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The diagnosis of invasive pulmonary aspergillosis (IPA) diseases in non-neutropenic patients remains challenging. It is essential to develop optimal non-invasive or minimally invasive detection methods for the rapid and reliable diagnosis of IPA. Metagenomic next-generation sequencing (mNGS) in bronchoalveolar lavage fluid (BALF) can be a valuable tool for identifying the microorganism. Our study aims to evaluate the performance of mNGS in BALF in suspected IPA patients and compare it with other detection tests, including serum/BALF galactomannan antigen (GM) and traditional microbiological tests (BALF fungal culture and smear and lung biopsy histopathology). Ninety-four patients with suspicion of IPA were finally enrolled in our study. Thirty-nine patients were diagnosed with IPA, and 55 patients were non-IPA. There was significance between the IPA and non-IPA groups, such as BALF GM (P < 0.001), history of glucocorticoid use (P = 0.004), and pulmonary comorbidities (P = 0.002), as well as no significance of the other demographic data including age, sex, BMI, history of cigarette, blood GM assay, T-SPOT. TB, and NEUT#/LYMPH#. The sensitivity of the BALF mNGS was 92.31%, which was higher than that of the traditional tests or the GM assays. The specificity of BALF mNGS was 92.73%, which was relatively similar to that of the traditional tests. The AUC of BALF mNGS was 0.925, which presented an excellent performance compared with other traditional tests or GM assays. Our study demonstrated the important role of BALF detection by the mNGS platform for pathogen identification in IPA patients with non-neutropenic states, which may provide an optimal way to diagnose suspected IPA disease. [ABSTRACT FROM AUTHOR]

Simple Summary: Macrophages are an integral part of the tumor microenvironment, playing a role in immunoregulation. We investigated the antigenic and cytokine macrophages profile derived from bronchoalveolar lavage fluid in lung affected by cancer (cBALF) and healthy lung (hBALF) of 36 patients. Macrophages markers: CD206, CD163, CD80, CD86, CD40, Arginase-1, and CD68 were evaluated by flow cytometry. Cytokines (IL-1 RA, IL-6, IL-10, TNF-α, IL-1β, IL-12, IL-23, and TGF-β) profile was analyzed. There was higher median proportion of macrophages in cBALF than in hBALF. The population of macrophages presented immunophenotype: cCD68+bright CD206+bright CD163+bright CD80+ CD86+ CD40+bright CD45+ cArginase+. We observed some trends in the expression of the analyzed antigens in clBALF and hlBLAF. High concentrations of IL-1RA and IL-6 and correlations between pro- and anti-inflammatory cytokines in cBALF and hBALF supernatants were found. We expanded knowledge of the macrophages polarization, their diversity and unique properties based on the antigenic pattern and cytokine profile. Macrophages play an important role in the suppression and activation of immune anti-cancer response, but little is known about dominant macrophage phenotype in the lung cancer environment, evaluated by bronchoalveolar lavage fluid (BALF). The aim of this study was to characterize macrophages in BALF from a lung affected by cancer (cBALF) and a healthy lung (hBALF) of the same patient regarding their individual macrophage polarization and selected cytokines profile. A total of 36 patients with confirmed lung cancer were investigated. Macrophages markers: CD206 CD163 CD80 CD86 CD40 CD45, Arginase-1, and CD68 were evaluated by flow cytometry. Cytokines (IL-1 RA, IL-6, IL-10, TNF-α, IL-1β, IL-12, IL-23, and TGF-β) profile was analyzed. There was higher median proportion of macrophages in Cbalf than in Hbalf. The population of macrophages presented immunophenotype: Ccd68+bright CD206+bright CD163+bright CD80+ CD86+ CD40+bright CD45+ cArginase+. We observed some trends in the expression of the analyzed antigens in clBALF and hlBLAF. The highest concentrations of IL-1RA and IL-6 were in Cbalf and Hbalf supernatant. There were the correlations between pro- and anti-inflammatory cytokines. The findings showed that macrophages include a diverse and plastic group with the presence of different antigens and cytokines, and determining the target phenotype is a complex and variable process. [ABSTRACT FROM AUTHOR]

Asthma is a chronic inflammatory disorder of the airway and is associated with pyroptosis. microRNAs (miRNAs) underlie pathogenic mechanism in asthma. This study is expected to evaluate the role of miR-20b in asthma-induced airway inflammation via regulating thioredoxin-interacting protein (TXNIP) and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. The asthmatic mouse model was established via ovalbumin (OVA) induction. Expressions of miR-20b, TXNIP, and NLRP3 in lung tissues were determined. Bronchial hyperresponsiveness was appraised, cells in bronchoalveolar lavage fluid were counted and categorized, and histopathological damage was observed. Levels of inflammatory and pyroptotic cytokines were measured. The binding relationship of miR-20b and TXNIP was testified. Co-location and interaction between TXNIP and NLRP3 were detected. Mice were infected with the lentivirus packaged with pcDNA3.1-TXNIP or pcDNA3.1-NLRP3 for joint experiments to observe the pathological changes of mice. miR-20b was poorly expressed, while TXNIP and NLRP3 were highly expressed in OVA-induced mice. miR-20b overexpression attenuated airway inflammation and pyroptosis, manifested by alleviation of histopathological damage, declined numbers of total cells and inflammatory cells, lowered bronchial hyperresponsiveness, decreased levels of pro-inflammatory and pyroptotic cytokines, and increased anti-inflammatory cytokines. miR-20b targeted TXNIP and inhibited TXNIP expression, and TXNIP can bind to NLRP3 and upregulated NLRP3 expression. Upregulation of TXNIP or NLRP3 could reverse the protecting role of miR-20b overexpression in OVA-induced mice. miR-20b inhibited TXNIP expression to reduce the binding of TXNIP and NLRP3, thus restricting pyroptosis and airway inflammation of asthmatic mice. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is a medical condition characterized by widespread inflammation in the lungs with consequent proportional loss of gas exchange function. ARDS is linked with severe pulmonary or systemic infection. Several factors, including secretory cytokines, immune cells, and lung epithelial and endothelial cells, play a role in the development and progression of this disease. The present study is based on Pubmed database information (1987–2022) using the words "Acute respiratory distress syndrome", "Interleukin", "Cytokines" and "Immune cells". Cytokines and immune cells play an important role in this disease, with particular emphasis on the balance between pro-inflammatory and anti-inflammatory factors. Neutrophils are one of several important mediators of Inflammation, lung tissue destruction, and malfunction during ARDS. Some immune cells, such as macrophages and eosinophils, play a dual role in releasing inflammatory mediators, recruitment inflammatory cells and the progression of ARDS, or releasing anti-inflammatory mediators, clearing the lung of inflammatory cells, and helping to improve the disease. Different interleukins play a role in the development or inhibition of ARDS by helping to activate various signaling pathways, helping to secrete other inflammatory or anti-inflammatory interleukins, and playing a role in the production and balance between immune cells involved in ARDS. As a result, immune cells and, inflammatory cytokines, especially interleukins play an important role in the pathogenesis of this disease Therefore, understanding the relevant mechanisms will help in the proper diagnosis and treatment of this disease. [ABSTRACT FROM AUTHOR]

Objective: Pneumocystis jirovecii pneumonia (PJP) can be a life-threatening opportunistic infection. We aimed to evaluate the diagnostic accuracy of metagenomic next-generation sequencing (mNGS) for PJP. Methods: A comprehensive electronic literature search of Web of Knowledge, PubMed, Cochrane Library, CNKI and Wanfang data was performed. Bivariate analysis was conducted to calculate the pooled sensitivity, specificity, diagnostic odds ratio (DOR), the area under the summary receiver operator characteristic (SROC) curve and the Q-point value (Q*). Results: The literature search resulted in 9 studies with a total of 1343 patients, including 418 cases diagnosed with PJP and 925 controls. The pooled sensitivity of mNGS for diagnosis of PJP was 0.974 [95% confidence interval (CI), 0.953–0.987]. The pooled specificity was 0.943 (95% CI, 0.926–0.957), the DOR was 431.58 (95% CI, 186.77-997.27), the area under the SROC curve was 0.987, and the Q* was 0.951. The I2 test indicated no heterogeneity between studies. The Deek funnel test suggested no potential publication bias. Subgroup analyses showed that the area under the SROC curve of mNGS for diagnosis of PJP in immunocompromised and non-HIV patients was 0.9852 and 0.979, respectively. Conclusions: Current evidence indicates that mNGS exhibits excellent accuracy for the diagnosis of PJP. The mNGS is a promising tool for assessment of PJP in both immunocompromised and non-HIV patients. [ABSTRACT FROM AUTHOR]

