Cystic Fibrosis Cell Lines Research Articles

Allergic contact dermatitis to parthenolide

Parthenolide is a sesquiterpene lactone that is found in the fruit and flowers of the plant feverfew (Tanacetum parthenium or Chrysanthemum parthenium). Parthenolide, comprising 85% of the total sesquiterpene content, is located in the superficial leaf glands of the plant. This plant is grown all over the world and is used for its anti-inflammatory effects to treat a wide range of conditions, including migraine headache and arthritis. Parthenolide also occurs in other common flowering plants of the family Asteraceae (Compositae), including daisies and magnolias. We present a case of contact dermatitis to parthenolide. A 76-year-old woman with a history of allergic rhinitis presented with a chronic hand dermatitis for many years. She denied any associated environmental changes, such as changes in detergents, soaps, conditioners, shampoos, or cosmetics. There was no association with any particular foods or specific medications, such as nonsteroidal anti-inflammatory medications or antibiotics. The dermatitis was extremely pruritic and appeared to worsen when she performed gardening activities, especially during the fall, spring, and summer months. She did not perform much gardening in the winter season, and the dermatitis improved during this time. She had previously seen dermatology and was prescribed multiple topical preparations, including clobetasol proprionate 0.05%, triamcinolone acetonide 0.1%, and pimecrolimus 1%, with only temporary benefit. On examination, there was erythema with thickening of the dorsal surface of the hands and fingers, as well as scaling erythema and lichenification of the palms. There were no vesicles noted. T.R.U.E. Test patch testing was performed. At 48 hours after application, there was a 1þ reaction (erythema and infiltration) to parthenolide. The patient cancelled the 96hour patch test read but reported increased pruritis and irritation at the testing site in the days following the 48-hour read. At the 7-day patch test read, there was a 2þ reaction (increased erythema with infiltration and papules) to parthenolide (Figure 1). Based on the history (working extensively with flowering plants), timing (dermatitis worsening with exposure to these plants), and positive patch test result, a diagnosis of allergic contact dermatitis to parthenolide was made. Protective handwear was recommended for the patient with improvement in the dermatitis. Allergic contact sensitization is a frequent cause of chronic hand dermatitis (24%). A “crescendo” (increase in reaction) effect over the timing of the patch test reading, which occurred in our patient, is common in allergic contact dermatitis. Although Alstroemeria (Peruvian lily) is the most common cause of hand dermatitis in floral workers in the United States, parthenolide is the most common cause of plant dermatitis in India. In one study in Denmark, the prevalence of parthenolide-induced contact dermatitis was estimated to be 2.25% of all patients with contact dermatitis. Although direct contact is thought to be the main method of sensitization, parthenolide dermatitis has also been shown to occur via airborne particles, occurring not only on the palmar surfaces of the hands but also on the upper eyelids, posterior ear, submandibular region, decolletage of neck, forearms, and chest. Other Compositae allergens, including ragweed, sunflower, and goldenrod, can also cause airborne contact dermatitis. In addition, patients with a past or present history of atopic dermatitis may become sensitized to parthenolide at any age, regardless of occupational exposures. Because of the purported anti-inflammatory effects of parthenolide, multiple studies have provided insight into possible mechanisms. One study demonstrated that parthenolide inhibited IkB kinase and nuclear factor kappa B activation in cystic fibrosis cell lines in mice. Another group studied the effect of parthenolide on human monocyteederived dendritic cells stimulated with LPS or TNF-a. Parthenolide inhibited the upregulation of costimulatory molecules (including CD 80, MHC class II, and CD 40) on dendritic cells induced by LPS but not by TNF-a, and parthenolide also prevented TNF-a and IL-12 secretion by LPS-stimulated dendritic cells. Parthenolide prevented nuclear factor kappa B nuclear translocation and IkB degradation in human dendritic cells induced by both LPS and TNF-a. Because of these potential effects, parthenolide is being used in various herbal remedies, including herbal teas, tinctures, tablets, and supplements. Topical products containing parthenolide have been shown to produce positive patch testing reactions in parthenolide-sensitive patients.

