TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium

Excessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs.     

In vitro 3D culture lung model from expanded primary cystic fibrosis human airway cells

BackgroundIn vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators.MethodsPrimary CF airway cells were expanded using PneumaCult^TM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor).ResultsCF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm² and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661.ConclusionsCF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.     

P. aeruginosa LPS stimulates calcium signaling and chloride secretion via CFTR in human bronchial epithelial cells

BackgroundPseudomonas aeruginosa airway infection is associated with a high mortality rate in cystic fibrosis. Lipopolysaccharide (LPS), a main constituent of the outer membrane of P. aeruginosa, is responsible for activation of innate immune response but its role on airway epithelium ion transport, is not well known. The aim of this study was to determine the role for P. aeruginosa LPS in modulating chloride secretion and intracellular calcium in the human bronchial epithelial cell line, 16HBE14o ?.MethodsWe used intracellular calcium imaging and short-circuit current measurement upon exposure of cells to P. aeruginosa LPS.ResultsApical LPS stimulated intracellular calcium release and calcium entry and enhanced chloride secretion. This latter effect was significantly inhibited by CFTR(inh)-172 and BAPTA-AM (intracellular Ca^2 + chelator).ConclusionsOur data provides evidence for a new role of P. aeruginosa LPS in stimulating calcium entry and release and a subsequent chloride secretion via CFTR in human bronchial epithelium.     

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

BackgroundChronic 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.MethodsPeripheral 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.Results51% 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.ConclusionsChronic 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.     

Modeling pulmonary cystic fibrosis in a human lung airway-on-a-chip

BackgroundCystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), which results in impaired airway mucociliary clearance, inflammation, infection, and respiratory insufficiency. The development of new therapeutics for CF are limited by the lack of reliable preclinical models that recapitulate the structural, immunological, and bioelectrical features of human CF lungs.MethodsWe leveraged organ-on-a-chip technology to develop a microfluidic device lined by primary human CF bronchial epithelial cells grown under an air-liquid interface and interfaced with pulmonary microvascular endothelial cells (CF Airway Chip) exposed to fluid flow. The responses of CF and healthy Airway Chips were analyzed in the presence or absence of polymorphonuclear leukocytes (PMNs) and the bacterial pathogen, Pseudomonas aeruginosa.ResultsThe CF Airway Chip faithfully recapitulated many features of the human CF airways, including enhanced mucus accumulation, increased cilia density, and a higher ciliary beating frequency compared to chips lined by healthy bronchial epithelial cells. The CF chips also secreted higher levels of IL-8, which was accompanied by enhanced PMN adhesion to the endothelium and transmigration into the airway compartment. In addition, CF Airway Chips provided a more favorable environment for Pseudomonas aeruginosa growth, which resulted in enhanced secretion of inflammatory cytokines and recruitment of PMNs to the airway.ConclusionsThe human CF Airway Chip may provide a valuable preclinical tool for pathophysiology studies as well as for drug testing and personalized medicine.     

Airway epithelial stem cell chimerism in cystic fibrosis lung transplant recipients

BackgroundThe conducting airway epithelium is repaired by tissue specific stem cells (TSC). In response to mild/moderate injury, each TSC repairs a discrete area of the epithelium. In contrast, severe epithelial injury stimulates TSC migration and expands the stem cell's reparative domain. Lung transplantation (LTx) can cause a moderate/severe airway injury and the remodeled airway contains a chimeric mixture of donor and recipient cells. These studies supported the hypothesis, LTx stimulates TSC migration resulting in epithelial chimerism. We tested this hypothesis in cystic fibrosis (CF) LTx patients.MethodsAirway mucosal injury was quantified using bronchoscopic imaging and a novel grading system. Bronchial brushing was used to recover TSC from 10 sites in the recipient and allograft airways. TSC chimerism was quantified by short tandem repeat analysis. TSC self-renewal and differentiation potential were assayed using the clone forming cell frequency and air-liquid-interface methods. Electrophysiology was used to determine if TSC chimerism altered epithelial ion channel activity.ResultsLTx caused a mild to moderate airway mucosal injury. Donor and recipient TSC were identified in 91% of anastomotic sites and 93% of bronchial airways. TSC chimerism did not alter stem cell self-renewal or differentiation potential. The frequency of recipient TSC was proportional to CF Transmembrane Conductance Regulator (CFTR)-dependent ion channel activity and 33% of allograft regions were at risk for abnormal CFTR activity.ConclusionsLTx in CF patients stimulates bidirectional TSC migration across the anastomoses. TSC chimerism may alter ion homeostasis and compromise the host defense capability of the allograft airway epithelium.     