Proteases such as neutrophil elastase cleave and activate the epithelial sodium channel (ENaC), causing airway dehydration. Our current study explores the impact of increased protease levels in vapers' airways on ENaC activity and airway dehydration. Human bronchial epithelial cultures (HBECs) were exposed to bronchoalveolar lavage fluid (BALF) from non-smokers, smokers and vapers. Airway surface liquid (ASL) height was measured by confocal microscopy as a marker of hydration. ENaC cleavage was measured by Western blotting. Human peripheral blood neutrophils were treated with a menthol-flavored e-liquid (Juul), and the resulting secretions were added to HBECs. BALF from smokers and vapers significantly and equally increased ENaC activity and decreased ASL height. The ASL height decrease was attenuated by protease inhibitors. Non-smokers' BALF had no effect on ENaC or ASL height. BALF from smokers and vapers, but not non-smokers, induced ENaC cleavage. E-liquid-treated neutrophil secretions cleaved ENaC and decreased ASL height. Our study demonstrated that elevated protease levels in vapers' airways have functional significance since they can activate ENaC, resulting in airway dehydration. Lung dehydration contributes to diseases like cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD) and asthma. Thus, our data predict that vaping, like smoking, will cause airway surface dehydration that likely leads to lung disease. [ABSTRACT FROM AUTHOR]

Omadacycline is a novel tetracycline antibiotic that exhibits good in vitro antibacterial activity against atypical pathogens such as Mycoplasma pneumoniae. It is approved for the treatment of adults with communityacquired bacterial pneumonia. However, the safety and efficacy of omadacycline in pediatric patients under 18 years of age have not yet been established. In the present paper, we report a case of pediatric communityacquired pneumonia in which initial empirical anti-infective therapy had failed. The patient received empirical anti-infective therapy with azithromycin and other antimicrobial agents upon admission but showed a poor clinical response and developed secondary tinnitus and liver dysfunction. After the confirmation of M. pneumoniae infection through metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid, an antibiotic switch to omadacycline was made. Thereafter, the patient's condition improved, and no adverse reactions were observed. These findings demonstrate that mNGS enables the identification of infection-causing pathogens in patients with unresponsive pneumonia. Omadacycline can be considered as an alternative option for antiinfective therapy in pediatric M. pneumoniae pneumonia, especially when the presence of bacterial resistance, adverse drug reactions, or organ failure are taken into consideration. [ABSTRACT FROM AUTHOR]

Worldwide, lower respiratory tract infections (LRTI) are an important cause of hospitalization in children. Due to the relative limitations of traditional pathogen detection methods, new detection methods are needed. The purpose of this study was to evaluate the value of metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid (BALF) samples for diagnosing children with LRTI based on the interpretation of sequencing results. A total of 211 children with LRTI admitted to the First Affiliated Hospital of Guangzhou Medical University from May 2019 to December 2020 were enrolled. The diagnostic performance of mNGS versus traditional methods for detecting pathogens was compared. The positive rate for the BALF mNGS analysis reached 95.48% (95% confidence interval [CI] 92.39% to 98.57%), which was superior to the culture method (44.07%, 95% CI 36.68% to 51.45%). For the detection of specific pathogens, mNGS showed similar diagnostic performance to PCR and antigen detection, except for Streptococcus pneumoniae, for which mNGS performed better than antigen detection. S. pneumoniae, cytomegalovirus and Candida albicans were the most common bacterial, viral and fungal pathogens. Common infections in children with LRTI were bacterial, viral and mixed bacterial-viral infections. Immunocompromised children with LRTI were highly susceptible to mixed and fungal infections. The initial diagnosis was modified based on mNGS in 29.6% (37/125) of patients. Receiver operating characteristic (ROC) curve analysis was performed to predict the relationship between inflammation indicators and the type of pathogen infection. BALF mNGS improves the sensitivity of pathogen detection and provides guidance in clinical practice for diagnosing LRTI in children. [ABSTRACT FROM AUTHOR]

The performance of lateral flow assay (LFA) in diagnosing invasive pulmonary aspergillosis (IPA) has not been well demonstrated. To address this, we conducted a meta-analysis assessing the overall accuracy of LFA in diagnosing IPA using bronchoalveolar lavage fluid (BALF). Over a systematical search and assessment of bias risk, we calculated the pooled specificity, sensitivity, and area under the receiver operating curve (AUC) to assess the diagnostic performance. Our meta-analysis included 11 studies. The combined total sensitivity and specificity for diagnosing IPA were 0.78 (95% confidence interval (CI): 0.71, 0.83) and 0.87 (95% CI: 0.81, 0.91), respectively. The AUC was 0.86 (95% CI: 0.82, 0.89). Our results demonstrate that LFA using galactomannan in BALF exhibits high sensitivity and specificity for diagnosing IPA. [ABSTRACT FROM AUTHOR]

Objective: To investigate prognostic value of cytokine levels in bronchoalveolar lavage fluid in patients with severe pneumonia. Methods: Clinical data of 44 patients with severe pneumonia who were admitted to Intensive Care Unit of Hospital of Chengdu University of Traditional Chinese Medicine from September 2020 to July 2022 were retrospectively analyzed. Patients' gender, age, acute physiology and chronic health scoreⅡ （APACHEⅡ）, oxygenation index, cytokine detection in bronchoalveolar lavage fluid and 28-day prognosis were collected. SPSS27.0 statistical software was used to analyze and process data. Results: IL-17A level［ 51.15（1.85, 125.20） pg/ml vs 9.65（0.01, 19.95） pg/ml］ in bronchoalveolar lavage fluid and APACHEⅡ（ 27.29±5.27 vs 21.81± 5.00） in death group were significantly higher than those in survival group （P<0.05）, oxygenation index ［（131.14±60.95） mmHg vs （188.69±56.85） mmHg］ was worse than survival group （P<0.05）. Binary Logistic regression analysis showed that IL-17A, APACHE Ⅱ and oxygenation index were independent risk factors affecting prognosis of patients with severe pneumonia ［OR: 1.033（95%CI= 1.010~1.056）, 1.228（95%CI=1.022~1.477） and 0.984（95%CI=0.969~1.000）, all P<0.05］. ROC curve analysis showed that IL-17A, APACHEⅡ and oxygenation index had predictive value for 28-day death in patients with severe pneumonia, and AUC were 0.735 （95%CI=0.567~0.904）, 0.741（95%CI=0.575~0.906）, 0.789（95%CI=0.634~0.944）（all P<0.01）. Conclusion: IL-17A, APACHE Ⅱ and oxygenation index are all risk factors affecting prognosis of patients with severe pneumonia, which can all predict 28-day prognosis of patients. [ABSTRACT FROM AUTHOR]

Background: Chronic obstructive pulmonary disease (COPD) is an airway‐associated lung disorder, resulting in airway inflammation. This article aimed to explore the role of the krüppel‐like factor 9 (KLF9)/microRNA (miR)‐494‐3p/phosphatase and tensin homolog (PTEN) axis in airway inflammation and pave a theoretical foundation for the treatment of COPD. Methods: The COPD mouse model was established by exposure to cigarette smoke, followed by measurements of total cells, neutrophils, macrophages, and hematoxylin and eosin staining. The COPD cell model was established on human lung epithelial cells BEAS‐2B using cigarette smoke extract. Cell viability was assessed by cell counting kit‐8 assay. miR‐494‐3p, KLF9, PTEN, and NLR family, pyrin domain containing 3 (NLRP3) levels in tissues and cells were measured by quantitative real‐time polymerase chain reaction or Western blot assay. Inflammatory factors (TNF‐α/IL‐6/IL‐8/IFN‐γ) were measured by enzyme‐linked immunosorbent assay. Interactions among KLF9, miR‐494‐3p, and PTEN 3′UTR were verified by chromatin immunoprecipitation and dual‐luciferase assays. Results: KLF9 was upregulated in lung tissues of COPD mice. Inhibition of KLF9 alleviated airway inflammation, reduced intrapulmonary inflammatory cell infiltration, and repressed NLRP3 expression. KLF9 bound to the miR‐494‐3p promoter and increased miR‐494‐3p expression, and miR‐494‐3p negatively regulated PTEN expression. miR‐494‐3p overexpression or Nigericin treatment reversed KLF9 knockdown‐driven repression of NLRP3 inflammasome and inflammation. Conclusion: KLF9 bound to the miR‐494‐3p promoter and repressed PTEN expression, thereby facilitating NLRP3 inflammasome‐mediated inflammation. [ABSTRACT FROM AUTHOR]

