Patrick Lebecque

Azithromycin reduces exaggerated cytokine production by M1 alveolar macrophages in cystic fibrosis

Macrophages phagocyte pathogenic microorganisms and orchestrate immune responses by producing a variety of inflammatory mediators. The cystic fibrosis (CF) transmembrane conductance regulator chloride channel has been reported to be of pivotal importance for macrophage functions. The exact phenotype and role of macrophages in CF is still unknown. Alveolar and peritoneal macrophages were monitored in CF mice homozygous for the F508 del mutation and in wild-type control animals. Classical (M1) and alternative (M2) macrophage polarization and responses to LPS from Pseudomonas aeruginosa were investigated, and the effect of azithromycin was examined in both cell populations. We show that alveolar macrophage counts were 1.7-fold higher in CF as compared with wild-type mice. The macrophage-related chemokine, chemokine C-C motif ligand (CCL)-2, was found to be at least 10-fold more abundant in the alveolar space of mutant mice. Cell count and CCL-2 protein levels were also increased in the peritoneal cavity of CF mice. Both M1 and M2 macrophage polarization were significantly enhanced in alveolar and peritoneal cells from F508del-CF mice as compared with control animals. LPS-stimulated expression of proinflammatory mediators, such as nitric oxide synthase-2, IL-1beta, and CCL-2, was increased, whereas anti-inflammatory IL-10 expression was decreased in CF macrophages. Azithromycin, added to cell cultures at 1 mg/liter, significantly reduced proinflammatory cytokine expression (IL-1beta, CCL-2, TNF-alpha) in M1-induced CF and wild-type alveolar macrophages. Our findings indicate that CF macrophages are ubiquitously accumulated, and that these cells are polarized toward classical and alternative activation status. Azithromycin down-regulates inflammatory cytokine production by M1-polarized CF alveolar macrophages.

Azithromycin reduces spontaneous and induced inflammation in ΔF508 cystic fibrosis mice

BackgroundInflammation plays a critical role in lung disease development and progression in cystic fibrosis. Azithromycin is used for the treatment of cystic fibrosis lung disease, although its mechanisms of action are poorly understood. We tested the hypothesis that azithromycin modulates lung inflammation in cystic fibrosis mice.MethodsWe monitored cellular and molecular inflammatory markers in lungs of cystic fibrosis mutant mice homozygous for the ΔF508 mutation and their littermate controls, either in baseline conditions or after induction of acute inflammation by intratracheal instillation of lipopolysaccharide from Pseudomonas aeruginosa, which would be independent of interactions of bacteria with epithelial cells. The effect of azithromycin pretreatment (10 mg/kg/day) given by oral administration for 4 weeks was evaluated.ResultsIn naive cystic fibrosis mice, a spontaneous lung inflammation was observed, characterized by macrophage and neutrophil infiltration, and increased intra-luminal content of the pro-inflammatory cytokine macrophage inflammatory protein-2. After induced inflammation, cystic fibrosis mice combined exaggerated cellular infiltration and lower anti-inflammatory interleukin-10 production. In cystic fibrosis mice, azithromycin attenuated cellular infiltration in both baseline and induced inflammatory condition, and inhibited cytokine (tumor necrosis factor-α and macrophage inflammatory protein-2) release in lipopolysaccharide-induced inflammation.ConclusionOur findings further support the concept that inflammatory responses are upregulated in cystic fibrosis. Azithromycin reduces some lung inflammation outcome measures in cystic fibrosis mice. We postulate that some of the benefits of azithromycin treatment in cystic fibrosis patients are due to modulation of lung inflammation.

Mutations in the Amiloride-Sensitive Epithelial Sodium Channel in Patients With Cystic Fibrosis-Like Disease

We investigated whether mutations in the genes that code for the different subunits of the amiloride‐sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)‐like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three‐fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.–55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R‐SCNN1A polymorphism was even found to result in a four‐fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R‐SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0–10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases. Hum Mutat 30:1–11, 2009.

Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

Phenotypic characterisation of patients with intermediate sweat chloride values: towards validation of the European diagnostic algorithm for cystic fibrosis

Background: In patients with symptoms suggestive of cystic fibrosis (CF) and intermediate sweat chloride values (30–60 mmol/l), extensive CFTR gene mutation analysis and nasal potential difference (NPD) measurement are used as additional diagnostic tests and a positive result in either test provides evidence of CFTR dysfunction. To define the phenotype of such patients and confirm the validity of grouping them, patients with intermediate sweat chloride values in whom either additional CF diagnostic test was abnormal were compared with subjects in whom this was not the case and patients with classic CF. Methods: The phenotypic features of four groups were compared: 59 patients with CFTR dysfunction, 46 with an intermediate sweat chloride concentration but no evidence of CFTR dysfunction (CF unlikely), 103 patients with CF and pancreatic sufficiency (CF-PS) and 62 with CF and pancreatic insufficiency (CF-PI). Results: The CFTR dysfunction group had more lower respiratory tract infections (p = 0.01), more isolation of CF pathogens (p<0.001) and clubbing (p = 0.001) than the CF unlikely group, but less frequent respiratory tract infections with CF pathogens than the CF-PS group (p = 0.05). Patients in the CF-PS group had a milder phenotype than those with PI. Many features showed stepwise changes through the patient groups. Conclusion: Patients with intermediate sweat chloride values and two CFTR mutations or an abnormal NPD measurement have a CF-like phenotype compatible with CFTR dysfunction and, as a group, differ phenotypically from patients with intermediate sweat chloride values in whom further CF diagnostic tests are normal as well as from CF-PS and CF-PI patients.

Does in utero exposure to heavy maternal smoking induce nicotine withdrawal symptoms in neonates

Maternal drug use during pregnancy is associated with fetal passive addiction and neonatal withdrawal syndrome. Cigarette smoking—highly prevalent during pregnancy—is associated with addiction and withdrawal syndrome in adults. We conducted a prospective, two-group parallel study on 17 consecutive newborns of heavy-smoking mothers and 16 newborns of nonsmoking, unexposed mothers (controls). Neurologic examinations were repeated at days 1, 2, and 5. Finnegan withdrawal score was assessed every 3 h during their first 4 d. Newborns of smoking mothers had significant levels of cotinine in the cord blood (85.8 ± 3.4 ng/mL), whereas none of the controls had detectable levels. Similar findings were observed with urinary cotinine concentrations in the newborns (483.1 ± 2.5 μg/g creatinine versus 43.6 ± 1.5 μg/g creatinine; p = 0.0001). Neurologic scores were significantly lower in newborns of smokers than in control infants at days 1 (22.3 ± 2.3 versus 26.5 ± 1.1; p = 0.0001), 2 (22.4 ± 3.3 versus 26.3 ± 1.6; p = 0.0002), and 5 (24.3 ± 2.1 versus 26.5 ± 1.5; p = 0.002). Neurologic scores improved significantly from day 1 to 5 in newborns of smokers (p = 0.05), reaching values closer to control infants. Withdrawal scores were higher in newborns of smokers than in control infants at days 1 (4.5 ± 1.1 versus 3.2 ± 1.4; p = 0.05), 2 (4.7 ± 1.7 versus 3.1 ± 1.1; p = 0.002), and 4 (4.7 ± 2.1 versus 2.9 ± 1.4; p = 0.007). Significant correlations were observed between markers of nicotine exposure and neurologic-and withdrawal scores. We conclude that withdrawal symptoms occur in newborns exposed to heavy maternal smoking during pregnancy.

