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MPB-07 Reduces the Inflammatory Response to Pseudomonas aeruginosa in Cystic Fibrosis Bronchial Cells

Laboratory of Molecular Pathology, Cystic Fibrosis Center, and Laboratory of Clinical Immunology, University Hospital of Verona; Department of Pathology, University of Verona, Verona, Italy; Institut de Physiologie et Biologie Cellulaires CNRS, Université de Poitiers, Poitiers, France; Department of Biochemistry and Molecular Biology. University of Ferrara, Ferrara, Italy

Correspondence and requests for reprints should be addressed to Maria Cristina Dechecchi, Laboratory of Molecular Pathology—Cystic Fibrosis Center, Azienda Ospedaliera di Verona-Piazzale Stefani 1, 37126, Verona, Italy. E-mail: cristina.dechecchi{at}azosp.vr.it

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Chronic lung inflammation in cystic fibrosis (CF) is specifically characterized by predominant endobronchial neutrophil infiltrates, colonization by Pseudomonas aeruginosa, and elevated levels of cytokines and chemokines, first of all IL-8. The extensive inflammatory process in CF lungs is the basis of progressive tissue damage and is largely considered detrimental, making antiinflammatory approaches a relevant therapeutic target. This neutrophil-dominated inflammation seems to be related to an excessive proinflammatory signaling, originating from the same surface epithelial cells expressing the defective CF transmembrane conductance regulator (CFTR) protein, although the underlying mechanisms have not been completely elucidated. To investigate the relationship between defective CFTR and the inflammatory response to P. aeruginosa in CF airway cells, we studied the effect of the F508 CFTR corrector, benzo(c)quinolizinium (MPB)-07 (Dormer et al., J Cell Science 2001;114:4073–4081). CF bronchial epithelial IB3-1 and CuFi-1 cells overproduced the inflammatory molecules, IL-8 and intercellular adhesion molecule (ICAM)-1, in response to P. aeruginosa, compared with the wild-type, CFTR-expressing bronchial cells, S9, and NuLi-1 cells. In both IB3-1 and CuFi-1 cells, the corrector MPB-07 dramatically reduces the IL-8 and ICAM-1 mRNA expression elicited by P. aeruginosa infection. Correction of CFTR-dependent Cl^– efflux was confirmed in MPB-07–treated IB3-1 and CuFi-1 cells. In conclusion, the F508 CFTR corrector MPB-07 produces an antiinflammatory effect in CF bronchial cells exposed to P. aeruginosa in vitro.

Key Words: cystic fibrosis • inflammation • Pseudomonas aeruginosa

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   MPB-07 is presented here as a useful tool to investigate the proinflammatory phenotype and to develop successful therapies targeted at the modulation of the detrimental effects of innate immunity observed in CF lungs.

 
Human airway epithelial cells represent the first line in the pulmonary defense against invading pathogens, having their own repertoire of innate immune functions. Once pathogens penetrate the initial barriers, a direct interaction with the epithelial cells occurs, which induces the expression of chemokines and cytokines to recruit and activate neutrophils, thus contributing to the overall inflammation within the lung (1).

Airway inflammation is a hallmark of cystic fibrosis (CF) lung disease, characterized by chronic infections with strikingly few pathogens, first of all Pseudomonas aeruginosa. Although controversial, a growing number of studies indicate that airway inflammation is excessive relative to the burden of infection. Increased numbers of neutrophils and elevated levels of cytokines have been revealed in patients with CF (2, 3). In response to similar levels of pulmonary infection (4), patients with CF and models of CF mice (5, 6) show higher inflammation compared with nonaffected individuals. CF airway epithelial cell lines have been shown to produce more IL-8 than that of CF conductance regulator (CFTR)–corrected cells in response to a variety of proinflammatory stimuli in vitro (7–9). Similarly, the exposure of CF cells to P. aeruginosa increased the production of IL-8 and other cytokines (10). Increased airway inflammation may occur in patients with CF, even in the absence of detectable infection (3, 11–13). It has also been reported that human fetal CF lung grafts develop progressive intraluminal inflammation before any infection (14, 15). These data support the hypothesis that the basic CFTR defect may itself initiate, or at least amplify, the pulmonary inflammatory response in patients with CF, although underlying mechanisms have not been completely elucidated. Attention is drawn to the nuclear transcription factor NF-B, which plays a central role in mediating the immune response. Stimulation of cells with proinflammatory agents, including bacteria, represents a danger signal to the cells, inducing NF-B activation (16). NF-B activating signals are also generated by intracellular stress situations, like the accumulation of proteins in the endoplasmic reticulum (ER), which is termed the ER-overload response (17). The most common mutation of the CF gene causes deletion of phenylalanine at residue 508 (F508), resulting in a CFTR protein that cannot be folded correctly, and is thus retained in the ER (18).