Objective To analyze the clinical characteristics of children with lobar pneumonia and the distribution of pathogens in bronchoalveolar lavage fluid (BALF) collected from these patients, hence providing a scientific basis for their precise diagnosis and treatment. Methods A total of 115 children diagnosed with lobar pneumonia from August 2019 to August 2022 at Suining Central Hospital were screened as the research subjects. The clinical manifestations and occurrence of complications in the patients were investigated. All the children underwent bronchoalveolar lavage after admission, and BALF samples were collected. Fluorescence quantitative PCR was adopted to detect and analyze the distribution and clinical characteristics of Streptococcus pneumoniae (SP) and other related pathogenic microorganisms in BALF specimens. Results Among the 115 pediatric patients with lobar pneumonia, the occurrence of manifestations or complications including involvement of ≥2 lung lobes, myocardial damage, pleural effusion, abnormal liver function, digestive system involvement, nervous system involvement, rash, renal function impairment, and lung atelectasis were observed in 46, 46, 39, 33, 18, 17, 11, 5, and 4 cases, respectively. The pathogen positivity rate in the BALF samples of the 115 patients was 87.0% (100/115), with 81 cases of single infection and 19 cases of mixed infection. A total of 121 strains of pathogens were isolated, including 83 strains of Mycoplasmal pneumonia (MP) (accounting for 68.6%) and SP(13.2%). The differences in the detection rates of HI, MP, RSV strains among different age groups were statistically significant (χ²=8.834, 19.454, 10.284, P<0.05), while the differences in the infection rates of SP, KP, CP, and ADV were not statistically significant (χ²=3.393, 2.67, 0.565, 0.097, P> 0.05). The MP pneumonia group showed significantly higher incidence of complications such as pleural effusion, nervous system involvement, and abnormal liver function than the non-MP pneumonia group (χ²=3.925, 4.195, and 4.513, P<0.05). The highest pathogen detection rate was in winter, accounting for 33.91%. Conclusions MP is the most common pathogen in BALF of children with lobar pneumonia. There is variation in the pathogen detection rate among different age groups and seasons. Those with combined infections were more prone to complications, which is worthy of attention by clinicians. [ABSTRACT FROM AUTHOR]

Background: There are relatively few studies investigating C-C motif chemokine ligand 2 (CCL2) level in bronchoalveolar lavage fluid (BALF) in children with Mycoplasma pneumoniae pneumonia (MPP), and the relationship between CCL2 level in BALF and refractory mycoplasma pneumoniae pneumonia (RMPP) is unclear. This study aims to explore the relationship between chemokine CCL2 level in BALF and clinical characteristics and clinical outcome in children with MPP. Methods: A total of 51 children with confirmed acute MPP and requiring bronchoalveolar lavage in Department of Pediatrics, Huanghe Sanmenxia Hospital and The First Clinical College of Xinxiang Medical University from October 2021 to February 2023 were selected as the study group. And 11 children with bronchial foreign body were selected as the control group. The study group was divided into the non-refractory mycoplasma pneumoniae pneumonia (NRMPP) group and the RMPP group based on the response to treatment. BALF and clinical data of the patients were collected. And CCL2 levels were tested in the patients. Differences in CCL2 level in BALF and clinical characteristics were tested and compared. Results: The CCL2 level in BALF of the study group was higher than that of the control group, with significant difference (P < 0.05). With ROC curve, the area under the curve (AUC) of CCL2 in BALF predicting RMPP was 0.94, the cut-off value was 0.645 ng/ml, the sensitivity was 85%, and the specificity was 94%, and the diagnostic value was better than that of serum CRP and LDH. Logistic regression analysis was used to build the RMPP prediction model, and CCL2 showed good predictive value. Conclusion: The level of CCL2 in BALF was high in children with MPP and had a high predictive value for RMPP. CCL2 can be used as one of the biomarkers for predicting RMPP. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a persistent and progressive respiratory disorder characterized by expiratory airflow limitation caused by chronic inflammation. Evidence has shown that COPD is correlated with neutrophil chemotaxis towards the airways, resulting in neutrophilic airway inflammation. This study aimed to evaluate neutrophil chemotaxis in bronchoalveolar lavage fluid (BALF) from COPD patients using a high-throughput nine-unit microfluidic platform and explore the possible correlations between neutrophil migratory dynamics and COPD development. The results showed that BALF from COPD patients induced stronger neutrophil chemotaxis than the Control BALF. Our results also showed that the chemotactic migration of neutrophils isolated from the blood of COPD patients was not significantly different from neutrophils from healthy controls, and neutrophil migration in three known chemoattractants (fMLP, IL-8, and LTB4) was not affected by glucocorticoid treatment. Moreover, comparison with clinical data showed a trend of a negative relationship between neutrophil migration chemotactic index (C. I.) in COPD BALF and patient's spirometry data, suggesting a potential correlation between neutrophil migration and the severity of COPD. The present study demonstrated the feasibility of using the microfluidic platform to assess neutrophil chemotaxis in COPD pathogenesis, and it may serve as a potential marker for COPD evaluation in the future. [ABSTRACT FROM AUTHOR]

The usefulness of bronchoalveolar lavage fluid (BALF) to support the diagnosis of acute cellular (ACR) rejection in lung transplant (LTX) recipients remains controversial. ACR has been associated with blood eosinophil counts (EOS) in other solid organ recipients, but there are few studies in relation to lung transplants. Our aim was to assess the usefulness of the combined analysis of BALF cellularity and EOS for the diagnosis of ACR in lung transplant recipients. This is a retrospective study of findings observed simultaneously in 887 transbronchial biopsies (TBB), BALF, and blood samples obtained from 363 LTx patients transplanted between 2014 and 2020. The variables collected were: demographics, ACR degree, BALF cellularity, and simultaneous blood EOS counts. The lymphocyte count in BALF was significantly higher in patients with ACR than in those without (11.35% vs. 6.11%; p < 0.001). In parallel, EOS counts were also significantly higher in patients with ACR than in the non-ACR group (EOS 213 ± 206/mm3 vs. 83 ± 129/mm3; p < 0.001). Increases in both parameters were associated with an increased risk of ACR (lymphocytes OR 1.100; 95% CI 1.080–1.131; EOS OR 1.460; 95% CI 1.350–1.580). The diagnostic specificity of ACR for a lymphocyte count > 12% was 71.1%, which increased to 95.8% when taking into account a simultaneous blood EOS count > 200/mm3. Simultaneous assessment of BALF lymphocyte counts and blood eosinophil counts may be useful for diagnosing ACR in patients with risk factors for TBB or in the presence of inconclusive histological samples. [ABSTRACT FROM AUTHOR]

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Lung myeloid cells are important in pulmonary immune homeostasis and in the pathogenesis of chronic obstructive pulmonary disease (COPD). Multiparameter immunophenotypic characterization of these cells is challenging because of their autofluorescence and diversity. We evaluated the immunophenotypic landscape of airway myeloid cells in COPD using time of flight mass cytometry. Cells from BAL, which were obtained from never-smokers (n = 8) and smokers with (n = 20) and without (n = 4) spirometric COPD, were examined using a 44-parameter time of flight mass cytometry panel. Unsupervised cluster analysis was used to identify cellular subtypes that were confirmed by manual gating. We identified major populations of CD68+ and CD68- cells with 22 distinct phenotypic clusters, of which 18 were myeloid cells. We found a higher abundance of putative recruited myeloid cells (CD68+ classical monocytes) in BAL from patients with COPD. CD68+ classical monocyte population had distinct responses to smoking and COPD that were potentially related to their recruitment from the interstitium and vasculature. We demonstrate that BAL cells from smokers and subjects with COPD have lower AXL expression. Also, among subjects with COPD, we report significant differences in the abundance of PDL1high and PDL2high clusters and in the expression of PDL1 and PDL2 across several macrophage subtypes suggesting modulation of inflammatory responses. In addition, several phenotypic differences in BAL cells from subjects with history of COPD exacerbation were identified that could inform potential disease mechanisms. Overall, we report several changes to the immunophenotypic landscape that occur with smoking, COPD, and past exacerbations that are consistent with decreased regulation and increased activation of inflammatory pathways.

Heavy metals released into the environment have a significant effect on respiratory health. Lung macrophages are important in mounting an inflammatory response to injury, but they are also involved in repair of injury. Macrophages develop mixed phenotypes in complex pathological conditions and polarize to a predominant phenotype depending on the duration and stage of injury and/or repair. Little is known about the reprogramming required for lung macrophages to switch between these divergent functions; therefore, understanding the mechanism(s) by which macrophages promote metabolic reprogramming to regulate lung injury is essential. Here, we show that lung macrophages polarize to a pro-inflammatory, classically activated phenotype after cadmium-mediated lung injury. Because metabolic adaptation provides energy for the diverse macrophage functions, these classically activated macrophages show metabolic reprogramming to glycolysis. RNA-Seq revealed up-regulation of glycolytic enzymes and transcription factors regulating glycolytic flux in lung macrophages from cadmium-exposed mice. Moreover, cadmium exposure promoted increased macrophage glycolytic function with enhanced extracellular acidification rate, glycolytic metabolites, and lactate excretion. These observations suggest that cadmium mediates the persistence of classically activated lung macrophages to exacerbate lung injury.

