Cystic Fibrosis Airway Fluids Research Articles

384 Impact of elexacaftor/tezacaftor/ivacaftor therapy on pathological reprogramming of lung-recruited neutrophils conditioned by cystic fibrosis airway fluid

384 Impact of elexacaftor/tezacaftor/ivacaftor therapy on pathological reprogramming of lung-recruited neutrophils conditioned by cystic fibrosis airway fluid

Pathological metabolic adaptation of neutrophils recruited to cystic fibrosis airway microenvironment can not be reversed by modulator therapy

Abstract Background Neutrophilic inflammation is a hallmark of cystic fibrosis (CF) lung disease. Prior studies from our group show that blood neutrophils undergo metabolic and functional adaptations upon entry into the CF lung lumen that causes them to become anabolic, while repressing bacterial killing function and enhancing degranulation. The recent advent of highly effective modulator therapy (HEMT) for the CFTR channel (mutated in CF) has improved patient outcomes, but it is unclear how HEMT impact CF lung neutrophilic inflammation. Method We used an organotypic model in which blood neutrophils are transmigrated through differentiated airway epithelial cells towards a chemoattractant control (leukotriene B4, LTB4), or airway supernatant from CF patients not on HEMT (CFASN) or on HEMT (CFMOD). We conducted phenotypic (FACS, CFU-assay) and metabolomic (metabolomics, 13C6-glucose tracing) analysis of the transmigrated neutrophils. Results CFMOD-recruited neutrophils contained ivacaftor (a HEMT drug), demonstrating exposure to the therapy. Compared to LTB4-recruited neutrophils, CFASN/CFMOD-recruited neutrophils showed increased AMP, GMP and intracellular and secreted metabolites related to citric acid cycle activity. 13C6-glucose flux indicated similar rates of extracellular glucose utilization in all transmigrated neutrophils, but higher total glycolysis to lactate in CFASN/CFMOD-transmigrated neutrophils. Conclusion Pathological metabolic adaptations of neutrophils following transmigration into CF airway fluid are not substantially altered by HEMT. Further studies are needed to assess the potential impact on functional adaptations by these cells. Supported by NIH (R56 HL150658), Cystic Fibrosis Foundation (CAMMAR21F0, TIROUV19G0), I3 Teams Research Award (Emory), and CF@LANTA, a component of Emory University and Children’s Healthcare of Atlanta.

Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro.

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

Myeloperoxidase oxidation of methionine associates with early cystic fibrosis lung disease.

Cystic fibrosis (CF) lung disease progressively worsens from infancy to adulthood. Disease-driven changes in early CF airway fluid metabolites may identify therapeutic targets to curb progression.CF patients aged 12-38 months (n=24; three out of 24 later denoted as CF screen positive, inconclusive diagnosis) received chest computed tomography scans, scored by the Perth-Rotterdam Annotated Grid Morphometric Analysis for CF (PRAGMA-CF) method to quantify total lung disease (PRAGMA-%Dis) and components such as bronchiectasis (PRAGMA-%Bx). Small molecules in bronchoalveolar lavage fluid (BALF) were measured with high-resolution accurate-mass metabolomics. Myeloperoxidase (MPO) was quantified by ELISA and activity assays.Increased PRAGMA-%Dis was driven by bronchiectasis and correlated with airway neutrophils. PRAGMA-%Dis correlated with 104 metabolomic features (p<0.05, q<0.25). The most significant annotated feature was methionine sulfoxide (MetO), a product of methionine oxidation by MPO-derived oxidants. We confirmed the identity of MetO in BALF and used reference calibration to confirm correlation with PRAGMA-%Dis (Spearman's ρ=0.582, p=0.0029), extending to bronchiectasis (PRAGMA-%Bx; ρ=0.698, p=1.5×10-4), airway neutrophils (ρ=0.569, p=0.0046) and BALF MPO (ρ=0.803, p=3.9×10-6).BALF MetO associates with structural lung damage, airway neutrophils and MPO in early CF. Further studies are needed to establish whether methionine oxidation directly contributes to early CF lung disease and explore potential therapeutic targets indicated by these findings.

