ALKBH5 Modulates Asthma Progression by Downregulating N6-methyladenosine Methylation.

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.

Assessment of a randomized controlled trial on the safety of pre-placing bronchial balloons in transbronchial lung cryobiopsy for diagnosing interstitial lung disease.

RATIONALE AND OBJECTIVES: Bleeding is a major complication of transbronchial lung cryobiopsy (TBLC), and pre-placing a bronchial balloon is one of the clinical practices used to prevent it, but with very weak evidence, which should be confirmed. This study aimed to conduct whether pre-placing a bronchial balloon in TBLC for diagnosing interstitial lung disease (ILD) is more safety. MATERIALS AND METHODS: In this prospective, single-center, randomized controlled trial, patients with suspected ILD were enrolled and randomly assigned to pre-placed balloon and none-pre-placed balloon groups. The primary outcome was incidence of moderate bleeding in each group. The secondary endpoints were the incidence of severe bleeding, pneumothorax, and other procedural complications. RESULTS: Exactly 250 patients were enrolled between August 2019 and March 2022, with 125 in each group. There were no significant differences in severe bleeding between the none-pre-placed balloon group and pre-placed balloon group (1.6% vs. 0.8%; adjusted p = 0.520), while more moderate bleeding occurred in the none-pre-placed balloon group (26.4% vs. 6.4%, adjusted p = 0.001), as well as more use of hemostatic drug (28.0% vs. 6.4%, adjusted p = 0.001). Three patients in the none-pre-placed balloon group used the bronchial balloon. More samples could be acquired in the pre-placed balloon group than in the none-pre-placed balloon group (3.8 ± 0.9 vs. 3.1 ± 0.9, p < 0.001). There were no significant differences in multidisciplinary discussion (MDD) between the two groups (89.6% vs. 91.2%, adjusted p = 0.182). CONCLUSION: A pre-placed bronchial balloon can reduce the incidence of moderate bleeding and increase the confidence of the bronchoscopists. However, it had no effect on increasing the diagnostic rate of MDD and reducing severe bleeding. REGISTRATION NUMBER: NCT04047667 ( www. CLINICALTRIALS: gov identifier).

Expression of glucocorticoid receptor and HDACs in airway smooth muscle cells is associated with response to steroids in COPD.

BACKGROUND: Steroid insensitivity in Chronic Obstructive Pulmonary Disease (COPD) presents a problem for controlling the chronic inflammation of the airways. The glucocorticoid receptor (GR) mediates the intracellular signaling of inhaled corticosteroids (ICS) by interacting with transcription factors and histone deacetylases (HDACs). The aim of this study was to assess if COPD patients' response to ICS in vivo, may be associated with the expression of GR, the complex of GR with transcription factors, and the expression of various HDACs in vitro. METHODS: Primary airway smooth muscle cells (ASMC) were established from endobronchial biopsies obtained from patients with asthma (n = 10), patients with COPD (n = 10) and subjects that underwent diagnostic bronchoscopy without pathological findings and served as controls (n = 6). ASMC were also established from 18 COPD patients, 10 responders and 8 non-responders to ICS, who participated in the HISTORIC study, an investigator-initiated and driven clinical trial that proved the hypothesis that COPD patients with high ASMC in their endobronchial biopsies respond better to ICS than patients with low ASMC. Expression of GR and its isoforms GRα and GRß and HDACs was investigated in primary ASMC in the absence or in the presence of dexamethasone (10- 8M) by western blotting. The complex formation of GR with transcription factors was assessed by co-immunoprecipitation. RESULTS: Expression of GR and its isoform GRα but not GRß was significantly reduced in ASMC from COPD patients as compared to controls. There were no significant differences in the expression of GR, GRα and GRß between responders and non-responders to ICS. However, treatment with dexamethasone upregulated the expression of total GR (p = 0.004) and GRα (p = 0.005) after 30 min in responders but not in non-responders. Τhe formation of the complex GR-c-Jun was increased 60 min after treatment with dexamethasone only in responders who exhibited significantly lower expression of HDAC3 (p = 0.005) and HDAC5 (p < 0.0001) as compared to non-responders. CONCLUSIONS: These data suggest that ASMC from COPD patients who do not respond to treatment with ICS, are characterized by reduced GR-c-Jun complex formation and increased expression of HDAC3 and HDAC5. TRIAL REGISTRATION: ISRCTN11017699 (Registration date: 15/11/2016).