Ruthenium-Modified Sensitive NO Sensors: Quantifying Nitric Oxide in the Pathobiology of Cystic Fibrosis

This is a preliminary work towards preparing a device capable to measure nitric oxide levels in a cystic fibrosis cell line model. It has been found that exhaled NO levels remains unchanged or reduced in cystic fibrosis patients unlike other inflammatory lung diseases like asthma where it increases. However it is not clear whether the lower NO levels in cystic fibrosis correlate with lowered production of this metabolite in the bronchial epithelium. We will present preliminary results of our ruthenium oxide modified combined electrodes and how they can be applied to the study of cystic fibrosis at the cellular level. In this work, we explore the performance of combined reference/working electrodes modified with ruthenium oxide and Poly(3,4-ethylenedioxythiophene) (PEDOT) in the detection of nitric oxide with the goal to measure nitric oxide at the level of single or collective cultured cells. The synergistic effect of the electrocatalytic activity of ruthenium oxide and the enhanced surface area for catalytic activity provided by the polymer greatly enhanced the analytical performance of our sensors in terms of sensitivity, selectivity and stability. With the incorporation of a layer by layer method of electrodeposition we attained a normalized sensitivity of 8.38E-4 pA/nM/μM2 and 1.16E-2 pA/nM/μM2 towards NO in the linear range of 0.2-1.6 mM and 2-16 nM respectively, at an applied potential of 0.8 V vs Ag/AgCl and a detection limit in the vicinity of 500 pM. In order to improve the selectivity of our sensors we coated the surface with nafion and also an ionic liquid composite and lowered the applied to 0.6 V vs. Ag/AgCl. We also measured the response to NO using Differential Pulse Amperometry to further decrease response from biological interferents especially nitrite and ascorbic acid.

Monitoring of Nitric Oxide Levels Released from NOS-Loaded Liposomes in Proximity of Cystic Fibrosis Cell Line Models

Nitric oxide (NO) is a biological free radical, associated with many physiological and pathological processes that include neuronal signaling, immune response and inflammatory response. In mammalian organisms, NO is produced from L-arginine in an NADPH-dependent reaction catalyzed by a family of nitric oxide synthase (NOS) enzymes. Fluctuations in NO levels generally accompany a number of inflammatory diseases. Cystic Fibrosis (CF) is an autosomal recessive genetic disease that chronically affects the lungs. It has been found that exhaled NO levels remains unchanged or reduced in cystic fibrosis patients unlike other inflammatory lung diseases like asthma where it increases. However it is not clear whether the lower NO levels in cystic fibrosis correlate with lowered production of this metabolite in the bronchial epithelium. Also, it has been proposed recently that nitric oxide can be used as a potent antimicrobial molecule to treat bacterial infection of CF tissue. Nitric oxide’s role in CF is mutlifaceted. In this work, we want to establish a basic system to understand how nitric oxide interacts with CF cells versus normal cells by monitoring levels of nitric oxide released from NOS-loaded liposomes on normal cells and CF cells. We will present work establishing the performance of combined reference/working electrodes modified with ruthenium oxide and poly(3,4-ethylenedioxythiophene) (PEDOT) for catalytic detection of nitric oxide. We will then show how we can use this nitric oxide sensitive probe to measure levels of nitric oxide from NOS-loaded liposomes. In this case, nitric oxide is triggered exogenously both in proximity of normal cells and near a CF cell line model. We will compare and contrast the observed amperometric levels of nitric oxide and discuss the results in light of potential biological implication of nitric oxide with the two cell models.

HLA-G expression and regulation during Pseudomonas aeruginosa infection in cystic fibrosis patients.

Deregulated immune response fails to control biofilm-forming bacteria, as Pseudomonas aeruginosa, in the lungs of cystic fibrosis (CF) patients. HLA-G is an immune-modulatory molecule involved in respiratory diseases and infections. HLA-G mRNA and protein were analyzed in plasma and exhaled breath condensate from CF patients undergoing intravenous antibiotic treatment, CF cell line and murine model. Therapy normalizes HLA-G plasmatic in CF patients suggesting a systemic anti-inflammatory role while in CF airway system, higher expression of HLA-G is associated with P. aeruginosa infection. CF cell line and murine model expressed higher HLA-G molecules in the presence of P. aeruginosa. Plasmatic and lung HLA-G expression suggest a role in reducing systemic inflammation and supporting P. aeruginosa infection.

