Pathogenesis Of Cystic Fibrosis Research Articles

Monitoring Neutrophil Elastase and Cathepsin G Activity in Human Sputum Samples.

Proteases are regulators of countless physiological processes and the precise investigation of their activities remains an intriguing biomedical challenge. Among the ~600 proteases encoded by the human genome, neutrophil serine proteases (NSPs) are thoroughly investigated for their involvement in the onset and progression of inflammatory conditions including respiratory diseases. Uniquely, secreted NSPs not only diffuse within extracellular fluids but also localize to plasma membranes. During neutrophil extracellular trap (NETs) formation, NSPs become an integral part of the secreted chromatin. Such complex behavior renders the understanding of NSPs pathophysiology a challenging task. Here, detailed protocols are shown to visualize, quantify and discriminate free and membrane-bound neutrophil elastase (NE) and cathepsin G (CG) activities in sputum samples. NE and CG are NSPs whose activities have pleiotropic roles in the pathogenesis of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD): they promote tissue remodeling, regulate downstream immune responses and correlate with lung disease severity. The protocols show how to separate fluid and cellular fraction, as well as the isolation of neutrophils from human sputum for enzymatic activity quantification via small-molecule Förster resonance energy transfer-based (FRET) reporters. To gather specific insights into the relative role of NE and CG activities, a FRET readout can be measured by different technologies: i) in vitro plate reader measurements allow for high-throughput and bulk detection of protease activity; ii) confocal microscopy spatiotemporally resolves membrane-bound activity at the cell surface; iii) small-molecule FRET flow cytometry enables for the rapid evaluation of anti-inflammatory treatments via single-cell protease activity quantification and phenotyping. The implementation of such methods opens the doors to explore NSPs pathobiology and their potential as biomarkers of disease severity for CF and COPD. Given their standardization potential, their robust readout and simplicity of transfer, the described techniques are immediately shareable for implementation across research and diagnostic laboratories.

Pseudomonas aeruginosa infection, but not mono or dual-combination CFTR modulator therapy affects circulating regulatory T cells in an adult population with cystic fibrosis

Chronic infection and an exaggerated inflammatory response are key drivers of the pathogenesis of cystic fibrosis (CF), especially CF lung disease. An imbalance of pro- and anti-inflammatory mediators, including dysregulated Th2/Th17 cells and impairment of regulatory T cells (Tregs), maintain CF inflammation. CF transmembrane conductance regulator (CFTR) modulator therapy might influence these immune cell abnormalities. Peripheral blood mononuclear cells and serum samples were collected from 108 patients with CF (PWCF) and 40 patients with non-CF bronchiectasis. Samples were analysed for peripheral blood lymphocytes subsets (Tregs; Th1-, Th1/17-, Th17- and Th2-effector cells) and systemic T helper cell-associated cytokines (interleukin [IL]-5, IL-13, IL-2, IL-6, IL-9, IL-10, IL-17A, IL-17F, IL-4, IL-22, interferon-γ, tumour necrosis factor-α) using flow cytometry. 51% of PWCF received CFTR modulators (ivacaftor, ivacaftor/ lumacaftor or tezacaftor/ ivacaftor). There were no differences in proportions of analysed T cell subsets or cytokines between PWCF who were versus were not receiving CFTR modulators. Additional analysis revealed lower percentages of Tregs in PWCF and chronic pulmonary Pseudomonas aeruginosa infection; this difference was also present in PWCF treated with CFTR modulators. Patients with non-CF bronchiectasis tended to have higher percentages of Th2- and Th17-cells and higher levels of peripheral cytokines versus PWCF. Chronic P. aeruginosa lung infection appears to impair Tregs in PWCF (independent of CFTR modulator therapy) but not those with non-CF bronchiectasis. Moreover, our data showed no statistically significant differences in major subsets of peripheral lymphocytes and cytokines among PWCF who were versus were not receiving CFTR modulators.

DNA Methylation Patterns Correlate with the Expression of SCNN1A, SCNN1B, and SCNN1G (Epithelial Sodium Channel, ENaC) Genes.

The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype–phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, β and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5′-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF.

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF–like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.

Targeting cystic fibrosis inflammation in the age of CFTR modulators: focus on macrophages.