Adult respiratory distress syndrome (ARDS) is the leading cause of death associated with SARS-CoV-2 infection and COVID-19. IGF-1 has been implicated in ARDS, yet its role in relation to COVID-19-related lung injury has not been investigated. We hypothesize that blockage of the IGF-1 receptor (IGF-1R) mitigates lung injury and decreases the risk of death in patients COVID-19-related ARDS. Patients with fibroproliferative ARDS have been shown to have increased IGF-1 and IGF-1R staining in lung tissue specimens. Rising levels of IGF-1 in bronchioalveolar fluid (BAL) and increased IGF-1 mRNA expression in lung tissues (but declining serum IGF-1 levels) have been found in late stage ARDS compared with early lung injury. Blockage of IGF-1R decreases lung tissue damage and increases survival in bleomycin-induced as well as H1N1 influenza-related lung injury in animal models. Teprotumumab is a monoclonal antibody directed against the IGF-1R that was FDA-approved in 2020 for the treatment of Graves' orbitopathy. In order to determine if teprotumumab may reduce lung injury and death related to ARDS in the setting of COVID-19, preliminary clinical data is needed. IGF-1 levels in serum and BAL fluid must be measured in patients with COVID-19-related ARDS. Histopathology from lung samples from patients with COVID-19-related ARDS must be examined for increased expression of the IGF-1R. Once these are ascertained, and if the data support IGF-1 involvement, a randomized, placebo-controlled phase 2A trial of teprotumumab therapy in the setting of COVID-19-related ARDS and non-COVID-19-related ARDS designed to generate initial data on short-term efficacy, safety, dosing and administration should be performed.

Ambient PM2.5 was collected during the winter season from Taiyuan, Shanxi, China; Jinan, Shandong, China; and Sacramento, California, USA, and used to create PMSX, PMSD, and PMCA extracts, respectively. Time-lag experiments were performed to explore the in vivo and in vitro toxicity of the PM extracts. In vivo inflammatory lung responses were assessed in BALB/c mice using a single oropharyngeal aspiration (OPA) of PM extract or vehicle (CTRL) on Day 0. Necropsies were performed on Days 1, 2, and 4 post-OPA, and pulmonary effects were determined using bronchoalveolar lavage (BAL) and histopathology. On Day 1, BAL neutrophils were significantly elevated in all PM- versus CTRL-exposed mice, with PMCA producing the strongest response. However, histopathological scoring showed greater alveolar and perivascular effects in PMSX-exposed mice compared to all three other groups. By Day 4, BAL neutrophilia and tissue inflammation were resolved, similar across all groups. In vitro effects were examined in human HepG2 hepatocytes, and U937 cells following 6, 24, or 48 h of exposure to PM extract or DMSO (control). Luciferase reporter and quantitative polymerase chain reaction assays were used to determine in vitro effects on aryl hydrocarbon receptor (AhR) activation and gene transcription, respectively. Though all three PM extracts activated AhR, PMSX produced the greatest increases in AhR activation, and mRNA levels of cyclooxygenase-2, cytochrome P450, interleukin (IL)-8, and interleukin (IL)-1β. These effects were assumed to result from a greater abundance of polycyclic aromatic hydrocarbons (PAHs) in PMSX compared to PMSD and PMCA.

Levels of iron and iron-related proteins including ferritin are higher in the lung tissue and lavage fluid of individuals with chronic obstructive pulmonary disease (COPD), when compared to healthy controls. Whether more iron in the extracellular milieu of the lung associates with distinct clinical phenotypes of COPD, including increased exacerbation susceptibility, is unknown. We measured iron and ferritin levels in the bronchoalveolar lavage fluid (BALF) of participants enrolled in the SubPopulations and InteRmediate Outcome Measures In COPD (SPIROMICS) bronchoscopy sub-study (n = 195). BALF Iron parameters were compared to systemic markers of iron availability and tested for association with FEV1 % predicted and exacerbation frequency. Exacerbations were modelled using a zero-inflated negative binomial model using age, sex, smoking, and FEV1 % predicted as clinical covariates. BALF iron and ferritin were higher in participants with COPD and in smokers without COPD when compared to non-smoker control participants but did not correlate with systemic iron markers. BALF ferritin and iron were elevated in participants who had COPD exacerbations, with a 2-fold increase in BALF ferritin and iron conveying a 24% and 2-fold increase in exacerbation risk, respectively. Similar associations were not observed with plasma ferritin. Increased airway iron levels may be representative of a distinct pathobiological phenomenon that results in more frequent COPD exacerbation events, contributing to disease progression in these individuals.

BackgroundIdiopathic inflammatory airway disease (IAD) in cats often is described as asthmatic (eosinophilic) or bronchitic (neutrophilic), but this designation requires collection of airway fluid and it fails to consider cats with mixed airway inflammation.ObjectiveTo identify clinical features that would differentiate inflammatory disease types.AnimalsForty-nine cats with nonspecific airway inflammation identified by bronchoscopic bronchoalveolar lavage (BAL) between 2011 and 2018 were evaluated.MethodsThis is a retrospective study. Cats were categorized by BAL differential cytology as having eosinophilic (eosinophils >20% with neutrophils 50%), mixed (eosinophils 20%-50% and neutrophils >14% or discordant inflammation from 2 BAL sites), or neutrophilic (neutrophils >14% and eosinophils

IL-33 and its receptor ST2 are contributing factors to airway inflammation and asthma exacerbation. The IL-33/ST2 signaling pathway is involved in both the onset and the acute exacerbations of asthma. In this study, we address the role of endogenous IL-33 and its autoamplification of the IL-33/ST2 pathway in Ag-dependent and Ag-independent asthma-like models. Wild-type, IL-33 knockout, ST2 knockout mice were either intratracheally administrated with 500 ng of rIL-33 per day for four consecutive days or were sensitized and challenged with OVA over 21 d. In wild-type mice, IL-33 or OVA induced similar airway hyperresponsiveness and eosinophilic airway inflammation. IL-33 induced its own mRNA and ST2L mRNA expression in the lung. IL-33 autoamplified itself and ST2 protein expression in airway epithelial cells. OVA also induced IL-33 and ST2 protein expression. In IL-33 knockout mice, the IL-33- and OVA-induced airway hyperresponsiveness and eosinophilic airway inflammation were both significantly attenuated, whereas IL-33-induced ST2L mRNA expression was preserved, although no autoamplification of IL-33/ST2 pathway was observed. In ST2 knockout mice, IL-33 and OVA induced airway hyperresponsiveness and eosinophilic airway inflammation were both completely diminished, and no IL-33/ST2 autoamplification was observed. These results suggest that endogenous IL-33 and its autoamplification of IL-33/ST2 pathway play an important role in the induction of asthma-like phenotype. Thus an intact IL-33/ST2 pathway is necessary for both Ag-dependent and Ag-independent asthma-like mouse models.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

Abstract Introduction Lung cancer is the most prevalent cancer with high mortality in China, and it is associated with the dysbiosis of the lung microbiome. This study attempted to screen for specific microorganisms as potential biomarkers for distinguishing benign lung disease from lung cancer. Methods Bronchoalveolar lavage fluid (BALF) sample was selected in the study instead of saliva to avoid contamination with oral microorganisms, and microbial taxonomic and functional differences in BALF samples from patients with lung cancer and those with those from patients with benign lung diseases were performed based on metagenomic next‐generation sequencing, for the first time, so that microorganisms other than bacteria could be included. Results The results showed that the intrasample diversity of malignant samples was different from benign samples, and the microbial differences among malignant samples were smaller, with lower microbial diversity, significantly changed microbial abundance and metabolic functions. Metabolic function analysis revealed amino acid‐related metabolism was more prevalent in benign samples, whereas carbohydrate‐related metabolism was more prevalent in malignant samples. By LEfSe, Metastat and Random Forest analysis, we identified a series of important differential microorganisms. Importantly, the model combining five key genera plus one tumor marker (neuron‐specific enolase) as indicators presented the optimal disease typing performance. Conclusion Thus results suggest the value of these differential microorganisms enriched in tumors in mechanism research and may be potential new targets for lung cancer therapy. More importantly, the biomarkers identified in this study can be conducive to improve the clinical diagnosis of lung cancer and have good application prospects.

