Cystic Fibrosis Lung Research Articles

Impact of antibiotic eradication therapy of Pseudomonas aeruginosa on long term lung function in cystic fibrosis

IntroductionWhile antibiotic eradication therapy (AET) of early Pseudomonas aeruginosa infection is considered standard of care, its long-term effect on the subsequent course of cystic fibrosis (CF) lung disease remains unclear. MethodsCF patients who were P. aeruginosa-free for at least a year and had a minimum of 10 years of pulmonary function measurements were included. Subjects were categorized as Never if they never had P. aeruginosa isolated from a respiratory tract sample. Subjects changed to the Eradicated group if they had a P. aeruginosa infection, were treated with AET, and subsequently cleared their infection. Subjects changed to the Chronic group if AET did not clear their P. aeruginosa infection. The primary outcome was absolute FEV1 decline over time, with age as the time variable. Mixed-effects linear regression models were used to account for the repeated lung function measurements over time within each patient. Results205 CF subjects (48% female) were included; the median (IQR) age at first infection was 9.6 (5.6, 14.6) years. The median (IQR) follow-up was 10.2 (5.7, 14.7) years for the Eradicated group, 8.8 (4.5, 14.9) years for the Chronic group and 2.8 (1.0, 5.7) years for the Never group was among those patients that had at least one P. aeruginosa infection over the study period, annual lung function decline of FEV1 was significantly less (-1.11% predicted/year; 95% CI: -1.18, -1.04) in the Eradication group compared to the Chronic group (-1.57%; -1.64, -1.50) (p<0.001). ConclusionsAET against P. aeruginosa improves lung function trajectory in CF patients.

Achromobacter spp. Adaptation in Cystic Fibrosis Infection and Candidate Biomarkers of Antimicrobial Resistance

Achromobacter spp. can establish occasional or chronic lung infections in patients with cystic fibrosis (CF). Chronic colonization has been associated with worse prognosis highlighting the need to identify markers of bacterial persistence. To this purpose, we analyzed phenotypic features of 95 Achromobacter spp. isolates from 38 patients presenting chronic or occasional infection. Virulence was tested in Galleria mellonella larvae, cytotoxicity was tested in human bronchial epithelial cells, biofilm production in static conditions was measured by crystal violet staining and susceptibility to selected antibiotics was tested by the disk diffusion method. The presence of genetic loci associated to the analyzed phenotypic features was evaluated by a genome-wide association study. Isolates from occasional infection induced significantly higher mortality of G. mellonella larvae and showed a trend for lower cytotoxicity than chronic infection isolates. No significant difference was observed in biofilm production among the two groups. Additionally, antibiotic susceptibility testing showed that isolates from chronically-infected patients were significantly more resistant to sulfonamides and meropenem than occasional isolates. Candidate genetic biomarkers associated with antibiotic resistance or sensitivity were identified. Achromobacter spp. strains isolated from people with chronic and occasional lung infection exhibit different virulence and antibiotic susceptibility features, which could be linked to persistence in CF lungs. This underlines the possibility of identifying predictive biomarkers of persistence that could be useful for clinical purposes.

Niche, not phylogeny, governs the response to oxygen availability among diverse Pseudomonas aeruginosa strains.

Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen, is a leading cause of chronic infection of airways in cystic fibrosis (CF) patients. Chronic infections typically arise from colonization by environmental strains, followed by adaptation of P. aeruginosa to the conditions within the CF airway. It has been suggested that oxygen availability can be an important source of selection causing trait changes associated with the transition to chronic infection, but little data exist on the response of P. aeruginosa to varying levels of oxygen. Here, we use a diverse collection of P. aeruginosa strains recovered from both CF patients and environmental sources to evaluate the role of oxygen availability in driving adaptation to the CF lung while also accounting for phylogenetic relatedness. While we can detect a signal of phylogeny in trait responses to oxygen availability, niche of origin is a far stronger predictor. Specifically, strains isolated from the lungs of CF patients are more sensitive to external oxidative stress but more resistant to antibiotics under anoxic conditions. Additionally, many, though not all, patho-adaptive traits we assayed are insensitive to oxygen availability. Our results suggest that inferences about trait expression, especially those associated with the transition to chronic infection, depend on both the available oxygen and niche of origin of the strains being studied.

