Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Proinflammatory Phenotype and Increased Caveolin-1 in Alveolar Macrophages with Silenced CFTR mRNA

  • Yaqin Xu,

    Affiliation Department of Pediatrics, Weill Cornell Medical College, New York, New York, United States of America

  • Anja Krause,

    Affiliation Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, United States of America

  • Hiroko Hamai,

    Affiliation Department of Pathology, Columbia University, New York, New York, United States of America

  • Ben-Gary Harvey,

    Affiliation Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, United States of America

  • Tilla S. Worgall,

    Affiliation Department of Pathology, Columbia University, New York, New York, United States of America

  • Stefan Worgall

    stw2006@med.cornell.edu

    Affiliations Department of Pediatrics, Weill Cornell Medical College, New York, New York, United States of America, Department of Genetic Medicine, Weill Cornell Medical College, New York, New York, United States of America

Proinflammatory Phenotype and Increased Caveolin-1 in Alveolar Macrophages with Silenced CFTR mRNA

  • Yaqin Xu, 
  • Anja Krause, 
  • Hiroko Hamai, 
  • Ben-Gary Harvey, 
  • Tilla S. Worgall, 
  • Stefan Worgall
PLOS
x

Abstract

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-κB, a positive regulator of IL-8 expression, and decreased expression of IκB-α, the inhibitory protein of NF-κB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1.

Introduction

CF lung disease is characterized by exaggerated inflammation even in the absence of detectable pathogens [1]. Studies related to inflammation in CF have mostly focused on defective CFTR in lung epithelial cells [2], but CFTR may also play an important role in immune cells [3][12].

Alveolar macrophages (AM) serve as first line defense within the respiratory tract, stimulate inflammation and recruit other cells of the immune system [13]. It is not known if AM play a primary role in CF lung disease. Increased numbers of AM were observed in the CF fetal airways [14] and recently in infants with CF [15], suggesting an involvement of AM in the early onset of inflammation. Studies in CF knockout mice suggested a role for CFTR in AM phagosomes and indicated that AM contribute directly to the exaggerated inflammatory response [16], [17]. Impaired clearance of apoptotic cells [18], [19], decreased antigen presentation, and T-cell stimulatory activity [20] have been described in CF lung disease, that could suggest potential functional abnormalities of AM in CF. However, studying the role of CFTR in AM derived from CF lungs is challenging as it is difficult to distinguish if the AM phenotype is primarily induced by the defective expression of CFTR in the AM or induced by the inflammatory milieu resulting from defective CFTR expression in epithelial or other cells [18], [19].

The enhanced inflammatory response in CF has been linked to apoptosis, but the exact mechanisms have been unclear and the results have been contradicting. Increased apoptosis was described in tracheal and pancreatic CF cells [21][23]. This was accompanied by an increase in inflammatory cytokines and NF-κB activation, which suggested a common pathway for apoptosis and inflammation in these cells. In contrast, a number of studies relate CFTR expression to apopotosis [24][29]. These have linked the lack of CFTR expression or expression of mutant CFTR in CF to a proinflammatory and antiapoptotic phenotype [24][29]. Others did not see differences in apoptosis in airway epithelial cells [30]. Furthermore, defective clearance of apoptotic cells in the CF airways was reported to be factor to further trigger the inflammation [18], [19]. The apparent inconsistencies of these findings could be related to the cell-type and apoptosis of AM in CF could play a role in the inflammatory response.

Both, inflammation and apoptosis in macrophages, are associated with caveolin 1 (Cav1) [31][33], a membrane protein that has been reported to colocalize with CFTR in epithelial cells [34]. Colocalization of CFTR and Cav1 has been proposed to constitute an “internalization platform”, necessary for appropriate immune response to infection [34]. Cav1 could be a macrophage-specific link between apoptosis and inflammation in CF. The regulation of Cav1 expression is through sterol regulatory element binding proteins (SREBPs), key transcription factors of cellular lipid homeostasis [35]. SREBP expression is primarily regulated by cellular cholesterol [36]. This relevant for CF as CFTR dysfunction has been shown to affect cellular cholesterol and SREBP [37][41]. Furthermore, alterations in cellular cholesterol may play a role in the inflammatory phenotype in CF [37], [40].

The goal of this study was (1) to analyze if decreased CFTR expression in human AM affects inflammation and apoptosis by using unstimulated AM derived from non-CF subjects with silenced CFTR expression; (2) to focus on Cav 1 and its regulation by SREBP as a potential factor. We found that silencing of CFTR induced an inflammatory phenotype and augmented apoptosis that were at least partially regulated by SREBP-mediated Cav1 expression. These findings suggest that defective CFTR in AM is relevant for the inflammation in CF lung disease.

Materials and Methods

Ethics Statement

Bronchoalveolar lavage (BAL) was performed in 42 clinical healthy volunteers (average 48±12 years, age range 24–79, 31 males and 11 females). Informed consent in written form was obtained from all volunteers according to Institutional Review Board (IRB) guidelines and was approved by the ethics committee of Weill Cornell Medical College (IRB protocol number: 0005004439).

Cells

Human alveolar macrophages (AM) were obtained by BAL [42] and were processed and cultured as described [13]. Briefly, the lavage fluid was filtered through one layer of gauze, centrifuged (400 g, 10 min) and washed three times in PBS, pH 7.4 (Invitrogen, Carlsbad, CA). Cells were then suspended in full RPMI 1640 medium containing 10% fetal bovine serum, 50 U/ml penicillin, 50 U/ml streptomycin and 2 mM glutamine (Invitrogen) and plated. Macrophage content (always >90%) was determined by Giemsa stain on cytospin preparations (Block Scientific, Inc., Bohemia, NY). Cell viability (always >90%) was determined by trypan blue exclusion. The cells were washed after 3 h to remove non-adherent cells.

CFTR Knockdown in AM

Five siRNAs specific pre-designed for CFTR were purchased (AM16708A, #1 ID: 145572, #2 ID: 104325, #3 ID: 104323, #4 ID: 4110, #5 ID: 3920; Applied Biosystems, Foster city, CA). To evaluate the siRNA transfection and knockdown efficacy, AM were transfected with different doses of pre-designed GAPDH gene specific siRNA (AM4390850, Applied Biosystems) in siPort as transfection reagent (Applied Biosystems). Transfection with a scrambled siRNA (AM4611, Applied Biosystems) was used as control. After the optimization of siRNA transfection, 100 nM pre-designed CFTR gene specific siRNA (#4 ID: 4110) was selected for the transfection to knockdown of CFTR in AM.

