Virginia De Rose

Genetic Deletion and Pharmacological Inhibition of PI3Kγ Reduces Neutrophilic Airway Inflammation and Lung Damage in Mice with Cystic Fibrosis-Like Lung Disease

Purpose. Neutrophil-dominated airway inflammation is a key feature of progressive lung damage in cystic fibrosis (CF). Thus, reducing airway inflammation is a major goal to prevent lung damage in CF. However, current anti-inflammatory drugs have shown several limits. PI3Kγ plays a pivotal role in leukocyte recruitment and activation; in the present study we determined the effects of genetic deletion and pharmacologic inhibition of PI3Kγ on airway inflammation and structural lung damage in a mouse model of CF lung disease. Methods. βENaC overexpressing mice (βENaC-Tg) were backcrossed with PI3Kγ-deficient (PI3Kγ KO) mice. Tissue damage was assessed by histology and morphometry and inflammatory cell number was evaluated in bronchoalveolar lavage fluid (BALF). Furthermore, we assessed the effect of a specific PI3Kγ inhibitor (AS-605240) on inflammatory cell number in BALF. Results. Genetic deletion of PI3Kγ decreased neutrophil numbers in BALF of PI3Kγ KO/βENaC-Tg mice, and this was associated with reduced emphysematous changes. Treatment with the PI3Kγ inhibitor AS-605240 decreased the number of neutrophils in BALF of βENaC-Tg mice, reproducing the effect observed with genetic deletion of the enzyme. Conclusions. These results demonstrate the biological efficacy of both genetic deletion and pharmacological inhibition of PI3Kγ in reducing chronic neutrophilic inflammation in CF-like lung disease in vivo.

Recurrent exacerbations affect FEV1 decline in adult patients with cystic fibrosis

Obstructive lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF). To identify risk factors contributing to FEV(1) decline in CF patients, we carried out a retrospective analysis of clinical and pulmonary function data in a population of CF patients followed up for 5 years and studied the correlation between clinical data and FEV(1) decline. Fifty-one adult CF patients were studied. The FEV(1) decline was related to the following clinical characteristics: CFTR genotype, age, gender, weight, height, age at diagnosis, baseline FEV(1), pancreatic function, presence of airway infection, pancreatic insufficiency and diabetes, number of exacerbations/year and intravenous (i.v.) antibiotic courses/year. Both the number of exacerbations/year and the number of i.v. antibiotic courses/year were strongly related to the FEV(1) decline. Patients with airway infection or with diabetes had significantly lower FEV(1) values during the study as compared with non-infected patients or patients without diabetes; however, both the presence of airway infection or diabetes did not affect the FEV(1) decline. These results suggest that the aggressive treatment of disease exacerbations is crucial for delaying lung function decline in CF.

IFN-γ inhibits the proliferation of allergen-activated T lymphocytes from atopic, asthmatic patients by inducing Fas/FasL-mediated apoptosis

The defect in interferon‐γ (IFN‐γ) production that results in a T helper cell type 2‐dominated response may be responsible for a decrease in the apoptosis of allergen‐activated T cells in asthma. We investigated the effect of recombinant IFN‐γ on proliferation, Fas/Fas ligand (FasL) expression, and apoptosis in allergen‐stimulated peripheral blood mononuclear cells obtained from atopic, asthmatic patients and nonatopic, control subjects. The addition of IFN‐γ at the start of cultures markedly inhibited the proliferative response to a specific allergen in cells from all asthmatic patients, whereas no change was observed in cells from nonatopic, control subjects. IFN‐γ induced an increase in the expression of Fas and FasL by allergen‐stimulated CD4+ T cells from asthmatic patients and caused the apoptosis of these cells. A Fas‐blocking monoclonal antibody prevented the inhibitory effect of IFN‐γ on allergen‐induced proliferation. These results suggest that IFN‐γ inhibits the proliferation of allergen‐stimulated CD4+ T cells from atopic, asthmatic patients by inducing the surface expression of Fas and FasL, which in turn triggers their apoptotic program. The defect in IFN‐γ production involved in the allergic, immune response may therefore be responsible for a decrease in apoptosis of allergen‐activated T lymphocytes in the airways of atopic, asthmatic patients.

Fcgamma receptor IIA genotype and susceptibility to P. aeruginosa infection in patients with cystic fibrosis.