Nitric oxide inhibits alginate biosynthesis in Pseudomonas aeruginosa and increases its sensitivity to tobramycin by downregulating algU gene expression

In the process of chronic cystic fibrosis (CF) infection, Pseudomonas aeruginosa (PA) is converted into a mucoid phenotype characterized by an overproduction of exopolysaccharide alginate. The alginate forms a thick mucus that causes difficulty in patient's breathing, drug resistance and contributes to both the morbidity and mortality of the patient. AlgU of PA, an extracytoplasmic function sigma factor, is responsible for the alginate overproduction and leads to mucoidy and chronic infection of CF patients. In this report, we found that endogenous and exogenous nitric oxide (NO) can significantly reduce algU expression, leading to down-regulation of a series of alginate synthesis-related genes (algD, alg8, algX, and algK), eventually down-regulated alginate synthesis. A fluorescent reporter strain was constructed to clarify the inhibitory effect of alginate synthesis through real-time monitoring in different conditions. The results showed that NO presented inhibitory effect on alginate synthesis in nine clinical PA isolates as in the PA reference strain, and the reduction of alginate was more significant in three mucoid strains (by about 51%, 70% and 61%, respectively, while 47% for the reference strain). In the co-culture system, effect of NO on PA fluorescence intensity is similar to that in monocultures, with the best effect at 10 μM NO donor sodium nitroprusside (SNP). Finally, we examined the changes in the antibiotic susceptibility of PA under NO-inhibited alginate conditions. In the presence of 10 μM SNP, the number of planktonic cells increased, and both adherent and planktonic PA cells showed increased susceptibility to tobramycin. We thus suggest that NO can potentially be employed as a therapeutic strategy to prevent cystic fibrosis lungs from PA infection.

Bicarbonate Effects on Antibacterial Immunity and Mucus Glycobiology in the Cystic Fibrosis Lung: A Review With Selected Experimental Observations.

The primary defect in cystic fibrosis (CF) is abnormal chloride and bicarbonate transport in the cystic fibrosis transmembrane conductance regulator (CFTR) epithelial ion channel. The apical surface of the respiratory tract is lined by an airway surface liquid layer (ASL) composed of mucin comprising mainly MUC5A and MUC5B glycoproteins. ASL homeostasis depends on sodium bicarbonate secretion into the airways and secretion deficits alter mucus properties leading to airway obstruction, inflammation, and infections. Downstream effects of abnormal ion transport in the lungs include altered intrinsic immune defenses. We observed that neutrophils killed Pseudomonas aeruginosa more efficiently when it had been exposed to sodium bicarbonate, and formation of neutrophil extracellular traps (NETs) by neutrophils was augmented in the presence of increasing bicarbonate concentrations. Physiological levels of bicarbonate sensitized P. aeruginosa to the antimicrobial peptide cathelicidin LL-37, which is present in both lung ASL and in NETs. Sodium bicarbonate has various uses in clinical medicine and in the care of CF patients, and could be further explored as a therapeutic adjunct against Pseudomonas infections.

Antibiotics Drive Expansion of Rare Pathogens in a Chronic Infection Microbiome Model.

