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Bronchial thermoplasty (BT) is a treatment for moderate-to-severe asthma in which the airway smooth muscle layer is targeted directly using thermal ablation. Although it has been shown to be safe and effective in long-term follow-up, questions remain about its mechanism of action, patient selection, and optimization of protocol based on structural phenotype. Using a cohort of 20 subjects who underwent thermoplasty and assessment by computed tomography (CT), we demonstrate that response to BT can be feasibly predicted based on pretreatment airway dimensions that inform a subject-specific computational model. Analysis revealed the need for CT assessment at total lung capacity, rather than functional residual capacity, which was less sensitive to the effects of BT. Final model predictions compared favorably with observed outcomes in terms of airway caliber and asthma control, suggesting that this approach could form the basis of improved clinical practice.

Organoids, which are self-organizing three-dimensional cultures, provide models that replicate specific cellular components of native tissues or facets of organ complexity. We describe a simple method to generate organoid cultures using isolated human tracheobronchial epithelial cells grown in mixed matrix components and supplemented at day 14 with the Wnt pathway agonist R-spondin 2 (RSPO2) and the bone morphogenic protein antagonist Noggin. In contrast to previous reports, our method produces differentiated tracheobronchospheres with externally orientated apical membranes without pretreatments, providing an epithelial model to study cilia formation and function, disease pathogenesis, and interaction of pathogens with the respiratory mucosa. Starting from 3 × 105 cells, organoid yield at day 28 was 1,720 ± 302. Immunocytochemistry confirmed the cellular localization of airway epithelial markers, including CFTR, Na+/K+ ATPase, acetylated-α-tubulin, E-cadherin, and ZO-1. Compared to native tissues, expression of genes related to bronchial differentiation and ion transport were similar in organoid and air-liquid interface (ALI) cultures. In matched primary cultures, mean organoid cilia length was 6.1 ± 0.2 µm, similar to that of 5.7 ± 0.1 µm in ALI cultures, and ciliary beating was vigorous and coordinated with frequencies of 7.7 ± 0.3 Hz in organoid cultures and 5.3 ± 0.8 Hz in ALI cultures. Functional measurement of osmotically induced volume changes in organoids showed low water permeability. The generation of numerous single testable units from minimal starting material complements prior techniques. This culture system may be useful for studying airway biology and pathophysiology, aiding diagnosis of ciliopathies, and potentially for high-throughput drug screening.

Aberrant anion secretion across the bronchial epithelium is associated with airway disease, most notably in cystic fibrosis. Although the cystic fibrosis transmembrane conductance regulator (CFTR) is recognized as the primary source of airway anion secretion, alternative anion transport mechanisms play a contributing role. An alternative anion transporter of growing interest is SLC26A9, a constitutively active chloride channel that has been shown to interact with CFTR and may also contribute to bicarbonate secretion. Interest in SLC26A9 has been fueled by genome-wide association studies that suggest it is a significant modifier of CF disease severity. Despite this growing evidence that SLC26A9 plays an important role in the airway, its presence and function in bronchial epithelia remain poorly understood, in part, because its activity is difficult to separate from the activity of CFTR. Here, we present results using primary human bronchial epithelia (HBE) from multiple patient sources to confirm that SLC26A9 mRNA is present in HBE and that its constitutive channel activity is unaffected by knockdown of CFTR. Furthermore, SLC26A9 and CFTR show differential responses to common inhibitors of anion secretion. Finally, we assess the impact of bicarbonate on the activity of SLC26A9 and CFTR. These results confirm that SLC26A9 is the primary source of constitutive anion secretion across HBE, and should inform future studies focused on activation of SLC26A9 as an alternative anion channel in CF. These results should provide a strong foundation to investigate how single-nucleotide polymorphisms in SLC26A9 modulate airway disease.

The inability to assess and measure changes to the airway smooth muscle (ASM) in vivo is a major challenge to evaluating asthma and its clinical outcomes. Bronchial thermoplasty (BT) is a therapy for asthma that aims to reduce the severity of excessive bronchoconstriction by ablating ASM. Although multiple long-term clinical studies of BT have produced encouraging results, the outcomes of BT treatment in practice have been variable, and questions remain regarding the selection of patients. Previously, we have demonstrated an imaging platform called orientation-resolved optical coherence tomography that can assess ASM endoscopically using an imaging catheter compatible with bronchoscopy. In this work, we present results obtained from a longitudinal BT study performed using a canine model (n = 8) and with the goal of investigating the use of orientation-resolved optical coherence tomography (OR-OCT) for measuring the effects of BT on ASM. We demonstrate that we are capable of accurately assessing ASM both before and in the weeks following the BT procedure using blinded matching to histological samples stained with Masson’s trichrome (P < 0.0001, r(2) = 0.79). Analysis of volumetric ASM distributions revealed significant decreases in ASM in treated airways (average cross-sectional ASM area: 0.245 ± 0.145 mm(2) pre-BT and 0.166 ± 0.112 mm(2) 6 wk following BT). These results demonstrate that OR-OCT can provide clinicians with the feedback necessary to better evaluate ASM and its response to BT, and may potentially play an important role in phenotyping asthma and predicting which patients are most likely to respond to BT treatment. NEW & NOTEWORTHY The inability to assess ASM in vivo is a significant hurdle in advancing our understanding of airway diseases such as asthma, as well as evaluating potential treatments and therapies. In this study, we demonstrate that endoscopic OR-OCT can be used to accurately measure changes to ASM structure following BT. Our results demonstrate how this technology could occupy an important role in asthma treatments targeting ASM.