The mRNA levels of CFTR in AM transfected with siRNAs were measured by real-time RT-PCR. RNA was extracted after 48 h from AM transfected with CFTR-siRNA or control-siRNA using TRIzol (Invitrogen). Following reverse transcription of 2 µg RNA, CFTR mRNA was amplified by real-time RT-PCR using a CFTR specific probe (Hs00357011_m1, Applied Biosystems). CFTR mRNA levels were quantified using the ΔΔCt method (Applied Biosystems) and normalized relative to 18s ribosomal RNA (Applied Biosystems). PCR reactions for CFTR and 18s ribosomal RNA were optimized to yield equal amplification efficiency. CFTR protein expression was determined by Western analysis. Total cellular fractions were prepared from AM transfected CFTR-siRNA and control-siRNA after 48 h. Following determination of protein concentration (Micro BCA™ Protein Assay Kit; PIERCE, IL), 100 µg protein was separated by electrophoresis on 4–12% Bis-Tris Gel (NuPAGE@Novex, Invitrogen), transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA) and incubated with a mouse anti-CFTR antibody (1∶1000, R&D System, Minneapolis, MN). A horseradish peroxidase-conjugated goat anti-mouse secondary antibody (1∶10000, Bio-Rad Laboratories) and Amersham ECL Plus Western Blotting System (GE Healthcare Bio-Sciences, Piscataway, NJ) were used for detection. Following stripping of the membrane, anti-β-tubulin antibody (1∶1000, Sigma-Aldrich, St. Louis, MO) was added to detect β-tubulin as endogenous control. Expression levels of CFTR relative to β-tubulin were quantified using Image J software [43].

Inflammatory Response

To evaluate if decreased expression of CFTR results in a pro-inflammatory phenotype, we analyzed secreted IL-8 levels and expression of NF-κB in AM transfected with CFTR-siRNA. IL-8 was determined in the culture medium at 0, 2, 4, 8, 24, 48, and 72 h after transfection by ELISA (R&D System), and three measurements were made for each time point. Protein expressions of phosphorylated NF-κB and IκB-α were quantified with CFTR knockdown after 48 h by Western analysis. Fifty µg protein was separated by electrophoresis on 4–12% Bis-Tris Gel (NuPAGE@Novex, Invitrogen), transferred to a PVDF membrane (Bio-Rad Laboratories) and incubated respectively with a mouse anti-phospho-NF-κB p65 antibody (1∶1000, Cell Signaling Technology, Inc., Boston, MA) and with a mouse anti-IκB-α antibody (1∶1000, Cell Signaling Technology, Inc.), and normalized to β-tubulin expression as outlined above.

Apoptosis

To analyze if AM with decreased CFTR are more susceptible to apoptosis, apoptosis was evaluated by situ terminal deoxynucleotidyltransferase (TUNEL) assay and cleavage of poly (ADP-ribose) polymerase (PARP). AM, plated on coverslip dishes at a density 5×105/well were transfected with CFTR-siRNA or control-siRNA. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) after 48 h for 15 min, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 5 min and DNA strand breaks were detected using flurorescein thiocyanate conjugated dUTP. Nuclei were counterstained with 4′-6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The number of apoptotic cells was determined by fluorescent microscopy (Nikon Instruments, NY) by counting 10 fields. Cleaved PARP protein expression was determined in the cell lysates by Western analysis. Fifty µg protein was separated by electrophoresis on 4–12% Bis-Tris Gel (NuPAGE@Novex, Invitrogen), transferred to a PVDF membrane (Bio-Rad Laboratories) and incubated with a rabbit anti-PARP antibody (1∶200, Santa Cruz Biotechnology, Inc.) and normalized to β-tubulin expression as outlined above.

Cav1 Expression

To analyze if Cav1, a membrane lipid raft protein, that has been postulated to be colocalized with CFTR in epithelial cells and that is related to inflammation and apoptosis in macrophages, is affected by decreased CFTR expression in AM, we evaluated Cav1 RNA and protein levels. AM were transfected by CFTR-siRNA or control-siRNA and protein and RNA extracted after 48 h as described above. The mRNA of Cav1 was amplified by real-time RT-PCR using a Cav1 specific probe (Hs00184697_m1, Applied Biosystems). Cav1 protein levels were measured by Western analysis. Fifty µg protein was separated by electrophoresis on 4–12% Bis-Tris Gel (NuPAGE@Novex, Invitrogen), transferred to a PVDF membrane (Bio-Rad Laboratories) and incubated with a rabbit anti-Cav1 antibody (1∶200, Santa Cruz Biotechnology, Inc.) and normalized to β-tubulin expression as outlined above.

SREBP Expression and Activity

As Cav1 is related to lipid metabolism in macrophages, and the SREBP is known to be a major regulator of Cav1 expression, we evaluated expression and activity of SREBP in the AM with CFTR knockdown. Protein expression of SREBP was assessed in AM transfected with CFTR-siRNA or control-siRNA by Western analysis. Fifty µg of total cellular protein was loaded on 4–12% Bis-Tris Gel (NuPAGE@Novex, Invitrogen), transferred to a PVDF membrane (Bio-Rad Laboratories) membrane and probed with rabbit anti-SREBP-1 antibody (1∶1000, BD-Pharmingen, San Diego, CA). Transcriptional activity of SRE was assessed using an adenovirus vector expressing the SRE-promoter of HMG-CoA synthase linked to a luciferase reporter gene and an unregulated nuclear β-galactosidase gene (AdZ-SRE-luc), used to control for transfection efficiency. AM were transfected with CFTR-siRNA and control-siRNA for 48 h, and then infected with AdZ-SRE-Luc at 104 pu/cell for 48 h. Luciferase and β-galactosidase activities were analyzed in the cell lysates by luminometric luciferase and β-galactosidase assays (both, Stratagene, La Jolla, CA). Luciferase was quantified in a luminometer (BD-Pharmingen). The β-galactosidase levels were determined in a microplate luminometer (Bio-Rad Laboratories). Data are expressed as luciferase activity (RLU) normalized to β-galactosidase activity.

Quantification of Cellular Cholesterol Levels

AM were transfected with CFTR-siRNA and control-siRNA for 48 h. Cell lysates of 5×105 cells were harvested in PBS. Analysis of free and total cholesterol was carried out with 2 injections per sample on a DB17 (0.53-mm ID x 15 mx1 µM) gas-chromatography column at 250°C installed in a Hewlett-Packard 5890 gas-chromatograph equipped with a flame ionization detector (Global Medical Instrumentation Inc., St. Paul, MN). Lipids were extracted with hexane:isopropanol (3∶2, v/v) containing β-sitosterol (5 µg/sample) as an internal standard (Sigma-Aldrich). One third of the extract was used for determination of free cholesterol. The aliquot was dried down, resuspended in hexane (50 µl) and injected into the gas chromatograph. Two thirds of the extract were used to determine total cholesterol after alkaline hydrolysis. To each sample 200 µl KOH (50%, w/v) and 3 ml methanol were added, the mixture was capped with argon, vortexed and incubated at 80°C for 1 h, followed by lipid extraction using 3 ml of water and 5 ml of HPLC grade hexane (Sigma-Aldrich). Following centrifugation at 500 g for 5 min the hexane phase was concentrated by evaporation, resuspended in hexane and injected into the gas chromatograph. The difference between total cholesterol and free cholesterol is esterified cholesterol, and the mass of cholesterol ester is calculated as 1.67 x esterified cholesterol.