Yirui Xie,1 Bohao Dai,1 Xiaotang Zhou,1 Huiting Liu,1 Wei Wu,2 Fei Yu,3 Biao Zhu1 1State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The Department of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, People’s Republic of China; 2State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, 310003, People’s Republic of China; 3Key Laboratory of Clinical in vitro Diagnostic Techniques of Zhejiang Province, Department of Laboratory Medicine, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310003, People’s Republic of ChinaCorrespondence: Yirui Xie; Biao Zhu, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The Department of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79, QingChun Road, Hangzhou, 310003, People’s Republic of China, Tel/Fax +86-571-87236416, Email 1312019@zju.edu.cn; zhubiao1207@zju.edu.cnBackground: To evaluate the value and challenges of real-world clinical application of metagenomic next-generation sequencing (mNGS) for bronchoalveolar lavage fluid (BALF) in HIV-infected patients with suspected multi-pathogenic pneumonia.Methods: Fifty-seven HIV-infected patients with suspected mixed pneumonia who were agreed to undergo the bronchoscopy were recruited and retrospectively reviewed the results of mNGS and conventional microbiological tests (CMTs) of BALF from July 2020 to June 2022.Results: 54 patients were diagnosed with pneumonia including 49 patients with definite pathogens and five patients with probable pathogens. mNGS exhibited a higher diagnostic accuracy for fungal detection than CMTs in HIV-infected patients with suspected pulmonary infection. The sensitivity of mNGS in diagnosis of pneumonia in HIV-infected patients was much higher than that of CMTs (79.6% vs 61.1%; P < 0.05). Patients with mixed infection had lower CD4 T-cell count and higher symptom duration before admitting to the hospital than those with single infection. The detection rate of mNGS for mixed infection was significantly higher than that of CMTs and more co-pathogens could be identified by mNGS. The most common pattern of mixed infection observed was fungi-virus (11/29, 37.9%), followed by fungi-virus-bacteria (6/29, 20.7%) coinfection in HIV-infected patients with multi-pathogenic pneumonia.Conclusion: mNGS improved the pathogens detection rate and exhibited advantages in identifying multi-pathogenic pneumonia in HIV-infected patients. Early performance of bronchoscopy and mNGS are recommended in HIV-infected patients with low CD4 T cell counts and long duration of symptoms. The most common pattern of mixed infection observed was fungi-virus, followed by fungi-virus-bacteria coinfection in HIV infected patients with multi-pathogenic pneumonia.Keywords: HIV, multi-pathogenic pneumonia, metagenomic next-generation sequencing, conventional microbiological tests, bronchoalveolar lavage fluid

Context Acute respiratory distress syndrome (ARDS) is a highly fatal, inflammatory condition of lungs with multiple causes. There is no adequate treatment.Objective Using the murine LPS-induced ARDS model, we investigate SPPCT-800 (a complex lipid) as treatment for ARDS.Materials and methods C57B16/N mice received 50 μg of Escherichia coli O111:B4 lipopolysaccharide (LPS). SPPCT-800 was given as either: (1) 20 or 200 mg/kg dose 3 h after LPS; (2) 200 mg/kg (prophylactically) 30 min before LPS; or (3) eight 200 mg/kg treatments over 72 h. Controls received saline installations.Results At 48 and 72 h, SpO2 was 94% and 90% in controls compared to 97% and 94% in treated animals. Expiration times, at 24 and 48 h, were 160 and 137 msec for controls, but 139 and 107 msec with SPPCT-800. In BALF (24 h), cell counts were 4.7 × 106 (controls) and 2.9 × 106 (treated); protein levels were 1.5 mg (controls) and 0.4 mg (treated); and IL-6 was 942 ± 194 pg/mL (controls) versus 850 ± 212 pg/mL (treated) [at 72 h, 4664 ± 2591 pg/mL (controls) versus 276 ± 151 pg/mL (treated)]. Weight losses, at 48 and 72 h, were 20% and 18% (controls), but 14% and 8% (treated). Lung injury scores, at 24 and 72 h, were 1.4 and 3.0 (controls) and 0.3 and 2.2 (treated).Discussion and conclusions SPPCT-800 was effective in reducing manifestations of ARDS. SPPCT-800 should be further investigated as therapy for ARDS, especially in longer duration or higher cumulative dose studies.

Abstract Background The application of clinical mNGS for diagnosing respiratory infections improves etiology diagnosis, however at the same time, it brings new challenges as an unbiased sequencing method informing all identified microbiomes in the specimen. Methods Strategy evaluation and metagenomic analysis were performed for the mNGS data generated between March 2017 and October 2019. Diagnostic strengths of four specimen types were assessed to pinpoint the more appropriate type for mNGS diagnosis of respiratory infections. Microbiome complexity was revealed between patient cohorts and infection types. A bioinformatic pipeline resembling diagnosis results was built based upon multiple bioinformatic parameters. Results The positive predictive values (PPVs) for mNGS diagnosing of non-mycobacterium, Nontuberculous Mycobacteria (NTM), and Aspergillus were obviously higher in bronchoalveolar lavage fluid (BALF) demonstrating the potency of BALF in mNGS diagnosis. Lung tissues and sputum were acceptable for diagnosis of the Mycobacterium tuberculosis (MTB) infections. Interestingly, significant taxonomy differences were identified in sufficient BALF specimens, and unique bacteriome and virome compositions were found in the BALF specimens of tumor patients. Our pipeline showed comparative diagnostic strength with the clinical microbiological diagnosis. Conclusions To achieve reliable mNGS diagnosis result, BALF specimens for suspicious common infections, and lung tissues and sputum for doubtful MTB infections are recommended to avoid the false results given by the complexed respiratory microbiomes. Our developed bioinformatic pipeline successful helps mNGS data interpretation and reduces manual corrections for etiology diagnosis.

Lower respiratory tract infections are common in children. Bronchoalveolar lavage fluid has long been established as the best biological sample for detecting respiratory tract infections; however, it is not easily collected in children. Sputum may be used as an alternative yet its diagnostic accuracy remains controversial. Therefore, this study sought to evaluate the diagnostic accuracy of sputum for detecting lower respiratory tract infections using metagenomic next-generation sequencing. Paired sputum and bronchoalveolar lavage fluid samples were obtained from 68 patients; pathogens were detected in 67 sputum samples and 64 bronchoalveolar lavage fluid samples by metagenomic next-generation sequencing, respectively. The combined pathogen-detection rates in the sputum and bronchoalveolar lavage fluid samples were 80.90% and 66.2%, respectively. For sputum, the positive predictive values (PPVs) and negative predictive values (NPVs) for detecting bacteria were 0.72 and 0.73, respectively, with poor Kappa agreement (0.30; 95% confidence interval: 0.218-0.578, P < 0.001). However, viral detection in sputum had good sensitivity (0.87), fair specificity (0.57), and moderate Kappa agreement (0.46; 95% confidence interval: 0.231-0.693, P < 0.001). The PPVs and NPVs for viral detection in sputum were 0.82 and 0.67, respectively. The consistency between the sputum and bronchoalveolar lavage fluid was poor for bacterial detection yet moderate for viral detection. Thus, clinicians should be cautious when interpreting the results of sputum in suspected cases of lower respiratory tract infections, particularly with regards to bacterial detection in sputum. Viral detection in sputum appears to be more reliable; however, clinicians must still use comprehensive clinical judgment. [ABSTRACT FROM AUTHOR]

Background: Metagenomic next-generation sequencing (mNGS) is a powerful method for pathogen detection in various infections. In this study, we assessed the value of mNGS in the pathogen diagnosis and microbiome analysis of pneumonia in pediatric intensive care units (PICU) using bronchoalveolar lavage fluid (BALF) samples. Methods: A total of 104 pediatric patients with pneumonia who were admitted into PICU between June 2018 and February 2020 were retrospectively enrolled. Among them, 101 subjects who had intact clinical information were subject to parallel comparison of mNGS and conventional microbiological tests (CMTs) for pathogen detection. The performance was also evaluated and compared between BALF-mNGS and BALF-culture methods. Moreover, the diversity and structure of all 104 patients' lung BALF microbiomes were explored using the mNGS data. Results: Combining the findings of mNGS and CMTs, 94.06% (95/101) pneumonia cases showed evidence of causative pathogenic infections, including 79.21% (80/101) mixed and 14.85% (15/101) single infections. Regarding the pathogenesis of pneumonia in the PICU, the fungal detection rates were significantly higher in patients with immunodeficiency (55.56% vs. 25.30%, P =0.025) and comorbidities (40.30% vs. 11.76%, P=0.007). There were no significant differences in the α-diversity either between patients with CAP and HAP or between patients with and without immunodeficiency. Regarding the diagnostic performance, the detection rate of DNA-based BALF-mNGS was slightly higher than that of the BALF-culture although statistically insignificant (81.82% vs.77.92%, P=0.677) and was comparable to CMTs (81.82% vs. 89.61%, P=0.211). The overall sensitivity of DNA-based mNGS was 85.14% (95% confidence interval [CI]: 74.96%-92.34%). The detection rate of RNA-based BALF-mNGS was the same with CMTs (80.00% vs 80.00%, P>0.999) and higher than BALF-culture (80.00% vs 52.00%, P=0.045), with a sensitivity of 90.91% (95%CI: 70.84%-98.88%). Conclusions: mNGS is valuable in the etiological diagnosis of pneumonia, especially in fungal infections, and can reveal pulmonary microecological characteristics. For pneumonia patients in PICU, the mNGS should be implemented early and complementary to CMTs. [ABSTRACT FROM AUTHOR]