It has been suggested that genes other than CFTR could modulate the severity of lung disease in cystic fibrosis (CF). Neutrophil Fcγ receptor II (FcγRII) is involved in host defense against microorganisms and in inflammatory response. We evaluated the association between genetic variability of this gene and both airway infection with Pseudomonas aeruginosa and severity of lung disease in patients with CF. We studied 167 Italian unrelated patients with CF and 50 control subjects. The distribution of FcγRIIA genotypes in CF patients was compared with that in control subjects and the different genotypes were related with the presence or absence of P. aeruginosa infection and markers of disease severity in CF patients. The distribution of FcγRIIA genotypes was not significantly different between CF patients and controls. We observed that in CF patients with the same CFTR genotype (ΔF508/ΔF508), those carrying the R allele of FcγRIIA had an increased risk of acquiring chronic P. aeruginosa infection (P=0.042, R.R.: 4.38; 95% CI: 1.17÷22.4). Moreover, the frequency of R/R genotype in patients with chronic P. aeruginosa infection seems to be higher than that of control subjects and patients without chronic infection. The observation that CF patients carrying the R allele of FcγRIIA are at higher risk of acquiring chronic P. aeruginosa infection suggests that the FcγRII loci genetic variation is contributing to this infection susceptibility.

Expression of vascular remodelling markers in relation to bradykinin receptors in asthma and COPD

Background Vascular remodelling plays a central role in asthma and chronic obstructive pulmonary disease (COPD). Bradykinin (BK) is a vasoactive proinflammatory peptide mediating acute responses in asthma. We investigated the role of angiogenic factors in relation to BK receptors in asthma and COPD. Methods Bronchial biopsies from 33 patients with COPD, 24 old (≥50 years) patients with (≥50 years) asthma, 18 old control smokers, 11 old control non-smokers, 15 young (≤40yrs) patients with (≤40 years) asthma and 10 young control non-smokers were immunostained for CD31, vascular endothelial growth factor-A (VEGF-A), angiogenin and BK receptors (B2R and B1R). Fibroblast and endothelial co-localisation of relevant molecules were performed by immunofluorescence. BK-induced VEGF-A and angiogenin release was studied (ELISA) in bronchial fibroblasts from subjects with asthma and COPD. Results In bronchial lamina propria of old patients with asthma, CD31 and VEGF-A+ cell numbers were higher than old control non-smokers (p<0.05). Angiogenin+, B2R+ and B1R+ cell numbers in old patients with asthma were higher than in old control non-smokers, control smokers and patients with COPD (p<0.01). Angiogenin+ cell numbers were higher in patients with COPD than both old control groups (p<0.05). In all patients with asthma the number of B2R+ cells was positively related to the numbers of B1R+ (rs=0.43), angiogenin+ (rs=0.42) and CD31 cells (rs=0.46) (p<0.01). Angiogenin+ cell numbers were negatively related to forced expiratory volume in 1 s (rs=−0.415, p=0.008). Double immunofluorescence revealed that CD31 cells of capillary vessels coexpressed B2R and that fibroblasts coexpressed B2R, VEGF-A and angiogenin. BK (10−6M) induced significant angiogenin release in fibroblasts from asthma and to a lesser extent in COPD. Conclusions Unlike COPD, this study suggests the involvement of BK receptors in bronchial vascular remodelling in asthma.

Treatment of low bone density in young people with cystic fibrosis: a multicentre, prospective, open-label observational study of calcium and calcifediol followed by a randomised placebo-controlled trial of alendronate.

BACKGROUND Long-term complications of cystic fibrosis include osteoporosis and fragility fractures, but few data are available about effective treatment strategies, especially in young patients. We investigated treatment of low bone mineral density in children, adolescents, and young adults with cystic fibrosis. METHODS We did a multicentre trial in two phases. We enrolled patients aged 5-30 years with cystic fibrosis and low bone mineral density, from ten cystic fibrosis regional centres in Italy. The first phase was an open-label, 12-month observational study of the effect of adequate calcium intake plus calcifediol. The second phase was a 12-month, double-blind, randomised, placebo-controlled, parallel group study of the efficacy and safety of oral alendronate in patients whose bone mineral apparent density had not increased by 5% or more by the end of the observational phase. Patients were randomly assigned to either alendronate or placebo. Both patients and investigators were masked to treatment assignment. We used dual x-ray absorptiometry at baseline and every 6 months thereafter, corrected for body size, to assess lumbar spine bone mineral apparent density. We assessed bone turnover markers and other laboratory parameters every 3-6 months. The primary endpoint was mean increase of lumbar spine bone mineral apparent density, assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT01812551. FINDINGS We screened 540 patients and enrolled 171 (mean age 13·8 years, SD 5·9, range 5-30). In the observational phase, treatment with calcium and calcifediol increased bone mineral apparent density by 5% or more in 43 patients (25%). 128 patients entered the randomised phase. Bone mineral apparent density increased by 16·3% in the alendronate group (n=65) versus 3·1% in the placebo group (n=63; p=0·0010). 19 of 57 young people (33·3%) receiving alendronate attained a normal-for-age bone mineral apparent density Z score. In the observational phase, five patients had moderate episodes of hypercalciuria, which resolved after short interruption of calcifediol treatment. During the randomised phase, one patient taking alendronate had mild fever versus none in the placebo group; treatment groups did not differ significantly for other adverse events. INTERPRETATION Correct calcium intake plus calcifediol can improve bone mineral density in some young patients with cystic fibrosis. In those who do not respond to calcium and calcifediol alone, alendronate can safely and effectively increase bone mineral density. FUNDING Telethon Foundation (Italy).