Cystic fibrosis (CF) is a life-shortening, multi-organ, autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most prominent clinical manifestation in CF is the development of progressive lung disease characterised by an intense, chronic inflammatory airway response that culminates in respiratory failure and, ultimately, death. In recent years, a new class of therapeutics that have the potential to correct the underlying defect in CF, known as CFTR modulators, have revolutionised the field. Despite the exciting success of these drugs, their impact on airway inflammation, and its long-term consequences, remains undetermined. In addition, studies querying the absolute requirement for infection as a driver of CF inflammation have challenged the traditional consensus on CF pathogenesis, and also emphasise the need to prioritise complementary anti-inflammatory treatments in CF. Macrophages, often overlooked in CF research despite their integral role in other chronic inflammatory pathologies, have increasingly become recognised as key players in the initiation, perpetuation and resolution of CF lung inflammation, perhaps as a direct result of CFTR dysfunction. These findings suggest that macrophages may be an important target for novel anti-inflammatory interventional strategies to effectively treat CF lung function decline. This review will consider evidence for the efficacy of anti-inflammatory drugs in the treatment of CF, the potential role of macrophages, and the significance of targeting these pathways at a time when rectifying the basic defect in CF, through use of novel CFTR modulator therapies, is becoming increasingly viable.

Early pathogenesis of cystic fibrosis gallbladder disease in a porcine model

AbstractHepatobiliary disease causes significant morbidity in people with cystic fibrosis (CF), yet this problem remains understudied. We previously found that newborn CF pigs have microgallbladders with significant luminal obstruction in the absence of infection and consistent inflammation. In this study, we sought to better understand the early pathogenesis of CF pig gallbladder disease. We hypothesized that loss of CFTR would impair gallbladder epithelium anion/liquid secretion and increase mucin production. CFTR was expressed apically in non-CF pig gallbladder epithelium but was absent in CF. CF pig gallbladders lacked cAMP-stimulated anion transport. Using a novel gallbladder epithelial organoid model, we found that Cl− or HCO3− was sufficient for non-CF organoid swelling. This response was absent for non-CF organoids in Cl−/HCO3−-free conditions and in CF. Single-cell RNA-sequencing revealed a single epithelial cell type in non-CF gallbladders that coexpressed CFTR, MUC5AC, and MUC5B. Despite CF gallbladders having increased luminal MUC5AC and MUC5B accumulation, there was no significant difference in the epithelial expression of gel-forming mucins between non-CF and CF pig gallbladders. In conclusion, these data suggest that loss of CFTR-mediated anion transport and fluid secretion contribute to microgallbladder development and luminal mucus accumulation in CF.In this study, the authors assess the early pathogenesis of cystic fibrosis (CF) pig gallbladder disease. The CF pig gallbladder epithelium lacks cAMP-stimulated anion and fluid transport. CF pig gallbladders also demonstrate increased luminal mucins MUC5AC and MUC5B accumulation without significant changes in the epithelial expression of gel-forming mucins compared to non-CF pigs.

Impact of a Gap Junction Protein Alpha 4 Variant on Clinical Disease Phenotype in F508del Homozygous Patients With Cystic Fibrosis.

BackgroundLung disease phenotype varies widely even in the F508del (homozygous) genotype. Leukocyte-driven inflammation is important for pulmonary disease pathogenesis in cystic fibrosis (CF). Blood cytokines correlate negatively with pulmonary function in F508del homozygous patients, and gap junction proteins (GJA) might be related to the influx of blood cells into the lung and influence disease course. We aimed to assess the relationship between GJA1/GJA4 genotypes and the clinical disease phenotype.MethodsOne-hundred-and-sixteen homozygous F508del patients (mean age 27 years, m/f 66/50) were recruited from the CF centers of Bonn, Frankfurt, and Amsterdam. Sequence analysis was performed for GJA1 and GJA4. The clinical disease course was assessed over 3 years using pulmonary function tests, body mass index, Pseudomonas aeruginosa colonization, diabetes mellitus, survival to end-stage lung disease, blood and sputum inflammatory markers.ResultsSequence analysis revealed one clinically relevant single nucleotide polymorphism. In this GJA4 variant (rs41266431), homozygous G variant carriers (n = 84/116; 72.4%) had poorer pulmonary function (FVC% pred: mean 78/86, p < 0.040) and survival to end-stage lung disease was lower (p < 0.029). The frequency of P. aeruginosa colonization was not influenced by the genotype, but in those chronically colonized, those with the G/G genotype had reduced pulmonary function (FVC% pred: mean 67/80, p < 0.049). Serum interleukin-8 (median: 12.4/6.7 pg/ml, p < 0.052) and sputum leukocytes (2305/437.5 pg/ml, p < 0.025) were higher for the G/G genotype.ConclusionsIn carriers of the A allele (27.6%) the GJA4 variant is associated with significantly better protection against end-stage lung disease and superior pulmonary function test results in F508del homozygous patients. This SNP has the potential of a modifier gene for phenotyping severity of CF lung disease, in addition to the CFTR genotype.Clinical Trial RegistrationThe study was registered with ClinicalTrials.gov, number NCT04242420, retrospectively on January 24th, 2020.

