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 ß1 and ß2 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 ß1 and ß2 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.

Electrophysiological evaluation of cystic fibrosis conductance transmembrane regulator (CFTR) expression in human monocytes.

BACKGROUND: Cystic fibrosis is caused by mutations of CFTR gene, a protein kinase A-activated anion channel, and is associated to a persistent and excessive chronic lung inflammation, suggesting functional alterations of immune cells. Leukocytes express detectable levels of CFTR but the molecule has not been fully characterized in these cells. METHODS: Freshly isolated monocytes from healthy individuals and CF patients were assessed by protein expression, single cell electrophysiological and membrane depolarization assays. RESULTS: We recorded chloride currents by patch clamp in healthy monocytes, after the administration of a CFTR stimulus. Currents were sensitive to a specific blocker of the CFTR channel, CFTRinh-172 and were absent in CF monocytes. Next, we evaluated the effects of ex vivo exposure of monocytes from cystic fibrosis patients carrying the F508del mutation to a chemical corrector, Vertex-325. We found an increase in CFTR expression by confocal microscopy and a recovery of CFTR function by both patch clamp and single cell fluorescence analysis. CONCLUSIONS: We confirm the expression of functional CFTR in human monocytes and demonstrate that blood monocytes can represent an adequate source of primary cells to assess new therapies and define diagnosis of CF. GENERAL SIGNIFICANCE: Tests to evaluate CFTR functional abnormalities in CF disease might greatly benefit from the availability of a convenient source of primary cells. This electrophysiological study promotes the use of monocytes as a minimally invasive tool to study and monitor CFTR function in individual patients.

Tackling recalcitrant Pseudomonas aeruginosa infections in critical illness via anti-virulence monotherapy.

Intestinal barrier derangement allows intestinal bacteria and their products to translocate to the systemic circulation. Pseudomonas aeruginosa (PA) superimposed infection in critically ill patients increases gut permeability and leads to gut-driven sepsis. PA infections are challenging due to multi-drug resistance (MDR), biofilms, and/or antibiotic tolerance. Inhibition of the quorum-sensing transcriptional regulator MvfR(PqsR) is a desirable anti-PA anti-virulence strategy as MvfR controls multiple acute and chronic virulence functions. Here we show that MvfR promotes intestinal permeability and report potent anti-MvfR compounds, the N-Aryl Malonamides (NAMs), resulting from extensive structure-activity-relationship studies and thorough assessment of the inhibition of MvfR-controlled virulence functions. This class of anti-virulence non-native ligand-based agents has a half-maximal inhibitory concentration in the nanomolar range and strong target engagement. Using a NAM lead in monotherapy protects murine intestinal barrier function, abolishes MvfR-regulated small molecules, ameliorates bacterial dissemination, and lowers inflammatory cytokines. This study demonstrates the importance of MvfR in PA-driven intestinal permeability. It underscores the utility of anti-MvfR agents in maintaining gut mucosal integrity, which should be part of any successful strategy to prevent/treat PA infections and associated gut-derived sepsis in critical illness settings. NAMs provide for the development of crucial preventive/therapeutic monotherapy options against untreatable MDR PA infections.

Ratiometric sweat secretion optical test in cystic fibrosis, carriers and healthy subjects.

We have simplified the published procedure (5) for measuring sweat rates in individual human sweat glands. Sweat secretion rates were obtained from sweat drops secreted on the forearm by multiple individual glands. We computed a ratio between CFTR-dependent (by intradermal microinjection of a ß adrenergic cocktail) and CFTR-independent (by methacoline as cholinergic stimulus) sweat secretion rates. We obtained a reproducible, approximately linear readout of CFTR function with measurements performed by two different independent teams. We considered three groups (CF subjects, CF carriers and non-CF controls, n=22 in each group); their mean ratios was respectively 0.000, 0.104 and 0.205 The average ratio of CF subjects was consistent with diagnosis in 3 additional cases clinically resembling CF. All groups were clearly discriminated, with sensibility and specificity ranging from 82% to 100%. A software was developed for detecting sweat droplets. This bioassay is suitabile for multicentre studies focusing on CFTR targeted therapies, controversial diagnosis and functional relevance of rare CFTR mutations.

New insights into the Shwachman-Diamond Syndrome-related haematological disorder: hyper-activation of mTOR and STAT3 in leukocytes.

Shwachman-Diamond syndrome (SDS) is an inherited disease caused by mutations of a gene encoding for SBDS protein. So far little is known about SBDS exact function. SDS patients present several hematological disorders, including neutropenia and myelodysplastic syndrome (MDS), with increased risk of leukemic evolution. So far, the molecular mechanisms that underlie neutropenia, MDS and AML in SDS patients have been poorly investigated. STAT3 is a key regulator of several cellular processes including survival, differentiation and malignant transformation. Moreover, STAT3 has been reported to regulate neutrophil granulogenesis and to induce several kinds of leukemia and lymphoma. STAT3 activation is known to be regulated by mTOR, which in turn plays an important role in cellular growth and tumorigenesis. Here we show for the first time, to the best of our knowledge, that both EBV-immortalized B cells and primary leukocytes obtained from SDS patients present a constitutive hyper-activation of mTOR and STAT3 pathways. Interestingly, loss of SBDS expression is associated with this process. Importantly, rapamycin, a well-known mTOR inhibitor, is able to reduce STAT3 phosphorylation to basal levels in our experimental model. A novel therapeutic hypothesis targeting mTOR/STAT3 should represent a significant step forward into the SDS clinical practice.