ABSTRACTChronic (long-lasting) infections are globally a major and rising cause of morbidity and mortality. Unlike typical acute infections, chronic infections are ecologically diverse, characterized by the presence of a polymicrobial mix of opportunistic pathogens and human-associated commensals. To address the challenge of chronic infection microbiomes, we focus on a particularly well-characterized disease, cystic fibrosis (CF), where polymicrobial lung infections persist for decades despite frequent exposure to antibiotics. Epidemiological analyses point to conflicting results on the benefits of antibiotic treatment yet are confounded by the dependency of antibiotic exposures on prior pathogen presence, limiting their ability to draw causal inferences on the relationships between antibiotic exposure and pathogen dynamics. To address this limitation, we develop a synthetic infection microbiome model representing CF metacommunity diversity and benchmark on clinical data. We show that in the absence of antibiotics, replicate microbiome structures in a synthetic sputum medium are highly repeatable and dominated by oral commensals. In contrast, challenge with physiologically relevant antibiotic doses leads to substantial community perturbation characterized by multiple alternate pathogen-dominant states and enrichment of drug-resistant species. These results provide evidence that antibiotics can drive the expansion (via competitive release) of previously rare opportunistic pathogens and offer a path toward microbiome-informed conditional treatment strategies.IMPORTANCE We develop and clinically benchmark an experimental model of the cystic fibrosis (CF) lung infection microbiome to investigate the impacts of antibiotic exposures on chronic, polymicrobial infections. We show that a single experimental model defined by metacommunity data can partially recapitulate the diversity of individual microbiome states observed across a population of people with CF. In the absence of antibiotics, we see highly repeatable community structures, dominated by oral microbes. Under clinically relevant antibiotic exposures, we see diverse and frequently pathogen-dominated communities, and a nonevolutionary enrichment of antimicrobial resistance on the community scale, mediated by competitive release. The results highlight the potential importance of nonevolutionary (community-ecological) processes in driving the growing global crisis of increasing antibiotic resistance.

Super resolution microscopy analysis reveals increased Orai1 activity in asthma and cystic fibrosis lungs

QuestionIn diseases such as asthma and cystic fibrosis (CF), the immune response is dysregulated and the lung is chronically inflamed. Orai1 activation is required for the initiation and persistence of inflammation. However, Orai1 expression in the lung is poorly understood. We therefore tested the hypothesis that Orai1 expression was upregulated in asthmatic and CF lungs. Materials and methodsWe used LungMAP to analyze single-cell RNAseq data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, asthma, and CF donors. We imaged sections by confocal and super resolution microscopy, and analyzed Orai1 and STIM1 expression in different pulmonary cell types. ResultsOrai1 was broadly-expressed, but expression was greatest in immune cells. At mRNA and protein levels, there were no consistent trends in expression levels between the three phenotypes. Orai1 must interact with STIM1 in order to activate and conduct Ca2+. We therefore used STIM1/Orai1 co-localization as a marker of Orai1 activity. Using this approach, we found significantly increased co-localization between these proteins in epithelia, interstitial and luminal immune cells, but not alveoli, from asthma and CF lungs. Orai1 also aggregates as part of its activation process. Using super resolution microscopy, we also found significantly increased Orai1 aggregation in immune cells from asthmatic and CF lungs. ConclusionWe found evidence that Orai1 was more active in asthma and CF than normal lungs. These data suggest that Orai1 is a relevant target for reducing pulmonary inflammation.

Ruthenium(II) Polypyridyl Complexes for Antimicrobial Photodynamic Therapy: Prospects for Application in Cystic Fibrosis Lung Airways.

Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.

An optimized protocol for assessment of sputum macrorheology in health and muco-obstructive lung disease

