Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

Abstract

BackgroundEvaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes.DescriptionWestern blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC2(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80–1.00).Results and SignificanceCFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials.

Antibiotic therapy affects functional behaviour in cystic fibrosis blood mononuclear cells

“…Furthermore, during acute exacerbations, CF patients' LMNs had a severely impaired expression of the mature CFTR band with respect to LMNs from healthy subjects [4]. Here, in confirmation of that study, figure 1a shows a typical Western blot in which we loaded LMN lysates derived from a healthy donor and from a representative CF patient pre-and post-antibiotic treatment.…”

“…Different factors related to the mortality of pneumococcal disease have been described, including host factors such as age, comorbidities or immunosuppressive conditions [4], and organism-related factors such as serotype, bacterial load or viral co-infection [3,[5][6][7]. These factors have all been primarily identified to predict overall mortality, but information regarding the determinants of early mortality is scarce.…”

Ivacaftor-Induced Proteomic Changes Suggest Monocyte Defects May Contribute to the Pathogenesis of Cystic Fibrosis

“…Although most research on cystic fibrosis (CF) disease pathogenesis has focused on epithelia (1), recent work shows that CF transmembrane conductance regulator (CFTR) is expressed on human and murine monocytes and macrophages (2,3) and that CFTR deficiency could alter these cells' functions (2,4,5). Although these findings raise the possibility that immune dysfunction contributes to CF disease pathogenesis, questions remain.…”

Macrophage Polarization and Function in Cystic Fibrosis

“…Depending on the post translational processing, three isoforms of CFTR proteins have been shown in earlier studies (176) human macrophages (160). Conversely, the presence of isoform B was detected in monocytes from patients with CF (176,180). Truncated forms of the protein appear to be loosely localized on the membrane.…”

“…However, the prevalence varies Mutation in CFTR gene causes reduced transport of Cl -and HCO3 -ions in epithelial cells and blood cells (161,176,180,181). In the lung, disturbed airway surface liquid homeostasis produces thick and viscous mucus that leads to mucus stasis, airway obstruction, persistence infection, chronic inflammation, a gradual decrease in lung function that ultimately results in limited life expectancy (168,182,183).…”