The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

Th17/Treg imbalance in murine cystic fibrosis is linked to indoleamine 2,3-dioxygenase deficiency but corrected by kynurenines.

RATIONALE: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator affect the innate epithelial immune function of the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pathogenic fungi. The appreciation of whether such fungi are primarily responsible for or a consequence of ineffective airway inflammation is important for future therapeutics development. OBJECTIVES: To characterize the impact of the tryptophan/kynurenine pathway on pathogenic airway inflammation preventing effective fungal clearance in CF. METHODS: We studied the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the kynurenine pathway of tryptophan degradation, in human and murine CF, the impact of IDO on lung inflammation and immunity in murine CF, and the potential role of tryptophan catabolism in pathogenesis and therapy of fungus-associated lung inflammation. MEASUREMENTS AND MAIN RESULTS: IDO was defective in murine and human CF. Genetic and transcriptional regulatory mechanisms contributed to dysfunctional IDO activity that, in turn, correlated with imbalanced Th17/Treg-cell responses to Aspergillus fumigatus in murine CF. Treatments enhancing IDO function or preventing pathogenic Th17-cell activation restored protective immunity to the fungus and improved lung inflammation in murine CF. CONCLUSIONS: This study provides a link between tryptophan catabolism and lung immune homeostasis in murine CF, representing a proof-of-concept that targeting pathogenic inflammation via IDO-mimetic drugs may benefit patients with CF.

Hypoxia promotes danger-mediated inflammation via receptor for advanced glycation end products in cystic fibrosis.

RATIONALE: Hypoxia regulates the inflammatory-antiinflammatory balance by the receptor for advanced glycation end products (RAGE), a versatile sensor of damage-associated molecular patterns. The multiligand nature of RAGE places this receptor in the midst of chronic inflammatory diseases. OBJECTIVES: To characterize the impact of the hypoxia-RAGE pathway on pathogenic airway inflammation preventing effective pathogen clearance in cystic fibrosis (CF) and elucidate the potential role of this danger signal in pathogenesis and therapy of lung inflammation. METHODS: We used in vivo and in vitro models to study the impact of hypoxia on RAGE expression and activity in human and murine CF, the nature of the RAGE ligand, and the impact of RAGE on lung inflammation and antimicrobial resistance in fungal and bacterial pneumonia. MEASUREMENTS AND MAIN RESULTS: Sustained expression of RAGE and its ligand S100B was observed in murine lung and human epithelial cells and exerted a proximal role in promoting inflammation in murine and human CF, as revealed by functional studies and analysis of the genetic variability of AGER in patients with CF. Both hypoxia and infections contributed to the sustained activation of the S100B-RAGE pathway, being RAGE up-regulated by hypoxia and S100B by infection by Toll-like receptors. Inhibiting the RAGE pathway in vivo with soluble (s) RAGE reduced pathogen load and inflammation in experimental CF, whereas sRAGE production was defective in patients with CF. CONCLUSIONS: A causal link between hyperactivation of RAGE and inflammation in CF has been observed, such that targeting pathogenic inflammation alleviated inflammation in CF and measurement of sRAGE levels could be a useful biomarker for RAGE-dependent inflammation in patients with CF.

Isolation and characterization of microparticles in sputum from cystic fibrosis patients.

BACKGROUND: Microparticles (MPs) are membrane vesicles released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. Cystic fibrosis (CF) lung disease is characterized by massive neutrophil granulocyte influx in the airways, their activation and eventually apoptosis. We investigated on the presence and phenotype of MPs in the sputum, a rich non-invasive source of inflammation biomarkers, of acute and stable CF adult patients. METHODS: Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 stable) and 7 patients with primary ciliary dyskinesia (PCD), was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, and identified in the supernatants of treated sputum by cytofluorimetry and immunolabelling for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens. RESULTS: Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria, confirming microbiological tests. CF sputa contained higher number of MPs in comparison with PCD sputa. Levels of CD11a+-and CD66b+-, but not CD11b+-MPs were significantly higher in CF than in PCD, without differences between acute and stable patients. CONCLUSIONS: In summary, MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. This novel isolation method for MPs from sputum opens a new opportunity for the study of lung pathology in CF.

Growth assessment of paediatric patients with CF comparing different auxologic indicators: A multicentre Italian study.