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

31 Sphingosine 1-phosphate receptor 3 is highly upregulated on human neutrophils upon migration to the airways

Objective Migration of polymorphonuclear neutrophils (PMNs) from blood into the airway lumen is accompanied by changes in surface receptors and responsiveness to environmental cues, such as sphingolipids. In particular, signaling via extracellular ceramide/sphingosine 1 phosphate (S1P) was suggested to contribute to human airway disease, such as cystic fibrosis (CF). We hypothesized that airway PMNs would alter expression of the S1P receptor, S1PR3, which was recently implicated in lung inflammation and fibrosis. Methods We collected blood and sputum from healthy control (HC) and CF subjects and analyzed S1PR3 expression in live PMNs gated by flow cytometry. In addition, we used a novel in vitro model to study the effect of transepithelial migration of blood PMNs on S1PR3 expression. Results We observed a 10-fold upregulation of surface S1PR3 expression on HC and CF airway PMNs, in vivo, compared to their blood counterparts. This major increase was recapitulated in vitro, when naive blood PMNs were induced to transmigrate through an airway epithelial layer upon exposure to apical CF airway fluid. S1PR3 expression increased over time, by 10-fold after 10 hours, and up to 20-fold at 18 hours. Conclusion Modulation of S1PR3 expression follows PMN migration to both HC and CF airways. Further studies will determine the importance of this receptor in mediating responses to ceramide/S1P signaling in these cells, including PMN migration, apoptosis and activation.

Human resistin, a neutrophil-derived metabolic and inflammatory mediator correlates with decreased lung function and NETosis in cystic fibrosis airway disease (HUM1P.261)

Abstract Resistin, a protein initially cloned from mouse adipocytes, is a strong antagonist of insulin signaling. In humans, it is located in both the azurophil and specific granules of PMNs and as a result can be elevated in metabolic and PMN-driven inflammatory disorders. Cystic fibrosis (CF) combines PMN-dominated airway inflammation with profound metabolic anomalies, but the presence of resistin in CF patients has not been studied thus far. Methods: We measured resistin levels using an ELISA on platelet-free plasma and airway fluid from CF patients and measured the presence of neutrophil extracellular traps (NETs) a marker of neutrophil activation in CF airway fluid. Results: Resistin levels in platelet-free plasma were significantly higher in CF subjects than in HC subjects. Resistin levels in expectorated CF sputum were 2 to 3 orders (100-500 fold) of magnitude higher than in plasma. Interestingly, we found a strong negative correlation between sputum resistin and lung function (Spearman Rho=-0.81, P&amp;lt;10-4). Similarly resistin was also shown to positively correlate with NETs in CF sputum. Conclusions: Resistin levels are abnormal in CF plasma and strikingly elevated in CF airway fluid during chronic disease. These results show, as also suggested by prior studies, that extracellular resistin is associated with PMN-driven CF airway inflammation, including NET release. Mechanistic studies looking at the precise impact of resistin on metabolism and inflammation in CF are warranted.

Sphingosine 1-phosphate receptor 3 is highly upregulated on human neutrophils upon migration to the airways (INC6P.315)

Abstract Background and hypothesis: Migration of polymorphonuclear neutrophils (PMNs) from blood into the airway lumen is accompanied by changes in surface receptors and responsiveness to environmental cues, such as sphingolipids. In particular, signaling via extracellular ceramide / sphingosine 1-phosphate (S1P) was suggested to contribute to human airway disease, such as cystic fibrosis (CF). We hypothesized that airway PMNs would alter expression of the S1P receptor, S1PR3, which was recently implicated in lung inflammation and fibrosis. Methods: We collected blood and sputum from healthy control (HC) and CF subjects and analyzed S1PR3 expression in live PMNs gated by flow cytometry. In addition, we used a novel in vitro model to study the effect of transepithelial migration of blood PMNs on S1PR3 expression. Results: We observed a 10-fold upregulation of surface S1PR3 expression on HC and CF airway PMNs, in vivo, compared to their blood counterparts. This major increase was recapitulated in vitro, when naive blood PMNs were induced to transmigrate through an airway epithelial layer toward apical CF airway fluid. S1PR3 expression increased over time, by 10-fold after 10 hours, and up to 20-fold at 18 hours. Conclusions: Modulation of S1PR3 expression follows PMN migration to both HC and CF airways. Further studies will determine the importance of this receptor in mediating responses to ceramide/S1P signaling in these cells, including PMN migration, apoptosis and activation.

