of PIV-positive cells present in CF HAE over time and show that the loss of PIV-positive ciliated cells paralleled the loss of Cl− transport (compare Figure 6C to 6A), suggesting that the magnitude of correction was indeed directly proportional to the number of CF ciliated cells expressing CFTR.
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),