[Comparison of ultrathin bronchoscopy with conventional bronchoscopy for the diagnostic value of peripheral pulmonary lesions].

Objective: To assess and compare the diagnostic efficacy of next-generation ultrathin bronchoscopy (UTB) and conventional bronchoscopy (CB), both combined with radial endobronchial ultrasound (r-EBUS), in the evaluation of peripheral pulmonary lesions (PPL). Methods: A cohort of 39 patients with PPL who underwent multimodal bronchoscopy at Dushu Lake Hospital, Soochow University, from June 1, 2021 to May 31, 2023 was consecutively enrolled. A single bronchoscopist performed multimodal bronchoscopies using CB (external diameter 4.9 mm or 5.9 mm, working channel diameter 2 or 3 mm, CB group) for transbronchial biopsy under r-EBUS guidance (rEBUS-TBLB), followed by UTB (external diameter 3 mm, working channel diameter 1.7 mm, UTB group) for transbronchial biopsy under r-EBUS guidance. Pathological findings and a 6-month clinical follow-up were used as the gold standard to compare the diagnostic yield of biopsy specimens, ultrasound characteristics, and localization rates of the two bronchoscope types. The aim was to evaluate the clinical application value of UTB combined with r-EBUS. Binary variables were analysed using the McNemar test for paired data. Continuous variables or ranked data were analysed using the Wilcoxon signed-rank test for paired data. Results: The diagnostic yields for UTB and CB groups were 66.67% (26/39) and 30.77% (12/39), respectively, with the UTB group significantly surpassing the CB group (χ2=10.56, P=0.001, 1-ß=0.968). r-EBUS with CB exhibited no visible lesion in 13 cases, adjacent to the lesion in 19 cases, and within the lesion in 7 cases.Substitution of UTB resulted in r-EBUS images changing from no visible lesion to adjacent to the lesion in 7 cases, from no visible lesion to within the lesion in 3 cases, and from adjacent to the lesion to within the lesion in 12 cases. The positioning of the r-EBUS probe in relation to the lesions improved significantly with UTB usage (Z=-4.46, P<0.001). Localization rates (number of patients with "within" or "adjacent to" the image/total number of patients) for UTB and CB were 92.30% (36/39) and 66.67% (26/39), respectively (χ2=8.10, P=0.002). UTB improved r-EBUS probe localization rates. The diagnostic yields of UTB were higher than CB for solid lesions, lesions>30 mm in diameter, non-upper lobar location, benign or malignant lesions and lesions with or without a bronchus sign. Conclusion: The UTB group demonstrated a significantly higher diagnostic yield than the CB group, providing superior r-EBUS probe images, and a significant diagnostic advantage for PPL.

[Diffuse thickening of bronchial walls with multiple nodular mucosal protrusions].

A 58-year-old woman presented with a six-month history of nasal congestion, sore throat and cough, and a five-month history of dyspnea. She had a history of xerostomia for one year. On examination, the bilateral submandibular gland and parotid glands were enlarged. Parotid and anterior cervical lymph nodes were palpable. There were rales in both lungs. The rest of the physical examination was unremarkable. Sialographic analysis showed normal caliber in the main duct, stenosis in secondary ducts, and dilation in the proximal ducts. Minor salivary gland biopsy demonstrated periductal lymphocytic infiltration. Chest computed tomography (CT) showed diffuse thickening of the tracheal and bilateral bronchial walls. Bronchoscopy revealed macroscopic multiple nodules mainly in the trachea and bilateral main bronchus. Endobronchial biopsy showed lymphocytic infiltration in the bronchial submucosa. She was diagnosed with Sjögren's syndrome and treated with glucocorticoids. The dose of prednisone was started at 30 mg/d and tapered gradually. Following treatment, the patient's clinical condition improved dramatically, with shrinkage of the enlarged lymph nodes, bilateral submandibular and parotid glands. A repeated chest CT scan revealed improvement of the tracheal and bilateral bronchial thickening. Multiple nodules in the airway regressed, as evidenced by repeated bronchoscopic examination. The final diagnosis was a large-airway disease associated with Sjögren's syndrome.Among airway diseases in Sjögren's syndrome, peripheral airway diseases including bronchiolitis and bronchiectasis are common; however, central airway lesions in Sjögren's syndrome, especially with macroscopic nodules, are rare. In this case, we demonstrated tracheal and endobronchial nodules in Sjögren's syndrome as determined by clinical features, CT scan, bronchoscopy, and response to therapy.

