Animals

This study was approved by the University of Adelaide animal ethics committee under application M-2019-038 and was conducted in accordance with ARRIVE guidelines. Male and female wild-type, Phe508del and CFTR knockout rats >8 week of age were employed. CF rats were maintained using previously described husbandry measures [5].

Rat tracheal epithelial cell isolation and air-liquid interface (ALI) culture

Rats were humanely killed by CO2 asphyxiation. Tracheal epithelial cells were isolated by pronase dissociation as previously described [22]. Briefly, tracheas were cannulated, excised, and infused with 1% pronase (Roche, Switzerland) solution in Ham’s F12 media (Sigma-Aldrich, MO, USA) supplemented with 1% PenStrep, 0.05 mg/mL Gentamicin, and 0.25 µg/mL Amphotericin B. Tracheas were incubated for 18-24 hours at 4 °C. On day two, epithelial cells were retrieved by flushing the tracheas with 30 mL Ham’s F12 media. Cells were collected by centrifugation (500 x g, 4 °C, and 10 mins) and incubated on ice with 0.5 mg/mL DNase (Sigma-Aldrich) and 10 mg/mL bovine serum albumin (BSA) (Sigma-Aldrich) to reduce cell clumping. Cells were spun down, resuspended in PneumaCult Ex-Plus media (#5040, StemCell Technologies, Canada) and assessed for number and viability. Cells were seeded directly onto 6-well Snapwell inserts (#CLS3407, Corning, NY, USA) pre-coated with 1:100 rat-tail collagen type I in phosphate-buffered saline (PBS) at a density of 300,000 cells per well. Once confluent, the growth media was removed from both apical and basolateral compartments and the basolateral compartment only was replaced with PneumaCult ALI media (#5001, StemCell Technologies). Media in the basolateral chamber was refreshed every 48 hours.

Immunohistochemistry (IHC)

Inserts were fixed in 10% neutral buffered formalin for 15 minutes, embedded in paraffin wax, and sectioned at 5 μm. Sections were deparaffinised and stained with hematoxylin and eosin (H&E), or processed for IHC as follows. Heat-mediated antigen retrieval was performed in sodium citrate buffer (pH 6.0) for 20 minutes. Sections were blocked with 1% BSA and 0.05% Tween-20 for one hour at room temperature. Primary antibodies were prepared in a dilution buffer consisting of 1% BSA and 0.1% Triton X-100 in PBS. Antibodies that were used included acetyl-alpha tubulin (5335, Cell Signalling Technology, MA, USA) at 1:500 dilution, cytokeratin 5 (ab52635, Abcam, United Kingdom) at 1:400 dilution and MUC5AC (MA5-12178, Invitrogen, MA, USA) at 1:200 dilution. Sections were incubated with primary antibodies overnight at 4 °C. Slides were washed thoroughly and incubated at room temperature for one hour with secondary antibodies donkey anti-rabbit (IgG) Alexa Fluor 488 (ab150073, Abcam) or goat anti-mouse (IgG) Alexa Fluor 568 (A-11004, Invitrogen) at a 1:200 dilution in antibody dilution buffer. The secondary antibody was washed and a DAPI nuclear counterstain was applied (4083S, Cell Signalling Technology). Slides were washed and mounted with ProLong Diamond Antifade mountant (P36970,  Invitrogen). Bright field images were captured on a Nikon Eclipse E400 microscope and fluorescence images on a Nikon Eclipse Ts2 with DS-Fi3 camera and NIS-elements D software version 4.20.02.

Transepithelial electrical resistance (TEER) measurements

At 7-10 days post air-lift, multilayered cells, mucus and cilia were all visible under light microscopy suggesting terminal differentiation of cultures. TEER measurements were then performed to ensure the appropriate formation of cell junctions and cell integrity. Using an epithelial voltammeter (Millicell-ERS voltmeter, Millipore) with silver chloride “chopstick” electrodes, triplicate measurements of TEER were obtained for each well followed by Ussing chamber measurements. Resistance obtained from a cell-free culture was subtracted from that measured across each culture and corrected for surface areas of inserts (1.1 cm2) to yield the TEER of the epithelial cells with values expressed in Ω/cm2.

Short-circuit current (Isc) measurements

Baseline CFTR function was assessed in wild-type (n = 14), Phe508del (n = 13) and KO (n = 12) rat ALI cultures that were generated from individual animals. At 7–10 days post air-lift the transepithelial short-circuit current (Isc) was measured using an Ussing chamber (VCC MC6, Physiologic Instruments, NV, USA) as previously described [23]. Cells grown on Snapwell inserts were mounted into sliders (P2302, Physiologic Instruments) in the Ussing chamber and submerged in Krebs Ringer Buffer (KRB) that was bubbled with carbogen (95% O2, 5% CO2). After compensating for voltage offsets, the transepithelial voltage was clamped at 0 mV and current and resistance were recorded with the Acquire and Analyze software (Physiologic Instruments). To assess chloride ion transport, sodium transport was blocked with the addition of amiloride bilaterally (10 µM final concentration) before stimulation of cAMP-mediated chloride transport with the addition of forskolin (10 µM final concentration) and blocking of CFTR transport using CFTRinh-172 (30 µM final concentration). Appropriate periods of equilibration between each addition were used. To calculate changes in Isc, the average current measured over the 60 seconds following amiloride addition was subtracted from the average current after forskolin measurement.

