Recombinant Slit2 attenuates tracheal fibroblast activation in benign central airway obstruction by inhibiting the TGF-ß1/Smad3 signaling pathway.

Airway fibrosis is among the pathological manifestations of benign central airway obstruction noted in the absence of effective treatments and requires new drug targets to be developed. Slit guidance ligand 2-roundabout guidance receptor 1 (Slit2-Robo1) is involved in fibrosis and organ development. However, its significance in airway fibrosis has not yet been reported. The study explored how the recombinant protein Slit2 functions in transforming growth factor-ß1 (TGF-ß1)-mediated airway fibrosis in vivo and in vitro. In this study, Slit2 expression initially increased in the tracheal granulation tissues of patients with tracheobronchial stenosis but decreased in the fibrotic tissue. In primary rat tracheal fibroblasts (RTFs), recombinant Slit2 inhibited the expression of extracellular matrices such as Timp1, α-SMA, and COL1A2, whereas recombinant TGF-ß1 promoted the expression of Robo1, α-SMA, and COL1A2. Slit2 and TGF-ß1 played a mutual inhibitory role in RTFs. Slit2 supplementation and Robo1 downregulation inhibited excessive extracellular matrix (ECM) deposition induced by TGF-ß1 in RTFs via the TGF-ß1/Smad3 pathway. Ultimately, exogenous Slit2 and Robo1 knockdown-mediated attenuation of airway fibrosis were validated in a trauma-induced rat airway obstruction model. These findings demonstrate that recombinant Slit2 alleviated pathologic tracheobronchial healing by attenuating excessive ECM deposition. Slit2-Robo1 is an attractive target for further exploring the mechanisms and treatment of benign central airway obstruction.

Elevated CXCL14 in Induced Sputum Was Associated with Eosinophilic Inflammation and Airway Obstruction in Patients with Asthma.

INTRODUCTION: CXCL14 involved in inflammatory processes was upregulated in the asthma expression profile datasets in our pilot study. However, the expression of CXCL14 in induced sputum and its potential clinical role in asthma were poorly reported. OBJECTIVE: We sought to detect CXCL14 expression in airway epithelium and induced sputum cells of asthma and explore its potential clinical implications. METHODS: The expression of CXCL14 in asthma was analyzed using R software based on multiple microarray datasets, including GSE43696, GSE63142, GSE67940, and GSE76262. Subsequent verification of the CXCL14 expression pattern in induced sputum and bronchial epithelium cells was performed by qRT-PCR and ELISA. Besides, the correlations between CXCL14 and eosinophilic inflammation indicators (FeNO, EOS#, and IgE), Th2 signature genes (SERPINB2, POSTN, and CLCA1), inflammatory cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, TSLP, IL-8, IL-17A, IFN-γ, and IL-2), and airway obstruction indicators (pulmonary function and mucin secretion) were further explored. RESULTS: The expression of CXCL14 in epithelium and sputum cells was upregulated in asthma and positively correlated with clinical eosinophilic indicators. The protein levels of CXCL14 were positively associated with Th2 signature genes (SERPINB2, POSTN, and CLCA1) and Th2 cytokines (IL-4, IL-5, IL-10, IL-13, IL-25, IL-33, and TSLP). Increased expression of CXCL14 was also observed in BEAS-2B cells stimulated by the cytokine IL-4. Furthermore, the expression of CXCL14 was positively correlated with MUC5AC secretion and negatively associated with pulmonary function. CONCLUSIONS: Upregulated CXCL14 in asthma was positively correlated with inflammatory indicators and negatively correlated with pulmonary function, which indicated that upregulated CXCL14 might act as a pathogenic gene through involvement in Th2 inflammation in asthma.

The affection of plasma exosomes on airway obstruction and endothelial function in OSA patients.

This study aimed to investigate the effects on airway obstruction and endothelial function by extracting and analyzing plasma exosomes with OSA patients. For this purpose, the clinical data and imaging data of 60 patients with OSA were retrospectively analyzed, who were admitted to the Central Hospital from Apr．2018 to Jul．2021．By using an electron microscope for the observation of exosomes, the degree of airway obstruction was compared by pulmonary function instrument, and HE staining was performed to analyze them. The results showed that the diameter of exosome particles was concentrated at 80.5 ~ 158.6 nm, the diameter of OSA exosomes was concentrated at about 121.9 nm, and the diameter of exosomes in the control group was concentrated at about 145.0 nm. Compared with the patients in the control group, the level of miRNA-33b-3p in the control group was significantly different (P < 0.05). The content of exosomal miRNA-33b-3p in OSA patients decreased significantly, and the corresponding airway obstruction increased. The results of HE staining showed that there were obvious atherosclerotic plaques in the arterial endothelium of the OSA group, and the atherosclerotic plaques were significantly reduced after miRNA-33b-3p injection (P < 0.05). In general, OSA patients can regulate airway obstruction and endothelial cell function by controlling the expression of Plasma exosomes and miRNA-33b-3p, resulting in increased airway obstruction and endothelial cell atherosclerosis.