Bronchoconstriction damages airway epithelia by crowding-induced excess cell extrusion.

Asthma is deemed an inflammatory disease, yet the defining diagnostic feature is mechanical bronchoconstriction. We previously discovered a conserved process called cell extrusion that drives homeostatic epithelial cell death when cells become too crowded. In this work, we show that the pathological crowding of a bronchoconstrictive attack causes so much epithelial cell extrusion that it damages the airways, resulting in inflammation and mucus secretion in both mice and humans. Although relaxing the airways with the rescue treatment albuterol did not affect these responses, inhibiting live cell extrusion signaling during bronchoconstriction prevented all these features. Our findings show that bronchoconstriction causes epithelial damage and inflammation by excess crowding-induced cell extrusion and suggest that blocking epithelial extrusion, instead of the ensuing downstream inflammation, could prevent the feed-forward asthma inflammatory cycle.

Clinical significance and potential pathogenesis of VCAN in adult non-cystic fibrosis bronchiectasis: a retrospective study.

BACKGROUND: The pathogenesis of adult non-cystic fibrosis (CF) bronchiectasis is complex, and the relevant molecular mechanism remains ambiguous. Versican (VCAN) is a key factor in inflammation through interactions with adhesion molecules. This study constructs a stable panoramic map of mRNA, reveals the possible pathogenesis of bronchiectasis, and provides new ideas and methods for bronchiectasis. METHODS: Peripheral blood and tissue gene expression data from patients with bronchiectasis and normal control were selected by bioinformatics analysis. The expression of VCAN in peripheral blood and bronchial tissues of bronchiectasis were obtained by transcriptome sequencing. The protein expression levels of VCAN in serums were verified by the enzyme-linked immunosorbent assay (ELISA). The mRNA expression levels of VCAN in co-culture of Pseudomonas aeruginosa and bronchial epithelial cells were verified by real-time quantitative polymerase chain reaction (RT-qPCR). In addition, the biological function of VCAN was detected by the transwell assay. RESULTS: The expression of VCAN was upregulated in the bronchiectasis group by sequencing analysis (P < 0.001). The expression of VCAN in the bronchial epithelial cell line BEAS-2B was increased in P. aeruginosa (P.a), which was co-cultured with BEAS-2B cells (P < 0.05). The concentration of VCAN protein in the serum of patients with bronchiectasis was higher than that in the normal control group (P < 0.05). Transwell experiments showed that exogenous VCAN protein induced the migration of neutrophils (P < 0.0001). CONCLUSIONS: Our findings indicate that VCAN may be involved in the development of bronchiectasis by increasing the migration of neutrophils and play an important role in bronchial pathogenesis.

Clinical characteristics and removal approaches of tracheal and bronchial foreign bodies in elders.

The symptoms of tracheobronchial foreign body in the elderly are not typical, so they are often missed or misdiagnosed. This study aims to depict the clinical characteristics of tracheobronchial foreign body inhalation in the elderly. We retrospectively analysed the clinical data of elder patients (age ≥ 65 years) diagnosed with tracheal and bronchial foreign bodies. The data included age, sex, clinical symptoms, type and location of foreign bodies, prehospital duration, Chest CT, bronchoscopic findings, and frequencies and tools for removing these elderly patients' tracheal and bronchial foreign bodies. All patients were followed up for a half year. Fifty-nine cases were included, of which only 32.2% had a definite aspiration history. Disease duration > 30 days accounted for 27.1% of the patients. 27.1% of the patients had a history of stroke, and 23.8% had Alzheimer's Disease. Regarding clinical symptoms, patients mainly experience cough and expectoration. The most common CT findings were abnormal density shadow (37.3%) and pulmonary infiltration (22.0%). Under bronchoscopy, purulent secretions were observed in 52.5% of patients, and granulation tissue hyperplasia was observed in 45.8%. Food (55.9%) was the most common foreign object, including seafood shells (5.1%), bones (20.3%), dentures (18.6%), and tablets (20.3%). The success rate of foreign body removal under a bronchoscope was 96.7%, 28.8% of the foreign bodies were on the left and 69.5% on the right. 5.1% of the elderly patients required rigid bronchoscopy, and 6.8% required two bronchoscopies. In elderly cohorts, tracheal foreign bodies are obscured by nonspecific clinical presentations and a paucity of aspiration history, challenging timely diagnosis. Predominantly constituted by food particles, with a notable predilection for the left bronchial tree, these cases demand skilled bronchoscopic management, occasionally requiring sophisticated approaches for successful extraction.

mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells.