CFTR modulator treatment of rat ALI cultures

Differentiated rat ALI cultures generated from individual animals (n = 8) were treated via the basolateral chamber with modulators 24 hours prior to short-circuit current measurements. Cells were treated with 3 µM elexacaftor (VX-445), 18 µM tezacaftor (VX-661), and 1 µM ivacaftor (VX-770) (Selleck Chemicals, TX, USA) as previously described [24]. A DMSO vehicle control was included with no differences observed to the untreated controls (data not shown). Following treatment, TEER and short-circuit current measurements were performed as described above.

LV-GFP vector transduction of rat airway epithelial cells

The VSV-G (vesicular stomatitis virus glycoprotein) pseudotyped EF1α-3XFLAG-fLuc-eGFP LV vector was produced in-house and was titered using flow cytometric detection of green fluorescent protein (GFP) as previously described [25]. Wild-type rat airway epithelial cells were seeded into collagen-coated 6-well plates (#152034, Thermo Fisher Scientific, MA, USA) at 25,000 cells per well and were maintained in F-medium containing ROCK inhibitor as previously described [23]. Following adhesion, cells were transduced at a multiplicity of infection (MOI) of 1, 10 or 100 with the appropriate volume of EF1α-3XFLAG-fLuc-eGFP vector at a titre of 1 ×109 transducing units (TU)/mL. Each MOI was tested in triplicate over two separate experiments (n = 6 per group in total). The following day, the media was refreshed. Three days post-transduction cells were imaged via fluorescent microscopy (Nikon Eclipse Ts2). Cells were harvested for flow cytometry to determine the proportion of GFP positive cells and to confirm their identity as basal cells. Cells were fixed in 4% paraformaldehyde, permeabilised in 0.1% saponin and stained with anti-cytokeratin 5 antibody (ab52635, Abcam) at a 1:100 dilution for 30 minutes. Cells were washed and stained with Goat anti-rabbit IgG Alexa Fluor 568 (A-11011, Invitrogen) at 1:500 dilution for 30 minutes. Cells were washed and stored overnight at 4 °C in PBS. Samples were measured using an LSRFortessa (BD biosciences, NJ, USA) and analysed using FlowJo software (v10).

LV-CFTR vector treatment of CF rat airway epithelial cells

The VSV-G pseudotyped EF1α-V5-CFTR LV vector was produced by the Functional Genomics South Australia Core Facility (FGSA) at the University of Adelaide. Vector titration was performed by transducing HEK 293T cells and detecting the V5 epitope tag by flow cytometry, as previously described [18]. The titre of the vector employed was 1.5 ×108 TU/mL. Tracheal cells were isolated from Phe508del (n = 5) and KO rats (n = 5) and were seeded onto inserts at a density of 300,000 cells per well. After 24 hours, reference wells were used to determine the average number of cells for calculating the MOI. An appropriate volume of LV vector was applied to each well to achieve an MOI of 1, and cells were incubated for approximately 24 hours. The following day the media was replaced, and the cells were airlifted. Following differentiation, TEER and short-circuit current measurements were performed as described above.

CFTR gene expression analyses by qPCR

Differentiated ALI cultures were lysed in PureLink lysis buffer (Thermo Fisher Scientific) and total RNA was extracted using the RNeasy® kit (Qiagen, Germany) following the manufacturer’s instructions. The quantification and evaluation of the purity of RNA samples was assessed using the NanoDrop™ Lite spectrophotometer (Thermo Fisher Scientific). Reverse transcription of RNA to synthesise complementary DNA was performed using the QuantiTect Reverse Transcription Kit (Qiagen) following the manufacturer’s guidelines. qPCR was performed using the Fast SYBR™ Green Master Mix (Thermo Fisher Scientific) following the manufacturer’s instructions, in line with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [26]. Primer sequences are included in the supplementary information (Table S1). The ability of the primers to specifically amplify either rat or human CFTR is shown in the supplementary figures (Figures S1 and S2). PCR samples were heated for 10 minutes at 50 °C followed by 3 minutes at 95 °C, then qPCR reactions were run for 40 cycles of 95 °C for 10 seconds (denaturation) and 56 °C or 60 °C for 60 seconds (combined annealing/extension) using a CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). Samples were run alongside a standard curve of known copy numbers for Cyclophilin A (CycA), rat CFTR and human LV-derived CFTR. The calculated copy number for rat CFTR and human CFTR was normalised to CycA copy number. PCR products were assessed by gel electrophoresis using a 1% agarose gel with a 100 bp DNA ladder (AXYM-DNA-100bp, Axygen, CA, USA) (Figure S3).

Statistics

Data was analysed using GraphPad Prism (v9). Where appropriate, data were assessed for normality using the Shapiro-Wilk test, and the Brown-Forsythe and Bartlett’s tests was used determine equal variances between groups. Data were analysed for statistically significant differences using one-way ANOVA with Tukey’s multiple comparisons test. A p-value of <0.05 was considered statistically significant. Graphs show the mean ± standard error of the mean (SEM) and reported in-text values are the mean ± standard deviation (SD).