Amphotericin B induces epithelial voltage responses in people with cystic fibrosis

BackgroundApproximately 10% of people with cystic fibrosis (CF) have mutations that result in little to no CFTR production and thus cannot benefit from CFTR modulators. We previously found that Amphotericin B (AmB), a small molecule that forms anion channels, restored HCO₃^? secretion and increased host defenses in primary cultures of CF airway epithelia. Further, AmB increased ASL pH in CFTR-null pigs, suggesting an alternative CFTR-independent approach to achieve gain-of-function. However, it remains unclear whether this approach can be effective in people.MethodsTo determine whether AmB can impact physiology in people with CF, we first tested whether Fungizone, a clinically approved AmB formulation, could cause electrophysiological effects consistent with anion secretion in primary cultures of CF airway epithelia. We then evaluated the capacity of AmB to change nasal potential difference (NPD), a key clinical biomarker, in people with CF not on CFTR modulators.ResultsAmB increased transepithelial Cl^? current and hyperpolarized calculated transepithelial voltage in primary cultures of CF airway epithelia from people with two nonsense mutations. In eight people with CF not on CFTR modulators, intranasal Fungizone treatment caused a statistically significant change in NPD. This change was similar in direction and magnitude to the effect of ivacaftor in people with a G551D mutation.ConclusionsOur results provide the first evidence that AmB can impact a clinical biomarker in people with CF. These results encourage additional clinical studies in people with CF to determine whether small molecule anion channels can provide benefit.     

Anti-inflammatory effects of lenabasum,a cannabinoid receptor type 2 agonist,on macrophages from cystic fibrosis

BackgroundLenabasum is an oral synthetic cannabinoid receptor type 2 agonist previously shown to reduce the production of key airway pro-inflammatory cytokines known to play a role in cystic fibrosis (CF). In a double-blinded, randomized, placebo-control phase 2 study, lenabasum lowered the rate of pulmonary exacerbation among patients with CF. The present study was undertaken to investigate anti-inflammatory mechanisms of lenabasum exhibits in CF macrophages.MethodsWe used monocyte-derived macrophages (MDMs) from healthy donors (n = 15), MDMs with CFTR inhibited with C-172 (n = 5) and MDMs from patients with CF (n = 4). Monocytes were differentiated to macrophages and polarized into classically activated (M1) macrophages by LPS or alternatively activated (M2) macrophages by IL-13 in presence or absence of lenabasum.ResultsLenabasum had no effect on differentiation, polarization and function of macrophages from healthy individuals. However, in CF macrophages lenabasum downregulated macrophage polarization into the pro-inflammatory M1 phenotype and secretion of the pro-inflammatory cytokines IL-8 and TNF-α in a dose-dependent manner. An improvement in phagocytic activity was also observed following lenabasum treatment. Although lenabasum did not restore the impaired polarization of anti-inflammatory M2 macrophage, it reduced the levels of IL-13 and enhanced the endocytic function of CF MDMs. The effects of lenabasum on MDMs with CFTR inhibited by C-172 were not as obvious.ConclusionIn CF macrophages lenabasum modulates macrophage polarization and function in vitro in a way that would reduce inflammation in vivo. Further studies are warranted to determine the link between activating the CBR2 receptor and CFTR.     