Research advances in molecular mechanisms underlying the pathogenesis of cystic fibrosis: From technical improvement to clinical applications (Review).

Cystic fibrosis (CF) is a chronic disease causing severe impairment to the respiratory system and digestive tracts. Currently, CF is incurable. As an autosomal recessive disorder, the morbidity of CF is significantly higher among Caucasians of European descent, whereas it is less pervasive among African and Asian populations. The disease is caused by identical mutations (homozygosity) or different mutations (heterozygosity) of an autosomal recessive mutation at position 7q31.2-q31.1 of chromosome 7. Diagnostic criteria and guidelines work concurrently with laboratory detection to facilitate precise CF detection. With technological advances, the understanding of CF pathogenesis has reached an unprecedented level, allowing for increasingly precise carrier screening, more effective early stage CF intervention and improved prognostic outcomes. These advances significantly increase the life quality and expectancy of patients with CF. Given the numerous improvements in the field of CF, the current review summarized the technical advances in the study of the molecular mechanisms underlying CF, as well as how these improvements facilitate the clinical outcomes of CF. Furthermore, challenges and obstacles to overcome are discussed.

INDICATORS OF CELL METABOLISM IN VITRO IN RESEARCHES OF ANTI-INFLAMMATORY AND CYTOTOXIC EFFECTS OF FULLEROPYRROLIDINES С60 AND THEIR INITIAL SUBSTRATES

The article considers data on the in vitro study of new fulleropyrrolidine compounds for anti-inflammatory and cytotoxic activity in cultures of human monocyte cell lines MonoMac-6 and THP-1Blue and also as inhibitors of human neutrophil elastase. This enzyme is a regulator of inflammation. In different situations, it can act both as a pro-inflammatory and as an anti-inflammatory agent. An imbalance in the regulation of elastase activity plays an important role in the pathogenesis of cystic fibrosis, acute respiratory distress syndrome, bronchiectasis, chronic obstructive pulmonary disease, type 2 diabetes mellitus, atherosclerosis and hypertension. In the future, such studies should lead to the creation of optimal in vitro models that most adequately reflect the situation in vivo and establish the relationship between the structure and activity of the studied drugs. It is noted that the presence of lipophilic properties in fullerene C60 derivatives is especially important in the development of pharmaceuticals for the control of pathogens of various infectious diseases. Fullerene C60 derivatives have the ability to easily penetrate lipid membranes, overcome the blood-brain barrier, and modulate ion transport. Compounds were tested for anti-inflammatory and cytotoxic activity (in vitro) on cultures of human monocytic cell lines MonoMac-6 and THP-1Blue. Modified fullerene compounds of various structures were tested for their inhibitory ability against neutrophil elastase enzyme (in vitro). Elastase activity was evaluated by the ability of fulleropyrrolidine compounds to hydrolyze the synthetic substrate N-methylsuccinyl-Ala-Ala-Pro-Val-7-amino-4-me-thylcoumarin (Calbiochem). The results of studies of fullerene compounds in relation to their anti-inflammatory and cytotoxic activity are obtained. The analysis of the fluorescence kinetics of the compounds was carried out. The cytotoxic activity of the samples was investigated in the Brine Schrimp test using Artemia salina. All compounds have cytotoxicity, which suggests a lack of selectivity of chemotherapeutic action. In general, the presence of a cytotoxic effect confirms the reality of antimicrobial action. The results of the study of the antibacterial and antifungal activity of the synthesized new fulleropyrrolidines and their starting substrates are described (S. aureus 505, Bacillus subtilis, Str.agalactiae, E. Coli M-17, Ps.aeruginosa, Candida аlbicans, Penicillium citrinum, Aspergillus niger, Aspergillus flavus, Trichophyton mentagraphytos, Epidermophyton fioccosum). As a result of the study of the potential antifungal activity of the compounds, it was found that only two drugs inhibit the growth of test cultures in vitro. All other studied samples have practically no activity against the yeast fungi Candida albicans. In general, the presence of a cytotoxic effect in the studied fullerene compounds confirms the reality of the antimicrobial action.