A randomized placebo-controlled trial of miglustat in cystic fibrosis based on nasal potential difference

BACKGROUND Preclinical data suggest that miglustat could restore the function of the cystic fibrosis transmembrane conductance regulator gene in cystic fibrosis cells. METHODS Single-center, randomized, double-blind, placebo-controlled, crossover Phase II study in 11 patients (mean±SD age, 26.3±7.7 years) homozygous for the F508del mutation received oral miglustat 200 mgt.i.d. or placebo for two 8-day cycles separated by a 14-day washout period. The primary endpoint was the change in total chloride secretion (TCS) assessed by nasal potential difference. RESULTS No statistically significant changes in TCS, sweat chloride values or FEV(1) were detected. Pharmacokinetic and safety were similar to those observed in patients with other diseases exposed to miglustat. CONCLUSIONS There was no evidence of a treatment effect on any nasal potential difference variable. Further studies with miglustat need to adequately address criteria for assessment of nasal potential difference.

Inhaled phosphodiesterase type 5 inhibitors restore chloride transport in cystic fibrosis mice

Sildenafil and vardenafil, two selective inhibitors of phosphodiesterase type 5 (PDE5) are able, when applied by intraperitoneal injection, to activate chloride transport in cystic fibrosis (CF) mice homozygous for the F508del mutation. Oral treatment with the drugs may be associated with adverse haemodynamic effects. We hypothesised that inhaled PDE5 inhibitors are able to restore ion transport in F508del CF airway epithelium. We developed a restraint-free mouse chamber for inhalation studies. PDE5 inhibitors were nebulised for 15 min at concentrations adjusted from recommended therapeutic oral doses for male erectile dysfunction. We measured in vivo nasal transepithelial potential difference 1 h after a single inhalation of sildenafil, vardenafil or tadalafil in F508del CF and normal homozygous mice. After nebulisation with the drugs in F508del mice, chloride transport, evaluated by perfusing the nasal mucosa with chloride-free buffer containing amiloride followed by forskolin, was normalised; the forskolin response was increased, with the largest values being observed with tadalafil and intermediate values with vardenafil. No detectable effect was observed on sodium conductance. Our results confirm the role of PDE5 inhibitors in restoring chloride transport function of F508del CF transmembrane conductance regulator protein and highlight the potential of inhaled sildenafil, vardenafil and tadalafil as a therapy for CF.

Omalizumab for Treatment of ABPA Exacerbations in CF Patients

Pediatric Pulmonology 2009;44(5):516/DOI 10.1002/ppul.21002 2009 Wiley-Liss, Inc.In this Letter to the Editor, IgE values should have been expressed in IU/ml. Inaddition,inthesixthparagraph,baselineIgEvalueofthefirstpatienthadbeenprintedas 261 UI/L erroneously. The correct value should be 4261 IU/ml. Please see the nextpage for the corrected article.The publisher regrets any inconvenience this has caused.

Azithromycin fails to reduce increased expression of neutrophil-related cytokines in primary-cultured epithelial cells from cystic fibrosis mice

BACKGROUND Beneficial effects of azithromycin in cystic fibrosis (CF) have been reported, however, its mechanism of action remains unclear. The present study aimed at investigating the effect of azithromycin on CF airway epithelial cells. METHODS Primary cultures of purified tracheal epithelial cells from F508del and normal homozygous mice were established. Responses to lipopolysaccharide from Pseudomonas aeruginosa (LPS, 0.1 microg/ml) on mRNA expression of neutrophil-related chemokines, pro- and anti-inflammatory cytokines were investigated in the presence or the absence of azithromycin (1 microg/ml). RESULTS CF airway epithelial cells showed upregulation of MIP-2 and KC responses to LPS, and azithromycin failed to downregulate these responses. In contrast, in CF cells, azithromycin increased KC and TNF-alpha expression under non-stimulated and LPS-stimulated conditions, respectively. In non-CF cells, the macrolide potentiated the LPS response on MIP-2 and on IL-10. CONCLUSIONS Airway epithelial cells contribute to the dysregulated immune processes in CF. Azithromycin rather stimulates cytokine expression in CF airway epithelial cells.