The growing interest in regulating flavored E-liquids must incorporate understanding of the “flavoring profile” of each E-liquid—which flavorings (flavoring chemicals) are present and at what concentrations not just focusing on the flavor on the label. We investigated the flavoring profile of 10 different flavored E-liquids. We assessed bronchial epithelial cell viability and apoptosis, phagocytosis of bacteria and apoptotic cells by macrophages after exposure to E-cigarette vapor extract (EVE). We validated our data in normal human bronchial epithelial cells (NHBE) and alveolar macrophages (AM) from healthy donors. We also assessed cytokine release and validated in the saliva from E-cigarette users. Increased necrosis/apoptosis (16.1–64.5% apoptosis) in 16HBE cells was flavor dependent, and NHBEs showed an increased susceptibility to flavors. In THP-1 differentiated macrophages phagocytosis was also flavor dependent, with AM also showing increased susceptibility to flavors. Further, Banana and Chocolate were shown to reduce surface expression of phagocytic target recognition receptors on alveolar macrophages. Banana and Chocolate increased IL-8 secretion by NHBE, whereas all 4 flavors reduced AM IL-1β secretion, which was also reduced in the saliva of E-cigarette users compared with healthy controls. Flavorant profiles of E-liquids varied from simple 2 compound mixtures to complex mixtures containing over a dozen flavorants. E-liquids with high benzene content, complex flavoring profiles, high chemical concentration had the greatest impacts. The Flavorant profile of E-liquids is key to disruption of the airway status quo by increasing bronchial epithelial cell apoptosis, causing alveolar macrophage phagocytic dysfunction, and altering airway cytokines.

Chloride secretion by airway epithelial cells is primordial for water and ion homeostasis and airways surface prevention of infections. This secretion is impaired in several human diseases, including cystic fibrosis, a genetic pathology due to CFTR gene mutations leading to chloride channel defects. A potential therapeutic approach is aiming at increasing chloride secretion either by correcting the mutated CFTR itself or by stimulating non-CFTR chloride channels at the plasma membrane. Here, we studied the role of phospholipase C in regulating the transepithelial chloride secretion in human airway epithelial 16HBE14o- and CFBE cells over-expressing wild type (WT)- or F508del-CFTR. Western blot analysis shows expression of the three endogenous phospholipase C (PLC) isoforms, namely, PLCδ1, PLCγ1, and PLCβ3 in 16HBE14o- cells. In 16HBE14o- cells, we performed Ussing chamber experiments after silencing each of these PLC isoforms or using the PLC inhibitor U73122 or its inactive analogue U73343. Our results show the involvement of PLCβ3 and PLCγ1 in CFTR-dependent short-circuit current activated by forskolin, but not of PLCδ1. In CFBE-WT CFTR and corrected CFBE-F508del CFTR cells, PLCβ3 silencing also inhibits CFTR-dependent current activated by forskolin and UTP-activated calcium-dependent chloride channels (CaCC). Our study supports the importance of PLC in maintaining CFTR-dependent chloride secretion over time, getting maximal CFTR-dependent current and increasing CaCC activation in bronchial epithelial cells.

The COVID-19 pandemic is an ongoing threat to public health. Since the identification of COVID-19, the disease caused by SARS-CoV-2, no drugs have been developed to specifically target SARS-CoV-2. To develop effective and safe treatment options, a better understanding of cellular mechanisms underlying SARS-CoV-2 infection is required. To fill this knowledge gap, researchers require reliable experimental systems that express the host factor proteins necessary for the cellular entry of SARS-CoV-2. These proteins include the viral receptor, angiotensin-converting enzyme 2 (ACE2), and the proteases, transmembrane serine protease 2 (TMPRSS2) and furin. A number of studies have reported cell-type-specific expression of the genes encoding these molecules. However, less is known about the protein expression of these molecules. We assessed the suitability of primary human bronchial epithelial (HBE) cells maintained in an air-liquid interface (ALI) as an experimental system for studying SARS-CoV-2 infection in vitro. During cellular differentiation, we measured the expression of ACE2, TMPRSS2, and furin over progressive ALI days by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunofluorescence staining. We also explored the effect of the fibrotic cytokine TGF-β on the expression of these proteins in well-differentiated HBE cells. Like ACE2, TMPRSS2 and furin proteins are localized in differentiated ciliated cells, as confirmed by immunofluorescence staining. These data suggest that well-differentiated HBE cells maintained in ALI are a reliable in vitro system for investigating cellular mechanisms of SARS-CoV-2 infection. We further identified that the profibrotic mediators, TGF-β1 and TGF-β2, increase the expression of furin, which is a protease required for the cellular entry of SARS-CoV-2.

In human airway smooth muscle (hASM), mitochondrial volume density is greater in asthmatic patients compared with normal controls. There is also an increase in mitochondrial fragmentation in hASM of moderate asthmatics associated with an increase in dynamin-related protein 1 (Drp1) and a decrease in mitofusin 2 (Mfn2) expression, mitochondrial fission, and fusion proteins, respectively. Proinflammatory cytokines such TNFα contribute to hASM hyperreactivity and cell proliferation associated with asthma. However, the involvement of proinflammatory cytokines in mitochondrial remodeling is not clearly established. In nonasthmatic hASM cells, mitochondria were labeled using MitoTracker Red and imaged in three dimensions using a confocal microscope. After 24-h TNFα exposure, mitochondria in hASM cells were more fragmented, evidenced by decreased form factor and aspect ratio and increased sphericity. Associated with increased mitochondrial fragmentation, Drp1 expression increased while Mfn2 expression was reduced. TNFα also increased mitochondrial biogenesis in hASM cells reflected by increased peroxisome proliferator-activated receptor-γ coactivator 1α expression and increased mitochondrial DNA copy number. Associated with mitochondrial biogenesis, TNFα exposure also increased mitochondrial volume density and porin expression, resulting in an increase in maximum O2 consumption rate. However, when normalized for mitochondrial volume density, O2 consumption rate per mitochondrion was reduced by TNFα exposure. Associated with mitochondrial fragmentation and biogenesis, TNFα also increased hASM cell proliferation, an effect mimicked by siRNA knockdown of Mfn2 expression and mitigated by Mfn2 overexpression. The results of this study support our hypothesis that in hASM cells exposed to TNFα mitochondria are more fragmented, with an increase in mitochondrial biogenesis and mitochondrial volume density resulting in reduced O2 consumption rate per mitochondrion.