Statistics

The results are presented as mean ± standard error of the mean (SEM). Significance was calculated using the student t-test.

Results

CFTR Knockdown in AM

Efficiency of CFTR knockdown in human AM following transfection with CFTR specific siRNA (CFTR-siRNA) was assessed by CFTR mRNA and protein expression. The siRNA transfection efficiency in AM could be achieved 95% with the transfection of GAPDH-siRNA labeled with green fluorescence and knockdown of GAPDH gene measured by real-time PCR was more than 90% compared to the scrambled control siRNA (control-siRNA) (data not shown). Forty eight hours after transfection, CFTR mRNA levels were reduced more than 100-fold in CFTR-siRNA treated cells compared to the cells transfected with control-siRNA (p = 0.001; Figure 1A). CFTR protein is known to have an immature form (band B) with the size of 150 kDa and a fully glycosylated mature form (band C) with the size of 170 kDa. Our data showed that significant decrease of immature band B of CFTR protein while the mature form band C remained unchanged after 48 h CFTR-siRNA transfection (Figure 1B). The quantification of CFTR protein showed no decrease of band C (Figure 1C) and a 5.2-fold reduction of band B (p = 0.01; Figure 1D) in AM transfected with CFTR-siRNA compared to the cells transfected with control-siRNA. The similar size of CFTR protein was observed in the positive control Calu-3 cell lysate and there was no band in negative control A549 cell lysate, confirming the specificity of the CFTR antibody (Figure 1B).

thumbnail
Figure 1. CFTR knockdown in AM.

Human AM, obtained by bronchoalveolar lavage from healthy adults, were transfected with CFTR-siRNA or control-siRNA and analyzed after 48 h for CFTR mRNA and protein by real-time RT-PCR and Western analysis. To confirm the specificity of CFTR antibody, A549 cells, which do not have intrinsic CFTR expression, and CFTR expressing cell line Calu-3 cells were used as negative and positive controls in the Western analysis. A. Real-time RT-PCR. Human 18s ribosomal RNA was used as normalization control. B. Western analysis. B-tubulin or GAPDH was used as control. CFTR protein was detected as an mmature form (band B) at the size of 150 kDa and a mature form (band C) at the size of 170 kDa. C. and D. Quantification of Western analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

https://doi.org/10.1371/journal.pone.0011004.g001

Increased Inflammatory Response in AM with CFTR Knockdown

To analyze if CFTR silencing in human AM affects inflammatory cytokine secretions, IL-8 levels were analyzed in cell culture supernatants. Compared to human AM transfected with control-siRNA, IL-8 levels were increased in the supernatant obtained from human AM transfected with CFTR-siRNA at 24, 36, 48, and 72 h post transfection (p = 0.03 at 24 h; p = 0.01 at 36 h; p = 0.001 at 48 h; p = 0.004 at 72 h; Figure 2A). As NF-κB is known to be a positive regulator of IL-8 secretion we analyzed phosphorylated NF-κB and IκB-α, one of three inhibitory proteins of NF-κB activation in the AM with silenced CFTR. Forty eight hours after transfection, phosphorylated NF-κB, detected at a size of 65 kDa (Figure 2B), was increased 3.7-fold, as estimated by densitometry quantification (p = 0.01) in human AM transfected with CFTR-siRNA (Figure 2C). Expression of IκB-α (Figure 2D) was decreased 1.7-fold (p = 0.03) in human AM with silenced CFTR (Figure 2E). The data suggest that reduced production of CFTR induces a proinflammatory phenotype in human AM.

thumbnail
Figure 2. Increased IL-8 secretion of AM with CFTR knockdown.

A. AM, transfected with CFTR-siRNA or control-siRNA, were analyzed after 6, 12, 18, 24, 36, 48, and 72 h for IL-8 in the cell culture supernatant by ELISA (* denotes p value: p = 0.03 at 24 h; p = 0.01 at 36 h; p = 0.001 at 48 h; p = 0.004 at 72 h). B. AM, transfected with CFTR-siRNA or control-siRNA after 48 h, were evaluated for phosphorylated NF-κB protein expression of by Western analysis. C. Quantification of phosphorylated NF-κB Western analysis. D. AM, transfected with CFTR-siRNA or control-siRNA after 48 h, were also evaluated for IκB-α protein expression of by Western analysis. E. Quantification of IκB-αWestern analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

https://doi.org/10.1371/journal.pone.0011004.g002

Apoptosis in AM with CFTR Knockdown

To assess if the inflammatory phenotype of the AM with silenced CFTR would affect apoptosis, apoptosis was evaluated by TUNEL assay and evaluation of caspase-3 mediated cleavage of poly (ADP-ribose) polymerase (PARP) in human AM 48 h after transfection with CFTR-siRNA or control-siRNA. Apoptotic cells were detected by TUNEL assay in AM transfected CFTR-siRNA (visualized as yellow fluorescence superimposed by blue fluorescence DAPI nuclear staining and green TUNEL staining; Figure 3A) and were increased compared to AM transfected with control-siRNA (p = 0.02; Figure 3B). To correlate the apoptosis with the proinflammatory phenotype, the proportion of IL-8 secretion to the percentage of non-apoptotic cells was measured in AM transfected with CFTR-siRNA or control-siRNA. At 48 h, higher IL-8 production was detected in AM with CFTR knockdown compared to the control (p = 0.04; Figure 3C). The expression of PARP, a well known apoptosis indicator, was evaluated by Western analysis. The cleaved form of PARP (89 kDa) was increased in human AM tranfected with CFTR-siRNA (Figure 3D). Quantification of PARP protein by densitometry revealed a 3.3-fold increase in human AM transfected with CFTR-siRNA compared to controls (p = 0.01; Figure 3E). The data suggest that knockdown of CFTR promotes apoptosis in human AM.

thumbnail
Figure 3. Increased apoptosis in AM with deficient CFTR.