Chitosan as a non-viral co-transfection system in a cystic fibrosis cell line

Successful gene therapy requires the development of suitable vehicles for the selective and efficient delivery of genes to specific target cells at the expense of minimal toxicity. In this work, we investigated a non-viral gene delivery system based on chitosan (CS) to specifically address cystic fibrosis (CF). Thus, electrostatic self-assembled CS-pEGFP and CS-pEGFP-siRNA complexes were prepared from high-pure fully characterized CS (Mw ∼20kDa and degree of acetylation ∼30%). The average diameter of positively-charged complexes (i.e. ζ∼+25mV) was ∼200nm. The complexes were found relatively stable over 14h in Opti-MEM. Cell viability study did not show any significant cytotoxic effect of the CS-based complexes in a human bronchial cystic fibrosis cell line (CFBE41o-). We evaluated the transfection efficiency of this cell line with both CS-pEGFP and co-transfected with CS-pEGFP-siRNA complexes at (N/P) charge ratio of 12. We reported an increase in the fluorescence intensity of CS-pEGFP and a reduction in the cells co-transfected with CS-pEGFP-siRNA. This study shows proof-of-principle that co-transfection with chitosan might be an effective delivery system in a human CF cell line. It also offers a potential alternative to further develop therapeutic strategies for inherited disease treatments, such as CF.

Bronchial epithelial cell lines and primary nasal epithelial cells from cystic fibrosis respond differently to cigarette smoke exposure

The effects of cigarette smoke extract (CSE) on airway epithelial cells (AECs) from cystic fibrosis (CF) and non-cystic fibrosis (non-CF) individuals are not fully understood. It has been suggested that CSE modulates inflammatory cytokine release from AECs by modulating the epidermal growth factor receptor (EGFR) pathway; these pathways could reveal novel therapeutic targets. We compared the effect of CSE pre-incubation on IL-8 release from CF and non-CF bronchial epithelial cell lines, and separately, with primary nasal epithelial cells (NECs) retrieved from CF and non-CF individuals. We also determined if the EGFR pathway regulates IL-8 release by LPS or cytomix in non-CF and CF AECs at baseline and following CSE exposure.CF and non-CF cell lines, NECs derived from both CF patients (R117H heterozygous and F508del homozygous), and from healthy subjects, were cultured in the presence or absence of CSE, and subsequently exposed to inflammatory stimuli. In cell lines CSE significantly reduced IL-8 release following inflammatory challenge. Conversely, CSE pre-treatment was pro-inflammatory in primary NECs. In NECs from control subjects, CSE increased cytomix and LPS induced IL-8 release, and for the R117H heterozygous NEC cultures, CSE enhanced basal IL-8 release. Cytomix and LPS induced IL-8 release from F508del homozygous NEC cultures was further heightened following CSE pre-treatment.EGFR inhibition mitigated IL-8 release from immortalised and primary non-CF and CF AECs, suggesting that constitutive and CSE elicited IL-8 release from AECs is partly regulated via the EGFR pathway.This study demonstrates the importance of the EGFR cascade in the regulation of constitutive and CSE induced inflammatory mediator release from immortalised and primary AECs. Moreover, it clearly highlights the significance of using primary cells to confirm results obtained from immortalised cell studies, as these model systems may respond very differently to the stimuli under investigation.

MiR-17 overexpression in cystic fibrosis airway epithelial cells decreases interleukin-8 production

Interleukin (IL)-8 levels are higher than normal in cystic fibrosis (CF) airways, causing neutrophil infiltration and non-resolving inflammation. Overexpression of microRNAs that target IL-8 expression in airway epithelial cells may represent a therapeutic strategy for cystic fibrosis. IL-8 protein and mRNA were measured in cystic fibrosis and non-cystic fibrosis bronchoalveolar lavage fluid and bronchial brushings (n=20 per group). miRNAs decreased in the cystic fibrosis lung and predicted to target IL-8 mRNA were quantified in βENaC-transgenic, cystic fibrosis transmembrane conductance regulator (Cftr)-/- and wild-type mice, primary cystic fibrosis and non-cystic fibrosis bronchial epithelial cells and a range of cystic fibrosis versus non-cystic fibrosis airway epithelial cell lines or cells stimulated with lipopolysaccharide, Pseudomonas-conditioned medium or cystic fibrosis bronchoalveolar lavage fluid. The effect of miRNA overexpression on IL-8 protein production was measured. miR-17 regulates IL-8 and its expression was decreased in adult cystic fibrosis bronchial brushings, βENaC-transgenic mice and bronchial epithelial cells chronically stimulated with Pseudomonas-conditioned medium. Overexpression of miR-17 inhibited basal and agonist-induced IL-8 protein production in F508del-CFTR homozygous CFTE29o(-) tracheal, CFBE41o(-) and/or IB3 bronchial epithelial cells. These results implicate defective CFTR, inflammation, neutrophilia and mucus overproduction in regulation of miR-17. Modulating miR-17 expression in cystic fibrosis bronchial epithelial cells may be a novel anti-inflammatory strategy for cystic fibrosis and other chronic inflammatory airway diseases.