Background: Airway mucus provides important protective functions in health and abnormal viscoelasticity is a hallmark of muco-obstructive lung diseases such as cystic fibrosis (CF). However, previous studies of sputum macrorheology from healthy individuals and patients with CF using different experimental protocols yielded in part discrepant results and data on a systematic assessment across measurement settings and conditions remain limited. Objectives: The aim of this study was to develop an optimized and reliable protocol for standardized macrorheological measurements of airway mucus model systems and native human sputum from healthy individuals and patients with muco-obstructive lung disease. Methods: Oscillatory rheological shear measurements were performed using bovine submaxillary mucin (BSM) at different concentrations (2% and 10% solids) and sputum samples from healthy controls (n = 10) and patients with CF (n = 10). Viscoelastic properties were determined by amplitude and frequency sweeps at 25°C and 37°C with or without solvent trap using a cone-plate geometry. Results: Under saturated atmosphere, we did not observe any temperature-dependent differences in 2% and 10% BSM macrorheology, whereas in the absence of evaporation control 10% BSM demonstrated a significantly higher viscoelasticity at 37°C. Similarly, during the measurements without evaporation control at 37°C we observed a substantial increase in the storage modulus G′ and the loss modulus G″ of the highly viscoelastic CF sputum but not in the healthy sputum. Conclusion: Our data show systematically higher viscoelasticity of CF compared to healthy sputum at 25°C and 37°C. For measurements at the higher temperature using a solvent trap to prevent evaporation is essential for macrorheological analysis of mucus model systems and native human sputum. Another interesting finding is that the viscoelastic properties are not much sensitive to the applied experimental deformation and yield robust results despite their delicate consistency. The optimized protocol resulting from this work will facilitate standardized quantitative assessment of abnormalities in viscoelastic properties of airway mucus and response to muco-active therapies in patients with CF and other muco-obstructive lung diseases.

Polyguluronate simulations shed light onto the therapeutic action of OligoG CF-5/20

Chronic mucoid P. aeruginosa cystic fibrosis (CF) lung infections are associated with the development of a biofilm composed of anionic acetylated exopolysaccharide (EPS) alginate, electrostatically stabilised by extracellular Ca2+ ions. OligoG CF-5/20, a low molecular weight guluronate rich oligomer, is emerging as a novel therapeutic capable of disrupting mature P. aeruginosa biofilms. However, its method of therapeutic action on the mucoid biofilm EPS is not definitively known at a molecular level. This work, utilising molecular dynamics (MD) and Density-Functional Theory (DFT), has revealed that OligoG CF-5/20 interaction with the EPS is facilitated solely through bridging Ca2+ ions, which are not liberated from their native EPS binding sites upon OligoG CF-5/20 dispersal, suggesting that OligoG CF-5/20 does not cause disruptions to mature P. aeruginosa biofilms through breaking EPS-Ca2+-EPS ionic cross-links. Rather it is likely that the therapeutic activity arises from sequestering free Ca2+ ions and preventing further Ca2+ induced EPS aggregation.

Early Effects of Elexacaftor-Tezacaftor-Ivacaftor Therapy on Magnetic Resonance Imaging in Patients with Cystic Fibrosis and Advanced Lung Disease.

This is a prospective, observational study involving three Cystic Fibrosis (CF) adult patients, evaluating the changes in chest magnetic resonance imaging (MRI) three months after the start of elexacaftor/tezacaftor and ivacaftor therapy. MRI showed a drastic reduction in mucus plugging and bronchial wall thickening, with an improvement in the diffusion-weighted MRI score. Similarly, a marked improvement in spirometric parameters, nutritional status, and sweat chloride was observed. Our preliminary data confirm that chest MRI could be a useful tool to assess disease progression in CF patients on modulatory drug therapy.

Macrophage Lysosomal Alkalinization Drives Invasive Aspergillosis in a Mouse Cystic Fibrosis Model of Airway Transplantation.

Cystic fibrosis (CF) lung transplant recipients (LTRs) exhibit a disproportionately high rate of life-threatening invasive aspergillosis (IA). Loss of the cystic fibrosis transmembrane conductance regulator (CFTR-/-) in macrophages (mφs) has been associated with lyosomal alkalinization. We hypothesize that this alkalinization would persist in the iron-laden post-transplant microenvironment increasing the risk of IA. To investigate our hypothesis, we developed a murine CF orthotopic tracheal transplant (OTT) model. Iron levels were detected by immunofluorescence staining and colorimetric assays. Aspergillus fumigatus (Af) invasion was evaluated by Grocott methenamine silver staining. Phagocytosis and killing of Af conidia were examined by flow cytometry and confocal microscopy. pH and lysosomal acidification were measured by LysoSensorTM and LysotrackerTM, respectively. Af was more invasive in the CF airway transplant recipient compared to the WT recipient (p < 0.05). CFTR-/- mφs were alkaline at baseline, a characteristic that was increased with iron-overload. These CFTR-/- mφs were unable to phagocytose and kill Af conidia (p < 0.001). Poly(lactic-co-glycolic acid) (PLGA) nanoparticles acidified lysosomes, restoring the CFTR-/- mφs’ ability to clear conidia. Our results suggest that CFTR-/- mφs’ alkalinization interacts with the iron-loaded transplant microenvironment, decreasing the CF-mφs’ ability to kill Af conidia, which may explain the increased risk of IA. Therapeutic pH modulation after transplantation could decrease the risk of IA.