A polymorphism in the 5' UTR of the DEFB1 gene is associated with the lung phenotype in F508del homozygous Italian cystic fibrosis patients

Abstract Background: The identification of cystic fibrosis (CF) patients who are at greater risk of lung damage could be clinically valuable. Thus, we attempted to replicate previous findings and verify the possible association between three single nucleotide polymorphisms (SNPs c.–52G>A, c.–44C>G and c.–20G>A) in the 5’ untranslated region (5′ UTR) of the β defensin 1 (DEFB1) gene and the CF pulmonary phenotype. Methods: Genomic DNA from 92 Italian CF patients enrolled in different regional CF centres was extracted from peripheral blood and genotyped for DEFB1 SNPs using TaqMan® allele specific probes. In order to avoid genetic confounding causes that can account for CF phenotype variability, all patients were homozygous for the F508del CFTR mutation, and were then classified on the basis of clinical and functional data as mild lung phenotype (Mp, n=50) or severe lung phenotype patients (Sp, n=42). Results: For the c.–20G>A SNP, the frequency of the A allele, as well as the AA genotype, were significantly more frequent in Mp than in Sp patients, and thus this was associated with a protective effect against severe pulmonary disease (OR=0.48 and 0.28, respectively). The effect of the c.–20G>A A allele is consistent with a recessive model, and the protective effect against Sp is exerted only when it is present in homozygosis. For the other two SNPs, no differences were observed as allelic and genotypic frequency in the two subgroups of CF patients. Conclusions: Our results, although necessary to be confirmed in larger and multiethnic populations, reinforce DEFB1 as a candidate modifier gene of the CF pulmonary phenotype.

Airway Epithelium Dysfunction in Cystic Fibrosis and COPD

Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene, whereas chronic obstructive pulmonary disease (COPD) is mainly caused by environmental factors (mostly cigarette smoking) on a genetically susceptible background. Although the etiology and pathogenesis of these diseases are different, both are associated with progressive airflow obstruction, airway neutrophilic inflammation, and recurrent exacerbations, suggesting common mechanisms. The airway epithelium plays a crucial role in maintaining normal airway functions. Major molecular and morphologic changes occur in the airway epithelium in both CF and COPD, and growing evidence suggests that airway epithelial dysfunction is involved in disease initiation and progression in both diseases. Structural and functional abnormalities in both airway and alveolar epithelium have a relevant impact on alteration of host defences, immune/inflammatory response, and the repair process leading to progressive lung damage and impaired lung function. In this review, we address the evidence for a critical role of dysfunctional airway epithelial cells in chronic airway inflammation and remodelling in CF and COPD, highlighting the common mechanisms involved in the epithelial dysfunction as well as the similarities and differences of the two diseases.

Airway Inflammatory/Immune Responses in COPD and Cystic Fibrosis

Department of Clinical and Biological Sciences, University of Torino, A.O.U. S.Luigi Gonzaga, Regione Gonzole 10, 10043 Orbassano, Torino, Italy Paris Descartes University and Cochin Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France University of Alabama at Birmingham, Birmingham, AL, USA Lung Biology Group, Department of Clinical Microbiology, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland

CFTR dysfunction in cystic fibrosis and chronic obstructive pulmonary disease

ABSTRACT Introduction: Obstructive lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are causes of high morbidity and mortality worldwide. CF is a multiorgan genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by progressive chronic obstructive lung disease. Most cases of COPD are a result of noxious particles, mainly cigarette smoke but also other environmental pollutants. Areas covered: Although the pathogenesis and pathophysiology of CF and COPD differ, they do share key phenotypic features and because of these similarities there is great interest in exploring common mechanisms and/or factors affected by CFTR mutations and environmental insults involved in COPD. Various molecular, cellular and clinical studies have confirmed that CFTR protein dysfunction is common in both the CF and COPD airways. This review provides an update of our understanding of the role of dysfunctional CFTR in both respiratory diseases. Expert commentary: Drugs developed for people with CF to improve mutant CFTR function and enhance CFTR ion channel activity might also be beneficial in patients with COPD. A move toward personalized therapy using, for example, microRNA modulators in conjunction with CFTR potentiators or correctors, could enhance treatment of both diseases.