Bronchomotor tone is regulated by contraction and relaxation of airway smooth muscle (ASM). A weakened ASM relaxation might be a cause of airway hyperresponsiveness (AHR), a characteristic feature of bronchial asthma. Pituitary adenylyl cyclase-activating polypeptide (PACAP) is known as a mediator that causes ASM relaxation. To date, whether or not the PACAP responsiveness is changed in asthmatic ASM is unknown. The current study examined the hypothesis that relaxation induced by PACAP is reduced in bronchial smooth muscle (BSM) of allergic asthma. The ovalbumin (OA)-sensitized mice were repeatedly challenged with aerosolized OA to induce asthmatic reaction. Twenty-four hours after the last antigen challenge, the main bronchial smooth muscle (BSM) tissues were isolated. Tension study showed a BSM hyperresponsiveness to acetylcholine in the OA-challenged mice. Both quantitative RT-PCR and immunoblot analyses revealed a significant decrease in PAC1receptor expression in BSMs of the diseased mice. Accordingly, in the antigen-challenged group, the PACAP-induced PAC1receptor-mediated BSM relaxation was significantly attenuated, whereas the relaxation induced by vasoactive intestinal polypeptide was not changed. These findings suggest that the relaxation induced by PACAP is impaired in BSMs of experimental asthma due to a downregulation of its binding partner PAC1receptor. Impaired BSM responsiveness to PACAP might contribute to the AHR in asthma.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide. While most develop a mild, self-limiting illness, some develop severe acute lower respiratory infection and persistent airway disease. Exposure to ambient particulate matter has been linked to asthma, bronchitis, and viral infection in multiple epidemiological studies. We hypothesized that coexposure to nanoparticles worsens RSV-induced airway epithelial barrier dysfunction. Bronchial epithelial cells were incubated with titanium dioxide nanoparticles (TiO2-NP) or a combination of TiO2-NP and RSV. Structure and function of epithelial cell barrier were analyzed. Viral titer and the role of reactive oxygen species (ROS) generation were evaluated. In vivo, mice were intranasally incubated with TiO2-NP, RSV, or a combination. Lungs and bronchoalveolar lavage (BAL) fluid were harvested for analysis of airway inflammation and apical junctional complex (AJC) disruption. RSV-induced AJC disruption was amplified by TiO2-NP. Nanoparticle exposure increased viral infection in epithelial cells. TiO2-NP induced generation of ROS, and pretreatment with antioxidant, N-acetylcysteine, reversed said barrier dysfunction. In vivo, RSV-induced injury and AJC disruption were augmented in the lungs of mice given TiO2-NP. Airway inflammation was exacerbated, as evidenced by increased white blood cell infiltration into the BAL, along with exaggeration of peribronchial inflammation and AJC disruption. These data demonstrate that TiO2-NP exposure exacerbates RSV-induced AJC dysfunction and increases inflammation by mechanisms involving generation of ROS. Further studies are required to determine whether NP exposure plays a role in the health disparities of asthma and other lung diseases, and why some children experience more severe airway disease with RSV infection.

Parametric response mapping (PRM) is a computed tomography (CT)-based method to phenotype patients with chronic obstructive pulmonary disease (COPD). It is capable of differentiating emphysema-related air trapping with nonemphysematous air trapping (small airway disease), which helps to identify the extent and localization of the disease. Most studies evaluating the gene expression in smokers and COPD patients related this to spirometric measurements, but none have investigated the relationship with CT-based measurements of lung structure. The current study aimed to examine gene expression profiles of brushed bronchial epithelial cells in association with the PRM-defined CT-based measurements of emphysema (PRMEmph) and small airway disease (PRMfSAD). Using the Top Institute Pharma (TIP) study cohort (COPD = 12 and asymptomatic smokers = 32), we identified a gene expression signature of bronchial brushings, which was associated with PRMEmph in the lungs. One hundred thirty-three genes were identified to be associated with PRMEmph. Among the most significantly associated genes, CXCL11 is a potent chemokine involved with CD8+ T cell activation during inflammation in COPD, indicating that it may play an essential role in the development of emphysema. The PRMEmph signature was then replicated in two independent data sets. Pathway analysis showed that the PRMEmph signature is associated with proinflammatory and notch signaling pathways. Together these findings indicate that airway epithelium may play a role in the development of emphysema and/or may act as a biomarker for the presence of emphysema. In contrast, its role in relation to functional small airways disease is less clear.

