Restoration of CFTR function in patients with cystic fibrosis carrying the F508del-CFTR mutation

<div><p>Restoration of BECN1/Beclin 1-dependent autophagy and depletion of SQSTM1/p62 by genetic manipulation or autophagy-stimulatory proteostasis regulators, such as cystamine, have positive effects on mouse models of human cystic fibrosis (CF). These measures rescue the functional expression of the most frequent pathogenic CFTR mutant, F508del, at the respiratory epithelial surface and reduce lung inflammation in <i>Cftr^F508del</i> homozygous mice. Cysteamine, the reduced form of cystamine, is an FDA-approved drug. Here, we report that oral treatment with cysteamine greatly reduces the mortality rate and improves the phenotype of newborn mice bearing the <i>F508del-CFTR</i> mutation. Cysteamine was also able to increase the plasma membrane expression of the F508del-CFTR protein in nasal epithelial cells from <i>F508del</i> homozygous CF patients, and these effects persisted for 24 h after cysteamine withdrawal. Importantly, this cysteamine effect after washout was further sustained by the sequential administration of epigallocatechin gallate (EGCG), a green tea flavonoid, both <i>in vivo</i>, in mice, and <i>in vitro</i>, in primary epithelial cells from CF patients. In a pilot clinical trial involving 10 <i>F508del-CFTR</i> homozygous CF patients, the combination of cysteamine and EGCG restored BECN1, reduced SQSTM1 levels and improved CFTR function from nasal epithelial cells <i>in vivo</i>, correlating with a decrease of chloride concentrations in sweat, as well as with a reduction of the abundance of <i>TNF/TNF-alpha (tumor necrosis factor)</i> and <i>CXCL8</i> (<i>chemokine [C-X-C motif] ligand 8</i>) transcripts in nasal brushing and TNF and CXCL8 protein levels in the sputum. Altogether, these results suggest that optimal schedules of cysteamine plus EGCG might be used for the treatment of CF caused by the <i>F508del-CFTR</i> mutation.</p></div

Adult Raphe-Specific Deletion of Lmx1b Leads to Central Serotonin Deficiency

The transcription factor Lmx1b is essential for the differentiation and survival of central serotonergic (5-HTergic) neurons during embryonic development. However, the role of Lmx1b in adult 5-HTergic neurons is unknown. We used an inducible Cre-LoxP system to selectively inactivate Lmx1b expression in the raphe nuclei of adult mice. Pet1-CreERT2 mice were generated and crossed with Lmx1bflox/flox mice to obtain Pet1-CreERT2; Lmx1bflox/flox mice (which termed as Lmx1b iCKO). After administration of tamoxifen, the level of 5-HT in the brain of Lmx1b iCKO mice was reduced to 60% of that in control mice, and the expression of tryptophan hydroxylase 2 (Tph2), serotonin transporter (Sert) and vesicular monoamine transporter 2 (Vmat2) was greatly down-regulated. On the other hand, the expression of dopamine and norepinephrine as well as aromatic L-amino acid decarboxylase (Aadc) and Pet1 was unchanged. Our results reveal that Lmx1b is required for the biosynthesis of 5-HT in adult mouse brain, and it may be involved in maintaining normal functions of central 5-HTergic neurons by regulating the expression of Tph2, Sert and Vmat2

Azithromycin reduces spontaneous and induced inflammation in ΔF508 cystic fibrosis mice

BACKGROUND: Inflammation 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. METHODS: We 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. RESULTS: In 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. CONCLUSION: Our 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

Variation in MSRA Modifies Risk of Neonatal Intestinal Obstruction in Cystic Fibrosis

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5′ to and within the MSRA gene were associated with MI (P = 1.99×10−5 to 1.08×10−6; Bonferroni P = 0.057 to 3.1×10−3). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53–0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2×10−4). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr−/− and Cftr−/−Msra−/− mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function

The Primary Folding Defect and Rescue of ΔF508 CFTR Emerge during Translation of the Mutant Domain

In the vast majority of cystic fibrosis (CF) patients, deletion of residue F508 from CFTR is the cause of disease. F508 resides in the first nucleotide binding domain (NBD1) and its absence leads to CFTR misfolding and degradation. We show here that the primary folding defect arises during synthesis, as soon as NBD1 is translated. Introduction of either the I539T or G550E suppressor mutation in NBD1 partially rescues ΔF508 CFTR to the cell surface, but only I539T repaired ΔF508 NBD1. We demonstrated rescue of folding and stability of NBD1 from full-length ΔF508 CFTR expressed in cells to isolated purified domain. The co-translational rescue of ΔF508 NBD1 misfolding in CFTR by I539T advocates this domain as the most important drug target for cystic fibrosis