Pseudomonas aeruginosa elastase LasB modulates CFTR expression and innate immune responses in airway epithelial cells

Introduction Pseudomonas aeruginosa (P.a.) is an opportunistic pathogen that is responsible for nocosomial infections and is the major bacterium found in the cystic fibrosis (CF) lungs. Our group has shown that host endogenous proteases such as neutrophil elastase are able to cleave and inactivate CFTR in the airways [1] . Here we hypothesize that the P.a. virulence factor elastase LasB could modulate CFTR expression and innate immune responses in airway epithelial cells. Methods The effect of recombinant LasB and secretomes from P.a. strains (WT and ΔLasB PAO1, referred as WT-SEC and ΔLasB-SEC) on CFTR integrity was evaluated in the alveolar epithelial cell line A549 infected with the adenovirus Ad GFP-WT-CFTR and in the Calu-3 cell line grown on semi-permeable supports. The P.a. elastase and secretomes were used to treat the corrected CF cell line CFBE41o-WT-CFTR for ion transport experiments in Ussing chambers and the NCI-H292 and the polarized Calu-3 cell lines for the measurement of secreted cytokines and that of elafin, an important mucosal anti-microbial molecule. Results SDS-PAGE analysis of the secretomes showed that LasB is the main secreted protein by WT PAO1. Recombinant LasB (10 nM) and the WT-SEC (10%) but not the ΔLasB-SEC, induced a slight decrease in CFTR expression in our CFTR overexpressing model. The recombinant elastase did not show any effect on the activity of ENaC or CFTR in Ussing chamber experiments. However, WT-SEC (10%) seemed to decrease CFTR activity as reported by the measurement of forskolin/ibmx induced short-circuit current (ΔIsc) and CFTR (Inh-172) -sensitive ΔIsc, when compared to the effect of the ΔLasB-SEC or the LB medium alone. Finally, recombinant LasB, as well as the WT-SEC but not ΔLasB-SEC decreased IL-6 and elafin secretion whereas it had no effect on IL-8 secretion. Conclusion Taken together, our results show that LasB, the major type 2 secretion system virulence factor, is able to modulate the expression of CFTR and its activity when used in the P.a. secretome. Moreover, it targets the immune response by regulating the expression of cytokines and anti-microbial peptides and we are currently investigating the molecular mechanisms and signalling pathways underlying these processes.

Pseudomonas Quinolone Signal Modulates Cystic Fibrosis Epithelial Cell Response through the Toll- Like Receptor 4

Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe infection in compromised individuals, including persons with Cystic Fibrosis (CF). The expression of many virulence factors in P. aeruginosa is controlled by Quorum Sensing Molecules (QSMs) that are synthesized and secreted by this bacterium. Recent studies suggest that QSMs are also capable of interspecies communication, with exposure of mammalian cells to Acyl-Homoserine Lactones (AHLs) and Pseudomonas Quinolone Signal (PQS) resulting in an immunomodulatory response. Although the initial immune response is intended to clear/contain the infection, this process is ineffective in CF lungs, and the persistent, excessive inflammation eventually leads to structural damage to the tissue. The goal of this study was to examine the response of IB3-1 CF airway epithelial cell line to PQS, alone and in conjunction with Pseudomonas-Derived Lipopolysaccharides (pLPS). In contrast to results obtained with other cell types suggesting that PQS is anti-inflammatory, PQS induced inflammation in the IB3- 1 CF cell line and exacerbated inflammation when administered simultaneously with LPS, as determined by ELISA for two markers of inflammation, interleukin-6 and interleukin-8. In addition, PQS was shown, for the first time, to act through toll-like receptor 4, a receptor that traditionally has only been associated with LPS. A better understanding of the role that QSMs play in the inflammatory response can potentially lead to new strategies to minimize airway destruction.