Hyperactivation of NF-B has been described both in unstimulated and in P. aeruginosa–infected airway cells from patients with CF (6, 19, 20), supporting a link between the mistrafficked F508 CFTR protein and the excessive inflammation that characterizes CF airway pathology. NF-B activation has also been reported in CF cell lines that lack CFTR Cl^– channel function, but do not accumulate mutant protein in the ER, consistent with excessive cytokine responses to P. aeruginosa infection as a consequence of impaired cAMP-dependent Cl^– channel (21). A study in mice with CF (22) demonstrates that any functional defect in CFTR may account for the increased airway inflammation. Importantly, the lack of CFTR activity has been recently shown to be responsible for the onset of the inflammatory cascade in CF (23). Therefore, the molecular bases of this intrinsic upregulation of proinflammatory mediators and appropriate strategies to control it remain unclear. Managing airway inflammation has been proven beneficial for patients with CF, so different therapeutic strategies aimed to minimize inflammation have been proposed (24). Rescue/correction of the CFTR defect is a long-term goal addressing multiple aspects of CF pathology, including inflammation. For example, 8-cyclopentyl-1,3-dipropylxanthine both activates mutant CFTR Cl^– channel activity and corrects the trafficking defect (25, 26). Suppression of IL-8 hypersecretion has also been reported in CF respiratory epithelial cell lines pretreated with 8-cyclopentyl-1,3-dipropylxanthine (27), indicating the feasibility of reducing the proinflammatory status through correction of the underlying defect. A potential advantage of pharmacotherapy for defective F508 CFTR processing and gating is that it minimizes concerns about treating the wrong cells or losing physiologic CFTR regulation. High-throughput screening of chemically heterogeneous compounds is now revealing a growing number of activators (28) and correctors of defective processing (29).

We previously demonstrated that the benzo(c)quinolizinium (MPB) compounds MPB-07 and MPB-91 increase F508 CFTR trafficking in CF airway cells (30, 31). In the present study, airway inflammatory response to P. aeruginosa was investigated by measuring the expression of IL-8 and intercellular adhesion molecule (ICAM)-1 induced by the P. aeruginosa laboratory strain, PAO1, in CF respiratory epithelial cell lines. Here, we demonstrate that CF bronchial epithelial IB3-1 and CuFi-1 cells overproduce inflammatory mediators in response to PAO1, compared with the corresponding wild-type (WT), CFTR-corrected S9 and non-CF NuLi-1 cells, respectively. Attempts to revert defective processing of F508 CFTR to that of the WT CFTR were performed by using the corrector, MPB-07. In both IB3-1 and CuFi-1 cells, we demonstrated that MPB-07 significantly reduces ICAM-1 and IL-8 mRNA expression elicited by P. aeruginosa infection. CFTR-dependent Cl^– channel activity was detected in MPB-07–treated IB3-1 and CuFi-1 cells, strengthening the relationship between correction of the trafficking defect and reduction of inflammatory response to P. aeruginosa infection in CF cells. Therefore, MPB-07 is suggested to have an antiinflammatory effect in CF cell lines.

	MATERIALS AND METHODS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Cell Lines and Bacteria
IB3-1 is a human bronchial epithelial cell line, immortalized with adeno-12/SV40, derived from a patient with CF with a F508/W1282X mutant genotype (32). Defective CFTR-dependent Cl^– channel in IB3-1 cells has been corrected in the S9 cell line by transfection with wild-type adeno-associated viral CFTR (33). IB3-1 and S9 cells, available through the Johns Hopkins/American Type Culture Collection, were obtained from LGC Promochem, Europe (Venice, Italy). Both cell lines were grown in LHC-8 basal medium (Biofluids Inc., Rockville, MO) supplemented with 5%fetal bovine serum. All culture flasks and plates were coated with a solution containing 35 µg/ml bovine collagen (Becton-Dickinson, Franklin Lakes, NJ), 1 µg/ml bovine serum albumin (Sigma, St. Louis, MO) and 1 µg/ml human fibronectin (Becton-Dickinson), as previously described (32). CuFi-1 and NuLi-1 cells, a generous gift of A. Klingelhutz, P. Karp, and J. Zabner (University of Iowa, Iowa City, IA), were derived from human bronchial epithelium from a patient with CF (CuFi-1, CFTR F508/F508 mutant genotype) or a subject without CF (NuLi-1, WT CFTR), and had been transformed by the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes (34). These cell lines were grown on human placental collagen type VI (Sigma)–coated flasks in BEGM (Cambrex Bio Science, Walkersville, MD) medium, as described (34). PAO1, a prototypic laboratory strain of P. aeruginosa kindly provided by A. Prince (Columbia University, New York, NY), were grown in trypticase soy broth or agar (TSA) (Difco, Detroit, MI).