Objective To investigate the application value of endobronchial ultrasound-guided transbronchial needle aspiration(EBUS-TBNA） for tissue culture in the diagnosis and treatment of pulmonary infectious diseases. Methods Twenty-nine patients with suspected pulmonary infectious disease due to treatment failure were selected and received EBUS-TBNA for tissue culture, including 14 cases who underwent bronchoalveolar lavage fluid culture. We recorded the microbial culture results of tissue specimens obtained by EBUS-TBNA and bronchoalveolar lavage fluid and evaluated whether the microorganisms cultured by EBUS-TBNA tissue specimens and bronchoalveolar lavage fluid were responsible pathogens for pulmonary infection, and then we calculated the clinical consistency rate, sensitivity and specificity. Based on the microbial culture results of EBUS-TBNA tissue specimens, treatment strategies were adjusted. We recorded out‐comes and effective rate of the patients and the occurrence of complications during and after EBUS-TBNA. Results Among 29 cases, 22 were finally diagnosed with pulmonary infectious diseases, of whom 17 cases had positive results for microbial culture by EBUS-TBNA, with the positive rate of 77. 3%(17/22). Five of 14 patients who underwent microbial culture by bronchoalveolar lavage fluid had positive results, with the positive rate of 35. 7%(5/14), which was lower than that of EBUS-TBNA(P<0. 05). Among the 17 patients with positive microbial culture of EBUS-TBNA, the consistency rate of clinically responsible pathogen was 70. 6%(12/17）, and the treatment response rate was 70. 6%(12/17). The clinical agreement rate of Streptococci as the responsible pathogens in patients with microbial culture results as streptococci by EBUS-TBNA was 87. 5%(7/8）, and the treatment response rate was 100%(7/7）. The sensitivity and specificity of EBUS-TBNA tissue specimens for microbial culture for the determination of responsible pathogens were 80. 0% and 28. 6%, respectively. Due to the confounding effects of tuberculosis and viral pneumonia, the clinical consistency rate of EBUS-TBNA tissue specimens in comparison with the final diagnosis of disease was 63. 6%(14 / 22）, which was higher than that of bronchoalveolar lavage fluid culture(P<0. 05）. The clinical consistency rate of 14 patients who underwent bronchoalveolar lavage fluid culture was 20. 0%(1/5）. All patients had no major hemoptysis during surgery and no emerging bloodstream infections after surgery. One case of pneumothorax occurred after surgery, and the chest tightness was improved after thoracic puncture and aspiration. After surgery, five cases showed mild blood in sputum, which stopped spontaneously after it is not treated. Conclusions EBUS-TBNA for microbial culture is easier to detect pathogens and has a higher clinical agreement rate than bronchoalveolar lavage fluid, which is helpful to improve the detection rate of clinically responsible pathogens, especially Streptococci. It is helpful to improve the detection rate of clinically responsible pathogens, especially Streptococcus, and guide clinical treatment with high safety. [ABSTRACT FROM AUTHOR]

Introduction: Unbiased metagenomic next-generation sequencing (mNGS) has been used for infection diagnosis. In this study, we explored the clinical diagnosis value of mNGS for pulmonary complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: From August 2019 to June 2021, a prospective study was performed to comparatively analyze the pathogenic results of mNGS and conventional tests for bronchoalveolar lavage fluid (BALF) from 134 cases involving 101 patients with pulmonary complications after allo-HSCT. Results: More pathogens were identified by mNGS than with conventional tests (226 vs 120). For bacteria, the diagnostic sensitivity (P = 0.144) and specificity (P = 0.687) were similar between the two methods. For fungus except Pneumocystis jirovecii (PJ), conventional tests had a significantly higher sensitivity (P = 0.013) with a similarly high specificity (P = 0.109). The sensitivities for bacteria and fungi could be increased with the combination of the two methods. As for PJ, both the sensitivity (100%) and specificity (99.12%) of mNGS were very high. For viruses, the sensitivity of mNGS was significantly higher (P = 0.021) and the negative predictive value (NPV) was 95.74% (84.27–99.26%). Pulmonary infection complications accounted for 90.30% and bacterium was the most common pathogen whether in single infection (63.43%) or mixed infection (81.08%). The 6-month overall survival (OS) of 88.89% in the early group (mNGS ≤ 7 days) was significantly higher than that of 65.52% (HR 0.287, 95% CI 0.101–0.819, P = 0.006) in the late group (mNGS > 7 days). Conclusions: mNGS for BALF could facilitate accurate and fast diagnosis for pulmonary complications. Early mNGS could improve the prognosis of patients with pulmonary complications after allo-HSCT. Trial Registration: ClinicalTrials.gov identifier, NCT 04051372. [ABSTRACT FROM AUTHOR]

Rationale: Sarcoidosis is a granulomatous interstitial lung disease involving a complex interplay among different cluster of differentiation 4 (CD4+) thymus cell (T-cell) subsets. Originally described as a type 1 T-helper (Th1) inflammatory disease, recent evidence suggests that both effector and regulatory T-cell subgroups play a critical role in sarcoidosis, but this remains controversial. Objectives: We aimed to investigate the distribution of CD4+ T-cell subpopulations in sarcoidosis patients and its potential associations with clinical disease activity and a radiographic fibrotic phenotype. Methods: We measured the frequencies of regulatory T cells (Tregs), Th1, Th17, and Th17.1 cells in the peripheral blood and/or bronchoalveolar lavage fluid (BALF) of 62 sarcoidosis patients, 66 idiopathic pulmonary fibrosis (IPF) patients, and 41 healthy volunteers using flow cytometry. We also measured the changes in these T-cell subpopulations in the blood at the follow-up visits of 11 sarcoidosis patients. Measurements and Results: An increased percentage of Tregs was observed in the peripheral blood of sarcoidosis patients, with a positive association to disease activity and a fibrotic radiographic phenotype. We found a higher frequency of Tregs, a lower proportion of Th17.1 cells, and a lower ratio of Th17.1 cells to total Tregs in the peripheral blood of both active and fibrotic sarcoidosis patients, compared with IPF patients or healthy donors. In contrast, a lower frequency of Tregs and a higher proportion of Th17.1 cells was found in the BALF of sarcoidosis patients than in that of IPF patients. There was an imbalance of Tregs and Th17.1 cells between the peripheral blood and BALF in sarcoidosis patients. Following immunoregulatory therapy, the proportion of circulating Tregs in sarcoidosis patients decreased. Conclusion: A higher proportion of Tregs in the peripheral blood of sarcoidosis patients was related to disease activity, fibrotic phenotype, and the need for immunoregulatory therapy. The imbalanced distribution of Tregs and Th17.1 cells in patients' peripheral blood and BALF suggests that the lung microenvironment has an effect on the immunological pathogenesis of sarcoidosis. Therefore, further studies on the functional analysis of Tregs and Th17.1 cells in sarcoidosis patients are warranted. [ABSTRACT FROM AUTHOR]