Tenacious mucus produced by tracheal and bronchial submucosal glands is a defining feature of several airway diseases, including cystic fibrosis (CF). Airway acidification as a driving force of CF airway pathology has been controversial. Here we tested the hypothesis that transient airway acidification produces pathologic mucus and impairs mucociliary transport. We studied pigs challenged with intra-airway acid. Acid had a minimal effect on mucus properties under basal conditions. However, cholinergic stimulation in acid-challenged pigs revealed retention of mucin 5B (MUC5B) in the submucosal glands, decreased concentrations of MUC5B in the lung lavage fluid, and airway obstruction. To more closely mimic a CF-like environment, we also examined mucus secretion and transport following cholinergic stimulation under diminished bicarbonate and chloride transport conditions ex vivo. Under these conditions, airways from acid-challenged pigs displayed extensive mucus films and decreased mucociliary transport. Pretreatment with diminazene aceturate, a small molecule with ability to inhibit acid detection through blockade of the acid-sensing ion channel (ASIC) at the doses provided, did not prevent acid-induced pathologic mucus or transport defects but did mitigate airway obstruction. These findings suggest that transient airway acidification early in life has significant impacts on mucus secretion and transport properties. Furthermore, they highlight diminazene aceturate as an agent that might be beneficial in alleviating airway obstruction.

The volume fraction of extracellular matrix (ECM) within the layer of airway smooth muscle (ASM) is increased in subjects with fixed airflow obstruction. We postulated that changes in ECM within the ASM layer will impact force transmission during induced contraction and/or in response to externally applied stresses like a deep inspiration (DI). Subjects were patients undergoing lung resection surgery who were categorized as unobstructed ( n = 12) or “fixed” obstructed ( n = 6) on the basis of preoperative spirometry. The response to a DI, assessed by the ratio of isovolumic flows from maximal and partial inspirations (M/P), was also measured preoperatively. M/P was reduced in the obstructed group ( P = 0.02). Postoperatively, bronchial segments were obtained from resected tissue, and luminal narrowing to acetylcholine and bronchodilation to simulated DI were assessed in vitro. Airway wall dimensions and the volume fraction of ECM within the ASM were quantified. Maximal airway narrowing to acetylcholine ( P = 0.01) and the volume fraction of ECM within the ASM layer ( P = 0.02) were increased in the obstructed group, without a change in ASM thickness. Whereas bronchodilation to simulated DI in vitro was not different between obstructed and unobstructed groups, it was correlated with increased M/P (bronchodilation/less bronchoconstriction) in vivo ( P = 0.03). The volume fraction of ECM was inversely related to forced expiratory volume in 1 s FEV1 %predicted ( P = 0.04) and M/P ( P = 0.01). Results show that in subjects with fixed airflow obstruction the mechanical behavior of the airway wall is altered and there is a contemporaneous shift in the structural composition of the ASM layer. NEW & NOTEWORTHY Cartilaginous airways from subjects with fixed airflow obstruction have an increase in the volume fraction of extracellular matrix within the airway smooth muscle layer. These airways are also intrinsically more reactive to a contractile stimulus, which is expected to contribute to airway hyperresponsiveness in this population, often attributed to geometric mechanisms. In view of these results, we speculate on how changes in extracellular matrix may impact airway mechanics.

Airway surface dehydration is a pathological feature of cystic fibrosis (CF) lung disease. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated Cl− channel controlled in part by the adenosine A2B receptor. An alternative CFTR-independent mechanism of fluid secretion is regulated by ATP via the P2Y2 receptor (P2Y2R) that activates Ca2+-regulated Cl− channels (CaCC/TMEM16) and inhibits Na+ absorption. However, due to rapid ATP hydrolysis, steady-state ATP levels in CF airway surface liquid (ASL) are inadequate to maintain P2Y2R-mediated fluid secretion. Therefore, inhibiting airway epithelial ecto-ATPases to increase ASL ATP levels constitutes a strategy to restore airway surface hydration in CF. Using [γ32P]ATP as radiotracer, we assessed the effect of a series of ATPase inhibitory compounds on the stability of physiologically occurring ATP concentrations. We identified the polyoxometalate [Co4(H2O)2(PW9O34)2]10− (POM-5) as the most potent and effective ecto-ATPase inhibitor in CF airway epithelial cells. POM-5 caused long-lasting inhibition of ATP hydrolysis in airway epithelia, which was reversible upon removal of the inhibitor. Importantly, POM-5 markedly enhanced steady-state levels of released ATP, promoting increased ASL volume in CF cell surfaces. These results provide proof of concept for ecto-ATPase inhibitors as therapeutic agents to restore hydration of CF airway surfaces. As a test of this notion, cell-free sputum supernatants from CF subjects were studied and found to have abnormally elevated ATPase activity, which was markedly inhibited by POM-5.

Fractal biological structures are pervasive throughout the plant and animal kingdoms, with the mammalian lung being a quintessential example. The lung airway and vascular trees are generated during embryogenesis from a small set of building codes similar to Turing mechanisms that create robust trees ideally suited to their functions. Whereas the blood flow pattern generated by these fractal trees has been shown to be genetically determined, the geometry of the trees has not. We explored a newly established repository providing high-resolution bronchial trees from the four most commonly studied laboratory mice (B6C3F1, BALB/c, C57BL/6 and CD-1). The data fit a fractal model well for all animals with the fractal dimensions ranging from 1.54 to 1.67, indicating that the conducting airway of mice can be considered a self-similar and space-filling structure. We determined that the fractal dimensions of these airway trees differed by strain but not sex, reinforcing the concept that airway branching patterns are encoded within the DNA. The observations also highlight that future study design and interpretations may need to consider differences in airway geometry between mouse strains. NEW & NOTEWORTHY Similar to larger mammals such as humans, the geometries of the bronchial tree in mice are fractal structures that have repeating patterns from the trachea to the terminal branches. The airway geometries of the four most commonly studied mice are different and need to be considered when comparing results that employ different mouse strains. This variability in mouse airway geometries should be incorporated into computer models exploring toxicology and aerosol deposition in mouse models.