OBJECTIVES: To evaluate growth in Italian patients with cystic fibrosis (CF). PATIENTS AND METHODS: A multicentre cross-sectional study was carried out on patients with CF attending Italian reference centres. Anthropometric data were evaluated using the Centers for Disease Control and Prevention 2000 reference data. Nutritional failure was defined as height-for-age percentile (HAP) <5th (all patients); weight-for-length percentile (WLP) <10th (patients <2 years); body mass index percentile (BMIp) <15th (patients between 2 and 18 years). The risk of malnutrition (defined as HAP, WLP, and BMIp <25th) and the proportion of patients below the "BMIp goal" (BMIp > or =50th) were also evaluated. Nutritional status was evaluated in the whole population and in relation to age, sex, pancreatic insufficiency, meconium ileus, and lung function. RESULTS: A total of 892 patients with CF (50.7% males, mean age 9.2 years, range 0.1-18 years) were enrolled. The proportion of children with HAP <5th, WLP<10th and BMIp<15th was 12.2%. 12.9%, 20.9%, respectively, and 54.4% did not fulfill the BMIp > or =50th goal. HAP <25th identified the highest proportion of children at risk of malnutrition, whereas BMIp <15th identified the highest proportion of children with nutritional failure. Whatever the criterion used to define malnutrition, the highest proportion of children with nutritional failure was found in adolescence (11-18 years). z scores for height, weight, and BMI were significantly associated with pancreatic status and lung function. Differences among centres for the auxologic parameters were not significant, except for BMIp. CONCLUSIONS: Nutritional failure is present in a minority of Italian patients with CF, particularly during adolescence. Different auxologic indicators should be used for identifying children at risk for or with actual malnutrition.

A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis.

T helper 9 (Th9) cells contribute to lung inflammation and allergy as sources of interleukin-9 (IL-9). However, the mechanisms by which IL-9/Th9 mediate immunopathology in the lung are unknown. Here we report an IL-9-driven positive feedback loop that reinforces allergic inflammation. We show that IL-9 increases IL-2 production by mast cells, which leads to expansion of CD25+ type 2 innate lymphoid cells (ILC2) and subsequent activation of Th9 cells. Blocking IL-9 or inhibiting CD117 (c-Kit) signalling counteracts the pathogenic effect of the described IL-9-mast cell-IL-2 signalling axis. Overproduction of IL-9 is observed in expectorates from cystic fibrosis (CF) patients, and a sex-specific variant of IL-9 is predictive of allergic reactions in female patients. Our results suggest that blocking IL-9 may be a therapeutic strategy to ameliorate inflammation associated with microbial colonization in the lung, and offers a plausible explanation for gender differences in clinical outcomes of patients with CF.

IL-1 receptor antagonist ameliorates inflammasome-dependent inflammation in murine and human cystic fibrosis.

Dysregulated inflammasome activation contributes to respiratory infections and pathologic airway inflammation. Through basic and translational approaches involving murine models and human genetic epidemiology, we show here the importance of the different inflammasomes in regulating inflammatory responses in mice and humans with cystic fibrosis (CF), a life-threatening disorder of the lungs and digestive system. While both contributing to pathogen clearance, NLRP3 more than NLRC4 contributes to deleterious inflammatory responses in CF and correlates with defective NLRC4-dependent IL-1Ra production. Disease susceptibility in mice and microbial colonization in humans occurs in conditions of genetic deficiency of NLRC4 or IL-1Ra and can be rescued by administration of the recombinant IL-1Ra, anakinra. These results indicate that pathogenic NLRP3 activity in CF could be negatively regulated by IL-1Ra and provide a proof-of-concept evidence that inflammasomes are potential targets to limit the pathological consequences of microbial colonization in CF.

Pro-inflammatory effect of cystic fibrosis sputum microparticles in the murine lung.

BACKGROUND: The role of microparticles (MPs) in the inflammatory process of cystic fibrosis (CF) airways is not known. Here, we have studied the pro-inflammatory potential of CF MPs in a model of acute lung injury. METHODS: Swiss mice were subjected to intratracheal administration of MPs obtained from CF and primary ciliary diskinesia (PCD) patients. Histopathology, total and differential cell counts in bronchoalveolar lavage fluid were used to evaluate the inflammatory reaction in the lung. Lipopolysaccharide (LPS)-like activity of MPs was studied by Limulus amebocyte lysate assay. RESULTS: MPs obtained from acute CF patients determined peribronchial and perivascular inflammatory infiltrates similar to those elicited by LPS. This inflammation was granulocyte-dominated and higher than that determined by MPs obtained from stable CF, whereas PCD MPs caused a macrophage-dominated inflammation. While LPS-activity was not found in circulating blood MPs prepared from CF patients, it was present in MPs obtained from CF sputum and sputum CD66b(+) neutrophils. Finally, LPS-like activity was only detected in circulating MPs after incubation with LPS as well as in MPs obtained from LPS-stimulated neutrophils obtained from healthy donors. CONCLUSIONS: These data suggest that the pro-inflammatory potential of neutrophil-derived MPs in the CF airways may be subsequent to the binding of shedded LPS.