The In Vitro Replication Cycle of Achromobacter xylosoxidans and Identification of Virulence Genes Associated with Cytotoxicity in Macrophages.

ABSTRACTAchromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized in vitro and in vivo model systems to identify and test bacterial gene products that promote a pathological response. We have previously identified macrophages as a target host cell for A. xylosoxidans-induced cytotoxicity. By optimizing our macrophage infection model, we determined that A. xylosoxidans enters macrophages and can reside within a membrane bound vacuole for extended periods of time. Intracellular replication appears limited with cellular lysis preceding an enhanced, mainly extracellular replication cycle. Using our optimized in vitro model system along with transposon mutagenesis, we identified 163 genes that contribute to macrophage cytotoxicity. From this list, we characterized a giant RTX adhesin encoded downstream of a type one secretion system (T1SS) that mediates bacterial binding and entry into host macrophages, an important first step toward cellular toxicity and inflammation. The RTX adhesin is encoded by other human isolates and is recognized by antibodies present in serum isolated from CF patients colonized by A. xylosoxidans, indicating this virulence factor is produced and deployed in vivo. This study represents the first characterization of A. xylosoxidans replication during infection and identifies a variety of genes that may be linked to virulence and human pathology.IMPORTANCE Patients affected by CF develop chronic bacterial infections characterized by inflammatory exacerbations and tissue damage. Advancements in sequencing technologies have broadened the list of opportunistic pathogens colonizing the CF lung. A. xylosoxidans is increasingly recognized as an opportunistic pathogen in CF, yet our understanding of the bacterium as a contributor to human disease is limited. Genomic studies have identified potential virulence determinants in A. xylosoxidans isolates, but few have been mechanistically studied. Using our optimized in vitro cell model, we identified and characterized a bacterial adhesin that mediates binding and uptake by host macrophages leading to cytotoxicity. A subset of serum samples from CF patients contains antibodies that recognize the RTX adhesion, suggesting, for the first time, that this virulence determinant is produced in vivo. This work furthers our understanding of A. xylosoxidans virulence factors at a mechanistic level.

Staphylococcus aureus and Pseudomonas aeruginosa Isolates from the Same Cystic Fibrosis Respiratory Sample Coexist in Coculture.

ABSTRACTRespiratory infections with bacterial pathogens remain the major cause of morbidity in individuals with the genetic disease cystic fibrosis (CF). Some studies have shown that CF patients that harbor both Staphylococcus aureus and Pseudomonas aeruginosa in their lungs are at even greater risk for more severe and complicated respiratory infections and earlier death. However, the drivers for this worse clinical condition are not well understood. To investigate the interactions between these two microbes that might be responsible for their increased pathogenic potential, we obtained 28 pairs of S. aureus and P. aeruginosa from the same respiratory samples from 18 individuals with CF. We compared the survival of each S. aureus CF isolate cocultured with its corresponding coinfecting CF P. aeruginosa to when it was cocultured with non-CF laboratory strains of P. aeruginosa. We found that the S. aureus survival was significantly higher in the presence of the coinfecting P. aeruginosa compared to laboratory P. aeruginosa strains, regardless of whether the coinfecting isolate was mucoid or nonmucoid. We also tested how a non-CF S. aureus strain, JE2, behaved with each P. aeruginosa CF isolate and found that its interaction was similar to how the CF S. aureus isolate interacted with its coinfecting P. aeruginosa. Altogether, our work suggests that interactions between S. aureus and P. aeruginosa that promote coexistence in the CF lung are isolate-dependent and that this interaction appears to be driven mainly by P. aeruginosa.IMPORTANCE Previous studies have shown that in laboratory settings, Pseudomonas aeruginosa generally kills Staphylococcus aureus. However, these bacteria are often found coinfecting the lungs of cystic fibrosis (CF) patients, which has been associated with worse patient outcomes. To investigate the interactions between these two bacteria, we competed 28 coinfection pairs obtained from the same lung samples of 18 different CF patients. We compared these results to those we previously reported of each CF S. aureus isolate against a non-CF laboratory strain of P. aeruginosa. We found that S. aureus survival against its corresponding coinfection P. aeruginosa was higher than its survival against the laboratory strain of P. aeruginosa. These results suggest that there may be selection for coexistence of these microbes in the CF lung environment. Further understanding of the interactions between P. aeruginosa and S. aureus will provide insights into the drivers of coexistence and their impact on the host.