An inhibitor of RORγ for chronic pulmonary obstructive disease treatment.

The role of RORγ as a transcription factor for Th17 cell differentiation and thereby regulation of IL-17 levels is well known. Increased RORγ expression along with IL-17A levels was observed in animal models, immune cells and BAL fluid of COPD patients. Increased IL-17A levels in severe COPD patients are positively correlated with decreased lung functions and increased severity symptoms and emphysema, supporting an urgency to develop novel therapies modulating IL-17 or RORγ for COPD treatment. We identified a potent RORγ inhibitor, PCCR-1 using hit to lead identification followed by extensive lead optimization by structure-activity relationship. PCCR-1 resulted in RORγ inhibition with a high degree of specificity in a biochemical assay, with > 300-fold selectivity over other isoforms of ROR. Our data suggest promising potency for IL-17A inhibition in human and canine PBMCs and mouse splenocytes with no significant impact on Th1 and Th2 cytokines. In vivo, PCCR-1 exhibited significant efficacy in the acute CS model with dose-dependent inhibition of the PD biomarkers that correlated well with the drug concentration in lung and BAL fluid, demonstrating an acceptable safety profile. This inhibitor effectively inhibited IL-17A release in whole blood and BALf samples from COPD patients. Overall, we identified a selective inhibitor of RORγ to pursue further development of novel scaffolds for COPD treatment.

VEGF receptor 2 (KDR) protects airways from mucus metaplasia through a Sox9-dependent pathway.

Mucus-secreting goblet cells are the dominant cell type in pulmonary diseases, e.g., asthma and cystic fibrosis (CF), leading to pathologic mucus metaplasia and airway obstruction. Cytokines including IL-13 are the major players in the transdifferentiation of club cells into goblet cells. Unexpectedly, we have uncovered a previously undescribed pathway promoting mucous metaplasia that involves VEGFa and its receptor KDR. Single-cell RNA sequencing analysis coupled with genetic mouse modeling demonstrates that loss of epithelial VEGFa, KDR, or MEK/ERK kinase promotes excessive club-to-goblet transdifferentiation during development and regeneration. Sox9 is required for goblet cell differentiation following Kdr inhibition in both mouse and human club cells. Significantly, airway mucous metaplasia in asthmatic and CF patients is also associated with reduced KDR signaling and increased SOX9 expression. Together, these findings reveal an unexpected role for VEGFa/KDR signaling in the defense against mucous metaplasia, offering a potential therapeutic target for this common airway pathology.

Neutrophil Extracellular Traps Increase Airway Mucus Viscoelasticity and Slow Mucus Particle Transit.

Mucus obstruction is a key feature of many inflammatory airway diseases. Neutrophil extracellular traps (NETs) are released upon neutrophil stimulation and consist of extracellular chromatin networks studded with cytotoxic proteins. When released in the airways, these NETs can become part of the airway mucus. We hypothesized that the extracellular DNA and/or oxidative stress (e.g., by the release of reactive oxygen species and myeloperoxidase during NETs formation in the airways) would increase mucus viscoelasticity. We collected human airway mucus from endotracheal tubes of healthy patients admitted for elective surgery and coincubated these samples with NETs from phorbol 12-myristate 13-acetate-stimulated neutrophils. Unstimulated neutrophils served as controls, and blocking experiments were performed with dornase alfa for extracellular DNA and the free radical scavenger dimethylthiourea for oxidation. Compared with controls, the coincubation of mucus with NETs resulted in 1) significantly increased mucus viscoelasticity (macrorheology) and 2) significantly decreased mesh pore size of the mucus and decreased movement of muco-inert nanoparticles through the mucus (microrheology), but 3) NETs did not cause visible changes in the microstructure of the mucus by scanning EM. Incubation with either dornase alfa or dimethylthiourea attenuated the observed changes in macrorheology and microrheology. This suggests that the release of NETs may contribute to airway mucus obstruction by increasing mucus viscoelasticity and that this effect is not solely due to the release of DNA but may in part be due to oxidative stress.

Laryngeal Chemoreflex in Health and Disease: A Review.