AM transfected with CFTR-siRNA or control-siRNA for 48 h were analyzed for apoptosis by TUNEL assay and for cleaved PARP protein expression by Western analysis. A. TUNEL assay. The nuclear staining of green fluorescence was shown as the positive apoptosis signal. DAPI served as normal nuclear staining control. B. Quantification of TUNEL assay. C. IL-8 secretion adjusted to the percentage of non-apoptotic cells in AM transfected with CFTR-siRNA or control-siRNA after 48 h. D. Western analysis of cleaved PARP protein expression using β-tubulin as loading control. E. Quantification of Western analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

https://doi.org/10.1371/journal.pone.0011004.g003

Cav1 is Increased in AM with CFTR Knockdown

We next evalaluated the expression of Cav1, a component of cellular membrane lipid rafts, that colocalizes with CFTR and is involved in regulation of inflammatory responses and apoptosis in macrophages. Forty eight hours after transfection Cav1 mRNA was increased 2.7-fold (p = 0.03) in human AM transfected with CFTR-siRNA compared to controls transfected with control-siRNA (Figure 4A). Consistently, protein expression of Cav1, detected at size of 22 kDa, was increased in human AM tranfected with CFTR-siRNA compared to controls (Figure 4B). Quantification of Cav1 protein expression by densitometry revealed a 2.2-fold (p = 0.02) increase in human AM transfected with CFTR-siRNA compared to controls (Figure 4C).

thumbnail
Figure 4. Cav1 expression is increased in AM with deficient CFTR.

AM transfected with CFTR-siRNA or control-siRNA were analyzed for Cav1 mRNA and protein expression after 48 h. A. Real-time RT-PCR. The human 18 s ribosome RNA served as the normalization control. B. Western analysis. B-tubulin was used as control. C. Quantification of Western analysis. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

https://doi.org/10.1371/journal.pone.0011004.g004

SREBP Cleavage and SRE Transcription Activity in AM with CFTR Knockdown

We next analyzed if the increased Cav1 expression could have been regulated by SREBP, a known negative regulator of its transcription and that has been found to be increased in CF epithelial cells. Expression and activity levels of SREBP in AM with CFTR knockdown were evaluated by Western analysis and promoter reporter gene expression. Western analysis, carried out 48 h after transfection, revealed decreased expression of mature SREBP (mSREBP) (68 kDa), the transcriptionally active protein that is cleaved, by a multi-step process, from the inactive precursor from, precursor SREBP (pSREBP) (125 kDa) in human AM transfected with CFTR-siRNA compared to human AM transfected with control-siRNA (Figure 5A). Concentration of mSREBP was decreased 2-fold in AM with silenced CFTR (p = 0.02; Figure 5B) as estimated by densitometry. To evaluate SRE-mediated gene transcription, human AM were transduced with AdZ-SRE-luc, an Ad vector expressing a luciferase reporter gene under the control of an SRE-promoter or AdNull, a control vector. Consistent with decreased mSREBP levels, transcriptional activity of SRE was reduced in CFTR-deficient human AM compared to human AM transfected with control-siRNA (p = 0.03; Figure 5C).

thumbnail
Figure 5. Decreased cleavage of the SREBP and transcription activity of SRE in AM with deficient CFTR.

A. AM transfected with CFTR-siRNA or control-siRNA were evaluated after 48 h for SREBP protein expression by Western analysis. B. Quantification of Western analysis. C. Transcription activity assay of SRE. AM, transfected with CFTR-siRNA for 48 h, were infected with AdZ-SRE-luc. The transcriptional activity of SRE was measured by luciferase assay using β-gal as normalization control. D. Total cellular cholesterol, free cholesterol, and cholesterol ester were measured by liquid chromatography. Shown is the mean ± SEM of three pairs of independent samples. This experiment is the representative of 6 studies.

https://doi.org/10.1371/journal.pone.0011004.g005

SREBP is a key transcription factor of cellular lipid homeostasis and is usually regulated by altered cellular cholesterol. An altered cellular cholesterol found in CF epithelial cells has been proposed to lead to SREBP activation, we determined cellular cholesterol levels in the AM with silenced CFTR. Total and free cholesterol mass were analyzed by gas chromatography and determined cholesteryl ester mass by substraction of free cholesterol from total cholesterol. Total cholesterol and free cholesterol mass were not different in human AM transfected with CFTR-siRNA or control-siRNA 48 h or 63 h (data not shown) after transfection (Figure 5D). We measured de novo synthesis of free cholesterol by evaluating incorporation of 3H-mevalonolactone into free cholesterol over 16 h and incorporation of 3H-oleate into cholesteryl-ester over 4 h and found no difference between both experimental groups (data not shown). The finding of unaffected cholesterol mass in the presence of increased Cav1 expression and decreased mSREBP are consistent with a model in which knockdown of CFTR in human AM induces a redistribution of regulatory pools of cholesterol.

Discussion

The contribution of AM to the hyperinflammatory milieu in the CF lung is not clear. The present study attempts to address this question by analyzing AM from healthy donors in which expression of CFTR is decreased by siRNA-mediated knockdown. AM with decreased CFTR expression exhibited an inflammatory phenotype as evidence by increased IL-8 and NF-κB, similar to the phenotype described in epithelial cells that express no or defective CFTR [44][46]. In contrast to data obtained in epithelial cells, our results demonstrated an inverse relationship between expression of CFTR and apoptosis as well as expression of mSREBP [39]. The data suggest an AM specific phenotype in which increased expression of Cav1 could be a consequence of decreased mSREBP and cause for increased apoptosis in this cell type.

A hyperinflammatory response is a hallmark of CF lung disease [47]. CFTR-deficient epithelial cells or epithelial cells treated with CFTR chloride channel inhibitors show increased secretion of IL-8 [44], [46]. Elevated levels of proinflammatory cytokines, such as IL-8 and IL-6, and decreased levels of the anti-inflammatory cytokine IL-10 are characteristic findings in the bronchioalveolar lavage fluid of CF patients even in the absence of pathogens [1], [48], [49]. These cytokines are thought to be primarily produced by the CFTR-deficient epithelial cells, however increased baseline levels of IL-8 have also been observed in blood monocytes of CF patients [12] and in AM derived from CF knockout mice [17]. Our results that demonstrate increased secretion of IL-8 and phosphorylated NF-κB, a positive regulator of IL-8 expression in human AM obtained from healthy donor in which CFTR expression was decreased by siRNA-medaited knockdown are consistent with this shifting paradigm [50]. Increased activation of NF-κB in CFTR-deficient epithelial cells has been thought to partially be a result of abnormal trafficking of mutant CFTR protein CFTRΔF508, where its accumulation in the ER induces intracellular stress resulting in NF-κB activation [45]. Our findings suggest that the lack of CFTR in AM alone triggers the hyperinflammatory response and the activation of NF-κB in AM and emphasize the potential significance of this cell type in CF lung disease.

