Human recombinant cell culture grade TNFα was purchased from Jena Bioscience GmbH (Jena, Germany); Brefeldin A (BFA; Sigma-Aldrich, St Quentin Falavier, France, B7651); a protein kinase C inhibitor ¹⁰ , GF109203X, (Selleckchem, USA, S7208); Forskolin (from Coleus forskohlii) (Sigma-Aldrich, F6886), Amiloride hydrochloride hydrate (Sigma-Aldrich, A7410-5G); inh-172 (Sigma-Aldrich, C2992-5MG); genistein (Sigma-Aldrich, G6649-5MG). Anti-CFTR antibodies (abs): MM13-4 mouse monoclonal ab against N-terminus of CFTR, (Millipore, France, 05-581); 24-1 mouse monoclonal ab against C-terminus of CFTR (R&D Systems, MAB, 25031). Anti-tubulin abs: rabbit polyclonal anti-tubulin ab (Ab4074) and anti-NaKATPAse mouse monoclonal ab (Ab7671) were from Abcam (Paris, France). For western blot analysis, the secondary polyclonal goat anti-mouse abs were IRDye ^® 800CW (Li-Cor, Bad Homburg, Germany, 926-32210), anti-keratin 8 mouse monoclonal abs (Progen, Biotechnik GmbH, Heildelberg, Germany, 61038), and anti-NHERF1 (rabbit polyclonal Santa-Cruz Biotechnology, B2107). For immunocytochemistry, anti–Zona occludens 1 (ZO-1) rabbit polyclonal abs were purchased from Santa Cruz Biotechnology, sc-10804; secondary anti-mouse (A-11001) and anti-rabbit (A-24923) Alexa-fluor (488 and 594) IgGs were from Life technologies (Fontenay s/Bois, France). For proximity ligation, the secondary abs were provided by OLINK in the kit (DUO92004 for Duolink ^® in Situ PLA ^® Probe anti-mouse MINUS, and DUO92002 for Duolink ^® in Situ PLA ^® Probe anti-rabbit PLUS, Bioscience, Uppsala, Sweden). In situ kits for proximity ligation assay were purchased from OLINK. Human primary bronchial epithelial (HBE) cells in air-liquid interface are cultivated on microporous filters purchased from Corning Incorporated (Transwell ^® polyester membrane cell culture inserts, 6.5mm diameter, New York, USA). IL1β was obtained from ENZO life sciences (ALX-520-001-C010, Villeurbanne, France).

HeLa cell culture. HeLa cells stably transfected with the wild type CFTR plasmid construct and the mutated F508del-CFTR were kindly provided by Pascale Fanen (INSERM U955, Créteil, France). Cells were cultured in Dulbecco’s modified Eagle medium, supplemented with 10% FCS, 2mM glutamine, 100g/ml streptomycin, 100 units/ml penicillin and 250g/ml Zeocin (all reagents were from Invitrogen) in an incubator at 37°C and 5% CO ₂.

Primary Human bronchial epithelial (HBE) cell culture. Primary HBE cells were isolated from bronchial explants of CF and non-CF patients after lung transplantation, with consent approved by Comité de protection des personnes Ile de France II, 2010-05-03 A3. Cells were isolated from bronchial tissue by enzyme digestion and were cultured in differentiation medium (DMEM/F12, supplemented with 15% of fetal calf serum) on type I collagen-coated filters. Briefly, bronchial explants were washed twice with washing medium (Eagle’s minimum essential medium + antibiotics (2.5µg/ml amphotericin B, 150µg/ml tazocillin, 25µg/ml ciprofloxacin) + dithiotheitol (DTT) + DNAse) and at least twice with (Eagle’s minimum essential medium MEM) containing only antibiotics (as above) to remove DTT. Bronchial explants were then incubated for 24h in a differentiation medium containing antibiotics (150µg/ml piperacillin plus tazobactam, 25µg/ml ciprofloxacin), Amphotericin B and protease, at 4°C with constant rotation (1500rpm for 5min). Next day, 15% fetal calf serum was added to neutralize proteases, and bronchia with medium were placed on a Petri dish. Epithelial cells were scraped with a curved scalpel from the inner surface of bronchia, centrifuged (1500rpm, 7min, 4°C) and re-suspended in trypsin (incubation 10min). After that, differentiation medium with serum (FCS) was added and cells were re-centrifuged. Cells were resuspended in an appropriate volume of FCS medium (DMEM/F12, 5% FCS, non-essential amino acids, appropriate antibiotics depending on the patient’s clinical status) and counted. The cells were plated with 10 ⁶ cells/cm ² to cover apical surface of each filter coated as described above. UG2% medium (DMEM/F12, supplemented with 2% Ultroser G, appropriate antibiotics (amphotericin B, tazocillin, ciprofloxacin, concentration as above) was added to the basal side of filters. The next day, apical medium (FCS) was aspirated and cells were gently washed (to remove cells other than epithelial) with PBS-antibiotics. Starting from the second day of culture, the basal medium was changed daily. Basal medium which passed to the apical compartment was removed daily. Cells were cultured at an air-liquid interface for at least 21 days and were differentiated to form polarized epithelium, after 2 weeks of growth ¹¹ . Cell differentiation was verified with immunofluorescent staining of markers: α-tubulin for ciliated cells, Zona-occludens (ZO-1) for tight junctions, keratin 8 (K8) for simple epithelia marker, mucin 5 AC (MUC5AC) for goblet cells and CFTR. The transepithelial resistance (R _T) of cultures was measured and short circuit current (Isc) experiments were performed on cultures with at least 800Ω/cm ².