Cell Infection
Cells were seeded at densities ranging from 50,000 to 100,000 cells/cm² at 24 h before infection. After adhesion, IB3-1 and S9 cells were starved in serum-free LHC-8 for 18 h. Before the experiment, bacteria from overnight cultures in TSA plates were grown in 20 ml trypticase soy broth at 37°C with shaking until achieving an optical density at 660 nm of 1 x 10⁹ cfu/ml, determined by dilution plating. Bacteria were washed twice by resuspension in PBS and then underwent a final dilution in cell culture medium. The doses of PAO1 were determined by plating aliquots of dilutions on TSA plates, and are expressed as cfu/cell. Unless otherwise indicated, monolayers of cells, in duplicate, were infected with PAO1 at 37°C for 4 h at doses indicated in the figures.

Real-Time qPCR
Monolayers of cells, infected as described above, were washed with PBS and detached. Total RNA was extracted using the High Pure RNA Isolation kit (Roche, Mannheim, Germany), following the manufacturer's instructions. Reverse transcription (RT) was performed using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA): 300–1,000 ng of total RNA were reverse transcribed in the presence of random hexamers (1x), dNTPs (2.5x), 100 U RNAse inhibitor, 250 U Multiscribe reverse transcriptase, and reverse transcriptase buffer (1x), in a total volume of 100 µl, for 10 min at 25°C and 120 min at 37°C. As negative control, the reaction was also performed in the absence of RNA. The cDNA was amplified in 25 µl reactions using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Carlsbad, CA), in the ABI Prism 5700 sequence detection system (Applied Biosystems). Human ICAM-1, IL-8, and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) were amplified in separate tubes: primers selected with Primer Express software (Applied Biosystems) are indicated below:

human ICAM-1 forward primer 5'-TATGGCAACGACTCCTTCTCG-3'

human ICAM-1 reverse primer 5'-CTCTGCGGTCACACTGACTGA-3'

or

human IL-8 forward primer 5'-GACCACACTGCGCCAACA-3'

human IL-8 reverse primer 5'-GCTCTCTTCCATCAGAAAGTTACA

TAATTT-3'

or

human GAPDH forward primer 5'-GTGGAGTCCACTGGCGTCTT-3'

human GAPDH reverse primer 5'-GCAAATGAGCCCAGCCTTC-3'

The real-time quantitative PCR (qPCR) reactions were performed in duplicate for both target (IL-8 and ICAM-1) and normalizer gene (GAPDH). Thermal protocol consisted of 2 min at 50°C, a denaturation step at 95°C for 2 min, then 50 cycles of a 15-s, 95°C denaturation step, and a 30-s annealing/extension step at 60°C. Relative quantification of gene expression was performed using the comparative threshold method, as described by the manufacturer's User Bulletin 2 (Applied Biosystems). The ratios obtained after normalization were expressed as folds of change over untreated samples.

Cell Surface ICAM-1 Expression by Flow Cytometry Analysis
Cells infected by PAO1 were washed three times in PBS containing 150 µg/ml gentamicin (2 h after infection) and incubated in cell culture medium plus 50 µg/ml gentamicin cells for 24 h. Cells were then detached with a solution of 1% EGTA in PBS without calcium and magnesium, and washed in PBS. Aliquots of cell suspensions (5 x 10⁵ cells) were stained by direct immunofluorescence methods. Briefly, cells were incubated on ice for 30 min with a mouse monoclonal antibody anti-human CD54 (ICAM-1) directly conjugated with phycoerythrin. Nonrelevant mouse isotype control antibody was used to determine background fluorescence level. Both antibodies were purchased from Becton Dickinson Biosciences. After washing, the cells were fixed with 1% paraformaldehyde and examined by a FACSCanto Flow Cytometer (Becton Dickinson Biosciences), equipped with a 488-nm Ar and a 633-nm HeNe laser. A total of 5,000 events were examined for each sample. The settings were used with linear amplification (262,144 channels) of the forward- and side-scatter signals (FS and SS), and logarithmic amplification (4 log decades) of the fluorescence FL2 signal. Data were analyzed after gating on (FS, SS) parameters using the FACSDiva software (Becton Dickinson Biosciences). Results were reported as percentage of CD54-positive cells.