The mechanistic target of rapamycin (RAPA) complex 1 (mTORC1) - transcription factor EB (TFEB) pathway plays a crucial role in response to nutritional status, energy and environmental stress for maintaining cellular homeostasis. But there is few reports on its role in the toxic effects of arsenic exposure and the related mechanisms. Here, we show that the exposure of bronchial epithelial cells (BEAS-2B) to sodium arsenite promoted the activation of mTORC1 (p-mTORC1) and the inactivation of TFEB (p-TFEB), the number and activity of lysosomes decreased, the content of reduced glutathione (GSH) and superoxide dismutase (SOD) decreased, the content of malondialdehyde (MDA) increased, the DNA and chromosome damage elevated. Further, when mTORC1 was inhibited with RAPA, p-mTORC1 and p-TFEB down-regulated, GSH and SOD increased, MDA decreased, the DNA and chromosome damage reduced significantly, as compared with the control group. Our data revealed for the first time that mTORC1 - TFEB pathway was involved in sodium arsenite induced lysosomal alteration, oxidative stress and genetic damage in BEAS-2B cells, and it may be a potential intervention target for the toxic effects of arsenic.

Misdiagnosed Asthma Turned Out to Be Bronchial Fibroepithelial Polyp: Case Report and Review of Literature.

Bronchial fibroepithelial polyps are exceedingly rare with few cases have been reported. They can manifest with a wide array of symptoms; ranging from being totally asymptomatic, cough, refractory dyspnea, and hemoptysis. In our case, our patient's condition was diagnosed and was managed as asthma. It is one of the rare benign conditions to be encountered, shares similar morphology with other tumors such as angiomyofibroblastoma, aggressive angiomyxoma, and cellular angiofibroma. These lesions have a slow growth pattern which may end up with obstruction. According to the tumor size and symptoms caused by it, treatment varies from observation to complete resection. This case describes an incidental finding of fibroepithelial polyp in the main bronchus for a patient with long-term refractory cough for 5 years, was misdiagnosed to have asthma. Diagnosis typically involves imaging and bronchoscopy, followed by appropriate therapeutic measures and careful monitoring to assess the prognosis.

Heat-Induced Secretion of Heat Shock Proteins 70 and 90 Does not Affect the Expression of the Glucocorticoid Receptor in Primary Airway Cells in COPD.

PURPOSE: The response to glucocorticoids is hampered in many COPD patients by a yet unknown mechanism. Earlier we reported that short-term heat exposure of primary human bronchial epithelial cells (BEC) and airway smooth muscle cells (ASMC) of asthma patients increased the expression and secretion of extracellular heat shock proteins (eHSPs) resulting in increased expression of glucocorticoid receptor (GR) in BEC and inhibition of ASMC remodeling. The aim of the present study was to assess if the same mechanism is also present in primary airway wall cells of COPD patients. METHODS: Primary BEC and ASMC were established from endobronchial biopsies obtained from COPD patients (n = 73), who participated in the HISTORIC study, an investigator-initiated and driven clinical trial. Secretion and protein expression of HSPs was assessed by ELISA and Western blotting. Expression of total GR, its isoforms GRα and GRß and toll-like receptor 4 (TLR4) was determined by Western-blotting. RESULTS: Short heat exposure (65 °C, 10 s) of BEC resulted in a significant increase of the secretion of eHSP70 and eHSP90, while the intracellular protein was not altered. Heat treatment or exposure to eHSP70 or eHSP90 had no effect on the expression of GR and GR-isoforms. However, eHSP70 and eHSP90 significantly reduced the expression of TLR4. CONCLUSIONS: The results of this study indicate that primary airway cells from COPD patients respond differently to heat exposure and extracellular HSP70 or HSP90 than cells from asthma patients regarding the expression of GR and this may explain the reduced response to glucocorticoids in patients with COPD. TRIAL REGISTRATION: ISRCTN11017699.

Bronchial thermoplasty decreases airway remodeling by inhibiting autophagy via the AMPK/mTOR signaling pathway.