Food web and metabolic interactions of the lung inhabitants Streptococcus pneumoniae and Pseudomonas aeruginosa.

Bacteria that successfully adapt to different substrates and environmental niches within the lung and overcome the immune defence can cause serious lung infections. Such infections are generally complex, and recognized as polymicrobial in nature. Both Pseudomonas aeruginosa and Streptococcus pneumoniae can cause chronic lung infections and were both detected in cystic fibrosis (CF) lung at different stages. In this study, single and dual species cultures of Pseudomonas aeruginosa and Streptococcus pneumoniae were studied under well-controlled planktonic growth conditions. Under pH-controlled conditions, both species apparently benefited from the presence of the other. In co-culture with P. aeruginosa, S. pneumoniae grew efficiently under aerobic conditions, whereas in pure S. pneumoniae culture, growth inhibition occurred in bioreactors with dissolved oxygen concentrations above the microaerobic range. Lactic acid and acetoin that are produced by S. pneumoniae were efficiently utilized by P. aeruginosa. In pH-uncontrolled co-cultures, the low pH triggered by S. pneumoniae assimilation of glucose and lactic acid production negatively affected the growth of both strains. Nevertheless, ammonia production improved significantly, and P. aeruginosa growth dominated at later growth stages. This study revealed unreported metabolic interactions of two important pathogenic microorganisms and shed new lights into pathophysiology of bacterial lung infection.

Insulin-Like Growth Factor Binding Protein (IGFBP-6) as a Novel Regulator of Inflammatory Response in Cystic Fibrosis Airway Cells.

Cystic Fibrosis (CF) patients are prone to contracting bacterial lung infections with opportunistic pathogens, especially Pseudomonas aeruginosa. Prolonged P. aeruginosa infections have been linked to chronic inflammation in the CF lung, whose hallmarks are increased levels of cytokines (i.e., TNF-α, IL-1β, IL-6) and neutrophil attraction by chemokines, like IL-8. Recently, insulin-like growth factor binding protein 6 (IGFBP-6) has been shown to play a putative role in the immune system and was found at higher levels in the sera and synovial tissue of rheumatoid arthritis patients. Moreover, it has been demonstrated that IGFBP-6 has chemoattractant properties towards cells of the innate (neutrophils, monocytes) and adaptive (T cells) immunity. However, it is not known whether IGFBP-6 expression is dysregulated in airway epithelial cells under infection/inflammatory conditions. Therefore, we first measured the basal IGFBP-6 mRNA and protein levels in bronchial epithelial cells lines (Wt and F508del-CFTR CFBE), finding they both are upregulated in F508del-CFTR CFBE cells. Interestingly, LPS and IL-1β+TNFα treatments increased the IGFBP-6 mRNA level, that was reduced after treatment with an anti-inflammatory (Dimethyl Fumarate) in CFBE cell line and in patient-derived nasal epithelial cultures. Lastly, we demonstrated that IGFBP-6 reduced the level of pro-inflammatory cytokines in both CFBE and primary nasal epithelial cells, without affecting rescued CFTR expression and function. The addition of a neutralizing antibody to IGFBP-6 increased pro-inflammatory cytokines expression under challenge with LPS. Together, these data suggest that IGFBP-6 may play a direct role in the CF-associated inflammation.