Cystic Fibrosis: Overview of the Current Development Trends and Innovative Therapeutic Strategies.

Cystic Fibrosis (CF), an autosomal recessive genetic disease, is caused by a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This mutation reduces the release of chloride ions (Cl−) in epithelial tissues, and hyperactivates the epithelial sodium channels (ENaC) which aid in the absorption of sodium ions (Na+). Consequently, the mucus becomes dehydrated and thickened, making it a suitable medium for microbial growth. CF causes several chronic lung complications like thickened mucus, bacterial infection and inflammation, progressive loss of lung function, and ultimately, death. Until recently, the standard of clinical care in CF treatment had focused on preventing and treating the disease complications. In this review, we have summarized the current knowledge on CF pathogenesis and provided an outlook on the current therapeutic approaches relevant to CF (i.e., CFTR modulators and ENaC inhibitors). The enormous potential in targeting bacterial biofilms using antibiofilm peptides, and the innovative therapeutic strategies in using the CRISPR/Cas approach as a gene-editing tool to repair the CFTR mutation have been reviewed. Finally, we have discussed the wide range of drug delivery systems available, particularly non-viral vectors, and the optimal properties of nanocarriers which are essential for successful drug delivery to the lungs.

Genotypic and Phenotypic Diversity of Staphylococcus aureus Isolates from Cystic Fibrosis Patient Lung Infections and Their Interactions with Pseudomonas aeruginosa.

Staphylococcus aureus has recently overtaken Pseudomonas aeruginosa as the most commonly recognized bacterial pathogen that infects the respiratory tracts of individuals with the genetic disease cystic fibrosis (CF) in the United States. Most studies of S. aureus in CF patient lung infections have focused on a few isolates, often exclusively laboratory-adapted strains, and how they are killed by P. aeruginosa Less is known about the diversity of S. aureus CF patient lung isolates in terms of both their virulence and their interaction with P. aeruginosa To begin to address this gap, we recently sequenced 64 clinical S. aureus isolates and a reference isolate, JE2. Here, we analyzed the antibiotic resistance genotypes, sequence types, clonal complexes, spa types, agr types, and presence/absence of other known virulence factor genes of these isolates. We hypothesized that virulence phenotypes of S. aureus, namely, toxin production and the mucoid phenotype, would be lost in these isolates due to adaptation in the CF patient lung. In contrast to these expectations, we found that most isolates can lyse both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), suggesting that these phenotypes were not lost during adaptation to the CF lung. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus isolates from CF patients, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.IMPORTANCEStaphylococcus aureus is now the most frequently detected recognized pathogen in the lungs of individuals who have cystic fibrosis (CF) in the United States, followed closely by Pseudomonas aeruginosa When these pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus Here, we present an in-depth study of 64 S. aureus clinical isolates from CF patients, for which we investigated genetic diversity utilizing whole-genome sequencing, virulence phenotypes, and interactions with P. aeruginosa We found that S. aureus isolated from CF lungs are phylogenetically diverse; most retain known virulence factors and vary in their interactions with P. aeruginosa (i.e., they range from being highly sensitive to P. aeruginosa to completely tolerant to it). Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

An Innovative Lipidomic Workflow to Investigate the Lipid Profile in a Cystic Fibrosis Cell Line.

Altered lipid metabolism has been associated to cystic fibrosis disease, which is characterized by chronic lung inflammation and various organs dysfunction. Here, we present the validation of an untargeted lipidomics approach based on high-resolution mass spectrometry aimed at identifying those lipid species that unequivocally sign CF pathophysiology. Of n.13375 mass spectra recorded on cystic fibrosis bronchial epithelial airways epithelial cells IB3, n.7787 presented the MS/MS data, and, after software and manual validation, the final number of annotated lipids was restricted to n.1159. On these lipids, univariate and multivariate statistical approaches were employed in order to select relevant lipids for cellular phenotype discrimination between cystic fibrosis and HBE healthy cells. In cystic fibrosis IB3 cells, a pervasive alteration in the lipid metabolism revealed changes in the classes of ether-linked phospholipids, cholesterol esters, and glycosylated sphingolipids. Through functions association, it was evidenced that lipids variation involves the moiety implicated in membrane composition, endoplasmic reticulum, mitochondria compartments, and chemical and biophysical lipids properties. This study provides a new perspective in understanding the pathogenesis of cystic fibrosis and strengthens the need to use a validated mass spectrometry-based lipidomics approach for the discovery of potential biomarkers and perturbed metabolism.