158 Prevalence of Pneumocystis jirovecii in a cross section of patients attending a large UK adult cystic fibrosis centre

Background and Objectives: Fungi, particularly Aspergillus and Candida species, are increasingly found in cystic fibrosis (CF) airway fluids. However, their association with other CF pathogens, medications and lung function, especially in CF children, remains elusive. We analyzed the relationship of fungal colonization to microbiological and clinical parameters of pediatric CF patients. Methods and Results: Fungal colonization and long-term clinical parameters like BMI and lung function were retrospectively studied in over 500 CF patients. Colonization was defined based on Chotirmall et al. (Chest 2010). Candida albicans (CA) was the most prevalent fungus detected in CF airway fluids, followed by C. non-albicans > Aspergillus fumigatus (AF) > A. non-fumigatus species. We found an association between fungal colonization and (among other parameters) bacterial co-colonization, lung function, BMI and antibiotic therapy. Conclusion: This study suggests that colonization with CA or AF is affected by bacterial co-colonization and may modulate the disease severity already in pediatric CF patients.

157 Long-term impact of fungi on pediatric cystic fibrosis lung disease

Background and Objectives: Fungi, particularly Aspergillus and Candida species, are increasingly found in cystic fibrosis (CF) airway fluids. However, their association with other CF pathogens, medications and lung function, especially in CF children, remains elusive. We analyzed the relationship of fungal colonization to microbiological and clinical parameters of pediatric CF patients. Methods and Results: Fungal colonization and long-term clinical parameters like BMI and lung function were retrospectively studied in over 500 CF patients. Colonization was defined based on Chotirmall et al. (Chest 2010). Candida albicans (CA) was the most prevalent fungus detected in CF airway fluids, followed by C. non-albicans > Aspergillus fumigatus (AF) > A. non-fumigatus species. We found an association between fungal colonization and (among other parameters) bacterial co-colonization, lung function, BMI and antibiotic therapy. Conclusion: This study suggests that colonization with CA or AF is affected by bacterial co-colonization and may modulate the disease severity already in pediatric CF patients.

WS19.5 Characterization of fungal colonization phenotypes in pediatric cystic fibrosis lung disease

Objectives: Fungi, particularly Aspergillus and Candida species, are increasingly found in cystic fibrosis (CF) airway fluids. However, their relationship to other CF pathogens, medications and lung function, especially in CF children, remains poorly understood. To address this question, we analyzed the associations of fungal colonization with microbiological and clinical parameters of pediatric CF patients. Methods: Fungal colonization was categorized in n = 128 CF patients based on Chotirmall et al. (Chest 2010). Candida albicans (CA) was the most prevalent fungus detected in CF airway fluids, followed by C. non-albicans > Aspergillus fumigatus (AF) > A. non-fumigatus species. Colonization with CA correlated positively with age, inhaled antibiotic use, and Aspergillus species, while an inverse correlation with Haemophilus and no association with S. aureus or Pseudomonas was found. Colonization with AF correlated positively with age, inhaled antibiotics, Pseudomonas, Stenotrophomonas, atypical mycobacteria and CF-related diabetes and inversely with Haemophilus. ABPA showed no correlation with AF colonization, but with non-fumigatus species and use of azithromycin. Colonization with AF or CA correlated inversely with cross-sectional FEV1, but not with MEF25. Colonization with AF correlated with longitudinal FEV1 decline. Conclusion: This study suggests that colonization with CA or AF is affected by bacterial co-colonization and may modulate lung function already in pediatric CF.

WS19.6 Changes in the weather and the respiratory health of adults with cystic fibrosis

Objectives: Fungi, particularly Aspergillus and Candida species, are increasingly found in cystic fibrosis (CF) airway fluids. However, their relationship to other CF pathogens, medications and lung function, especially in CF children, remains poorly understood. To address this question, we analyzed the associations of fungal colonization with microbiological and clinical parameters of pediatric CF patients. Methods: Fungal colonization was categorized in n = 128 CF patients based on Chotirmall et al. (Chest 2010). Candida albicans (CA) was the most prevalent fungus detected in CF airway fluids, followed by C. non-albicans > Aspergillus fumigatus (AF) > A. non-fumigatus species. Colonization with CA correlated positively with age, inhaled antibiotic use, and Aspergillus species, while an inverse correlation with Haemophilus and no association with S. aureus or Pseudomonas was found. Colonization with AF correlated positively with age, inhaled antibiotics, Pseudomonas, Stenotrophomonas, atypical mycobacteria and CF-related diabetes and inversely with Haemophilus. ABPA showed no correlation with AF colonization, but with non-fumigatus species and use of azithromycin. Colonization with AF or CA correlated inversely with cross-sectional FEV1, but not with MEF25. Colonization with AF correlated with longitudinal FEV1 decline. Conclusion: This study suggests that colonization with CA or AF is affected by bacterial co-colonization and may modulate lung function already in pediatric CF.