Nobiletin Stimulates Chloride Secretion in Human Bronchial Epithelia via a cAMP/PKA-Dependent Pathway

Background/Aims: Nobiletin, a citrus flavonoid isolated from tangerines, alters ion transport functions in intestinal epithelia, and has antagonistic effects on eosinophilic airway inflammation of asthmatic rats. The present study examined the effects of nobiletin on basal short-circuit current (I_SC) in a human bronchial epithelial cell line (16HBE14o-), and characterized the signal transduction pathways that allowed nobiletin to regulate electrolyte transport. Methods: The I_SC measurement technique was used for transepithelial electrical measurements. Intracellular calcium ([Ca²⁺]_i) and cAMP were also quantified. Results: Nobiletin stimulated a concentration-dependent increase in I_SC, which was due to Cl^- secretion. The increase in I_SC was inhibited by a cystic fibrosis transmembrane conductance regulator inhibitor (CFTR_inh-172), but not by 4,4'-diisothiocyano-stilbene-2,2'-disulphonic acid (DIDS), Chromanol 293B, clotrimazole, or TRAM-34. Nobiletin-stimulated I_SC was also sensitive to a protein kinase A (PKA) inhibitor, H89, and an adenylate cyclase inhibitor, MDL-12330A. Nobiletin could not stimulate any increase in I_SC in a cystic fibrosis (CF) cell line, CFBE41o-, which lacked a functional CFTR. Nobiletin stimulated a real-time increase in cAMP, but not [Ca²⁺]_i. Conclusion: Nobiletin stimulated transepithelial Cl^- secretion across human bronchial epithelia. The mechanisms involved activation of adenylate cyclase- and cAMP/PKA-dependent pathways, leading to activation of apical CFTR Cl^- channels.

The low PLC-δ1 expression in cystic fibrosis bronchial epithelial cells induces upregulation of TRPV6 channel activity

Increase of Ca2+ influx in Cystic Fibrosis (CF) cells has been reported to be related to Transient Receptor Potential Canonical (TRPC6) channel, which is implicated in a functional coupling with Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Several members of the Transient Receptor Potential Vanilloid (TRPV) channels family have already been described as emerging target for respiratory diseases. Two specific isoforms, TRPV5 and TRPV6 are of particular interest in the context of CF Ca2+ homeostasis as they are highly selective toward Ca2+ and constitutively activated. Thus, we investigated the involvement of these channels in Ca2+ influx in CF and non-CF human bronchial epithelial cell lines. 16HBE14o-, CFBE41o- cell lines, primary human airway epithelial cells (hAEC) and freshly isolated human airway epithelial cells from CF and non-CF individuals were used. We showed that both channels are expressed in CF and non-CF cells and constitutive Ca2+ influx was significantly higher (85%) in cells from CF individuals compared to cells from non-CF ones. Using the selective inhibitor of TRPV6 channel SOR-C27 and a siRNA strategy, our results revealed that TRPV6 was mostly involved in the increase of Ca2+ influx. TRPV6 channel is negatively regulated by the PLC-PIP2 pathway. We measured the Ca2+ influx in the presence of the non-specific PLC inhibitor, U73122, in non-CF human bronchial epithelial cells. Ca2+ influx was increased by 33% with U73122 and this increase was largely reduced in the presence of SOR-C27. PLC inhibition in CF cells by U73122 had no effect on Ca2+ influx. These results showed that PLC-PIP2 pathway is dysregulated in CF cells and leads to the increase of TRPV6 activity. The regulation of TRPV6 by PLC-PIP2 pathway implicates the specific PLC isoform, PLC-δ1. Immunoblot experiments revealed that expression of PLC-δ1 was decreased by 70% in CF cells. TRPV6 activity was normalized but not the level of expression of PLC-δ1 protein after F508del-CFTR rescue by low temperature for 48h or treated for 24h by 10μM VX-809 in CF cells. This study revealed TRPV6 and PLC-δ1 as critical actor of Ca2+ homeostasis in CF human bronchial epithelial cells.