TNFα was added to the culture medium for periods of time indicated in the Results section without fetal calf serum except for path-clamp experiments.

Protein sample preparation for CFTR immunoblotting. Sample preparation protocols are described in detail elsewhere ^{12, 13} . Briefly, cells were washed on ice twice with phosphate-buffered saline (PBS) solution containing 0.1mM CaCl ₂ and 1mM MgCl ₂. PBS (Mg ²⁺- and Ca ²⁺-free) was added to scraped cells. A first centrifugation was done at 1500g at 4°C. Cells were resuspended in a hypotonic solution containing 10mM KCl, 10mM TRIS at pH7.4, 1.5mM MgCl ₂ and homogenized with a mini Potter-Elvehjem tissue grinder. Cells were centrifuged at 15000g at 4°C for 15min. The resulting supernatant was re-centrifuged for 1h at 100000g at 4°C. The pellet was resuspended in a hypotonic solution. The total amount of protein was quantified by Lowry-Folin assay ¹⁴ .

Western blot analysis. Western blot analyses were performed as described elsewhere with slight modifications ¹⁵ . Briefly, equal amounts of proteins/lane were electrophoresed on an 8% SDS-PAGE and electrotransferred onto nitrocellulose membranes over 2h at 4°C in Tris-glycine buffer (Biorad) at 200 mA. Next, nitrocellulose membranes were incubated in PBS + 0.1% tween20 (Sigma, 9005-64-5) containing 5% milk (Regilait Bio, Supermarket Simply, Paris) saturation solution for 1h. Proteins were immunoblotted for 2h with the MM-13-4 ab (1/1000). After extensive washing, the nitrocellulose membranes were incubated with anti-tubulin (1/5000) or anti-NaKATPase (1/5000) abs. Next, the last washes (three times for 30min in washing buffer: PBS plus 0.1% Tween20) were done. CFTR, tubulin or NaK-ATPase were detected using Odyssey detection system (Li-Cor, Bad Homburg, Germany). The relative protein expression was assessed using the ImageJ 1.47v software ( http://imagej.nih.gov/ij/index.html).

ELISA analysis. Analysis of interleukin-8 (IL-8) secretion was performed on basolateral culture media (UG2% medium) of primary HBE cells grown at an air-liquid interface after 24h incubation time at 37°C. Basal, non-induced, levels of IL-8 secretion were measured in the regular basolateral culture media after 24h incubation at 37°C. The effect of TNF-α on IL-8 secretion was determined on the same culture filters after 10min, 3h, and 24h incubation with 50ng/ml TNF-α. Stimulation after 10min and 3h of incubation at 37°C with TNF-α were followed by washings and incubation with regular media for 24h, which was used to measure IL-8 secretion. IL-8 secretion levels were determined using ELISA immunoassay (Human CXCL8/IL-8 Quantikine ELISA Kit from R&D Systems) following the manufacturer's instructions.