Expression and Regulation of Interferon-Related Development Regulator–1 in Cystic Fibrosis Neutrophils

A genome-wide association study identified interferon-related development regulator-1 (IFRD1), a protein expressed by neutrophils, as a key modifier gene in cystic fibrosis (CF) lung disease. Here, we investigated the expression and regulation of IFRD1 in CF neutrophils. IFRD1 expression was quantified in peripheral blood and airway neutrophils from patients with CF, patients with non-CF lung disease, and healthy control subjects. The regulation of IFRD1 expression was analyzed using isolated neutrophils and ex vivo stimulation assays with CF airway fluids. IFRD1 single-nucleotide polymorphisms (SNPs) were analyzed in a CF cohort (n = 572) and correlated with longitudinal lung function and IFRD1 expression. Patients with CF expressed higher protein levels of IFRD1 in peripheral blood neutrophils compared with healthy or non-CF disease control subjects. Within patients with CF, IFRD1 protein expression levels in neutrophils were lower in airway fluids compared with peripheral blood. High IFRD1 expression was positively associated with the production of reactive oxygen species (ROS) in CF neutrophils. In vitro regulation studies showed that CF airway fluid and the CF-characteristic chemokines CXCL8 and CXCL2 down-regulated IFRD1 expression in neutrophils, an effect that was mediated through CXCR2. Genetic analyses showed that three IFRD1 SNPs were associated with longitudinal declines in lung function, and modulated IFRD1 expression. These studies demonstrate that IFRD1 expression is systemically up-regulated in human CF neutrophils, is linked to the production of ROS, and is modulated by chemokines in CF airway fluids, depending on the IFRD1 genotype. Understanding the regulation of IFRD1 may pave the way for novel therapeutic approaches to target neutrophilic inflammation in CF.

The Chitinase-Like Protein YKL-40 Modulates Cystic Fibrosis Lung Disease

The chitinase-like protein YKL-40 was found to be increased in patients with severe asthma and chronic obstructive pulmonary disease (COPD), two disease conditions featuring neutrophilic infiltrates. Based on these studies and a previous report indicating that neutrophils secrete YKL-40, we hypothesized that YKL-40 plays a key role in cystic fibrosis (CF) lung disease, a prototypic neutrophilic disease. The aim of this study was (i) to analyze YKL-40 levels in human and murine CF lung disease and (ii) to investigate whether YKL-40 single-nucleotide polymorphisms (SNPs) modulate CF lung disease severity. YKL-40 protein levels were quantified in serum and sputum supernatants from CF patients and control individuals. Levels of the murine homologue BRP-39 were analyzed in airway fluids from CF-like βENaC-Tg mice. YKL-40SNPs were analyzed in CF patients. YKL-40 levels were increased in sputum supernatants and in serum from CF patients compared to healthy control individuals. Within CF patients, YKL-40 levels were higher in sputum than in serum. BRP-39 levels were increased in airways fluids from βENaC-Tg mice compared to wild-type littermates. In both CF patients and βENaC-Tg mice, YKL-40/BRP-39 airway levels correlated with the severity of pulmonary obstruction. Two YKL-40 SNPs (rs871799 and rs880633) were found to modulate age-adjusted lung function in CF patients. YKL-40/BRP-39 levelsare increased in human and murine CF airway fluids, correlate with pulmonary function and modulate CF lung disease severity genetically. These findings suggest YKL-40 as a potential biomarker in CF lung disease.