ILC2 Lung-Homing in Cystic Fibrosis Patients: Functional Involvement of CCR6 and Impact on Respiratory Failure.

Cystic fibrosis patients suffer from a progressive, often fatal lung disease, which is based on a complex interplay between chronic infections, locally accumulating immune cells and pulmonary tissue remodeling. Although group-2 innate lymphoid cells (ILC2s) act as crucial initiators of lung inflammation, our understanding of their involvement in the pathogenesis of cystic fibrosis remains incomplete. Here we report a marked decrease of circulating CCR6+ ILC2s in the blood of cystic fibrosis patients, which significantly correlated with high disease severity and advanced pulmonary failure, strongly implicating increased ILC2 homing from the peripheral blood to the chronically inflamed lung tissue in cystic fibrosis patients. On a functional level, the CCR6 ligand CCL20 was identified as potent promoter of lung-directed ILC2 migration upon inflammatory conditions in vitro and in vivo using a new humanized mouse model with light-sheet fluorescence microscopic visualization of lung-accumulated human ILC2s. In the lung, blood-derived human ILC2s were able to augment local eosinophil and neutrophil accumulation and induced a marked upregulation of pulmonary type-VI collagen expression. Studies in primary human lung fibroblasts additionally revealed ILC2-derived IL-4 and IL-13 as important mediators of this type-VI collagen-inducing effect. Taken together, the here acquired results suggest that pathologically increased CCL20 levels in cystic fibrosis airways induce CCR6-mediated lung homing of circulating human ILC2s. Subsequent ILC2 activation then triggers local production of type-VI collagen and might thereby drive extracellular matrix remodeling potentially influencing pulmonary tissue destruction in cystic fibrosis patients. Thus, modulating the lung homing capacity of circulating ILC2s and their local effector functions opens new therapeutic avenues for cystic fibrosis treatment.

Blood co-expression modules identify potential modifier genes of diabetes and lung function in cystic fibrosis.

Cystic fibrosis (CF) is a rare genetic disease that affects the respiratory and digestive systems. Lung disease is variable among CF patients and associated with the development of comorbidities and chronic infections. The rate of lung function deterioration depends not only on the type of mutations in CFTR, the disease-causing gene, but also on modifier genes. In the present study, we aimed to identify genes and pathways that (i) contribute to the pathogenesis of cystic fibrosis and (ii) modulate the associated comorbidities. We profiled blood samples in CF patients and healthy controls and analyzed RNA-seq data with Weighted Gene Correlation Network Analysis (WGCNA). Interestingly, lung function, body mass index, the presence of diabetes, and chronic P. aeruginosa infections correlated with four modules of co-expressed genes. Detailed inspection of networks and hub genes pointed to cell adhesion, leukocyte trafficking and production of reactive oxygen species as central mechanisms in lung function decline and cystic fibrosis-related diabetes. Of note, we showed that blood is an informative surrogate tissue to study the contribution of inflammation to lung disease and diabetes in CF patients. Finally, we provided evidence that WGCNA is useful to analyze-omic datasets in rare genetic diseases as patient cohorts are inevitably small.

Autophagy and Others Respiratory Diseases.

Besides COPD, pulmonary fibrosis, and asthma, autophagy also participates in the development of many other respiratory diseases. Cystic fibrosis is an innate lung disease. Unlike idiopathic pulmonary fibrosis, cystic fibrosis has unique pathogenesis. Autophagy is an essential biological mechanism for the removal of misfolded proteins and damaged organelles in cells. Abnormal autophagy activity is involved in the pathogenesis of cystic fibrosis. Various studies have demonstrated that abnormalities or impaired autophagy are associated with cardiovascular diseases including pulmonary vascular disease. Autophagy plays a key role in maintaining normal vascular biological functions and vascular cell tissue homeostasis, and also plays an important role in the pathogenesis of various vascular diseases. For example, recent studies have found that autophagy participates in the occurrence and development of pulmonary hypertension. In addition, autophagy plays a central role in both innate and adaptive immune responses in immune cells or other cells with immune function. Thus, autophagy is the important cellular biological mechanism which causes cell fighting against pathogenic microorganisms including viruses, bacteria, and parasites. In this chapter, we discuss the work related to autophagy and other lung diseases.

Slowing ribosome velocity restores folding and function of mutant CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.