Background: The discovery of immune checkpoint inhibitors (ICIs) is a breakthrough in the field of cancer therapy. However, ICIs may cause immune‐related adverse reactions, including checkpoint inhibitor‐related pneumonitis (CIP). The aim of this study was to investigate cytokines in bronchoalveolar lavage fluid (BALF) of patients with CIP compared with patients with pulmonary infection and patients with cancer. Methods: We retrospectively analyzed 34 cytokine levels and T cell subsets in BALF supernatant samples from ICI‐treated patients with CIP (n = 13), pulmonary infection (n = 10), and progressive cancer (n = 12). Cytokine levels and T cell subsets were compared among the three groups of patients. Results: We observed significantly higher levels of IFN‐γ‐induced protein 10(IP‐10) (p = 0.002), and percentage of CD3 + CD8 + T cells (p = 0.020) in BALF of patients with CIP compared with the other two groups. However, we found significantly lower levels of interleukin‐21 (p = 0.008) in BALF of patients with progressive disease compared with the other two groups. Conclusions: Cytokine profile and character of cell subsets in BALF was helpful for the differential diagnosis of CIP. IP‐10 may play an important role in pathophysiology for CIP and also be a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Objective: The aim of this study was to explore the predictive value of the ratio of procalcitonin (PCT) in serum to Simpson's dominance index (SDI) in bronchoalveolar lavage fluid (BALF), in short-term prognosis of patients with severe bacterial pneumonia (SBP). Methods: This is a retrospective review of case materials of 110 patients with SBP who selected BALF metagenomic next-generation sequencing technique in the intensive care unit (ICU) of the Affiliated Hospital of Yangzhou University from January 2019 and July 2022. Based on the acute physiology and chronic health status score II, within 24 h after admission to the ICU, patients were divided into a non-critical group (n = 40) and a critical group (n = 70). Taking death caused by bacterial pneumonia as the endpoint event, the 28-day prognosis was recorded, and the patients were divided into a survival group (n = 76) and a death group (n = 34). The SDI, PCT, C-reactive protein (CRP), PCT/SDI, and CRP/SDI were compared and analyzed. Results: Compared with the non-critical group, the critical group had a higher PCT level, a greater PCT/SDI ratio, a longer ventilator-assisted ventilation time (VAVT), and more deaths in 28 days. Compared with the survivors, the death group had a higher PCT level, a lower SDI level, and a greater PCT/SDI ratio. The SDI level was significantly negatively correlated with the VAVT (r = 0.675, p < 0.05), while the PCT level, ratio of PCT/SDI, and ratio of CRP/SDI were remarkably positively correlated with VAVT (r = 0.669, 0.749, and 0.718, respectively, p < 0.05). The receiver operating characteristic (ROC) curves analysis showed that the area under ROC curves of PCT/SDI predicting patient death within 28 days was 0.851, followed by PCT + SDI, PCT, SDI, and CRP/SDI (0.845, 0.811, 0.778, and 0.720, respectively). The sensitivity and specificity of PCT/SDI for predicting death were 94.1% and 65.8%, respectively, at the optimal value (11.56). Cox regression analysis displayed that PCT/SDI (HR = 1.562; 95% CI: 1.271 to 1.920; p = 0.039) and PCT (HR = 1.148; 95% CI: 1.105 to 1.314; p = 0.015) were independent predictors of death in patients. Conclusion: The ratio of PCT/SDI was a more valuable marker in predicting the 28-day prognosis in patients with SBP. [ABSTRACT FROM AUTHOR]

Objectives: To compare clinical outcomes in patients with severe pneumonia according to the diagnostic strategy used. Methods: In this retrospective, nested, case–control study, patients with severe pneumonia who had undergone endotracheal aspirate (ETA) metagenomic next-generation sequencing of (mNGS) testing (n = 53) were matched at a ratio of 1 to 2 (n = 106) by sex, age, underlying diseases, immune status, disease severity scores, and type of pneumonia with patients who had undergone bronchoalveolar lavage fluid (BALF) mNGS. The microbiological characteristics and patient's prognosis of the two groups were compared. Results: An overall comparison between the two groups showed no significant differences in bacterial, fungal, viral, or mixed infections. However, subgroup analysis of 18 patients who received paired ETA and BALF mNGS showed a complete agreement rate for the two specimens of 33.3%. There were more cases for whom targeted treatment was initiated (36.79% vs. 22.64%; P = 0.043) and fewer cases who received no clinical benefit after mNGS (5.66% vs. 15.09%; P = 0.048) in the BALF group. The pneumonia improvement rate in the BALF group was significantly higher than in the ETA group (73.58% vs. 87.74%, P = 0.024). However, there were no significant differences in ICU mortality or 28-day mortality. Conclusions: We do not recommend using ETA mNGS as the first-choice method for analyzing airway pathogenic specimens from severe pneumonia patients. [ABSTRACT FROM AUTHOR]

Chronic lung allograft dysfunction (CLAD) increases morbidity and mortality for lung transplant recipients. Club cell secretory protein (CCSP), produced by airway club cells, is reduced in the bronchoalveolar lavage fluid (BALF) of lung recipients with CLAD. We sought to understand the relationship between BALF CCSP and early posttransplant allograft injury and determine if early posttransplant BALF CCSP reductions indicate later CLAD risk. We quantified CCSP and total protein in 1606 BALF samples collected over the first posttransplant year from 392 adult lung recipients at 5 centers. Generalized estimating equation models were used to examine the correlation of allograft histology or infection events with protein-normalized BALF CCSP. We performed multivariable Cox regression to determine the association between a time-dependent binary indicator of normalized BALF CCSP level below the median in the first posttransplant year and development of probable CLAD. Normalized BALF CCSP concentrations were 19% to 48% lower among samples corresponding to histological allograft injury as compared with healthy samples. Patients who experienced any occurrence of a normalized BALF CCSP level below the median over the first posttransplant year had a significant increase in probable CLAD risk independent of other factors previously linked to CLAD (adjusted hazard ratio 1.95; p = 0.035). We discovered a threshold for reduced BALF CCSP to discriminate future CLAD risk; supporting the utility of BALF CCSP as a tool for early posttransplant risk stratification. Additionally, our finding that low CCSP associates with future CLAD underscores a role for club cell injury in CLAD pathobiology. [ABSTRACT FROM AUTHOR]

Purpose: Metagenomic next-generation sequencing (mNGS) has been extensively used in the diagnosis of infectious diseases but has rarely been applied in non-tuberculous mycobacterial pulmonary disease (NTMPD). This study analyzed the diagnostic performance of mNGS in bronchoalveolar lavage fluid (BALF) samples to identify non-tuberculous mycobacteria (NTM). Patients and Methods: A total of 231 patients with suspected NTMPD were recruited from the First Affiliated Hospital, School of Medicine, Zhejiang University, from March 2021 to October 2022. A total of 118 cases were ultimately included. Of these patients, 61 cases were enrolled in the NTMPD group, 23 cases were enrolled in the suspected-NTMPD group, and 34 cases were enrolled in the non-NTMPD group. The diagnostic performance of traditional culture, acid-fast staining (AFS), and mNGS for NTMPD was assessed. Results: Patients in the NTMPD group had a higher proportion of bronchiectasis (P=0.007). Among mNGS-positive samples in the NTMPD group, a significantly higher reads number of NTM was observed in AFS-positive patients [61.50 (22.00, 395.00) vs 15.50 (6.00, 36.25), P=0.008]. Meanwhile, mNGS demonstrated a sensitivity of 90.2%, which was far superior to AFS (42.0%) and culture (77.0%) (P< 0.001). The specificity of mNGS in detecting NTM was 100%, which was the same as that of traditional culture. The area under the receiver operating characteristic curve of mNGS was 0.951 (95% CI 0.906– 0.996), which was higher than that of culture (0.885 [95% CI 0.818– 0.953]) and AFS (0.686 [95% CI 0.562– 0.810]). In addition to NTM, other pulmonary pathogens were also found by mNGS. Conclusion: mNGS using BALF samples is a rapid and effective diagnostic tool for NTMPD, and mNGS is recommended for patients with suspected NMTPD or NTM coinfected pneumonia. [ABSTRACT FROM AUTHOR]

Objective: Thisstudy was conducted to establish a rat model of acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) using the combination of bleomycin (BLM) and lipopolysaccharides (LPS). Materials and Method: Twenty-four male Sprague Dawley rats were allocated into two equal groups: the sham or the bleomycin and lipopolysaccharides-induced AE-IPF group (BLM-LPS). On Day 7, BLM intratracheally and LPS intraperitoneally were both used to administer AE-IPF. The BLM-LPS group and its respective sham group were terminated on Days 8, 14, or 21. Samples of bronchoalveolar lavage fluid (BALF) and lungs were taken and investigated for cell count and histopathology. Results: On Day 8, histological analysis revealed inflammatory cell infiltration with edema and hyaline membrane, and the BALF differential cell count revealed high neutrophil counts. By having a higher collagen density area and Ashcroft modified score than the sham group on Day 14, the BLM-LPS group displayed significantly lower oxygen saturation, alveolar air area, and a fibrotic appearance. However, there was a spontaneous resolution in inflammation and fibrotic appearance on Day 21 after the BLM administration. Conclusions: By combining BLM and LPS, it was possible to create a successful rat model of AE-IPF. The present modelshowed the peak exacerbation on Day 8 and the fibrotic peak on Day 14, which gradually improved. The optimal time for the new AE-IPF therapeutic intervention was determined to be between Days 8 and 14. [ABSTRACT FROM AUTHOR]

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Omadacycline is a novel tetracycline antibiotic that exhibits good in vitro antibacterial activity against atypical pathogens such as Mycoplasma pneumoniae. It is approved for the treatment of adults with community-acquired bacterial pneumonia. However, the safety and efficacy of omadacycline in pediatric patients under 18 years of age have not yet been established. In the present paper, we report a case of pediatric community-acquired pneumonia in which initial empirical anti-infective therapy had failed. The patient received empirical anti-infective therapy with azithromycin and other antimicrobial agents upon admission but showed a poor clinical response and developed secondary tinnitus and liver dysfunction. After the confirmation of M. pneumoniae infection through metagenomic next-generation sequencing (mNGS) of bronchoalveolar lavage fluid, an antibiotic switch to omadacycline was made. Thereafter, the patient’s condition improved, and no adverse reactions were observed. These findings demonstrate that mNGS enables the identification of infection-causing pathogens in patients with unresponsive pneumonia. Omadacycline can be considered as an alternative option for anti-infective therapy in pediatric M. pneumoniae pneumonia, especially when the presence of bacterial resistance, adverse drug reactions, or organ failure are taken into consideration.