Although expression of CFTR in human AM is relatively low compared to epithelial cells [11], knockdown by transfection of CFTR specific siRNA significantly decreased protein expression. As the half-life of plasma membrane CFTR exceeds 48 h [51], we did not observe a decrease of the mature form of CFTR. Other studies using CFTR siRNA also demonstrated the decrease of single band of CFTR protein [38], [52][54], which presumably also reflects the 150 kDa immature form. Because we could not keep AM with silenced CFTR in culture for extended periods to see decreased expression of the mature form of the protein, the observed effects could reflect the effect of intracellular trafficking rather than reduced CFTR expression on the cell membrane. In addition, our model using silencing of CFTR expression for short term culture may not reflect long term phenotypes of AM in the lower airways in the CF lung and also does not predict the phenotype induced by CFTR trafficking mutations such as ΔF508.

The relationship between CFTR and apoptosis is currently not clear. Several studies that investigated susceptibility to apoptosis in cell lines and tissue obtained from CF patients have yielded inconclusive results [21][30]. On one hand, increased apoptosis was observed in small intestine biopsies from CF patients [23]. Pancreatic apoptosis, associated with over-expression of IL-8 and activation of NF-κB pathway, was proposed as a possible mechanism for CF-related diabetes [21], [22]. Other studies however, did not observe increased baseline apoptosis in respiratory epithelial cells expressing mutant CFTR except after exposure of the cells to P. aeruginosa [30].

In contrast, several other studies reported that expression of defective CFTR or knockdown of CFTR protects against apoptosis phenotype [24][29]. CF knockout mice exhibited a baseline proinflammatory state and an anti-apoptotic phenotype in the pancreas [24] and showed failed induction of apoptosis in response to P. aeruginosa [28]. Recently, delayed apoptosis has been described in polymorphonuclear neutrophil (PMN) from CF patients, which might explain PMN persistence in the CF lung [29].

Our study showed increased baseline apoptosis in AM with CFTR knockdown. The increased apoptosis of inflammatory cells such as macrophages and neutrophils has been considered as an anti-inflammatory phenotype, however our study showed both augmented apoptosis and increased proinflammatory in AM with CFTR knockdown. Altered apoptosis has not been described for disruption of other epithelial chloride channels, including members of volume-regulated chloride channel CLC family [55], [56]. We suggested that as a consequence of more proinflammatory cytokine production the cells are prone to undergo apoptosis. Consistent with our findings, increased Cav1 has been observed in murine peritoneal macrophages undergoing apoptosis [31]. These contradictory observations could be related to cell type-specific responses and/or the agents used to induce apoptosis. Again, as outlined above, the phenotype of AM in the CF lung may not be accurately predicted by the short term culture results of our study.

Defective or absent CFTR is known to be associated with abnormalities in the cellular lipid metabolism [40], [57], [58]. The mSREBP and membrane free cholesterol levels have been shown to be increased in epithelial cells that express defective CFTR [39]. It is, however not clear whether these changes in cholesterol metabolism are cell-type or CFTR mutation specific, i.e. only found in cells that express ΔF508 CFTR protein [37]. Notably, knockdown of CFTR in human AM did not affect total and free cellular cholesterol levels in our study. On one hand, the lack of difference in cholesterol mass could be related to differences in cell type. In epithelial cells, cholesterol is mainly found in the plasma membrane, in macrophages, a significant amount of cholesterol can be found stored in intracellular compartments. Notably, failure to detect differences in total and free cholesterol mass does not exclude increased free cholesterol in the plasma membrane, but is rather supported by the finding of increased Cav1 mass, a protein known to bind free cholesterol in caveolae.

Increased expression of Cav1 in the present study following decrease of CFTR expression in AM supports that CFTR and Cav1 interact in this cell type. Potential explanation for increased SREBP in CF epithelial cells and decreased SREBP in AM macrophages that lack CFTR could be that intracellular cholesterol pools to the decreased mSREBP expression and decreased SRE activity seen in the AM with decreased CFTR expression, as Cav1 expression is negatively regulated by SRE in macrophages. Furthermore, it could also explicate the unchanged cellular cholesterol levels seen in the present study.

In summary, the present study points to a role in CFTR in AM, that may be similar to what is known for epithelial cells, as decreased CFTR expression resulted in an inflammatory cellular phenotype. In addition, AM with decreased CFTR expression showed augmented apoptosis, increased expression of Cav1, and decreased activation of SRE, which may be specific for this cell type. Lack of CFTR expression in AM may play a role in CF lung disease and the study of the abnormalities associated with lack of CFTR expression in this cell type may aid in understanding the complex cellular functions that CFTR is involved in.

Acknowledgments

We thank R.G. Crystal for insightful discussions and support with providing the alveolar macrophages. We are grateful to D Dang and M Teater for technical assistance and N Mohamed for help in preparing this manuscript.

Author Contributions

Conceived and designed the experiments: YX SW. Performed the experiments: YX HH. Analyzed the data: YX AK HH TSW. Contributed reagents/materials/analysis tools: YX AK BGH TSW. Wrote the paper: YX SW.