This study aimed to explore the value of synchrotron-based phase-contrast microcomputed tomography (micro-CT) in pulmonary vascular pathobiology. The microanatomy of the lung is complex with intricate branching patterns. Tissue sections are therefore difficult to interpret. Recruited intrapulmonary bronchopulmonary anastomoses (IBAs) have been described in several forms of pulmonary hypertension, including alveolar capillary dysplasia with misaligned pulmonary veins (ACD/MPV). Here, we examine paraffin-embedded tissue using this nondestructive method for high-resolution three-dimensional imaging. Blocks of healthy and ACD/MPV lung tissue were used. Pulmonary and bronchial arteries in the ACD/MPV block had been preinjected with dye. One section per block was stained, and areas of interest were marked to allow precise beam-alignment during image acquisition at the X02DA TOMCAT beamline (Swiss Light Source). A ×4 magnifying objective coupled to a 20-µm thick scintillating material and a sCMOS detector yielded the best trade-off between spatial resolution and field-of-view. A phase retrieval algorithm was applied and virtual tomographic slices and video clips of the imaged volumes were produced. Dye injections generated a distinct attenuation difference between vessels and surrounding tissue, facilitating segmentation and three-dimensional rendering. Histology and immunohistochemistry post-imaging offered complementary information. IBAs were confirmed in ACD/MPV, and the MPVs were positioned like bronchial veins/venules. We demonstrate the advantages of using synchrotron-based phase-contrast micro-CT for three-dimensional characterization of pulmonary microvascular anatomy in paraffin-embedded tissue. Vascular dye injections add additional value. We confirm intrapulmonary shunting in ACD/MPV and provide support for the hypothesis that MPVs are dilated bronchial veins/venules.

The shortening of airway smooth muscle (ASM) is greatly affected by time. This is because stimuli affecting ASM shortening, such as bronchoactive molecules or the strain inflicted by breathing maneuvers, not only alter quick biochemical processes regulating contraction but also slower processes that allow ASM to adapt to an ever-changing length. Little attention has been given to the effect of time on ASM shortening. The present study investigates the effect of changing the time interval between simulated deep inspirations (DIs) on ASM shortening and its responsiveness to simulated DIs. Excised tracheal strips from sheep were mounted in organ baths and either activated with methacholine or relaxed with isoproterenol. They were then subjected to simulated DIs by imposing swings in distending stress, emulating a transmural pressure from 5 to 30 cmH2O. The simulated DIs were intercalated by 2, 5, 10, or 30 min. In between simulated DIs, the distending stress was either fixed or oscillating to simulate tidal breathing. The results show that although shortening was increased by prolonging the interval between simulated DIs, the bronchodilator effect of simulated DIs (i.e., the elongation of the strip post- vs. pre-DI) was not affected, and the rate of re-shortening post-simulated DIs was decreased. As the frequency with which DIs are taken increases upon bronchoconstriction, our results may be relevant to typical alterations observed in asthma, such as an increased rate of re-narrowing post-DI. NEW & NOTEWORTHY The frequency with which patients with asthma take deep inspirations (DIs) increases during bronchoconstriction. This in vitro study investigated the effect of changing the time interval between simulated DIs on airway smooth muscle shortening. The results demonstrated that decreasing the interval between simulated DIs not only decreases shortening, which may be protective against excessive airway narrowing, but also increases the rate of re-shortening post-simulated DIs, which may contribute to the increased rate of re-narrowing post-DI observed in asthma.

The transient receptor potential polycystin-2 (TRPP2) is encoded by the Pkd2 gene, and mutation of this gene can cause autosomal dominant polycystic kidney disease (ADPKD). Some patients with ADPKD experience extrarenal manifestations, including radiologic and clinical bronchiectasis. We hypothesized that TRPP2 may regulate airway smooth muscle (ASM) tension. Thus, we used smooth muscle- Pkd2 conditional knockout ( Pkd2SM-CKO) mice to investigate whether TRPP2 regulated ASM tension and whether TRPP2 deficiency contributed to bronchiectasis associated with ADPKD. Compared with wild-type mice, Pkd2SM-CKOmice breathed more shallowly and faster, and their cross-sectional area ratio of bronchi to accompanying pulmonary arteries was higher, suggesting that TRPP2 may regulate ASM tension and contribute to the occurrence of bronchiectasis in ADPKD. In a bioassay examining isolated tracheal ring tension, no significant difference was found for high-potassium-induced depolarization of the ASM between the two groups, indicating that TRPP2 does not regulate depolarization-induced ASM contraction. By contrast, carbachol-induced contraction of the ASM derived from Pkd2SM-CKOmice was significantly reduced compared with that in wild-type mice. In addition, relaxation of the carbachol-precontracted ASM by isoprenaline, a β-adrenergic receptor agonist that acts through the cAMP/adenylyl cyclase pathway, was also significantly attenuated in Pkd2SM-CKOmice compared with that in wild-type mice. Thus, TRPP2 deficiency suppressed both contraction and relaxation of the ASM. These results provide a potential target for regulating ASM tension and for developing therapeutic alternatives for some ADPKD complications of the respiratory system or for independent respiratory disease, especially bronchiectasis.