Whole-cell patch-clamp recordings. The technique for patch-clamp recordings in the whole-cell configuration has been described elsewhere ^{16, 17} . Stably transfected cells were plated in 35-mm glass bottom plates that were mounted on the stage of an inverted microscope. Patch experiments were performed at room temperature with an Axopatch 200A amplifier controlled by a computer via a digidata 1440 interface (Axon Instruments, USA). Pipettes were pulled from hard glass (Kimax 51) using a Sutter micropipette puller, and the tips were fire-polished. Current recordings were performed using the nystatin-perforated patch-clamp configuration ¹⁶ . The nystatin stock solution (50 mg/ml) was prepared daily in DMSO. The stock solution was diluted (1:250) with the internal solution, which was sonicated for 1min. The internal solution contained the following (in mM): 131 NaCl, 2 MgCl ₂ and 10 Hepes, pH 7.3 adjusted with NaOH. The bath solution contained (in mM): 150 NaCl, 1 CaCl ₂, 1 MgCl ₂, 35 sucrose and 10 Hepes-Na ⁺, pH 7.3, adjusted with NaOH.

Currents were recorded by application of regular pulses of -60 mV for 1s, with a holding potential of 0 mV and an interval of 3 s.

To establish the I-V curves, regular voltage pulses were interrupted by a series of 9 voltage jumps (1-s duration each) toward membrane potentials between -100 and +80 mV. CFTR Cl ^- currents, I _CFTR, were activated using 400 µM 8-(4-chlorophenylthio)-cAMP sodium salt (CPT-cAMP) and 100 µM 3-isobutyl-1-methylxanthine (IBMX).

When maximal stimulation was reached, cells were bathed with 5 to 50ng/ml of TNFα in the presence of CPT-cAMP and IBMX TNFα solution, and steady-state was achieved after 7 to 10 minutes.

Then 5 µM of the CFTR inhibitor, CFTR _inh172, was added to the CPT-cAMP containing perfusion solution (solution +/- TNFα). I _CFTR, defined CFTR currents as a difference in current amplitude recorded during maximum stimulation with solution +/- TNFα and maximum inhibition with CFTR _inh172. Data were analyzed using the Student’s t-test (Origin Pro 9.1 software, RITME, France); results were considered to be statistically significant if the p value was less than 0.05 (for non-parametric tests, the Mann-Whitney U test was used).

Short-circuit current experiments. For short-circuit current measurements, primary human bronchial epithelial cells (HBE) were grown on permeable filters (0.33-cm ² surface area) at an air-liquid interface for differentiation and then inserts were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA). For all measurements, a Cl ^- gradient was applied by differential composition of basal and apical Ringer solutions. The basal Ringer solution contains: 145mM NaCl, 3.3mM K ₂HPO ₄, 10mM HEPES, 10mM D-Glucose, 1.2mM MgCl ₂, and 1.2mM CaCl ₂; and apical solution contains: 145mM Na-Gluconate, 3.3mM K ₂HPO ₄, 10mM HEPES, 10mM D-Glucose, 1.2mM MgCl ₂, 1.2mM CaCl ₂. Cells were washed for a 30-min stabilization period in Ringer solutions and aerated with 95% O ₂/5% CO ₂ at 37°C. Transepithelial resistance (R _T) was measured by applying a 15mV pulse and calculating R _T by Ohm’s Law. Isc was measured with an EVC4000 Precision V/I Clamp (World Precision Instruments) and registered using a PowerLab 4/30 workstation (AD Instruments, Castle Hill, Australia). During continuous recording of Isc (in voltage-clamp mode) various inhibitors and activators were added. After stabilization of baseline Isc, amiloride (100µM) was added to the apical side of inserts to inhibit the apical epithelial sodium channel (ENaC). Then Forskolin (10µM) and IBMX (100µM) were added to apical and basolateral compartments, followed by Genistein (50µM) and then CFTR inhibitor Inh-172, added apically at a 5µM concentration.

Immunocytochemistry. HeLa cells and polarized epithelial monolayers of HBE cells were fixed with ice-cold acetone for 5min, then rinsed twice with PBS. Permeabilization was done with PBS containing 0.1% Triton X100 for 15min (PBS-T). Cells were then incubated in blocking solution (3% BSA in PBS-T) for 20min. CFTR immuno-detection by confocal microscopy (see below) was performed with p.24-1 antibody diluted 1/300 (for HeLa cells) or 1/100 (for primary HBE cells) in blocking solution, during overnight incubation at 4°C. Accompanying K8 or ZO-1 staining were done simultaneously. Following this, cells were washed four times for 5min each in PBS-T 0.1% and blocked in 10% goat serum (in PBS-T). Goat secondary IgGs conjugated to Alexa 488 and 594 were added for 30min at 1/1000 dilution in 10% goat serum. After a final four washes for 5min each, Vectashield mounting medium containing DAPI (Vector Laboratories, H-1200) was used to mount cells on microscope slides.