IL-8 Secretion
Cells were infected as previously described here. Growth media were collected at the end of incubation, centrifuged, and stored at –80°C. Released IL-8 was determined in the same experimental conditions as mRNA measurements, using an ELISA kit (Biosource Europe, Nivelles, Belgium). According to the manufacturer, the sensitivity of the assay is less than 1 pg/ml.

Manipulations to Increase CFTR Trafficking to the Cell Membrane
Attempts were made to revert defective processing of CFTR F508 to that of the WT CFTR by incubating IB3-1, CuFi-1, and NuLi-1 cells in the presence of 250 µM MPB-07, dissolved in water, at 37°C, both before and after infection with PAO1. ICAM-1 and IL-8 mRNA and protein levels were then assayed as described previously here.

CFTR Function Assay
CFTR function was assessed by single-cell fluorescence imaging, using the potential-sensitive probe, bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC2(3); Molecular Probes, Eugene, OR), as previously reported (35), with minor changes. Briefly, cells grown on coated, round, glass coverslips were washed in a Cl^–-containing solution (101 mM Na⁺, 114 mM Cl^–, 5 mM K⁺, 2 mM Ca²⁺, 2 mM Mg²⁺, 50 mM mannitol, 5 mM glucose, 5 mM Hepes-Tris, pH 7.4), mounted in the perfusion chamber (Medical Systems, Greenvale, NY) and perfused for 10–15 min at 25°C with a Cl^–-free solution (101 mM Na⁺, 106 mM gluconate, 14 mM acetate, 5 mM K⁺, 2 mM Ca²⁺, 2 mM Mg²⁺, 50 mM mannitol, 5 mM glucose, and 5 mM Hepes-Tris, pH 7.4), containing 100 nM DiSBAC2(3). The time courses were performed at 25°C, and a baseline signal was acquired for 3 min before the addition of the stimulus. CFTR-dependent Cl^– channel was stimulated by a cAMP-elevating cocktail (20 µM forskolin plus 100 µM 3-isobutyl-1-methyl-xanthine [IBMX] and 50 µM genistein), added to the chamber at the time indicated in the figures. The thiazolidinone CFTR inhibitor, CFTR_inh-172, kindly provided by A. Verkman (University of California, San Francisco) (36), was added to a final concentration of 10 µM. Fluorescence was measured with a Nikon TMD inverted microscope through a Nikon Fluor 40x objective (Nikon Europe, Firenze, Italy). The signal was acquired with a Hamamatsu C2400–97 CCD intensified videocamera (Hamamatsu City, Japan) at a rate of 1 frame/30 s, with an integration time ranging from 0.1 to 1.0 s. Fluorescence coming from each single cell was analyzed by customized software (Spin, Vicenza, Italy). Results are presented as transformed data to obtain the percentage signal variation (Fx) relative to the time of addition of the stimulus, according to the equation: Fx = ([Ft – Fo]/Fo) x 100, where Ft and Fo are the fluorescence values at the time t and at the time of addition of the stimulus, respectively.

Electrophoretic Mobility Shift Assay
Cells were seeded on coated Petri dishes (5-cm diameter) at a density of 100,000/cm², 24 h before infection, infected as described above for 2 h, and detached by trypsin. DNA-binding proteins were extracted by hypotonic lysis, followed by high-salt extraction of nuclei (37). Electrophoretic mobility shift assay (EMSA) was performed as previously described (38). Briefly, double-stranded synthetic oligodeoxynucleotides mimicking the NF-B– and the AP-1–binding site were employed (NF-B, sense: 5'-AATCGTGGAATTTCCTCT-3'; AP-1, sense: 5'-TGTGATGACTCAGGTTTG-3'). Oligodeoxynucleotides were labeled with ³²-P-ATP using 10 U of T4-polinucleotide-kinase (MBI Fermentas GmbH, St. Leon-Rot, Germany) in 500 mM Tris–HCl, pH 7.6, 100 mM MgCl₂, 50 mM DTT, 1 mM spermidine, and 1 mM EDTA, in the presence of 50 µCi ³²-P-ATP, in a volume of 20 µl for 45 min at 37°C. The reaction was brought to 150 mM NaCl, and 150 ng complementary oligodeoxynucleotide was added. Reaction temperature was increased to 100°C for 5 min, and left to decrease to room temperature overnight. Nuclear extracts from IB3-1 and CuFi-1 cells were used at concentrations ranging from 0.5 to 2 µg/reaction in the presence of poly(dI:dC) (1 mg/reaction) to abolish nonspecific binding. After 25 min binding at room temperature, the samples were run at constant voltage (200 V) under low ionic strength conditions (0.25x Tris–borate–EDTA buffer: 22 mM Tris–borate and 0.4 mM EDTA) on 6% polyacrylamide gels. Gels were dried and subjected to standard autoradiographic procedures.