Sensitive Electrochemical NO Sensors: Towards Measurement of Nitric Oxide on Primary Epithelial Cell Models

Nitric oxide is an important physiologic metabolite implicated in both health and disease states. Accurate determination of NO in tissues and cells is of paramount importance. The major challenges with NO measurement are its short half-life and often low nanomolar concentrations in tissues. Electrochemical tools provide a mean to accurately measure this analyte in confined environments using miniaturized probes. In the past, our lab explored electrodeposited alternate layers of ruthenium oxide nanoparticles and a conductive sulfur-containing polymer on carbon microelectrode fibers for catalytic determination of NO.In this work, we explore the performance of combined reference/working electrodes modified with ruthenium oxide in the detection of nitric oxide with the goal to measure nitric oxide at the level of single or collective cultured cells. This is a preliminary work towards preparing a device capable to measure nitric oxide levels in a cystic fibrosis cell line model. It has been found that exhaled NO levels remains unchanged or reduced in cystic fibrosis patients unlike other inflammatory lung diseases like asthma where it increases. However it is not clear whether the lower NO levels in cystic fibrosis correlate with lowered production of this metabolite in the bronchial epithelium. It was shown that levels of nitrite and nitrate, the primary stable end products of NO metabolism increases in the breath condensate of patients with cystic fibrosis. We will present preliminary results of our ruthenium oxide modified combined electrodes and how they can be applied to the study of cystic fibrosis at the cellular level.

Functional and pharmacological characterization of volume-regulated anion channels in human normal and cystic fibrosis bronchial and nasal epithelial cells

Volume-regulated anion channels (VRACs) are widely present in various cell types and have important functions ranging from regulatory volume decrease to control of cell proliferation and apoptosis. Here we aimed to compare the biophysical features and pharmacological profiles of VRAC currents in healthy and cystic fibrosis (CF) respiratory epithelial cells in order to characterize these currents both functionally and pharmacologically. Whole-cell electrophysiology was used to characterize the VRAC current in normal (16HBE14o−; HBE) and CF cell lines (CFBE14o−; CFBE), as well as in native human nasal epithelial cells. Application of hypotonic solution produced current responses of similar sizes in both HBE and CFBE cells. Biophysical properties of VRACs, such as instantaneous activation and deactivation upon voltage step, some inactivation at potentials positive to 40mV and outwardly-rectifying I–V curves, were indistinguishable in both cell types. Extensive pharmacological analysis of the currents revealed a similar pharmacological profile in response to three blockers – NPPB, DCPIB and DIDS. Native primary human nasal epithelial cells from both healthy and CF volunteers also showed typical VRAC responses of comparable sizes. VRACs in these cells were more sensitive to external solution hypotonicity compared to HBE and CFBE cells. In all cell types studied robust VRAC currents could be induced at constant cell volume by G-protein activation with GTPγS infusion. This study provides the first extensive comparative functional and pharmacological analysis of VRAC currents in normal and CF airway epithelial cells and shows that VRACs are unimpaired molecularly or functionally in CF.

Expression of Pro-inflammatory Interleukin-8 is Reduced by Ayurvedic Decoctions

Eleven decoctions, obtained from indian plants widely used in ayurvedic medicine, have been investigated as a possible source of molecules exhibiting biological activity on the interaction between DNA and NF-kB, a transcription factor involved in the expression of proinflammatory genes. Cystic fibrosis (CF) cell line stimulated by TNF-α has been used as inflammatory cellular model to determinate interleukin-8 (IL-8), one of the most relevant pro-inflammatory mediator in CF regulated by the NF-kB. The chemical characterization of these 11 decoctions by spectrophotometric analysis and NMR fingerprinting highlighted that sugars and polyphenols seemed to be the main compounds. Our results demonstrated that Azadirachta indica, Terminalia bellerica, Terminalia chebula, Hemidesmus indicus, Emblica officinalis and Swertia chirata are the most active decoctions in inhibiting NF-kB/DNA interactions by EMSA assay and in reducing pro-inflammatory IL- 8 expression in CF cells at IC50 concentrations by Real-Time and Bio-plex analyses. Finally, we observed the increase of all inhibitory activities with the rise of total polyphenols, procyanidins and flavonoids, except for the levels of IL-8 mRNA accumulation, that were as high as flavonoid content grown up by the statistical multivariate analyses. In conclusion, these six decoctions might be interesting to explore new anti-inflammatory treatments for diseases, such as CF.