References

  1. 1. Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, et al. (1995) Early pulmonary inflammation in infants with cystic fibrosis. Am J Respir Crit Care Med 151: 1075–1082.TZ KhanJS WagenerT. BostJ. MartinezFJ Accurso1995Early pulmonary inflammation in infants with cystic fibrosis.Am J Respir Crit Care Med15110751082
  2. 2. Jacquot J, Tabary O, Le Rouzic P, Clement A (2008) Airway epithelial cell inflammatory signalling in cystic fibrosis. Int J Biochem Cell Biol 40: 1703–1715.J. JacquotO. TabaryP. Le RouzicA. Clement2008Airway epithelial cell inflammatory signalling in cystic fibrosis.Int J Biochem Cell Biol4017031715
  3. 3. Brennan S (2008) Innate immune activation and cystic fibrosis. Paediatr Respir Rev 9: 271–280.S. Brennan2008Innate immune activation and cystic fibrosis.Paediatr Respir Rev9271280
  4. 4. del Fresno C, Gomez-Pina V, Lores V, Soares-Schanoski A, Fernandez-Ruiz I, et al. (2008) Monocytes from cystic fibrosis patients are locked in an LPS tolerance state: down-regulation of TREM-1 as putative underlying mechanism. PLoS ONE 3: e2667.C. del FresnoV. Gomez-PinaV. LoresA. Soares-SchanoskiI. Fernandez-Ruiz2008Monocytes from cystic fibrosis patients are locked in an LPS tolerance state: down-regulation of TREM-1 as putative underlying mechanism.PLoS ONE3e2667
  5. 5. McDonald TV, Nghiem PT, Gardner P, Martens CL (1992) Human lymphocytes transcribe the cystic fibrosis transmembrane conductance regulator gene and exhibit CF-defective cAMP-regulated chloride current. J Biol Chem 267: 3242–3248.TV McDonaldPT NghiemP. GardnerCL Martens1992Human lymphocytes transcribe the cystic fibrosis transmembrane conductance regulator gene and exhibit CF-defective cAMP-regulated chloride current.J Biol Chem26732423248
  6. 6. Moss RB, Hsu YP, Olds L (2000) Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes. Clin Exp Immunol 120: 518–525.RB MossYP HsuL. Olds2000Cytokine dysregulation in activated cystic fibrosis (CF) peripheral lymphocytes.Clin Exp Immunol120518525
  7. 7. Moss RB (2004) Lymphocytes in cystic fibrosis lung disease: a tale of two immunities. Clin Exp Immunol 135: 358–360.RB Moss2004Lymphocytes in cystic fibrosis lung disease: a tale of two immunities.Clin Exp Immunol135358360
  8. 8. Painter RG, Valentine VG, Nicholas A, Leidal K, Zhang Q, et al. (2006) CFTR expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis. Biochemistry 45: 10260–10269.RG PainterVG ValentineA. NicholasK. LeidalQ. Zhang2006CFTR expression in human neutrophils and the phagolysosomal chlorination defect in cystic fibrosis.Biochemistry451026010269
  9. 9. Painter RG, Bonvillain RW, Valentine VG, Lombard GA, LaPlace SG, et al. (2008) The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils. J Leukoc Biol 83: 1345–1353.RG PainterRW BonvillainVG ValentineGA LombardSG LaPlace2008The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils.J Leukoc Biol8313451353
  10. 10. Xu Y, Tertilt C, Krause A, Quadri L, Crystal R, et al. (2009) Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells. Respir Res 10: 26.Y. XuC. TertiltA. KrauseL. QuadriR. Crystal2009Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells.Respir Res1026
  11. 11. Yoshimura K, Nakamura H, Trapnell BC, Chu CS, Dakemans W, et al. (1991) Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin. Nucleic Acids Res 19: 5417–5423.K. YoshimuraH. NakamuraBC TrapnellCS ChuW. Dakemans1991Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin.Nucleic Acids Res1954175423
  12. 12. Zaman MM, Gelrud A, Junaidi O, Regan MM, Warny M, et al. (2004) Interleukin 8 secretion from monocytes of subjects heterozygous for the ΔF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered. Clin Diagn Lab Immunol 11: 819–824.MM ZamanA. GelrudO. JunaidiMM ReganM. Warny2004Interleukin 8 secretion from monocytes of subjects heterozygous for the ΔF508 cystic fibrosis transmembrane conductance regulator gene mutation is altered.Clin Diagn Lab Immunol11819824
  13. 13. Russi TJ, Crystal RG (1997) Use of bronchoalveolar lavage and airway brushing to investigate the human lung. In: Crystal RG, West JB, Barnes PJ, Weibel E, editors. The Lung: Scientific Foundations. Philadelphia: Lippincott-Raven Publishers. pp. 371–382.TJ RussiRG Crystal1997Use of bronchoalveolar lavage and airway brushing to investigate the human lung.RG CrystalJB WestPJ BarnesE. WeibelThe Lung: Scientific FoundationsPhiladelphiaLippincott-Raven Publishers371382
  14. 14. Hubeau C, Puchelle E, Gaillard D (2001) Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis. J Allergy Clin Immunol 108: 524–529.C. HubeauE. PuchelleD. Gaillard2001Distinct pattern of immune cell population in the lung of human fetuses with cystic fibrosis.J Allergy Clin Immunol108524529
  15. 15. Brennan S, Sly PD, Gangell CL, Sturges N, Winfield K, et al. (2009) Alveolar macrophages and CC chemokines are increased in children with cystic fibrosis. Eur Respir J 34: 655–661.S. BrennanPD SlyCL GangellN. SturgesK. Winfield2009Alveolar macrophages and CC chemokines are increased in children with cystic fibrosis.Eur Respir J34655661
  16. 16. Di A, Brown ME, Deriy LV, Li C, Szeto FL, et al. (2006) CFTR regulates phagosome acidification in macrophages and alters bactericidal activity. Nat Cell Biol 8: 933–944.A. DiME BrownLV DeriyC. LiFL Szeto2006CFTR regulates phagosome acidification in macrophages and alters bactericidal activity.Nat Cell Biol8933944
  17. 17. Bruscia EM, Zhang PX, Ferreira E, Caputo C, Emerson JW, et al. (2009) Macrophages directly contribute to the exaggerated inflammatory response in cystic fibrosis transmembrane conductance regulator-/- mice. Am J Respir Cell Mol Biol 40: 295–304.EM BrusciaPX ZhangE. FerreiraC. CaputoJW Emerson2009Macrophages directly contribute to the exaggerated inflammatory response in cystic fibrosis transmembrane conductance regulator-/- mice.Am J Respir Cell Mol Biol40295304
  18. 18. Vandivier RW, Fadok VA, Hoffmann PR, Bratton DL, Penvari C, et al. (2002) Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis. J Clin Invest 109: 661–670.RW VandivierVA FadokPR HoffmannDL BrattonC. Penvari2002Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis.J Clin Invest109661670
  19. 19. Vandivier RW, Fadok VA, Ogden CA, Hoffmann PR, Brain JD, et al. (2002) Impaired clearance of apoptotic cells from cystic fibrosis airways. Chest 121: 89S.RW VandivierVA FadokCA OgdenPR HoffmannJD Brain2002Impaired clearance of apoptotic cells from cystic fibrosis airways.Chest12189S