Inhalation of organic dust (OD) from swine confinement facilities leads to pulmonary inflammation, airway hyperresponsiveness, and oxidative stress. In mice, pretreatment with a hydroxyl radical scavenger prevents airway inflammation and airway hyperresponsiveness (AHR) induced by OD exposure. We sought to determine a mechanism by which OD could induce oxidative stress in bronchial epithelial cells. Human bronchial epithelial cells (BEAS-2B or NHBE) were treated with various concentrations of OD, followed by evaluation of intracellular oxidative stress using 2′,7′–dichlorofluorescein diacetate (DCFDA). After stimulation with OD, gene expression of antioxidant genes was assessed by real-time quantitative PCR followed by quantification of Nrf2 nuclear translocation using a luciferase reporter assay. Phagocytic markers (CD36 and CD68) were analyzed by FACS. Cells were treated with an actin inhibitor, cytochalasin D, before OD exposure and evaluated for Nrf2 nuclear translocation and DCFDA. Mice were pretreated with sulforaphane, the Nrf2 activator, before OD exposure and evaluated for pulmonary inflammation and airway reactivity. OD induced a time- and concentration-dependent increase in DCFDA. mRNA expression levels of Nrf2-dependent genes and Nrf2 nuclear translocation were increased after OD exposure. OD exposure increased the expression of CD68 and CD36. Cytochalasin D prevented oxidative stress and Nrf2 nuclear translocation after OD. Pretreatment with sulforaphane prevented OD-induced inflammation and AHR while increasing the uptake of OD in bronchial epithelial cells. Bronchial epithelial cells can phagocytose OD, resulting in an increase in endogenous oxidative stress. Nrf2-dependent mechanisms mediate the antioxidant response to OD.

Patients with frequent exacerbations represent a chronic obstructive pulmonary disease (COPD) subgroup requiring better treatment options. The aim of this study was to determine the innate immune mechanisms that underlie susceptibility to frequent exacerbations in COPD. We measured sputum expression of immune mediators and bacterial loads in samples from patients with COPD at stable state and during virus-associated exacerbations. In vitro immune responses to rhinovirus infection in differentiated primary bronchial epithelial cells (BECs) sampled from patients with COPD were additionally evaluated. Patients were stratified as frequent exacerbators (≥2 exacerbations in the preceding year) or infrequent exacerbators (

Serine peptidase inhibitor, clade B, member 10 (SERPINB10) expression is increased in IL-13-stimulated human bronchial epithelial cells and in a murine model of allergic airway inflammation. However, the role of SERPINB10 in asthma remains unknown. We examined the association between epithelial SERPINB10 expression and airway eosinophilia in subjects with asthma and the role of Serpinb10 in allergic airway inflammation in an animal model. Epithelial SERPINB10 mRNA and protein expression were markedly increased in subjects with asthma ( n = 60) compared with healthy controls ( n = 25). Epithelial SERPINB10 mRNA levels were significantly correlated with airway hyperresponsiveness (AHR) and three parameters reflecting airway eosinophilia including the percentage of sputum eosinophils, the number of eosinophils in bronchial submucosa, and fraction of exhaled nitric oxide in subjects with asthma. Moreover, epithelial SERPINB10 expression was strongly correlated with the epithelial gene signature ( CLCA1, POSTN, and SERPINB2) for type 2 status. In normal human bronchial epithelial cells cultured at air-liquid interface, knockdown of SERPINB10 suppressed IL-13-stimulated periostin (encoded by POSTN) and CCL26 (eotaxin-3) expression by inhibiting the activation of p38 MAPK. Epithelial CCL26 mRNA levels were correlated with SERPINB10 expression in subjects with asthma. Airway knockdown of Serpinb10 alleviated AHR, airway eosinophilia and the expression of periostin and Ccl26 in a murine model of allergic airway disease. Taken together, epithelial SERPINB10 is a novel marker for airway eosinophilia in asthma. Epithelial SERPINB10 contributes to allergic airway eosinophilic inflammation, at least in part, by regulating the expression of periostin and CCL26.

Supplemental O2 (hyperoxia) is necessary for preterm infant survival but is associated with development of bronchial airway hyperreactivity and childhood asthma. Understanding early mechanisms that link hyperoxia to altered airway structure and function are key to developing advanced therapies. We previously showed that even moderate hyperoxia (50% O2) enhances intracellular calcium ([Ca2+]i) and proliferation of human fetal airway smooth muscle (fASM), thereby facilitating bronchoconstriction and remodeling. Here, we introduce cellular clock biology as a novel mechanism linking early oxygen exposure to airway biology. Peripheral, intracellular clocks are a network of transcription-translation feedback loops that produce circadian oscillations with downstream targets highly relevant to airway function and asthma. Premature infants suffer circadian disruption whereas entrainment strategies improve outcomes, highlighting the need to understand relationships between clocks and developing airways. We hypothesized that hyperoxia impacts clock function in fASM and that the clock can be leveraged to attenuate deleterious effects of O2 on the developing airway. We report that human fASM express core clock machinery ( PER1, PER2, CRY1, ARNTL/BMAL1 , CLOCK) that is responsive to dexamethasone (Dex) and altered by O2. Disruption of the clock via siRNA-mediated PER1 or ARNTL knockdown alters store-operated calcium entry (SOCE) and [Ca2+]i response to histamine in hyperoxia. Effects of O2 on [Ca2+]i are rescued by driving expression of clock proteins, via effects on the Ca2+ channels IP3R and Orai1. These data reveal a functional fASM clock that modulates [Ca2+]i regulation, particularly in hyperoxia. Harnessing clock biology may be a novel therapeutic consideration for neonatal airway diseases following prematurity.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondria appear to play an essential role in HPV, the impact of mitochondrial dysfunction on HPV remains incompletely defined. Mice lacking the mitochondrial complex I (CI) subunit Ndufs4 ( Ndufs4−/−) develop a fatal progressive encephalopathy and serve as a model for Leigh syndrome, the most common mitochondrial disease in children. Breathing normobaric 11% O2 prevents neurological disease and improves survival in Ndufs4−/− mice. In this study, we found that either genetic Ndufs4 deficiency or pharmacological inhibition of CI using piericidin A impaired the ability of left mainstem bronchus occlusion (LMBO) to induce HPV. In mice breathing air, the partial pressure of arterial oxygen during LMBO was lower in Ndufs4−/− and in piericidin A-treated Ndufs4+/+ mice than in respective controls. Impairment of HPV in Ndufs4−/− mice was not a result of nonspecific dysfunction of the pulmonary vascular contractile apparatus or pulmonary inflammation. In Ndufs4-deficient mice, 3 wk of breathing 11% O2 restored HPV in response to LMBO. When compared with Ndufs4−/− mice breathing air, chronic hypoxia improved systemic oxygenation during LMBO. The results of this study show that, when breathing air, mice with a congenital Ndufs4 deficiency or chemically inhibited CI function have impaired HPV. Our study raises the possibility that patients with inborn errors of mitochondrial function may also have defects in HPV.