Significant functional differences in differentiated Conditionally Reprogrammed (CRC)- and Feeder-free Dual SMAD inhibited-expanded human nasal epithelial cells

BackgroundPatient-derived airway cells differentiated at Air Liquid Interface (ALI) are valuable models for Cystic fibrosis (CF) precision therapy. Different culture expansion methods have been established to extend expansion capacity of airway basal cells, while retaining functional airway epithelium physiology. Considerable variation in response to CFTR modulators is observed in cultures even within the same CFTR genotype and despite the use of similar ALI culture techniques. We aimed to address culture expansion method impact on differentiation.MethodsNasal epithelial brushings from 14 individuals (CF=9; non-CF=5) were collected, then equally divided and expanded under conditional reprogramming culture (CRC) and feeder-serum-free “dual-SMAD inhibition” (SMADi) methods. Expanded cells from each culture were differentiated with proprietary PneumaCult?-ALI media. Morphology (Immunofluorescence), global proteomics (LC-MS/MS) and function (barrier integrity, cilia motility, and ion transport) were compared in CRC^ALI and SMADi^ALI under basal and CFTR corrector treated (VX-809) conditions.ResultsNo significant difference in the structural morphology or baseline global proteomics profile were observed. Barrier integrity and cilia motility were significantly different, despite no difference in cell junction morphology or cilia abundance. Epithelial Sodium Channels and Calcium-activated Chloride Channel activity did not differ but CFTR mediated chloride currents were significantly reduced in SMADi^ALI compare to their CRC^ALI counterparts.ConclusionAlteration of cellular physiological function in vitro were more prominent than structural and differentiation potential in airway ALI. Since initial expansion culture conditions significantly influence CFTR activity, this could lead to false conclusions if data from different labs are compared against each other without specific reference ranges.     

CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts

Background

Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector?+?VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions.

Macrophage PD-1 associates with neutrophilia and reduced bacterial killing in early cystic fibrosis airway disease

BackgroundMacrophages are the major resident immune cells in human airways coordinating responses to infection and injury. In cystic fibrosis (CF), neutrophils are recruited to the airways shortly after birth, and actively exocytose damaging enzymes prior to chronic infection, suggesting a potential defect in macrophage immunomodulatory function. Signaling through the exhaustion marker programmed death protein 1 (PD-1) controls macrophage function in cancer, sepsis, and airway infection. Therefore, we sought to identify potential associations between macrophage PD-1 and markers of airway disease in children with CF.MethodsBlood and bronchoalveolar lavage fluid (BALF) were collected from 45 children with CF aged 3 to 62 months and structural lung damage was quantified by computed tomography. The phenotype of airway leukocytes was assessed by flow cytometry, while the release of enzymes and immunomodulatory mediators by molecular assays.ResultsAirway macrophage PD-1 expression correlated positively with structural lung damage, neutrophilic inflammation, and infection. Interestingly, even in the absence of detectable infection, macrophage PD-1 expression was elevated and correlated with neutrophilic inflammation. In an in vitro model mimicking leukocyte recruitment into CF airways, soluble mediators derived from recruited neutrophils directly induced PD-1 expression on recruited monocytes/macrophages, suggesting a causal link between neutrophilic inflammation and macrophage PD-1 expression in CF. Finally, blockade of PD-1 in a short-term culture of CF BALF leukocytes resulted in improved pathogen clearance.ConclusionTaken together, these findings suggest that in early CF lung disease, PD-1 upregulation associates with airway macrophage exhaustion, neutrophil takeover, infection, and structural damage.     