  20. 20. Knight RA, Kollnberger S, Madden B, Yacoub M, Hodson ME (1997) Defective antigen presentation by lavage cells from terminal patients with cystic fibrosis. Clin Exp Immunol 107: 542–547.RA KnightS. KollnbergerB. MaddenM. YacoubME Hodson1997Defective antigen presentation by lavage cells from terminal patients with cystic fibrosis.Clin Exp Immunol107542547
  21. 21. Ali BR (2009) Is cystic fibrosis-related diabetes an apoptotic consequence of ER stress in pancreatic cells? Med Hypotheses 72: 55–57.BR Ali2009Is cystic fibrosis-related diabetes an apoptotic consequence of ER stress in pancreatic cells?Med Hypotheses725557
  22. 22. Rottner M, Kunzelmann C, Mergey M, Freyssinet JM, Martinez MC (2007) Exaggerated apoptosis and NF-κB activation in pancreatic and tracheal cystic fibrosis cells. FASEB J 21: 2939–2948.M. RottnerC. KunzelmannM. MergeyJM FreyssinetMC Martinez2007Exaggerated apoptosis and NF-κB activation in pancreatic and tracheal cystic fibrosis cells.FASEB J2129392948
  23. 23. Maiuri L, Raia V, De Marco G, Coletta S, de Ritis G, et al. (1997) DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis? FEBS Lett 408: 225–231.L. MaiuriV. RaiaG. De MarcoS. ColettaG. de Ritis1997DNA fragmentation is a feature of cystic fibrosis epithelial cells: a disease with inappropriate apoptosis?FEBS Lett408225231
  24. 24. Dimagno MJ, Lee SH, Hao Y, Zhou SY, McKenna BJ, et al. (2005) A proinflammatory, antiapoptotic phenotype underlies the susceptibility to acute pancreatitis in cystic fibrosis transmembrane regulator (-/-) mice. Gastroenterology 129: 665–681.MJ DimagnoSH LeeY. HaoSY ZhouBJ McKenna2005A proinflammatory, antiapoptotic phenotype underlies the susceptibility to acute pancreatitis in cystic fibrosis transmembrane regulator (-/-) mice.Gastroenterology129665681
  25. 25. Gottlieb RA, Dosanjh A (1996) Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis. Proc Natl Acad Sci U.S.A 93: 3587–3591.RA GottliebA. Dosanjh1996Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C127 cells: possible relevance to cystic fibrosis.Proc Natl Acad Sci U.S.A9335873591
  26. 26. L'hoste S, Chargui A, Belfodil R, Duranton C, Rubera I, et al. (2009) CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells. Free Radical Biol Med 46: 1017–1031.S. L'hosteA. CharguiR. BelfodilC. DurantonI. Rubera2009CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells.Free Radical Biol Med4610171031
  27. 27. Noe J, Petrusca D, Rush N, Deng P, VanDemark M, et al. (2009) CFTR regulation of intracellular pH and ceramides is required for lung endothelial cell apoptosis. Am J Respir Cell Mol Biol 41: 314–323.J. NoeD. PetruscaN. RushP. DengM. VanDemark2009CFTR regulation of intracellular pH and ceramides is required for lung endothelial cell apoptosis.Am J Respir Cell Mol Biol41314323
  28. 28. Cannon CL, Kowalski MP, Stopak KS, Pier GB (2003) Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator. Am J Respir Cell Mol Biol 29: 188–197.CL CannonMP KowalskiKS StopakGB Pier2003Pseudomonas aeruginosa-induced apoptosis is defective in respiratory epithelial cells expressing mutant cystic fibrosis transmembrane conductance regulator.Am J Respir Cell Mol Biol29188197
  29. 29. Moriceau S, Lenoir G, Witko-Sarsat V (2010) In cystic fibrosis homozygotes and heterozygotes, neutrophil apoptosis is delayed and modulated by diamide or roscovitine: evidence for an innate neutrophil disturbance. J Innate Immun 2: 260–266.S. MoriceauG. LenoirV. Witko-Sarsat2010In cystic fibrosis homozygotes and heterozygotes, neutrophil apoptosis is delayed and modulated by diamide or roscovitine: evidence for an innate neutrophil disturbance.J Innate Immun2260266
  30. 30. Rajan S, Cacalano G, Bryan R, Ratner AJ, Sontich CU, et al. (2000) Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells. Analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors. Am J Respir Cell Mol Biol 23: 304–312.S. RajanG. CacalanoR. BryanAJ RatnerCU Sontich2000Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells. Analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors.Am J Respir Cell Mol Biol23304312
  31. 31. Gargalovic P, Dory L (2003) Cellular apoptosis is associated with increased caveolin-1 expression in macrophages. J Lipid Res 44: 1622–1632.P. GargalovicL. Dory2003Cellular apoptosis is associated with increased caveolin-1 expression in macrophages.J Lipid Res4416221632
  32. 32. Gargalovic P, Dory L (2003) Caveolins and macrophage lipid metabolism. J Lipid Res 44: 11–21.P. GargalovicL. Dory2003Caveolins and macrophage lipid metabolism.J Lipid Res441121
  33. 33. Wang XM, Kim HP, Song R, Choi AMK (2006) Caveolin-1 confers antiinflammatory effects in murine macrophages via the MKK3/p38 MAPK pathway. Am J Physiol Respir Cell Mol Biol 34: 434–442.XM WangHP KimR. SongAMK Choi2006Caveolin-1 confers antiinflammatory effects in murine macrophages via the MKK3/p38 MAPK pathway.Am J Physiol Respir Cell Mol Biol34434442
  34. 34. Bajmoczi M, Gadjeva M, Alper SL, Pier GB, Golan DE (2009) Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa. Am J Physiol Cell Physiol 297: C263–C277.M. BajmocziM. GadjevaSL AlperGB PierDE Golan2009Cystic fibrosis transmembrane conductance regulator and caveolin-1 regulate epithelial cell internalization of Pseudomonas aeruginosa.Am J Physiol Cell Physiol297C263C277
  35. 35. Bist A, Fielding PE, Fielding CJ (1997) Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol. Proc Natl Acad Sci USA 94: 10693–10698.A. BistPE FieldingCJ Fielding1997Two sterol regulatory element-like sequences mediate up-regulation of caveolin gene transcription in response to low density lipoprotein free cholesterol.Proc Natl Acad Sci USA941069310698
  36. 36. Brown MS, Goldstein JL (1997) The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89: 331–340.MS BrownJL Goldstein1997The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor.Cell89331340
  37. 37. Gentzsch M, Choudhury A, Chang XB, Pagano RE, Riordan JR (2007) Misassembled mutant ΔF508 CFTR in the distal secretory pathway alters cellular lipid trafficking. J Cell Sci 120: 447–455.M. GentzschA. ChoudhuryXB ChangRE PaganoJR Riordan2007Misassembled mutant ΔF508 CFTR in the distal secretory pathway alters cellular lipid trafficking.J Cell Sci120447455
  38. 38. Mailhot G, Ravid Z, Barchi S, Moreau A, Rabasa-Lhoret R, et al. (2009) CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells. Am J Physiol Gastrointest Liver Physiol 297: G1239–G1249.G. MailhotZ. RavidS. BarchiA. MoreauR. Rabasa-Lhoret2009CFTR knockdown stimulates lipid synthesis and transport in intestinal Caco-2/15 cells.Am J Physiol Gastrointest Liver Physiol297G1239G1249