High-Density Branched PEGylation for Nanoparticle Drug Delivery.

The surface modification of nanoparticles (NP) with a dense layer of polyethylene glycol (PEG) has been widely used to improve NP circulation time, bioavailability, and diffusion through biological barriers [e.g. extracellular matrix (ECM), mucus]. While linear PEG coatings are commonly used, branched PEG coatings have not been widely explored as a design parameter for NP drug delivery systems. NPs were densely coated with either linear 2, 5, 10kDa linear PEG or with 10kDa star-shaped, 4-arm branched PEG. NP cellular uptake was evaluated in HEK-293T and A549 cells. NP stability was evaluated in fetal bovine serum over 24h using dynamic light scattering. Diffusion of NPs within a Matrigel ECM model and sputum (mucus) collected from individuals with cystic fibrosis (CF) lung disease were analyzed through multiple particle tracking. PEG-coated NPs appeared more stable in serum compared to uncoated NPs, but the reduction in total protein adsorbed was most significant for branched PEG coated NP. All PEGylated NPs had similar cellular uptake in HEK-293T and A549 cells. Interestingly, branched-PEG coated NPs had the largest diffusion coefficient and moved most rapidly through Matrigel. However in CF mucus, linear 2 and 5 kDa PEG coated NPs had the largest fraction of rapidly diffusing particles while branched PEG coated NPs had less hindered mobility compared to linear 10 kDa PEG coated NPs. Branched PEGylation may have the potential to increase NP efficiency in reaching target cells based on an apparent increase in diffusion through an ECM model while maintaining NP stability and uptake in target cells comparable to their linear PEG counterparts.

The Use of Thiocyanate Formulations to Create Manganese Porphyrin Antioxidants That Supplement Innate Immunity.

The innate immune response to infection results in inflammation and oxidative damage, creating a paradox where most anti-inflammatory and antioxidant therapies can further suppress an already inadequate immune response. We have previously reported the beneficial effects of the exogenous supplementation of innate immunity with small pseudohalide thiocyanate (−SCN) in a mouse model of a cystic fibrosis (CF) lung infection and inflammation. The object of this study was to evaluate the use of −SCN as a counter anion for cationic manganese porphyrin (MnP) catalytic antioxidants, which could increase the parent compound’s antioxidant spectrum against hypohalous acids while supplementing innate immunity. The antioxidant activities of the parent compound were examined, as its chloride salt was compared with the −SCN-anion exchanged compound, (MnP(SCN) versus MnP(Cl)). We measured the superoxide dismutase activity spectrophotometrically and performed hydrogen peroxide scavenging using oxygen and hydrogen peroxide electrodes. Peroxidase activity was measured using an amplex red assay. The inhibition of lipid peroxidation was assessed using a thiobarbituric acid reactive species (TBARS) assay. The effects of the MnP compounds on macrophage phagocytosis were assessed by flow cytometry. The abilities of the MnP(Cl) formulations to protect human bronchiolar epithelial cells against hypochlorite (HOCl) and glycine chloramine versus their MnP(SCN) formulations were assessed using a cell viability assay. We found that anions exchanging out the chloride for −SCN improved the cellular bioavailability but did not adversely affect the cell viability or phagocytosis and that they switched hydrogen-peroxide scavenging from a dismutation reaction to a peroxidase reaction. In addition, the −SCN formulations improved the ability of MnPs to protect human bronchiolar epithelial cells against hypochlorous acid (HOCl) and glycine chloramine toxicity. These novel types of antioxidants may be more beneficial in treating lung disease that is associated with chronic infections or acute infectious exacerbations.

Defining and identifying early-onset lung disease in cystic fibrosis with cumulative clinical characteristics.