The larynx plays a key role in airway protection via the laryngeal chemoreflex (LCR). This involuntary reflex can be evoked when hazardous substances activate mucosal receptors, which send signals to be processed within the brainstem. Although the LCR is meant to be protective, the reflex can become hyperstimulated, even to benign stimuli, which can result in pathological disorders, such as chronic cough and inducible laryngeal obstruction. In this review, we will outline the mechanism of the LCR and its associated pathological disorders.

Oil red O staining for lipid-laden macrophage index of bronchoalveolar lavage: interobserver agreement and challenges to interpretation.

INTRODUCTION: Oil Red O (ORO) staining on cytologic specimens with calculation of the lipid-laden macrophage index (LLMI) is used as a part of the workup in a number of clinical settings, particularly when aspiration is of concern. As a part of ongoing internal quality improvement measures, the objective of the present study was to evaluate the interobserver agreement of the LLMI calculation and to identify factors that affect the variability of the calculation. MATERIALS AND METHODS: There were 9 study participants, which included 3 trainees, 3 cytotechnologists, and 3 cytopathologists. Each participant reviewed 100 ORO-stained bronchoalveolar lavage slides and assigned an LLMI score to each case. The scores were categorized into 3 groups according to the associated aspiration risk: low, LLMI <40; intermediate, LLMI 40 to 90; and high, LLMI >90. The participants were also requested to note any challenges to the calculation for each case. RESULTS: The interobserver agreement among all participants was fair (κ = 0.23). Stratified by participant group, the interobserver agreement among the trainees was fair (κ = 0.24), among cytotechnologists was fair (κ = 0.32), and among cytopathologists was moderate (κ = 0.60). In 70 cases, at least one participant scored the case at least one category higher than the other participants; in 47 cases there was a two category difference. A primary diagnostic challenge reported by participants was macrophage pigmentation (hemosiderin, anthracosis). CONCLUSIONS: We found only fair interobserver agreement among all 9 participants in the study. Hemosiderin and anthracotic pigmentation was a major factor impeding LLMI calculation resulting in overestimation of the LLMI.

TcCO2 changes correlate with partial obstruction in children suspected of sleep disordered breathing.

INTRODUCTION: Pediatric sleep disordered breathing (SDB) is characterized by long periods of partial upper airway obstruction (UAO) with low apnea-hypopnea indices (AHI). By measuring snoring and stertor, Sonomat studies allow quantification of these periods of partial UAO. AIM: To determine whether transcutaneous CO2 (TcCO2 ) levels correlate with increasing levels of partial UAO and to examine patterns of ΔTcCo2 in the transitions from (a) wakefulness to sleep and (b) non-rapid eye movement (NREM) to rapid eye movement (REM) sleep. METHODS: This was a retrospective review of sleep studies in seven asymptomatic controls aged 7 to 12 years and 62 symptomatic children with suspected SDB and no comorbidities, aged 2 to 13 years. Both groups underwent overnight polysomnography, including continuous TcCO2 , at one of two pediatric hospitals in Sydney. Changes in carbon dioxide levels between wake to NREM (sleep onset) and NREM to REM sleep were evaluated using an all-night TcCO2 trace time-linked to a hypnogram. Paired Sonomat recordings were used to quantify periods of UAO in the symptomatic group. RESULTS: The ΔTcCO2 at sleep onset was greater in SDB children than controls and ΔTcCO2 with sleep onset correlated with the duration of partial obstruction (r = .60; P < .0001). Children with an increase in TcCO2 from NREM to REM had a higher number of snoring and stertor events compared to those in whom TcCO2 decreased from NREM to REM (91 vs 30 events/h; P = < .0001). CONCLUSIONS: In children without comorbidities, the measurement of TcCO2 during sleep correlates with indicators of partial obstruction.

Acid exposure disrupts mucus secretion and impairs mucociliary transport in neonatal piglet airways.

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.

Immunopathology of Airway Surface Liquid Dehydration Disease.