Bronchial thermoplasty (BT), an effective treatment for severe asthma, requires heat to reach the airway to reduce the mass of airway smooth muscle cells (ASMCs). Autophagy is involved in the pathological process of airway remodeling in patients with asthma. However, it remains unclear whether autophagy participates in controlling airway remodeling induced by BT. In this study, we aim to elucidate the autophagy-mediated molecular mechanisms in BT. Our study reveal that the number of autophagosomes and the level of alpha-smooth muscle actin (α-SMA) fluorescence are significantly decreased in airway biopsy tissues after BT. As the temperature increased, BT causes a decrease in cell proliferation and a concomitant increase in the apoptosis of human airway smooth muscle cells (HASMCs). Furthermore, increase in temperature significantly downregulates cellular autophagy, autophagosome accumulation, the LC3II/LC3I ratio, and Beclin-1 expression, upregulates p62 expression, and inhibits the AMPK/mTOR pathway. Furthermore, cotreatment with AICAR (an AMPK agonist) or RAPA (an mTOR antagonist) abolishes the inhibition of autophagy and attenuates the increase in the apoptosis rate of HASMCs induced by the thermal effect. Therefore, we conclude that BT decreases airway remodeling by blocking autophagy induced by the AMPK/mTOR signaling pathway in HASMCs.

Eosinophil peroxidase promotes bronchial epithelial cells to secrete asthma-related factors and induces the early stage of airway remodeling.

Asthma is a heterogeneous disease characterized by chronic airway inflammation, reversible airflow limitation, and airway remodeling. Eosinophil peroxidase (EPX) is the most abundant secondary granule protein unique to activated eosinophils. In this study, we aimed to illustrate the effect of EPX on the epithelial-mesenchymal transition (EMT) in BEAS-2B cells. Our research found that both EPX and ADAM33 were negatively correlated with FEV1/FVC and FEV1%pred, and positively correlated with IL-5 levels. Asthma patients had relatively higher levels of ADAM33 and EPX compared to the healthy control group. The expression of TSLP, TGF-ß1 and ADAM33 in the EPX intervention group was significantly higher. Moreover, EPX could promote the proliferation, migration and EMT of BEAS-2B cells, and the effect of EPX on various factors was significantly improved by the PI3K inhibitor LY294002. The findings from this study could potentially offer a novel therapeutic target for addressing airway remodeling in bronchial asthma, particularly focusing on EMT.

Advances in co-pathogenesis of the united airway diseases.

According to the concept of "united airway diseases", the airway is a single organ in which upper and lower airway diseases are commonly comorbid. A range of inflammatory factors have been found to play an important role in the chain reaction of upper and lower airway diseases. However, the amount of research on this concept remains limited. The underlying mechanism of the relationship between typical diseases of the united airway, such as asthma, allergic rhinitis, and chronic sinusitis, also needs to be further explored. This review highlights the interaction between upper and lower respiratory diseases gathered from epidemiological, histoembryology, neural mechanistic, microbiological, and clinical studies, revealing the relationship between the upper and lower respiratory tracts.

Virtual bronchoscopic navigation with intraoperative cone-beam CT for the diagnosis of peripheral pulmonary nodules.

OBJECTIVE: Transbronchial biopsy is a safe manner with fewer complications than percutaneous transthoracic needle biopsy; however, the current diagnostic yield is still necessitating further improvement. We aimed to evaluate the diagnostic yield of using virtual bronchoscopic navigation (VBN) and cone-beam CT (CBCT) for transbronchial biopsy and to investigate the factors that affected the diagnostic sensitivity. METHODS: We retrospectively investigated 255 patients who underwent VBN-CBCT-guided transbronchial biopsy at our two centers from May 2021 to April 2022. A total of 228 patients with final diagnoses were studied. Patient characteristics including lesion size, lesion location, presence of bronchus sign, lesion type and imaging tool used were collected and analyzed. Diagnostic yield was reported overall and in groups using different imaging tools. RESULTS: The median size of lesion was 21 mm (range of 15.5-29 mm) with 46.1% less than 2 cm in diameter. Bronchus sign was present in 87.7% of the patients. The overall diagnostic yield was 82.1%, and sensitivity for malignancy was 66.3%. Patients with lesion > 2 cm or with bronchus sign were shown to have a significantly higher diagnostic yield. Four patients had bleeding and no pneumothorax occurred. CONCLUSION: Guided bronchoscopy with VBN and CBCT was an effective diagnostic method and was associated with a high diagnostic yield in a safe manner. In addition, the multivariant analysis suggested that lesion size and presence of bronchus sign could be a predictive factor for successful bronchoscopic diagnosis.

Tobacco use, self-reported professional dental cleaning habits, and lung adenocarcinoma diagnosis are associated with bronchial and lung microbiome alpha diversity.