SerpinB1 in cystic fibrosis airway fluids: quantity, molecular form and mechanism of elastase inhibition

Neutrophil serine proteases (NSPs), especially elastase, are major agents of lung destruction in cystic fibrosis (CF) patients. This study investigated SerpinB1, a highly efficient inhibitor of NSPs, in CF lung disease. Bronchoalveolar lavage fluid (BALF) from 31 children with CF and 24 control children was examined for amount and molecular species of SerpinB1, and its mechanism of action was studied. CF BALF had more SerpinB1 than control BALF (geometric mean 3.9 (95% CI 2.60-5.62) versus 1.37 (1.20-1.55) μg·mL⁻¹; p<0.001). BALF levels of SerpinB1 were higher for infected versus uninfected CF subjects (5.5 versus 2.7 μg·mL⁻¹; p<0.04) and substantially higher for elastase-positive versus -negative CF subjects (8.41 versus 1.89 μg·mL⁻¹; p<0.001). Most SerpinB1 in CF BALF had been cleaved. Adding recombinant SerpinB1 to CF BALF stoichiometrically inhibited endogenous elastase, indicating that the inhibitor functions in the CF microenvironment. In vitro simulations comparing SerpinB1 and α₁-antitrypsin (SerpinA1) showed that both rapidly form irreversible inhibitory covalent complexes with elastase and that these differed in survival time. The SerpinB1-elastase complex survived only briefly due to fragmentation of bound elastase, releasing cleaved SerpinB1, the molecular form in CF BALF. The findings define an innate role for SerpinB1 in CF airways.

Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs

Cystic fibrosis (CF) patients undergo progressive airway destruction caused in part by chronic neutrophilic inflammation. While opportunistic pathogens infecting CF airways can cause inflammation, we hypothesized that host-derived metabolic and stress signals would also play a role in this process. We show that neutrophils that have entered CF airways have increased phosphorylation of the eukaryotic initiation factor 4E and its partner the 4E-binding protein 1; 2 key effectors in the growth factor- and amino acid-regulated mammalian target of rapamycin (mTOR) pathway. Furthermore CF airway neutrophils display increased phosphorylation of the cAMP response element binding protein (CREB), a major transcriptional coactivator in stress signaling cascades. These active intracellular pathways are associated with increased surface expression of critical adaptor molecules, including the growth factor receptor CD114 and the receptor for advanced glycation end-products (RAGE), a CREB inducer and sensor for host-derived damage-associated molecular patterns (DAMPs). Most CF airway fluids lack any detectable soluble RAGE, an inhibitory decoy receptor for DAMPs. Concomitantly, CF airway fluids displayed high and consequently unopposed levels of S100A12; a potent mucosa- and neutrophil-derived DAMP. CF airway neutrophils also show increased surface levels of 2 critical CREB targets, the purine-recycling enzyme CD39 and the multifunctional, mTOR-inducing CXCR4 receptor. This coordinated set of events occurs in all patients, even in the context of minimal airway inflammation and well-preserved lung function. Taken together, our data demonstrate an early and sustained activation of host-responsive metabolic and stress pathways upon neutrophil entry into CF airways, suggesting potential targets for therapeutic modulation.

High susceptibility for cystic fibrosis human airway gland cells to produce IL-8 through the I kappa B kinase alpha pathway in response to extracellular NaCl content.

Increasing evidence suggests that in airways from cystic fibrosis (CF) patients, inflammation may precede bacterial infection and be related to an endogenous dysregulation of proinflammatory cytokines in airway epithelial cells. Several investigators have reported that, in CF airway fluids, elevated NaCl concentrations may also contribute to the diseased state by inhibiting the bactericidal properties of airway fluid. Because many proinflammatory cytokines are transcriptionally regulated by the NF-kappa B, we investigated whether an elevated extracellular NaCl content in airway fluids significantly impaired the regulation of the NF-kappa B/I kappa B alpha complex and the chemokine IL-8 production in primary non-CF and CF human bronchial gland epithelial cells. Exposure of non-CF gland cells to hypotonic (85 mM) NaCl solution, compared with isotonic (115 mM) NaCl and hypertonic (170 mM) NaCl solutions, resulted in a significant decrease in IL-8 production that was paralleled by a strong inhibition of activated NF-kappa B associated with an increased cytosolic expression of I kappa B alpha and a decrease in the I kappa B kinase alpha protein level. In CF gland cells, we demonstrated that, compared with the high IL-8 in an hypertonic solution, the release of IL-8 was significantly reduced 2-fold in an isotonic solution and 5-fold in a hypotonic solution. Strikingly, exposure of CF bronchial gland cells to either hypotonic or isotonic milieu did not result in a marked inhibition of the activated NF-kappa B/I kappa B alpha system. This is the first demonstration that primary human CF bronchial gland cells exhibit abnormally high IL-8 production through constitutively activated NF-kappa B and high I kappa B kinase alpha level, whatever the hypo-, iso-, and hypertonic NaCl milieu.