The primary purpose of pulmonary ventilation is to supply oxygen (O2) for sustained aerobic respiration in multicellular organisms. However, a plethora of abiotic insults and airborne pathogens present in the environment are occasionally introduced into the airspaces during inhalation, which could be detrimental to the structural integrity and functioning of the respiratory system. Multiple layers of host defense act in concert to eliminate unwanted constituents from the airspaces. In particular, the mucociliary escalator provides an effective mechanism for the continuous removal of inhaled insults including pathogens. Defects in the functioning of the mucociliary escalator compromise the mucociliary clearance (MCC) of inhaled pathogens, which favors microbial lung infection. Defective MCC is often associated with airway mucoobstruction, increased occurrence of respiratory infections, and progressive decrease in lung function in mucoobstructive lung diseases including cystic fibrosis (CF). In this disease, a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in dehydration of the airway surface liquid (ASL) layer. Several mice models of Cftr mutation have been developed; however, none of these models recapitulate human CF-like mucoobstructive lung disease. As an alternative, the Scnn1b transgenic (Scnn1b-Tg+) mouse model overexpressing a transgene encoding sodium channel nonvoltage-gated 1, beta subunit (Scnn1b) in airway club cells is available. The Scnn1b-Tg+ mouse model exhibits airway surface liquid (ASL) dehydration, impaired MCC, increased mucus production, and early spontaneous pulmonary bacterial infections. High morbidity and mortality among mucoobstructive disease patients, high economic and health burden, and lack of scientific understanding of the progression of mucoobstruction warrants in-depth investigation of the cause of mucoobstruction in mucoobstructive disease models. In this review, we will summarize published literature on the Scnn1b-Tg+ mouse and analyze various unanswered questions on the initiation and progression of mucobstruction and bacterial infections.

Evaluation of metabolic profile and C-reactive protein concentrations in brachycephalic dogs with upper airway obstructive syndrome.

BACKGROUND: Brachycephalic dogs have abnormal breathing patterns similar to those in humans with obstructive sleep apnea syndrome. Obstructive sleep apnea syndrome is associated with dyslipidemia, hyperglycemia, and insulin resistance. Despite the fact that anatomic and functional alterations are well described in brachycephalic dogs, little is known about the consequences of upper airway obstruction on systemic inflammatory response and metabolic profile. OBJECTIVES: To describe history, clinical presentation, and anatomic abnormalities; to evaluate systemic inflammatory response and metabolic profile; and to identify possible associations among clinical signs, anatomic abnormalities, inflammatory response, and metabolic profile in brachycephalic dogs with airway obstruction. ANIMALS: Thirty purebred brachycephalic dogs with brachycephalic airway obstructive syndrome (BAOS). METHODS: Prospective study. The following information was recorded and studied: respiratory and digestive signs, airway and digestive endoscopic anomalies, presence or absence of tracheal hypoplasia, histologic evaluation of gastrointestinal tract biopsy specimens, serum concentrations of C-reactive protein (CRP), fructosamine, insulin, glucose, triglyceride, cholesterol, and plasma concentrations of lipoprotein classes. RESULTS: A high proportion of dogs (76.7%) had gastrointestinal signs. Esophageal deviation, atony of the cardia of the stomach, and distal esophagitis were the most common endoscopic anomalies detected. Twenty-six (86.6%) dogs had different degrees of laryngeal collapse. Gastrointestinal histologic evaluation identified mostly chronic inflammation. Glucose, fructosamine, triglycerides, cholesterol, CRP, pre-beta, beta lipoproteins, and chylomicrons were increased to a variable extent. Significant associations among clinical signs, anatomic abnormalities, CRP, and metabolic profile were not found. CONCLUSION AND CLINICAL IMPORTANCE: Despite the presence of inflammation and some mild metabolic derangements, the clinicopathological variables evaluated did not offer valuable information in dogs with BAOS.

Heterogeneous Pulmonary Response After Tracheal Occlusion: Clues to Fetal Lung Growth.

BACKGROUND: Understanding inconsistent clinical outcomes in infants with severe congenital diaphragmatic hernia (CDH) after tracheal occlusion (TO) is a crucial step for advancing neonatal care. The objective of this study is to explore the heterogeneous airspace morphometry and the metabolic landscape changes in fetal lungs after TO. METHODS: Fetal lungs on days 1 and 4 after TO were examined using mass spectrometry-based metabolomics, fluorescence lifetime imaging microscopy (FLIM), the number of airspaces, and tissue-to-airspace ratio (TAR). RESULTS: Two morphometric areas were identified in TO lungs compared with controls (more small airspaces at day 1 and a higher number of enlarged airspaces at day 4). Global metabolomics analysis revealed a significant upregulation of glycolysis and a suppression of the tricarboxylic acid cycle in day 4 TO lungs compared with day 1 TO lungs. In addition, there was a significant increase in polyamines involved in cell growth and proliferation. Locally, FLIM analysis on day 1 TO lungs demonstrated two types of heterogeneous zones-similar to control and with increased oxidative phosphorylation. FLIM on day 4 TO lungs demonstrated appearance of zones with enlarged airspaces and a metabolic shift toward glycolysis, accompanied by a decrease in the FLIM "lipid-surfactant" signal. CONCLUSIONS: In normal fetal lungs, we report a novel temporal pattern of varied morphometric and metabolic changes. Initially, there is formation of zones with small airspaces, followed by airspace enlargement over time. Metabolically day 1 TO lungs have zones with increased oxidative phosphorylation, whereas day 4 TO lungs have a shift toward glycolysis in the enlarged airspaces. Based on our observations, we speculate that the "best responders" to tracheal occlusion should have bigger lungs with small airspaces and normal surfactant production.