The function and cell surface phenotype of lung macrophages vary within the respiratory tract. Alterations in the bioenergetic profile of macrophages may also be influenced by their location within the respiratory tract. This study sought to characterize the bioenergetic profile of macrophages sampled from different locations within the respiratory tract at baseline and in response to ex vivo xenobiotic challenge. Surface macrophages recovered from healthy volunteers by induced sputum and by bronchial and bronchoalveolar lavage were profiled using extracellular flux analyses. Oxygen consumption and extracellular acidification rates were measured at rest and after stimulation with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or 1,2-naphthoquinone (1,2-NQ). Oxygen consumption and extracellular acidification rates were highly correlated for all macrophage samples. Induced sputum macrophages had relatively higher oxygen consumption and extracellular acidification rates and were largely reliant on glycolysis. In contrast, bronchial fraction and bronchoalveolar macrophages depended more heavily on mitochondrial respiration. Bronchoalveolar macrophages showed elevated LPS-induced cytokine responses. Unlike their autologous peripheral blood monocytes, lung macrophages from any source did not display bioenergetic changes following LPS stimulation. The protein kinase C activator PMA did not affect mitochondrial respiration, whereas the air pollutant 1,2-NQ induced marked mitochondrial dysfunction in bronchoalveolar and bronchial fraction macrophages. The bioenergetic characteristics of macrophages from healthy individuals are dependent on their location within the respiratory tract. These findings establish a regional bioenergetic profile for macrophages from healthy human airways that serves as a reference for changes that occur in disease.

In asthma, it is unclear if the airway smooth muscle cells proliferate more or are increased at the onset of asthma and remain stable. This study aimed to compare smooth muscle cell proliferation in individuals with and without asthma and correlate proliferation rates with cell size and number and with granulocytic airway inflammation. Postmortem airway sections were labeled with proliferating cell nuclear antigen (PCNA) and percent positive muscle cells calculated. On the same sections, smooth muscle cell size and number and the number of eosinophils and neutrophils were estimated and compared in cases of nonfatal ( n = 15) and fatal ( n = 15) asthma and control subjects ( n = 15). The %PCNA+ muscle cells was not significantly different in fatal (29.4 ± 7.7%, mean ± SD), nonfatal asthma (28.6 ± 8.3%), or control subjects (24.6 ± 6.7%) and not related to mean muscle cell size ( r = 0.09), number ( r = 0.36), thickness of the muscle layer ( r = 0.05), or eosinophil numbers ( r = 0.04) in the asthma cases. These data support the hypothesis that in asthma the increased thickness of the smooth muscle layer may be present before or at the onset of asthma and independent of concurrent granulocytic inflammation or exacerbation. NEW & NOTEWORTHY There is debate regarding the origins of the increased airway smooth muscle in asthma. It may be independent of inflammation or arise as a proliferative response to inflammation. The present study found no increase in the proportion of proliferating smooth muscle cells in asthma and no relation of proliferation to numbers of airway smooth muscle cells or inflammation. These results support a stable increase in smooth muscle in asthma that is independent of airway inflammation.

The electronic cigarette (e-cig) has been suggested as a safer alternative to tobacco cigarettes. However, the health effects of e-cigs on the airways have not been fully investigated. Nicotine, the primary chemical constituent of the e-cig aerosol, has been shown to stimulate vagal bronchopulmonary C-fiber sensory nerves, which upon activation can elicit vigorous pulmonary defense reflexes, including airway constriction. In this study, we investigated the bronchomotor response to e-cig inhalation challenge in anesthetized guinea pigs and the mechanisms involved in regulating these responses. Our results showed that delivery of a single puff of e-cig aerosol into the lung triggered immediately a transient bronchoconstriction that sustained for >2 min. The increase in airway resistance was almost completely abolished by a pretreatment with either intravenous injection of atropine or inhalation of aerosolized lidocaine, suggesting that the bronchoconstriction was elicited by cholinergic reflex mechanism and stimulation of airway sensory nerves was probably involved. Indeed, electrophysiological recording further confirmed that inhalation of e-cig aerosol exerted a pronounced stimulatory effect on vagal bronchopulmonary C-fibers. These effects on airway resistance and bronchopulmonary C-fiber activity were absent when the e-cig aerosol containing zero nicotine was inhaled, indicating a critical role of nicotine. Furthermore, a pretreatment with nicotinic acetylcholine receptor antagonists by inhalation completely prevented the airway constriction evoked by e-cig aerosol inhalation. In conclusion, inhalation of a single puff of e-cig aerosol caused a transient bronchoconstriction that was mediated through cholinergic reflex and triggered by a stimulatory effect of nicotine on vagal bronchopulmonary C-fiber afferents.