Statistical Analysis
Results are expressed as mean (± SEM). Comparisons between groups were made by using Student's t test and a one-way ANOVA. Statistical significance was defined at P < 0.05.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Inflammatory Response to PAO1 Infection in CF Bronchial Epithelial Cell Lines
At first, we set up the experimental model in the widely used IB3-1 CF bronchial cell line. As a relatively brief exposure to P. aeruginosa has previously been shown to be sufficient to activate NF-B translocation in IB3-1 cells (19), we infected cells with PAO1 for 4 h before analyzing the inflammatory response. In the experimental design that we developed, host–bacterial interaction was investigated by comparing the transcription of ICAM-1 and IL-8 in IB3-1– and IB3-1–corrected S9 cells after infection with PAO1. IL-8 is a key factor in the regulation of airway inflammation in CF (2), being released from respiratory epithelial cells in response to bacteria, to regulate transepithelial migration and activation of neutrophils (1). ICAM-1 is constitutively expressed on the cell surface of a wide variety of cells, and it is modulated in response to a number of inflammatory mediators, including bacteria (39, 40). The expression of ICAM-1 on the membrane of human airway epithelial cells intervenes in the recruitment of neutrophils at sites of infection (40). Interestingly, it has been recently demonstrated that CF IB3-1 cells express more ICAM-1 than do IB3-1–corrected cells (41). Thus, ICAM-1 likely contributes, together with IL-8 and other chemokines, to the peculiarly excessive neutrophilia observed in the bronchial lumen of patients with CF. Dose responses of the stimulated transcription of ICAM-1 and IL-8 are shown in Figure 1. ICAM-1 and IL-8 mRNA are upregulated by PAO1 in both cell lines. Significantly higher mRNA levels are induced by PAO1 in IB3-1 than in S9 cells (P < 0.03 for ICAM-1 and P < 0.007 for IL-8, by ANOVA). To determine whether changes in mRNA were reflected in protein expression and release, the PAO1-stimulated ICAM-1 expression and IL-8 secretion were measured under the same experimental conditions. Results are shown in Figures 1C and 1D. Increased ICAM-1 and IL-8 transcription observed in IB3-1 cells was correlated with a significantly higher increase of both ICAM-1 expression on the cell membrane and IL-8 secretion (P < 0.04 for ICAM-1 and P < 0.008 for IL-8, by ANOVA). We extended the analysis to the recently developed CuFi-1 and NuLi-1 cells, which are useful respiratory cell models to study ion physiology and innate immunity (34). As shown by the results of dose–response experiments reported in Figures 2A and 2B, ICAM-1 and IL-8 mRNA expression is upregulated by PAO1 in both cell lines. Significantly higher induction is observed in CF CuFi-1 than in non-CF NuLi-1 cells, both for ICAM-1 and for IL-8 (P < 0.007 and P < 0.001, respectively, by Student's t test). These results, obtained in two different CF respiratory cell lines, provide further evidence that P. aeruginosa induces a much more elevated proinflammatory response in CF than in non-CF bronchial cells.

View larger version (17K): [in this window] [in a new window]   Figure 1. Increased inflammatory response to PAO1 infection in CF bronchial epithelial cells. IB3-1 and S9 cell lines were incubated for 4 h at 37°C with ranging doses of PAO1, and ICAM-1 (A) and IL-8 mRNA (B) levels were measured. The mRNA induction (relative to noninfected cells) was obtained by comparing the ratios ICAM-1:GADH and IL-8:GADH between noninfected and infected cells. A representative of seven independent experiments is shown. Results are expressed as mean (± SEM) of duplicate wells. ICAM-1 and IL-8 are upregulated by infection both in IB3-1 and S9 cells. Significantly higher mRNA levels were induced by PAO1 in IB3-1 than in S9 cells. (C) Percentage of IB3-1 and S9 cells expressing ICAM-1 protein after PAO1 infection. FACS analysis was performed, 24 h after the infection, in IB3-1 and S9 cells infected by 10 cfu/cell PAO1. A representative of two independent experiments is shown. Values are expressed as mean (± SEM) of duplicate wells. A total of 5,000 cells were analyzed by FACS. IB3-1 cells expressed higher ICAM-1 than S9 on the cell membrane. (D) PAO1-stimulated IL-8 secretion in IB3 and S9 cell lines. Cells were infected for 4 h by 10 cfu/cell of PAO1 and growth media collected at the end of incubation, centrifuged, and stored at –80°C. A representative of five independent experiments is shown. Results are expressed as mean (± SEM) of duplicate wells. Increased IL-8 transcription observed in IB3 cells is correlated with a highly significant increase of IL-8 secretion.