RATIONALE: The lung microbiome is an inflammatory stimulus whose role in the development of lung malignancies is incompletely understood. We hypothesized that the lung microbiome associates with multiple clinical factors, including the presence of a lung malignancy. OBJECTIVES: To assess associations between the upper and lower airway microbiome and multiple clinical factors including lung malignancy. METHODS: We conducted a prospective cohort study of upper and lower airway microbiome samples from 44 subjects undergoing lung lobectomy for suspected or confirmed lung cancer. Subjects provided oral (2), induced sputum, nasopharyngeal, bronchial, and lung tissue (3) samples. Pathologic diagnosis, age, tobacco use, dental care history, lung function, and inhaled corticosteroid use were associated with upper and lower airway microbiome findings. MEASUREMENTS AND MAIN RESULTS: Older age was associated with greater Simpson diversity in the oral and nasopharyngeal sites (p = 0.022 and p = 0.019, respectively). Current tobacco use was associated with greater lung and bronchus Simpson diversity (p < 0.0001). Self-reported last profession dental cleaning more than 6 months prior (vs. 6 or fewer months prior) was associated with lower lung and bronchus Simpson diversity (p < 0.0001). Diagnosis of a lung adenocarcinoma (vs. other pathologic findings) was associated with lower bronchus and lung Simpson diversity (p = 0.024). Last professional dental cleaning, dichotomized as ≤ 6 months vs. >6 months prior, was associated with clustering among lung samples (p = 0.027, R2 = 0.016). Current tobacco use was associated with greater abundance of pulmonary pathogens Mycoplasmoides and Haemophilus in lower airway samples. Self-reported professional dental cleaning ≤ 6 months prior (vs. >6 months prior) was associated with greater bronchial Actinomyces and lung Streptococcus abundance. Lung adenocarcinoma (vs. no lung adenocarcinoma) was associated with lower Lawsonella abundance in lung samples. Inhaled corticosteroid use was associated with greater abundance of Haemophilus among oral samples and greater Staphylococcus among lung samples. CONCLUSIONS: Current tobacco use, recent dental cleaning, and a diagnosis of adenocarcinoma are associated with lung and bronchial microbiome α-diversity, composition (ß-diversity), and the abundance of several respiratory pathogens. These findings suggest that modifiable habits (tobacco use and dental care) may influence the lower airway microbiome. Larger controlled studies to investigate these potential associations are warranted.

Endoplasmic reticulum stress enhances the expression of TLR3-induced TSLP by airway epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

Downregulation of protein phosphatase 2Aα in asthmatic airway smooth muscle.

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca2+]i), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca2+]i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca2+]i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma.NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

RATIONALE: Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. OBJECTIVES: To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. METHODS: In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. RESULTS: GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. CONCLUSIONS: GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

Utility of auto fluorescence-guided biopsy in suspected lung cancer patients with bronchial mucosal lesions.

BACKGROUND: Bronchoscopy is currently the most common technique for lung cancer diagnosis. Patients suspected of malignancy often undergo bronchoscopic examination, and biopsy is routinely used in patients with visible bronchial lesions. However, it is difficult to differentially diagnose lung cancer in patients with bronchial mucosal lesions. Thus, this study was conducted to investigate the utility of fluorescence-guided biopsy in suspected lung cancer patients with bronchial mucosal lesions. METHODS: We conducted a retrospective study in a single screening center to assess the sensitivity and specificity of fluorescence-guided biopsy compared with white light bronchoscopy (WLB) in patients with bronchial mucosal lesions. RESULTS: A total of 301 patients with bronchial mucosal lesions were enrolled in this study. The sensitivity for patients with fluorescence-guided biopsy was 60.3 % (95 % confidence interval [CI]: 53.1 %-67.1 %), which was higher than that of patients with WLB alone (45.2 %, 95 % CI: 38.2-52.4 %) (P = 0.0026). Additionally, compared with the WLB group, the fluorescence -guided biopsy group was found to have a significantly higher specificity (100 %, 95 % CI: 95.5-100 % versus 69.6 %, 95 % CI: 59.6-78.1 %), positive predictive value (100 %, 95 % CI: 96.1-100 % versus 74.3 %, 95 % CI: 65.5-81.7 %) and negative predictive value (56.3 %, 95 % CI: 48.8-63.6 % versus 39.4 %, 95 % CI: 32.3-47.0 %). CONCLUSION: Fluorescence-guided biopsy can serve as an important adjunct to WLB for the differential diagnosis of lung cancer in patients with bronchial mucosal lesions.