Confocal microscopy. Cells were visualized and images captured using Leica TCS SP5 AOBS confocal microscope (Heidelberg, Germany), equipped with 63x/1.4 oil differential interference contrast λ blue PL APO objective. Typically we performed multiple optical xy sections over the cell culture to reconstitute using the ImageJ software v.147, and the 3D reconstitution of polarized epithelia of HBE cells was performed with 3D Viewer plugin in ImageJ.

DNA proximity ligation assay. Cells were grown on round microscopy cover slips and fixed with ice-cold acetone for 5min, then rinsed twice with PBS. In the first step of the proximity ligation assay (PLA) procedure, cells were incubated in bovine serum albumin solution (blocking solution provided by O-link) for 30min at 37°C and then with either two primary anti-keratin-8 mouse monoclonal abs, or mouse monoclonal anti-NHERF1 ab and rabbit polyclonal anti-CFTR ab for 1h at 37°C. After three washes with PBS-T 0.1%, cells were incubated for 1h at 37°C with the PLA probes (secondary abs provided in the kit) specific to mouse and rabbit IgGs, coupled to the oligonucleotides. Cells were then washed three times and incubated with a mixture of ligase and oligonucleotide-connectors (sequences homologous to the oligonucleotides conjugated to PLA probes). Connectors hybridize with PLA probes only when the distance is <40 nm and form a circle which is enzymatically ligated. Following this, polymerase and nucleotides coupled to fluorochromes were added for amplification of circular oligonucleotides as a template, using the PLA probe sequences as primers. Each step of this protocol is separated by washing with PBS-0.1% tween20 solution to remove non-specific interactions. At the end of this procedure, cells were mounted on microscope slides with Vectashield mounting medium containing DAPI and signal was detected as fluorescent orange spots. PLA results are quantitative and presented as number of spots per cell.

Statistical analysis. Experiments were repeated at least three times and analyzed using the unpaired non-parametric Student’s t-test (Mann-Whitney U test) using Graphpad Prism 5 or Origin (see in patch-clamp section).

We first investigated the effect of acute TNFα treatment on HeLa cells stably transfected with F508del-CFTR as a function of time. A representative immunoblot is shown in ( Figure 1A) and the relative quantification in Figure 1B. The analysis of microsomal proteins showed that the fully glycosylated mature CFTR (band C) could be detected after a 10–30min treatment with 50ng/ml of TNF α. The effect persisted for 3–6h and decreased after 24h of treatment, suggesting that TNFα might have a very rapid correcting effect on misfolded F508del-CFTR. The same treatment performed on HeLa stably expressing WT-CFTR was without effect ( Figure 1C). We then tested if the effect of TNFα was concentration-dependent. Figure 1D shows the quantification of immunoblot analysis of proteins derived from F508del-CFTR HeLa cells treated with different concentrations of TNFα, ranging from to 50ng/ml for 3–6 h. The relative quantification of mature (band C) vs. core-glycosylated F508del-CFTR (band B) showed a maximal effect at 0.5ng/ml TNFα, which did not increased significantly at higher concentrations ( Figure 1D).

Immunoblot analysis data were supported by immunocytochemistry experiments. The treatment of F508del-CFTR expressing HeLA cells with 50ng/ml of TNFα for 3–6h resulted in a marked increase of CFTR, staining suggesting an increase in F508del-CFTR expression and a possible relocalization of F508del-CFTR to the plasma membrane ( Figure 2, white arrows).

In the next series of experiments, we tested whether TNFα-induced delivery of F508del-CFTR to the plasma membrane was associated with CFTR-Cl ^- channel function. Using the nystatin-perforated patch-clamp configuration, we observed the activation of a cAMP-dependent Cl ^- current, which was sensitive to a CFTR inhibitor (inh ₁₇₂, 5μM) attesting to the presence of a CFTR current (I _CFTR; Figure 3A, B and C) within 10–30min after addition of 5 or 50ng/ml TNFα to the solution ( Figure 3A, B and D). Non-treated control cells did not display I _CFTR ( Figure 3A). These experiments are in concordance with the biochemical data showing that acute TNFα translocates functional F508del-CFTR to the plasma membrane and therefore behaves like a corrector. Application of the same protocol to HeLa cells expressing WT-CFTR did not change the amplitude of I _CFTR (data not shown).