S103 Neutrophil trans-epithelial migration in cystic fibrosis airways

Background Lung injury in cystic fibrosis (CF) is caused by recurrent airway infection and inflammation partially due to the massive infiltration of neutrophils in airways. The processes regulating neutrophil migration...

S104 Targeting the bacterial cytoskeleton of CF pathogens for antimicrobial development–A cautionary tale?

BackgroundBurkholderia cepacia complex (BCC) bacteria are opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult due to the inherent multidrug resistance of...

Whey protein hydrolysates decrease IL-8 secretion in lipopolysaccharide (LPS)-stimulated respiratory epithelial cells by affecting LPS binding to Toll-like receptor 4

Whey proteins (WP) exert anti-inflammatory and antioxidant effects. Hyperbaric pressurisation of whey increases its digestibility and changes the spectrum of peptides released during digestion. We have shown that dietary supplementation with pressurised whey improves nutritional status and systemic inflammation in patients with cystic fibrosis (CF). Both clinical indices are largely affected by airway processes, to which respiratory epithelial cells actively contribute. Here, we tested whether peptides released from the digestion of pressurised whey can attenuate the inflammatory responses of CF respiratory epithelial cells. Hydrolysates of pressurised WP (pWP) and native WP (nWP, control) were generated in vitro and tested for anti-inflammatory properties judged by the suppression of IL-8 production in CF and non-CF respiratory epithelial cell lines (CFTE29o- and 1HAEo-, respectively). We observed that, in both cell lines, pWP hydrolysate suppressed IL-8 production stimulated by lipopolysaccharide (LPS) to a greater magnitude compared with nWP hydrolysate. Neither hydrolysate suppressed IL-8 production induced by TNF-α or IL-1β, suggesting an effect on the Toll-like receptor (TLR) 4 pathway, the cellular sensor for LPS. Further, neither hydrolysate affected TLR4 expression or neutralised LPS. Both pWP and nWP hydrolysates similarly reduced LPS binding to surface TLR4, while pWP tended to more potently increase extracellular antioxidant capacity. (1) anti-inflammatory properties of whey are enhanced by pressurisation; (2) suppression of IL-8 production may contribute to the clinical effects of pressurised whey supplementation on CF; (3) this effect may be partly explained by a combination of reduced LPS binding to TLR4 and enhanced extracellular antioxidant capacity.

The Mitochondrial Complex I Activity Is Reduced in Cells with Impaired Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Function

Cystic fibrosis (CF) is a frequent and lethal autosomal recessive disease. It results from different possible mutations in the CFTR gene, which encodes the CFTR chloride channel. We have previously studied the differential expression of genes in CF and CF corrected cell lines, and found a reduced expression of MTND4 in CF cells. MTND4 is a mitochondrial gene encoding the MTND4 subunit of the mitochondrial Complex I (mCx-I). Since this subunit is essential for the assembly and activity of mCx-I, we have now studied whether the activity of this complex was also affected in CF cells. By using Blue Native-PAGE, the in-gel activity (IGA) of the mCx-I was found reduced in CFDE and IB3-1 cells (CF cell lines) compared with CFDE/6RepCFTR and S9 cells, respectively (CFDE and IB3-1 cells ectopically expressing wild-type CFTR). Moreover, colon carcinoma T84 and Caco-2 cells, which express wt-CFTR, either treated with CFTR inhibitors (glibenclamide, CFTR(inh)-172 or GlyH101) or transfected with a CFTR-specific shRNAi, showed a significant reduction on the IGA of mCx-I. The reduction of the mCx-I activity caused by CFTR inhibition under physiological or pathological conditions may have a profound impact on mitochondrial functions of CF and non-CF cells.

Effect of allergy and inflammation on eicosanoid gene expression in CFTR deficiency

BackgroundAllergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2–15% of patients. We hypothesized that sensitization/challenge of CFTR−/− mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF. MethodsFABP-hCFTR+/−-CFTR−/− mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines. ResultsPla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR−/− mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells. ConclusionsThe distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF.

Iron induced HIF destabilization and altered Cellular Homeostatic Mechanisms in Cystic Fibrosis Cell Lines

Iron induced HIF destabilization and altered Cellular Homeostatic Mechanisms in Cystic Fibrosis Cell Lines