The effects of tracheal occlusion on Wnt signaling in a rabbit model of congenital diaphragmatic hernia.

PURPOSE: Tracheal occlusion (TO) reverses pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH), but its mechanism of action remains poorly understood. Wnt signaling plays a critical role in lung development, but few studies exist. The purpose of our study was to a) confirm that our CDH rabbit model produced PH which was reversed by TO and b) determine the effects of CDH +/- TO on Wnt signaling. METHODS: CDH was created in fetal rabbits at 23 days, TO at 28 days, and lung collection at 31 days. Lung body weight ratio (LBWR) and mean terminal bronchiole density (MTBD) were determined. mRNA and miRNA expression was determined in the left lower lobe using RT-qPCR. RESULTS: Fifteen CDH, 15 CDH + TO, 6 sham CDH, and 15 controls survived and were included in the study. LBWR was low in CDH, while CDH + TO was similar to controls (p = 0.003). MTBD was higher in CDH fetuses and restored to control levels in CDH + TO (p < 0.001). Reference genes TOP1, SDHA, and ACTB were consistently expressed within and between treatment groups. miR-33 and MKI67 were increased, and Lgl1 was decreased in CDH + TO. CONCLUSION: TO reversed pulmonary hypoplasia and stimulated early Wnt signaling in CDH fetal rabbits. TYPE OF STUDY: Basic science, prospective. LEVEL OF EVIDENCE: II.

Targeting of cathepsin S reduces cystic fibrosis-like lung disease.

Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS-/-) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of ßENaC-Tg mice compared with wild-type (WT) littermates. CatS-/-ßENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with ßENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of ßENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in ßENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target.

Changes in surface tension of saliva in Down syndrome.

OBJECTIVE: Surface tension in saliva might contribute to the maintenance of upper airway patency. The present study aimed to determine whether salivary surface tension is altered in patients with Down syndrome who are predisposed to upper airway collapse. PATIENTS AND METHODS: We used the pull-off force technique to measure surface tension in samples (100 µL) of saliva collected from twenty-three male patients with Down syndrome and twenty-three healthy males (controls). p < 0.05 was considered to indicate significance. RESULTS: Salivary surface tension was significantly lower in the patients than in the controls (57.3 ± 4.9 vs. 60.3 ± 4.7 mN/m; p = 0.039). Age and surface tension positively correlated in the patients (p = 0.001). CONCLUSIONS: The lower surface tension of saliva in patients with Down syndrome might compensate for an anatomical predisposition towards upper airway collapsibility and other risk factors. The function of surface tension in saliva might be altered due to aging in such patients.

Proteomic profiling of tracheal fluid in an ovine model of congenital diaphragmatic hernia and fetal tracheal occlusion.

Congenital diaphragmatic hernia (CDH) occurs in ~1:2,000 pregnancies and is associated with substantial morbidity and mortality. Fetal tracheal occlusion (TO) is an emerging therapy that improves lung growth and reduces mortality, although substantial respiratory compromise persists in survivors. In this study, we used tracheal fluid in a fetal sheep model of CDH with TO for proteomic analysis with subsequent validation of findings in sheep lung tissue. We found that the proteomic profiles of CDH tracheal fluid was most similar to control lung and CDH/TO lung most similar to TO lung. Among 118 proteins altered in CDH, only 11 were reciprocally regulated in CDH/TO. The most significantly altered pathways and processes were cell proliferation, phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin signaling, inflammation, and microtubule dynamics. CDH suppressed and TO promoted cell proliferation and AKT-related signaling cascades. By Western blot analysis and immunohistochemistry, epithelial PCNA and phosphorylated AKT were decreased in CDH and increased in TO and CDH/TO lungs. The Wnt target Axin2 was decreased threefold in CDH lung compared with control without a significant increase in CDH/TO lung. Cilia-related pathways were among the most dysregulated with CDH lung having a nearly twofold increase in acetylated α-tubulin and a relative increase in the number of ciliated cells. While TO improves lung growth and patient survival in CDH, the procedure substantially alters many processes important in lung development and cell differentiation. Further elucidation of these changes will be critical to improving lung health in infants with CDH treated with TO.