  39. 39. White NM, Jiang D, Burgess JD, Bederman IR, Previs SF, et al. (2007) Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis. Am J Physiol Lung Cell Mol Physiol 292: L476–L486.NM WhiteD. JiangJD BurgessIR BedermanSF Previs2007Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis.Am J Physiol Lung Cell Mol Physiol292L476L486
  40. 40. Worgall TS (2009) Lipid metabolism in cystic fibrosis. Curr Opin Clin Nutr Metab Care 12: 105–109.TS Worgall2009Lipid metabolism in cystic fibrosis.Curr Opin Clin Nutr Metab Care12105109
  41. 41. White NM, Corey DA, Kelley TJ (2004) Mechanistic similarities betweencultured cell models of cystic fibrosis and Niemann-Pick type C. Am J Respir Cell Mol Biol 31: 538–543.NM WhiteDA CoreyTJ Kelley2004Mechanistic similarities betweencultured cell models of cystic fibrosis and Niemann-Pick type C.Am J Respir Cell Mol Biol31538543
  42. 42. Worgall S, Heguy A, Luettich K, O'Connor TP, Harvey BG, et al. (2005) Similarity of gene expression patterns in human alveolar macrophages in response to Pseudomonas aeruginosa and Burkholderia cepacia. Infect Immun 73: 5262–5268.S. WorgallA. HeguyK. LuettichTP O'ConnorBG Harvey2005Similarity of gene expression patterns in human alveolar macrophages in response to Pseudomonas aeruginosa and Burkholderia cepacia.Infect Immun7352625268
  43. 43. Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11: 36–42.MD AbramoffPJ MagelhaesSJ Ram2004Image processing with ImageJ.Biophotonics Int113642
  44. 44. Perez A, Issler AC, Cotton CU, Kelley TJ, Verkman AS, et al. (2007) CFTR inhibition mimics the cystic fibrosis inflammatory profile. Am J Physiol Lung Cell Mol Physiol 292: L383–L395.A. PerezAC IsslerCU CottonTJ KelleyAS Verkman2007CFTR inhibition mimics the cystic fibrosis inflammatory profile.Am J Physiol Lung Cell Mol Physiol292L383L395
  45. 45. Weber AJ, Soong G, Bryan R, Saba S, Prince A (2001) Activation of NF-κB in airway epithelial cells is dependent on CFTR trafficking and Cl channel function. Am J Physiol Lung Cell Mol Physiol 281: L71–L78.AJ WeberG. SoongR. BryanS. SabaA. Prince2001Activation of NF-κB in airway epithelial cells is dependent on CFTR trafficking and Cl channel function.Am J Physiol Lung Cell Mol Physiol281L71L78
  46. 46. Tabary O, Escotte S, Couetil JP, Hubert D, Dusser D, et al. (2000) High susceptibility for cystic fibrosis human airway gland cells to produce IL-8 through the IκB kinase α pathway in response to extracellular NaCl content. J Immunol 164: 3377–3384.O. TabaryS. EscotteJP CouetilD. HubertD. Dusser2000High susceptibility for cystic fibrosis human airway gland cells to produce IL-8 through the IκB kinase α pathway in response to extracellular NaCl content.J Immunol16433773384
  47. 47. Puchelle E, Bajolet O, Abely M (2002) Airway mucus in cystic fibrosis. Paediatr Respir Rev 3: 115–119.E. PuchelleO. BajoletM. Abely2002Airway mucus in cystic fibrosis.Paediatr Respir Rev3115119
  48. 48. Dakin CJ, Numa AH, Wang HE, Morton JR, Vertzyas CC, et al. (2002) Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis. Am J Respir Crit Care Med 165: 904–910.CJ DakinAH NumaHE WangJR MortonCC Vertzyas2002Inflammation, infection, and pulmonary function in infants and young children with cystic fibrosis.Am J Respir Crit Care Med165904910
  49. 49. Muhlebach MS, Stewart PW, Leigh MW, Noah TL (1999) Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients. Am J Respir Crit Care Med 160: 186–191.MS MuhlebachPW StewartMW LeighTL Noah1999Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients.Am J Respir Crit Care Med160186191
  50. 50. Viatour P, Merville MP, Bours V, Chariot A (2005) Phosphorylation of NF-κB and IκB proteins: implications in cancer and inflammation. Trends Biochem Sci 30: 43–52.P. ViatourMP MervilleV. BoursA. Chariot2005Phosphorylation of NF-κB and IκB proteins: implications in cancer and inflammation.Trends Biochem Sci304352
  51. 51. Heda GD, Marino CR (2000) Surface expression of the cystic fibrosis transmembrane conductance regulator mutant ΔF508 is markedly upregulated by combination treatment with sodium butyrate and low temperature. Biochem Biophys Res Commun 271: 659–664.GD HedaCR Marino2000Surface expression of the cystic fibrosis transmembrane conductance regulator mutant ΔF508 is markedly upregulated by combination treatment with sodium butyrate and low temperature.Biochem Biophys Res Commun271659664
  52. 52. Li J, Allen KT, Sun XC, Cui M, Bonanno JA (2008) Dependence of cAMP meditated increases in Cl and HCO3 permeability on CFTR in bovine corneal endothelial cells. Exp Eye Res 86: 684–690.J. LiKT AllenXC SunM. CuiJA Bonanno2008Dependence of cAMP meditated increases in Cl and HCO3 permeability on CFTR in bovine corneal endothelial cells.Exp Eye Res86684690
  53. 53. Li T, Folkesson HG (2006) RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo. Am J Physiol Lung Cell Mol Physiol 290: L649–L660.T. LiHG Folkesson2006RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo.Am J Physiol Lung Cell Mol Physiol290L649L660
  54. 54. Zaidi T, Bajmoczi M, Zaidi T, Golan DE, Pier GB (2008) Disruption of CFTR-dependent lipid rafts reduces bacterial levels and corneal disease in a murine model of Pseudomonas aeruginosa keratitis. Invest Ophthalmol Vis Sci 49: 1000–1009.T. ZaidiM. BajmocziT. ZaidiDE GolanGB Pier2008Disruption of CFTR-dependent lipid rafts reduces bacterial levels and corneal disease in a murine model of Pseudomonas aeruginosa keratitis.Invest Ophthalmol Vis Sci4910001009
  55. 55. Blaisdell CJ, Morales MM, Andrade ACO, Bamford P, Wasicko M, et al. (2004) Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts. Am J Physiol Lung Cell Mol Physiol 286: L420–L426.CJ BlaisdellMM MoralesACO AndradeP. BamfordM. Wasicko2004Inhibition of CLC-2 chloride channel expression interrupts expansion of fetal lung cysts.Am J Physiol Lung Cell Mol Physiol286L420L426
  56. 56. Jentsch TJ, Poët M, Fuhrmann JC, Zdebik AA (2005) Physiological functions of CLC Chloride channels gleaned from human genetic disease and mouse models. Annu Rev Physiol 67: 779–807.TJ JentschM. PoëtJC FuhrmannAA Zdebik2005Physiological functions of CLC Chloride channels gleaned from human genetic disease and mouse models.Annu Rev Physiol67779807
  57. 57. Hamai H, Keyserman F, Quittell LM, Worgall TS (2009) Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling. J Lipid Res 50: 1101–1108.H. HamaiF. KeysermanLM QuittellTS Worgall2009Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling.J Lipid Res5011011108
  58. 58. Anderson RGW, Jacobson K (2002) A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296: 1821–1825.RGW AndersonK. Jacobson2002A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains.Science29618211825