View larger version (9K): [in this window] [in a new window]   Figure 2. ICAM-1 and IL-8 transcription response to PAO1 in human airway epithelial cell lines. CuFi-1 and NuLi-1 cell lines were incubated for 4 h at 37°C with ranging doses of PAO1, and ICAM-1 (A) and IL-8 (B) mRNA levels were measured. The mRNA induction (relative to noninfected cells) was obtained by comparing the ratios ICAM-1:GADH and IL-8:GADH between noninfected and infected cells. A representative of four independent experiments is shown. Results are expressed as mean (± SEM) of duplicate wells. ICAM-1 and IL-8 mRNA are upregulated by infection in both the cell lines. Significantly higher mRNA levels are induced by PAO1 in CuFi-1 than in NuLi-1 cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. MPB-07 reduces the PAO1-stimulated inflammatory response in IB3-1 cells. (A and B) Cells were incubated overnight at 37°C in the presence of MPB-07 (250 µM) and then infected with ranging doses of PAO1 for 4 h. A representative of four independent experiments is shown. Results are expressed as mean (± SEM) of duplicate wells. The mRNA induction (relative to noninfected cells) was obtained by comparing the ratios ICAM-1:GADH and IL-8:GADH between noninfected and infected cells. MPB-07 significantly reduces both ICAM-1 (A) and IL-8 (B) mRNA expression elicited by PAO1 infection in CF cells. (C and D) Cells, incubated overnight with MPB-07 (250 µM), were infected by 10 cfu/cell PAO1, and MPB-07 removed. After 24 h, IL-8 secretion in the growth media and ICAM-1 expression on the cell membrane were measured. A representative of two independent experiments is shown. Data reported are mean (± SEM) of duplicate wells, and are expressed as percentage of PAO1-stimulated increase of ICAM-1 protein expression (C) and IL-8 secretion (D). MPB-07 treatment of IB3-1 cells decreased PAO1-stimulated ICAM-1 protein expression and IL-8 secretion.

View larger version (15K): [in this window] [in a new window]   Figure 4. MPB-07 decreases the PAO1-stimulated inflammatory response in CF cells, but not in non-CF airway epithelial cell lines. Cells were incubated overnight at 37°C in the presence of MPB-07 (250 µM) and then infected with ranging doses of PAO1 for 4 h. (A and B) CuFi-1 cells. A representative of three independent experiments is shown. Results are expressed as mean (± SEM) of duplicate wells. The mRNA induction (relative to noninfected cells) was obtained by comparing the ratios ICAM-1:GADH and IL-8:GADH between noninfected and infected cells. MPB-07 significantly reduced both ICAM-1 and IL-8 mRNA expression elicited by PAO1 infection in CuFi-1 cells. (C and D) NuLi-1 cells. A representative of four independent experiments is shown. Results are expressed as mean (± SEM) of duplicate wells. MPB-07 did not reduce the PAO1-stimulated inflammatory mediator expression in non-CF NuLi-1 cells.

View larger version (23K): [in this window] [in a new window]   Figure 5. MPB-07, added after proinflammatory stimulus with PAO1, reduced the expression of proinflammatory mediators. IB3-1 cells (A and B) and CuFi-1 cells (C and D) were infected by 10 cfu/cell PAO1; MPB-07 (250 µM) was applied simultaneously and incubated at 37°C for 4 h. Representative experiments are shown (n = 4 for IB3-1 cells; n = 5 for CuFi-1 cells). Data are expressed as mean (± SEM) of duplicate wells. The mRNA induction (relative to noninfected cells) was obtained by comparing the ratios ICAM-1:GADH and IL-8:GADH between noninfected and infected cells. The PAO1-stimulated inflammatory response was still reduced when MPB-07 was added after PAO1.