To test whether other pro-inflammatory cytokines induce F508del-CFTR function, we tested the effects of different concentrations of IL-1β on F508del-CFTR-expressing HeLa cells. Treatment of cells with 10 ng IL-1β for 10–30min did not induce I _CFTR ( Figure 4A). Treatment of the same cells with 1 or 10 ng of IL-1β for 24h did not change the maturation pattern of F508del-CFTR ( Figure 4B).

To investigate whether the acute effects of TNFα on F508del-CFTR maturation may have physiological consequences, we performed experiments on primary human bronchial epithelial cells from CF patients homozygous for the F508del mutation, cultured at an air-liquid interface. Confocal microscopy analysis of F508del-CFTR distribution in reconstituted epithelium was performed. Figure 5 shows representative images obtained in HBE cell cultures from three different patients bearing F508del/F508del mutations. Green fluorescence, corresponding to the presence of CFTR protein, increased in cell preparations treated with TNFα (50ng/ml) compared to control, suggesting an increase in F508del-CFTR expression. Furthermore, in TNFα treated cells, F508del-CFTR appeared in the same plane as ZO-1, indicating its apical localization, in contrast to lighter and diffuse cytoplasmic staining in control conditions. The redistribution of F508del-CFTR to the apical side of epithelium occurred within 10min of TNFα 50ng/ml treatment and was sustained over 24h of treatment.

We investigated the functional consequences of F508del-CFTR insertion in the plasma membrane upon TNFα exposure using Isc measurements. Representative Isc recordings are shown in Figure 6: TNFα treatments of CF HBE cells enhanced the cAMP-sensitive Isc, which is consistent with increased activity of CFTR, as compared to non-treated control cells. Increased responses to Forskolin ( Figure 6A and B) as well as potentiation by genistein or inhibition by Inh-172 ( Figure 6B) were observed. In these cells, the effect was still visible after 24h of incubation with TNFα ( Figure 6). Altogether, these experiments suggest that TNFα exerts a correcting effect during the acute and resolving phases of inflammation, by promoting rapid insertion of F508del-CFTR into the apical membrane of primary HBE cells derived from CF patients.

To understand how TNFα enables the exit of F508del-CFTR from the ER, we investigated the role of several possible TNFα targets and signaling pathways, including ER to Golgi vesicular transport, keratin 8 and PKC-related signaling.

In a first set of experiments, cells were pre-treated with BFA, (35µM for 3–4h), an inhibitor of protein transport from the ER to the Golgi apparatus ¹⁸ . Pretreatment with BFA abolished I _CFTR induced by exposure of cells to 50ng/ml of TNFα ( Figure 7) indicating that vesicular trafficking is involved in TNFα action.

In a second set of experiments, the keratin-8 proximity to F508del-CFTR in HeLa cells +/- TNFα was evaluated. Using the PLA assay, we tested whether the number of K8-F508del-CFTR pairs that are closer than 40nm was changed by TNFα treatment. The results indicate that incubation with 50ng/ml TNFα for 30min or 3 h had no effect on the number K8-F508del-CFTR pairs ( Figure 8A and B) suggesting that the interaction between keratin 8 and F508del-CFTR was not a target of TNFα.

A third series of experiments were designed to investigate the possible role of protein kinase C (PKC) in TNFα action. TNFα has been reported to induce within 30min the insertion of the leptin B receptor into the plasma membrane in a PKC-dependent manner ¹⁰ . To test if this is also the case for F508delCFTR, cells were pre-treated for 2–4h with a PKC inhibitor, GF109203X. GF109203X prevented the TNFα-induced changes on I _CFTR, suggesting that a PKC-dependent signaling pathway is involved in this process ( Figure 9A and B).

As rescued F508del-CFTR still carries a misfolding mutation, it will be recognized by the peripheral quality control ¹⁹ . Therefore, we wanted to evaluate the effect of TNFα on the stability of rescued F508delCFTR. Because it was shown that the adaptor protein NHERF1 stabilizes CFTR at the plasma membrane ²⁰ , we investigated whether the number of NHERF1–F508delCFTR protein pairs was modified by TNFα treatment. Using the PLA assay, we observed that the former did not significantly change under TNFα treatment. These results indicate that, while TNFα enables the exit of F508delCFTR from the ER, it does not alter the peripheral quality control ( Supplementary Figure S1).