Oxidative stress and abnormal bioactive lipids in early cystic fibrosis lung disease

BackgroundClinical data indicate that airway inflammation in children with cystic fibrosis (CF) arises early, is associated with structural lung damage, and predicts progression. In bronchoalveolar lavage fluid (BALF) from CFTR mutant mice, several aspects of lipid metabolism are abnormal that contributes to lung disease. We aimed to determine whether lipid pathway dysregulation is also observed in BALF from children with CF, to identify biomarkers of early lung disease and potential therapeutic targets.MethodsA comprehensive panel of lipids that included Sphingolipids, oxylipins, isoprostanes and lysolipids, all bioactive lipid species known to be involved in inflammation and tissue remodeling, were measured in BALF from children with CF (1–6 years, N = 33) and age-matched non-CF patients with unexplained inflammatory disease (N = 16) by HPLC-MS/MS. Lipid data were correlated with chest CT scores and BALF inflammation biomarkers.ResultsThe ratio of long chain to very long chain ceramide species (LCC/VLCC) and lysolipid levels were enhanced in CF compared to non-CF patients, despite comparable neutrophil counts and bacterial load. In CF patients both LCC/VLCC and lysolipid levels correlated with inflammation and chest CT scores. The ceramide precursors Sphingosine, Sphinganine, Sphingomyelin, correlated with inflammation, whilst the oxidative stress marker isoprostane correlated with inflammation and chest CT scores. No correlation between lipids and current bacterial infection in CF (N = 5) was observed.ConclusionsSeveral lipid biomarkers of early CF lung disease were identified, which point toward potential disease monitoring and therapeutic approaches that can be used to complement CFTR modulators.     

Lack of CFTR alters the ferret pancreatic ductal epithelial secretome and cellular proteome: Implications for exocrine/endocrine signaling

BackgroundCystic fibrosis (CF) related diabetes is the most common comorbidity for CF patients and associated with islet dysfunction. Exocrine pancreas remodeling in CF alters the microenvironment in which islets reside. Since CFTR is mainly expressed in pancreatic ductal epithelium, we hypothesized altered CF ductal secretions could impact islet function through paracrine signals.MethodWe evaluated the secretome and cellular proteome of polarized WT and CF ferret ductal epithelia using quantitative ratiometric mass spectrometry. Differentially secreted proteins (DSPs) or expressed cellular proteins were used to mine pathways, upstream regulators and the CFTR interactome to map candidate CF-associated alterations in ductal signaling and phenotype. Candidate DSPs were evaluated for their in vivo pancreatic expression patterns and their functional impact on islet hormone secretion.ResultsThe secretome and cellular proteome of CF ductal epithelia was significantly altered relative to WT and implicated dysregulated TGFβ, WNT, and BMP signaling pathways. Cognate receptors of DSPs from CF epithelia were equally distributed among endocrine, exocrine, and stromal pancreatic cell types. IGFBP7 was a downregulated DSP in CF ductal epithelia in vitro and exhibited reduced CF ductal expression in vivo. IGFBP7 also altered WT islet insulin secretion in response to glucose. Many CFTR-associated proteins, including SLC9A3R1, were differentially expressed in the CF cellular proteome. Upstream regulators of the differential CF ductal proteome included TGFβ, PDX1, AKT/PTEN, and INSR signaling. Data is available via ProteomeXchange with identifier PXD025126.ConclusionThese findings provide a proteomic roadmap for elucidating disturbances in autocrine and paracrine signals from CF pancreatic ducts and how they may alter islet function and maintenance.     

Strong toll-like receptor responses in cystic fibrosis patients are associated with higher lung function

BackgroundCystic fibrosis (CF) airways disease varies widely among patients with identical cystic fibrosis transmembrane conductance regulator (CFTR) genotypes. Robust airway inflammation is thought to be deleterious in CF; inter-individual variation in Toll-like receptor (TLR)-mediated innate immune inflammatory responses (TMIIR) might account for a portion of the phenotypic variation. We tested if TMIIR in people with CF are different than those of healthy controls, and whether higher TMIIR in people with CF are associated with reduced lung function.MethodsWe cultured whole blood from clinically stable subjects with CF (n = 76) and healthy controls (n = 45) with TLR agonists, and measured cytokine production and expression of TLR-associated genes. We tested for differences in TLR-stimulated cytokine levels between subjects with CF and healthy subjects, and for associations between cytokine and gene expression levels with baseline lung function (forced expiratory volume in one second percent predicted (FEV₁%)) and decline in FEV₁% over time.ResultsTMIIR in blood from subjects with CF were lower than in healthy controls. Expression of TLR regulators SARM1, TOLLIP, and AKT1 were downregulated in CF. In subjects with CF we found that lower TLR4-agonist-induced IL-8 was associated with lower FEV₁% at enrollment (p<0.001) and with greater five year FEV₁% decline (p<0.001).ConclusionsTMIIR were lower in people with CF relative to healthy controls; however, unexpectedly, greater whole blood TMIIR were positively associated with lung function in people with CF. These findings suggest a complex interaction between inflammation and disease in people with CF.     