Alginate/Chitosan Particle-Based Drug Delivery Systems for Pulmonary Applications

Cystic fibrosis (CF) is a complex, potentially life-threatening disease that is most effectively treated through the administration of antibiotics (e.g., colistimethate sodium). Chronic infection with Pseudomonas aeruginosa is one of the most significant events in the pathogenesis of cystic fibrosis, and tobramycin is the treatment of choice for those patients with chronic P. aeruginosa infection who are deteriorating despite regular administration of colistimethate sodium. Effective treatment can be challenging due to the accumulation of thickened mucus in the pulmonary environment, and here we describe the results of our investigation into the development of alginate/chitosan particles prepared via precipitation for such environments. Tobramycin loading and release from the alginate/chitosan particles was investigated, with evidence of both uptake and release of sufficient tobramycin to inhibit P. aeruginosa in vitro. Functionalisation of the alginate/chitosan particles with secretory leukocyte protease inhibitor (SLPI) was shown to help inhibit the inflammatory response associated with lung infections (via inhibition of neutrophil elastase activity) and enhance their interaction with cystic fibrosis mucus (assayed via reduction of the depth of particle penetration into the mucus) in vitro, which have prospects to enhance their efficacy in vivo.

Airway Surface Liquid Has Innate Antiviral Activity That Is Reduced in Cystic Fibrosis.

Although chronic bacterial infections and inflammation are associated with progressive lung disease in patients with cystic fibrosis (CF), much less is known regarding the contributions of respiratory viral infections to this process. Clinical studies suggest that antiviral host defenses may be compromised in individuals with CF, and CF airway epithelia exhibit impaired antiviral responses in vitro. Here, we used the CF pig model to test the hypothesis that the antiviral activity of respiratory secretions is reduced in CF. We developed an in vitro assay to measure the innate antiviral activity present in airway surface liquid (ASL) from CF and non-CF pigs. We found that tracheal and nasal ASL from newborn non-CF pigs exhibited dose-dependent inhibitory activity against several enveloped and encapsidated viruses, including Sendai virus, respiratory syncytial virus, influenza A, and adenovirus. Importantly, we found that the anti-Sendai virus activity of nasal ASL from newborn CF pigs was significantly diminished relative to non-CF littermate controls. This diminution of extracellular antiviral defenses appears to be driven, at least in part, by the differences in pH between CF and non-CF ASL. These data highlight the novel antiviral properties of native airway secretions and suggest the possibility that defects in extracellular antiviral defenses contribute to CF pathogenesis.

Domain-interface dynamics of CFTR revealed by stabilizing nanobodies

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.

BLOOD MATRIX METALLOPROTEINASES LEVELS IN CYSTIC FIBROSIS CHILDREN (TEN YEARS OBSERVATION)

Introduction. Destructive fibrotic changes in lung tissue play a key role in the pathogenesis of cystic fibrosis (CF) in children. The development of pulmonary fibrosis may be caused by a violation of the pattern of matrix metalloproteinases (MMPs) and elevated production of profibrogenic growth factors (TGF-β1). Aim of the study. To compare the peculiarities of MMP patterns and transforming growth factor TGF-β1 with the data of the visualisation of airways features in cystic fibrosis (CF) children. Patients and Methods. The study included 80 inpatients aged of from 3 months to 18 years suffered from СF with the involvement of the lungs and digestive system observed for ten years. All patients were administered antibiotics (cefoperazone/sulbactam, ceftazidime, tienam, meropenem, amikacin) and inhalation (colisthmethate sodium, tobramycin) intravenously for a long time period. The reference group consisted of 16 children without pulmonary pathology. Blood serum concentrations of transforming growth factor-β1 (TGF-β1), matrix metalloproteinases (MMP-2, MMP-8, MMP-9) and tissue inhibitor of matrix metalloproteinases (TIMP-1) were determined by ELISA method. The morphological features of airways were evaluated by means of computer tomography (CT) with (GE Discovery CT750 HD). Results. In CF children patients blood serum MMP9 levels were significantly higher whereas TIMP-1 and MMP-2 appeared to be less than in children with intact airways. TGF-β1 levels in CF children were 9.8 times more than in cases from the reference group. CT data showed the pronounced changes in the airways structure as multiple bronchoectasias and pneumofibrosis. Conclusion. The revealed morphologic signs of the deterioration in airways’ structure in СF children patients can be related to the elevation of the rate of the fibrosis development due to the violation in the MMP and profibrogenic factors patterns and transforming growth factor TGF-β1.