Role of IL-1β in experimental cystic fibrosis upon P. aeruginosa Infection

Cystic fibrosis is associated with increased inflammatory responses to pathogen challenge. Here we revisited the role of IL-1β in lung pathology using the experimental F508del-CFTR murine model on C57BL/6 genetic background (Cftrtm1eur or d/d), on double deficient for d/d and type 1 interleukin-1 receptor (d/d X IL-1R1-/-), and antibody neutralization. At steady state, young adult d/d mice did not show any signs of spontaneous lung inflammation. However, IL-1R1 deficiency conferred partial protection to repeated P. aeruginosa endotoxins/LPS lung instillation in d/d mice, as 50% of d/d mice succumbed to inflammation, whereas all d/d x IL-1R1-/- double mutants survived with lower initial weight loss and less pulmonary collagen and mucus production, suggesting that the absence of IL-1R1 signaling is protective in d/d mice in LPS-induced lung damage. Using P. aeruginosa acute lung infection we found heightened neutrophil recruitment in d/d mice with higher epithelial damage, increased bacterial load in BALF, and augmented IL-1β and TNF-α in parenchyma as compared to WT mice. Thus, F508del-CFTR mice show enhanced IL-1β signaling in response to P. aeruginosa. IL-1β antibody neutralization had no effect on lung homeostasis in either d/d or WT mice, however P. aeruginosa induced lung inflammation and bacterial load were diminished by IL-1β antibody neutralization. In conclusion, enhanced susceptibility to P. aeruginosa in d/d mice correlates with an excessive inflammation and with increased IL-1β production and reduced bacterial clearance. Further, we show that neutralization of IL-1β in d/d mice through the double mutation d/d x IL-1R1-/- and in WT via antibody neutralization attenuates inflammation. This supports the notion that intervention in the IL-1R1/IL-1β pathway may be detrimental in CF patients

Vaccine strategies against cystic fibrosis pathogens

A great number of cystic fibrosis (CF) pathogens such as Pseudomonas aeruginosa, the Burkholderia cepacia and the Mycobacterium abscessus complex raised difficult therapeutic problems due to their intrinsic multi-resistance to numerous antibiotics. Vaccine strategies represent one of the key weapons against these multi-resistant bacteria in a number of clinical settings like CF. Different strategies are considered in order to develop such vaccines, linked either to priming the host response, or by exploiting genomic data derived from the bacterium. Interestingly, virulence factors synthesized by various pathogens might serve as targets for vaccine development and have been, for example, evaluated in the context of CF

Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency

Rationale: Cystic fibrosis (CF) is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Persistent lung inflammation, characterized by increasing polymorphonuclear leukocyte recruitment, is a major cause of the decline in respiratory function in patients with CF and is a leading cause of morbidity and mortality. CFTR is expressed in various cell types, including leukocytes, but its involvement in the regulation of leukocyte recruitment is unknown. Objectives: We evaluated whether CF leukocytes might present with alterations in cell adhesion and migration, a key process governing innate and acquired immune responses. Methods: We used ex vivo adhesion and chemotaxis assays, flow cytometry, immunofluorescence, and GTPase activity assays in this study. Measurements and Main Results: We found that chemoattractant-induced activation of β1 and β2 integrins and of chemotaxis is defective in mononuclear cells isolated from patients with CF. In contrast, polymorphonuclear leukocyte adhesion and chemotaxis were normal. The functionality of β1 and β2 integrins was restored by treatment of CF monocytes with the CFTR-correcting drugs VRT325 and VX809. Moreover, treatment of healthy monocytes with the CFTR inhibitor CFTR(inh)-172 blocked integrin activation by chemoattractants. In a murine model of lung inflammation, we found that integrin-independent migration of CF monocytes into the lung parenchyma was normal, whereas, in contrast, integrin-dependent transmigration into the alveolar space was impaired. Finally, signal transduction analysis showed that, in CF monocytes, chemoattractant-triggered activation of RhoA and CDC42 Rho small GTPases (controlling integrin activation and chemotaxis, respectively) was strongly deficient. Conclusions: Altogether, these data highlight the critical regulatory role of CFTR in integrin activation by chemoattractants in monocytes and identify CF as a new, cell type–selective leukocyte adhesion deficiency disease, providing new insights into CF pathogenesis