Microbial interaction: Prevotella spp. reduce P. aeruginosa induced inflammation in cystic fibrosis bronchial epithelial cells

BackgroundIn Cystic Fibrosis (CF) airways, the dehydrated, thick mucus promotes the establishment of persistent polymicrobial infections and drives chronic airways inflammation. This also predisposes the airways to further infections, the vicious, self-perpetuating cycle causing lung damage and progressive lung function decline. The airways are a poly-microbial environment, containing both aerobic and anaerobic bacterial species. Pseudomonas aeruginosa (P. aeruginosa) infections contribute to the excessive inflammatory response in CF, but the role of anaerobic Prevotella spp., frequently found in CF airways, is not known.MaterialsWe assessed innate immune signalling in CF airway epithelial cells in response to clinical strains of P. histicola, P. nigresens and P. aeruginosa. CFBE41o- cells were infected with P. aeruginosa (MOI 100, 2h) followed by infection with P. histicola or P. nigrescens (MOI 100, 2h). Cells were incubated under anaerobic conditions for the duration of the experiments.ResultsOur study shows that P. histicola and P. nigresens can reduce the growth of P. aeruginosa and dampen the inflammatory response in airway epithelial cells. We specifically illustrate that the presence of the investigated Prevotella spp. reduces Toll-like-receptor (TLR)-4, MAPK, NF-κB(p65) signalling and cytokine release (Interleukin (IL)-6, IL-8) in mixed infections.ConclusionOur work, for the first time, strongly indicates a relationship between P. aeruginosa and anaerobic Prevotella spp.. The observed modified NF-κB and MAPK signalling indicates some mechanisms underlying this interaction that could offer a novel therapeutic approach to combat chronic P. aeruginosa infection in people with CF.     

Reclassifying inconclusive diagnosis after newborn screening for cystic fibrosis. Moving forward

BackgroundNewborn screening for Cystic Fibrosis (CF) is associated with situations where the diagnosis of CF or CFTR related disorders (CFTR-RD) cannot be clearly ruled out.Materials/patients and methodsWe report a case series of 23 children with unconclusive diagnosis after newborn screening for CF and a mean follow-up of 7.7 years (4–13). Comprehensive investigations including whole CFTR gene sequencing, in vivo intestinal current measurement (ICM), nasal potential difference (NPD), and in vitro functional studies of variants of unknown significance, helped to reclassify the patients.ResultsExtensive genetic testing identified, in trans with a CF causing mutation, variants with varying clinical consequences and 3 variants of unknown significance (VUS). Eighteen deep intronic variants were identified by deep resequencing of the whole CFTR gene in 13 patients and were finally considered as non-pathogenic. All patients had normal CFTR dependent chloride transport in ICM. NPD differentiated 3 different profiles: CF-like tracings qualifying the patients as CF, such as F508del/D1152H patients; normal responses, suggesting an extremely low likelihood of developing a CFTR-RD such as F508del/TG11T5 patients; partial CFTR dysfunction above 20% of the normal, highlighting a remaining risk of developing CFTR-RD such as F508del/F1052V patients. The 3 VUS were reclassified as variant with defective maturation (D537N), defective expression (T582I) or with no clinical consequence (M952T).ConclusionThis study demonstrates the usefulness of combining genetic and functional investigations to assess the possibility of evolving to CF or CFTR-RD in babies with inconclusive diagnosis at neonatal screening.