Because of the heterogeneity in cystic fibrosis (CF) lung disease among young children, a clinical method to identify early-onset lung disease is needed. To develop a CF early-onset lung disease (CFELD) scoring system by utilizing prospectively collected longitudinal data on manifestations in the first 3 years of life. We studied 145 infants born during 2012-2017, diagnosed through newborn screening by age 3 months, and followed to 36 months of age. Cough severity, pulmonary exacerbations (PEx), respiratory cultures, and hospitalizations were collected at each CF center visit (every 1-2 months in infancy and quarterly thereafter). These data were used to construct the CFELD system and to classify lung disease into five categories: asymptomatic, minimal, mild, moderate, and severe. The most frequent manifestation of CF early lung disease was MD-reported PEx episodes, PEx hospitalizations, and positive Pseudomonas aeruginosa cultures. Parent-reported cough severity was correlated with the number of respiratory hospitalizations (r = 0.48, p < 0.0001). The distribution of CFELD categories was 10% asymptomatic, 17% minimal, 29% mild, 33% moderate, and 12% severe. The moderate and severe categories occurred threefold higher in pancreatic insufficient (PI, 49%) versus sufficient subjects (16%), p < 0.0001. In addition to PI, gastrointestinal and nutrition-related hospitalizations, plasma cytokines interleukin (IL)-6 and IL-10, duration of CFTR modulator therapy, and type of health insurance were significant predictors of CFELD scores. The CFELD scoring system is novel, allows systematic evaluation of lung disease prognosis early, and may aid in therapeutic decision-making particularly in the initiation of CFTR modulator therapy.

Activity of N-Acetylcysteine Alone and in Combination with Colistin against Pseudomonas aeruginosa Biofilms and Transcriptomic Response to N-Acetylcysteine Exposure.

ABSTRACTChronic colonization by Pseudomonas aeruginosa is critical in cystic fibrosis (CF) and other chronic lung diseases, contributing to disease progression. Biofilm growth and a propensity to evolve multidrug resistance phenotypes drastically limit the available therapeutic options. In this perspective, there has been growing interest in evaluating combination therapies, especially for drugs that can be administered by nebulization, which allows high drug concentrations to be reached at the site of infections while limiting systemic toxicity. Here, we investigated the potential antibiofilm activity of N-acetylcysteine (NAC) alone and in combination with colistin against a panel of P. aeruginosa strains (most of which are from CF patients) and the transcriptomic response of a P. aeruginosa CF strain to NAC exposure. NAC alone (8,000 mg/L) showed a limited and strain-dependent antibiofilm activity. Nonetheless, a relevant antibiofilm synergism of NAC-colistin combinations (NAC at 8,000 mg/L plus colistin at 2 to 32 mg/L) was observed with all strains. Synergism was also confirmed with the artificial sputum medium model. RNA sequencing of NAC-exposed planktonic cultures revealed that NAC (8,000 mg/L) mainly induced (i) a Zn2+ starvation response (known to induce attenuation of P. aeruginosa virulence), (ii) downregulation of genes of the denitrification apparatus, and (iii) downregulation of flagellar biosynthesis pathway. NAC-mediated inhibition of P. aeruginosa denitrification pathway and flagellum-mediated motility were confirmed experimentally. These findings suggested that NAC-colistin combinations might contribute to the management of biofilm-associated P. aeruginosa lung infections. NAC might also have a role in reducing P. aeruginosa virulence, which could be relevant in the very early stages of lung colonization.IMPORTANCE Pseudomonas aeruginosa biofilm-related chronic lung colonization contributes to cystic fibrosis (CF) disease progression. Colistin is often a last-resort antibiotic for the treatment of such P. aeruginosa infections, and it has been increasingly used in CF, especially by nebulization. N-acetylcysteine (NAC) is a mucolytic agent with antioxidant activity, commonly administered with antibiotics for the treatment of lower respiratory tract infections. Here, we show that NAC potentiated colistin activity against in vitro biofilms models of P. aeruginosa strains, with both drugs tested at the high concentrations achievable after nebulization. In addition, we report the first transcriptomic data on the P. aeruginosa response to NAC exposure.