© 2018 the American Physiological Society. Background: Understanding effects of acute smoke exposure (ASE) on airway epithelial gene expression and their relationship with the effects of chronic smoke exposure may provide biological insights into the development of smoking-related respiratory diseases. Methods: Bronchial airway epithelial cell brushings were collected from 63 individuals without recent cigarette smoke exposure and before and 24 h after smoking three cigarettes. RNA from these samples was profiled on Affymetrix Human Gene 1.0 ST microarrays. Results: We identified 91 genes differentially expressed 24 h after ASE (false discovery rate < 0.25). ASE induced genes involved in xenobiotic metabolism, oxidative stress, and inflammation and repressed genes related to cilium morphogenesis and cell cycle. While many genes altered by ASE are altered similarly in chronic smokers, metallothionein genes are induced by ASE and suppressed in chronic smokers. Metallothioneins are also suppressed in current and former smokers with lung cancer relative to those without lung cancer. Conclusions: Acute exposure to as little as three cigarettes and chronic smoking induce largely concordant changes in airway epithelial gene expression. Differences in short-term and long-term effects of smoking on metallothionein expression and their relationship to lung cancer requires further study given these enzymes’ role in the oxidative stress response.

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome-editing tools. Recently, conditional reprogramming of cells (CRC) with a Rho-associated protein kinase (ROCK) inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the life span of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial cell growth medium, instead of F medium, and 2% O2, instead of 21% O2, that extend HBEC life span while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell, and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof-of-concept, CRISPR/Cas9 genome editing and cloning were used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Inhaled cigarette smoke stimulated vagal bronchopulmonary C fibers via an action of nicotine on neuronal nicotinic acetylcholine receptor (nAChR). Recent studies have reported that nicotine at high concentrations can also activate the transient receptor potential ankyrin 1 receptor (TRPA1) expressed in these sensory nerves. This study was performed to characterize the airway response to inhaled nicotine aerosol and to investigate the relative roles of nAChR and TRPA1 in this response. Guinea pigs were anesthetized and mechanically ventilated; one tidal volume of nicotine aerosol (2% solution) was diluted by an equal volume of air and delivered directly into the lung via a tracheal cannula in a single breath. Our results showed the following: 1) Inhalation of nicotine aerosol triggered an immediate and pronounced bronchoconstriction; the increase in total pulmonary resistance reached a peak of 588 ± 205% (mean ± SE) in 10–40 s, which gradually returned to baseline after 1–5 min. 2) Pretreatment with either atropine (iv) or mecamylamine (aerosol) almost completely abolished the nicotine-induced bronchoconstriction; the mecamylamine pretreatment did not block the bronchoconstriction and bradycardia evoked by electrical stimulation of the distal end of one sectioned vagus nerve, indicating its minimal systemic effects. 3) Pretreatment with HC-030031, a selective TRPA1 antagonist, abolished the bronchoconstriction induced by allyl isothiocyanate, a selective TRPA1 agonist, but did not attenuate the nicotine-evoked bronchoconstriction. In conclusion, inhalation of a single breath of nicotine aerosol evoked acute bronchoconstriction mediated through the cholinergic reflex pathway. This reflex response was triggered by activation of nAChR, but not TRPA1, located in airway sensory nerves. NEW & NOTEWORTHY Recent reports revealed that nicotine at high concentration activated transient receptor potential ankyrin 1 receptor (TRPA1) expressed in vagal bronchopulmonary sensory nerves. This study showed that inhalation of a single breath of nicotine aerosol consistently evoked acute bronchoconstriction that was mediated through the cholinergic reflex pathway and triggered by activation of nicotinic acetylcholine receptor, but not TRPA1, located in these nerves. This is new and important information considering the recent rapid and alarming rise in the prevalence of e-cigarette use for nicotine inhalation.

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year round, but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore, in the current study, we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next, we determined the effect of GDF15 on viral replication, antiviral responses, and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally, we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein overexpression in mice led to exaggerated inflammatory responses to HRV, increased infectious particle release, and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover, GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly, Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation, which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e., HRV) infection in cigarette smoke-exposed airways with GDF15 overproduction.

Progranulin (PGRN) is a growth factor with multiple biological functions and has been suggested as an endogenous inhibitor of Tumor necrosis factor-α (TNF-α)-mediated signaling. TNF-α is believed to be one of the important mediators of the pathogenesis of asthma, including airway hyperresponsiveness (AHR). In the present study, effects of recombinant PGRN on TNF-α-mediated signaling and antigen-induced hypercontractility were examined in bronchial smooth muscles (BSMs) both in vitro and in vivo. Cultured human BSM cells (hBSMCs) and male BALB/c mice were used. The mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also received intranasal administrations of recombinant PGRN into the airways 1 h before each antigen inhalation. In hBSMCs, PGRN inhibited both the degradation of IκB-α (an index of NF-κB activation) and the upregulation of RhoA (a contractile machinery-associated protein that contributes to the BSM hyperresponsiveness) induced by TNF-α, indicating that PGRN has an ability to inhibit TNF-α-mediated signaling also in the BSM cells. In BSMs of the repeatedly antigen-challenged mice, an augmented contractile responsiveness to acetylcholine with an upregulation of RhoA was observed: both the events were ameliorated by pretreatments with PGRN intranasally. Interestingly, a significant decrease in PGRN expression was found in the airways of the repeatedly antigen-challenged mice rather than those of control animals. In conclusion, exogenously applied PGRN into the airways ameliorated the antigen-induced BSM hyperresponsiveness, probably by blocking TNF-α-mediated response. Increasing PGRN levels might be a promising therapeutic for AHR in allergic asthma.