View larger version (16K): [in this window] [in a new window]   Figure 6. A cAMP-activating cocktail plus genistein activate Cl– efflux in MPB-07–treated IB3-1 cells. (A) Functional evaluation of CFTR by DiSBAC2(3) assay in IB3-1 and S9 cell lines. Cells grown on round, glass coverslips were mounted on the perfusion chamber and perfused with Cl–-free solution containing DiSBAC2(3) to allow the equilibration of the dye within cell membranes. Fluorescence coming from each single cell was analyzed. Typical time courses are shown. Data represent the mean (± SEM) of the relative fluorescence collected from all the cells of the field (from 8 to 20). Forskolin and IBMX activated Cl– efflux only in IB3-1–corrected S9 cells. The CFTR potentiator, genistein, had a large response in S9 but not in IB3-1 cells. (B) Effect of MPB-07 on CFTR function in IB3-1 cells. Cells grown on round, glass coverslips were incubated overnight in the presence of MPB-07 (250 µM). CFTR function was evaluated by DiSBAC2(3) assay. Typical time courses are shown. Data represent the mean (± SEM) of the relative fluorescence collected from all the cells of the field (from 8 to 20). In MPB07-treated IB3-1 cells, forskolin and IBMX plus genistein activated Cl– efflux. No CFTR functional activity was observed in control IB3-1 cells.

View larger version (18K): [in this window] [in a new window]   Figure 7. MPB-07 restores CFTR function in CuFi-1 cells. (A) Functional evaluation of CFTR by DiSBAC2(3) assay in CuFi-1 and NuLi-1 cell lines. Cells grown on round, glass coverslips were mounted on the perfusion chamber and perfused with Cl–-free solution containing DiSBAC2(3) to allow the equilibration of the dye within cell membranes. Fluorescence coming from each single cell was analyzed. Typical time courses are shown. Data represent the mean (± SEM) of the relative fluorescence collected from all the cells of the field (from 8 to 20). Forskolin- and IBMX-activated Cl– efflux only in non-CF NuLi-1 cells. The CFTR potentiator, genistein, has a large response in NuLi-1 cells, inhibited by CFTRinh-172. No activation of CFTR function was detected in CuFi-1 cells. (B) Effect of MPB-07 on CFTR function in CuFi-1 cells. Cells grown on round, glass coverslips were incubated overnight in the presence of MPB07 (250 µM). CFTR function was evaluated by DiSBAC2(3) assay. Typical time courses are shown. Data represent the mean (± SEM) of the relative fluorescence collected from all the cells of the field (from 8 to 20). MPB-07–treated CuFi-1 cells show activation of CFTR function. No CFTR function was detected in control CuFi-1 cells.

Effects of MPB-07 on NF-B– and AP-1–Binding Activity

View larger version (53K): [in this window] [in a new window]   Figure 8. MPB-07 had no effect on the direct binding of the transcription factors, NF-kB (A) and AP-1 (B), to DNA. Nuclear extracts (2 µg) from IB3-1 cells were incubated with target NF-kB and AP-1 32P-labeled oligodeoxynucleotides in the presence of increasing amounts of MPB-07 (30–500 µM), and EMSA was performed.

View larger version (72K): [in this window] [in a new window]   Figure 9. MPB-07 has no effect on NF-kB and AP-1 overall binding activity. CuFi-1 and IB3-1 cells were cultured in the absence (A and C) or in the presence (B and D) of MPB-07 for 24 h, and then infected (C and D), or not (A and B), with of PAO1 for 2 h. Nuclear extracts were isolated, increasing amounts were mixed with NF-kB and AP-1 target 32P-labeled oligodeoxynucleotides, and EMSA was performed.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

Different evidence suggests that inflammatory response is constitutively activated in the airways of patients with CF, even before bacterial infection, as shown by the presence of an elevated concentration of proinflammatory cytokines in the lungs of infants with CF without lung bacterial infection (3, 11–13), and in xenografts reconstituting the CF surface respiratory epithelium in strict sterile conditions (14, 15). The question arises on the mechanism(s) underlying CF proinflammatory status. An increasing number of reports indicate that diseases due to mutations, causing protein misfolding with accumulation in the ER, are accompanied by inflammation (for review, see Ref. 44). In these cases, it has been proposed that protein overloading in the ER may activate the transcription factor, NF-B (17). Among the different genes involved in the innate immune response, which are activated by NF-B, a key role in CF is usually assigned to the chemokine, IL-8, which drives the intrabronchial recruitment of neutrophils. Within the different adhesion molecules cooperating in transepithelial migration of neutrophils, studies on CF converge on the importance of the adhesion molecule, ICAM-1, which was found to be increased in CF respiratory epithelial cells (41, 45). As far as CF is concerned, constitutive activation of NF-B through an ER-overload stress signaling has been demonstrated in the CF epithelial respiratory cells bearing the F508 CFTR allele (46). Thus, the proinflammatory circuitry in CF airways seems to be initiated from those surface epithelial cells expressing the mutated CFTR protein. Consistent with this hypothesis, reduction of the constitutive NF-B activation has been observed by growing CF cells at a permissive temperature, allowing partial escape of the mutant, misfolded F508 CFTR from the ER (19). Moreover, very recent results demonstrate that selective inhibition of the ER-associated degradation rescues the functional mutant CFTR to the cell surface and suppresses NF-B–mediated IL-8 levels (47). However, CF mouse models have recently suggested that the F508 CFTR protein misfolding–dependent ER-overloading mechanism is not the only cause of the exaggerated activation of the proinflammatory circuitry in CF in vivo. For instance, recent studies performed in human tracheal epithelial cells and in mice with CF report hyperinflammatory response to P. aeruginosa challenge, independent of misprocessing of CFTR (22, 23). Moreover, a CF-like lung disease accompanied by inflammation has been reproduced by overexpressing the epithelial sodium channel in mice bearing the wild-type CF gene (48). Thus, any other condition resulting in alteration of chloride and sodium transport on airway surface could lead to activation of proinflammatory mediators, independently of CFTR misfolding. Because MPB-07 allows recovery of Cl^– efflux function by correction of F508 CFTR misprocessing (30, 31), we cannot exclude the possibility that the antiinflammatory mechanism of this compound is mediated by restoration of Cl^– efflux, although correction of F508 CFTR misprocessing seems to be the most likely explanation in our experimental model in vitro. In favor of this hypothesis is the fact that we observed antiinflammatory effects in MPB-07–corrected CF cells in the absence of forskolin/genistein (i.e., without activation of CFTR-dependent Cl^– efflux).

We report here that MPB-07 has an antiinflammatory effect in CF cell lines infected by PAO1, opening the possibility that, besides correction of F508 CFTR mislocation (31) or function (30), MPB-07 could inhibit the signaling between the receptors recognized by P. aeruginosa and the transcription of IL-8 and ICAM-1. At the initial stages of respiratory infections, airway epithelial cells sense P. aeruginosa through Toll-like receptors (TLR)-5 and AsialoGM1, which recognizes its flagellin protein (49, 43). This interaction seems to be crucial in the initiation of the host inflammatory response to clear the invading pathogen (50). Molecules involved in TLR-dependent signaling have been identified, although the networks of the pathway that results in an integrated host response to bacteria have not been completely delineated. In respiratory epithelial cells, TLR-5 transduces the signal into the nucleus, mainly using the classical pathway involving MyD88, IRAK, and TRAF6, thus leading to downstream activation of NF-B and production of chemokines (49, 50). Other transcription factors, such as AP-1, are also activated by TLR signaling (43). In this respect, we did not observe any direct inhibition of MPB-07 in the activation of the transcription factors, NF-B and AP-1 (Figure 9). Therefore, MPB-07 is expected to retain low activity on NF-B– and AP-1–regulated housekeeping genes, suggesting low overall toxicity of this compound. However, aside from these transcription factors, MPB-07 could interfere with other pathways in signal transduction, resulting in the reduction of the inflammatory response through a mechanism that requires further investigation. Based on the present results, MPB-07 may be proposed as a potential therapeutic strategy for modulation of innate immunity in CF lungs.

	Acknowledgments

 
The authors are grateful to A. Tamanini for helpful discussions, A. Prince for the P. aeruginosa laboratory strain PAO1, M.G. Giri for statistical analysis, F. Quiri for excellent technical assistance, In Vitro Model and Cell Culture Care of the University of Iowa for providing NuLi-1 and CuFi-1 cells, and A.S. Verkman for CFTR_inhib-172.

	Footnotes

 
^* These authors contributed equally to this work.

This work was supported by Italian Cystic Fibrosis Research Foundation (FFC 14/2004), Associazione Veneta per la lotta contro la Fibrosi Cistica, Azienda Ospedaliera di Verona, Legge 548/93, Finanziamento Ricerca Fibrosi Cistica 2004 (to M.C.D.), Fondazione Cariverona—Bando 2005—Malattie rare e della povertà (to G.C.), and by Vaincre la Mucoviscidose (to Y.M. and F.B.).

Originally Published in Press as DOI: 10.1165/rcmb.2006-0200OC on December 29, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 5, 2006

Accepted in final form December 13, 2006

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