This is the first study reporting the presence of airborne nano-sized plastic particles in the bronchoalveolar lavage fluid (BALF) samples of patients undergoing diagnostic bronchoscopy. The results represent the plastic pollution content in the lower airways of the residents of Northern Europe. Airborne micro- and nanoplastic particles (MP/NPs) are widely dispersed worldwide and intrude on human organisms to various extents, with the respiratory tract being the first line of exposure. The amounts of inhaled MP/NPs, their fate in the human respiratory tract, and the effects on the health of human airways and other exposed organs remain largely unknown.In this clinical study, human BALF samples were assessed by means of optical and transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy (TEM-EDX). Results show that MP/NPs levels vary in the interval of 0.14–12.8 particles per 100 ml of BALF and are present in all samples tested, mainly in a fragmented form. External pollution by MP/NPs was excluded by carefully choosing methodology and equipment. This finding is a timely addition of valuable information and stimulates further research into the biological effects of inhaled MP/NPs.

No targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K2P2.1 (TREK-1) K+ channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca2+ channels (CaV) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa2+ concentrations. In addition, CCL-2 secretion was decreased after L-type CaV inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.

Background: Interferon-gamma (IFN-γ) is a key mediator of sarcoidosis-related granulomatous inflammation. Previous findings of IFN-γ-producing Th17 cells in bronchoalveolar lavage fluid from sarcoidosis patients invokes the transition of Th17.0 cells to Th17.1 cells in the disease's pathogenesis. Since the T-bet transcription factor is crucial for this transition, the goal of this study was to determine if T-bet expression in Th17.0 cells reflects the extent of granulomatous inflammation in sarcoidosis patients as assessed by clinical outcomes. Methods: Using a case-control study design, we identified two groups of sarcoidosis subjects (total N = 43) with pulmonary function tests (PFTs) that either (1) changed (increased or decreased) longitudinally or (2) were stable. We used flow cytometry to measure the transcription factors T-bet and RORγt in Th1, Th17.0, and Th17.1 cell subsets defined by CCR6, CCR4 and CXCR3 in blood samples. We compared the percentages of T-bet+ cells in RORγt+Th17.0 cells (defined as CCR6+CCR4+CXCR3-) based on subjects' PFT group. We also assessed the relationship between the direction of change in PFTs with the changes in %T-bet+ frequencies using mixed effects modeling. Results: We found that T-bet expression in subjects' RORγt+Th17.0 cells varied based on clinical outcome. The T-bet+ percentage of RORγt+Th17.0 cells was higher in the cases (subject group with PFT changes) as compared to controls (stable group) (27 vs. 16%, p = 0.0040). In comparisons before and after subjects' PFT changes, the T-bet+ frequency of RORγt+Th17.0 cells increased or decreased in the opposite direction of the PFT change. The percentage of these T-bet+ cells was also higher in those with greater numbers of involved organs. Serum levels of interferon-γ-induced chemokines, CXCL9, CXCL10, and CXCL11, and whole blood gene expression of IFN-γ-related genes including GBP1, TAP1, and JAK2 were independently positively associated with the T-bet+ frequencies of RORγt+Th17.0 cells. Conclusions: These data suggest that expression of T-bet in Th17.0 cells could reflect the extent of granulomatous inflammation in sarcoidosis patients because they represent a transition state leading to the Th17.1 cell phenotype. These findings indicate that Th17 plasticity may be part of the disease paradigm.

Human mesenchymal stem/stromal cells (hMSCs) are a promising therapy for acute respiratory distress syndrome (ARDS) and other inflammatory conditions. While considerable research has focused on paracrine effects and mitochondrial transfer that improve lung fluid balance, hMSCs are well known to have immunomodulatory properties as well. Some of these immunomodulatory properties have been related to previously reported paracrine effectors such as indoleamine-2,3-dioxygenase (IDO), but these effects cannot fully account for cell-contact dependent immunomodulation. Here, we report that CD40 is upregulated on hMSCs under the same conditions previously reported to induce IDO. Further, CD40 transcription is also upregulated on hMSCs by ARDS pulmonary edema fluid but not by hydrostatic pulmonary edema fluid. Transcription of CD40, as well as paracrine effectors TSG6 and PTGS2 remained significantly upregulated for at least 12 hours after withdrawal of cytokine stimulation. Finally, induction of this immune phenotype altered the transdifferentiation of hMSCs, one of their hallmark properties. CD40 may play an important role in the immunomodulatory effects of hMSCs in ARDS and inflammation.

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+ cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.

BackgroundEosinophilic lung disease is a poorly understood inflammatory airway disease that results in substantial morbidity.ObjectiveTo describe clinical findings in dogs with eosinophilic lung disease defined on the basis of radiographic, bronchoscopic, and bronchoalveolar lavage fluid (BAL) analysis. Categories included eosinophilic bronchitis (EB), eosinophilic granuloma (EG), and eosinophilic bronchopneumopathy (EBP).AnimalsSeventy-five client owned dogs.MethodsMedical records were retrospectively reviewed for dogs with idiopathic BAL fluid eosinophilia. Information abstracted included duration and nature of clinical signs, bronchoscopic findings, and laboratory data. Thoracic radiographs were evaluated for the pattern of infiltrate, bronchiectasis, and lymphadenomegaly.ResultsThoracic radiographs were normal or demonstrated a bronchial pattern in 31 dogs assigned a diagnosis of EB. Nine dogs had intraluminal mass lesions and were bronchoscopically diagnosed with EG. The remaining 35 dogs were categorized as having EBP based on radiographic changes, yellow green mucus in the airways, mucosal changes, and airway collapse. Age and duration of cough did not differ among groups. Dogs with EB were less likely to have bronchiectasis or peripheral eosinophilia, had lower total nucleated cell count in BAL fluid, and lower percentage of eosinophils in BAL fluid compared to dogs in the other 2 groups. In contrast to previous reports, prolonged survival (>55 months) was documented in dogs with EG.Conclusions and clinical importanceDogs with eosinophilic lung disease can be categorized based on imaging, bronchoscopic and BAL fluid cytologic findings. Further studies are needed to establish response to treatment in these groups.

BackgroundChronic lung allograft dysfunction (CLAD) is the main limitation to long-term survival after lung transplantation. Because effective therapies are lacking, early identification and mitigation of risk factors is a pragmatic approach to improve outcomes. Acute cellular rejection (ACR) is the most pervasive risk factor for CLAD, but diagnosis requires transbronchial biopsy, which carries risks. We hypothesized that gene expression in the bronchoalveolar lavage (BAL) cell pellet (CP) could replace biopsy and inform on mechanisms of CLAD.MethodsWe performed RNA sequencing on BAL CPs from 219 lung transplant recipients with A-grade ACR (n = 61), lymphocytic bronchiolitis (n = 58), infection (n = 41), or no rejection/infection (n = 59). Differential gene expression was based on absolute fold difference >2.0 and Benjamini-adjusted p-value ≤0.05. We used the Database for Annotation, Visualization and Integrated Discovery Bioinformatics Resource for pathway analyses. For classifier modeling, samples were randomly split into training (n = 154) and testing sets (n = 65). A logistic regression model using recursive feature elimination and 5-fold cross-validation was trained to optimize area under the curve (AUC).ResultsDifferential gene expression identified 72 genes. Enriched pathways included T-cell receptor signaling, natural killer cell-mediated cytotoxicity, and cytokine-cytokine receptor interaction. A 4-gene model (AUC = 0.72) and classification threshold defined in the training set exhibited fair performance in the testing set; accuracy was 76%, specificity 82%, and sensitivity 60%. In addition, classification as ACR was associated with worse CLAD-free survival (hazard ratio = 2.42; 95% confidence interval = 1.29-4.53).ConclusionsBAL CP gene expression during ACR is enriched for immune response pathways and shows promise as a diagnostic tool for ACR, especially ACR that is a precursor of CLAD.

BackgroundAllergic asthma causes morbidity in many subjects, and novel precision-directed treatments would be valuable.ObjectiveWe sought to examine the role of a novel innate molecule, repulsive guidance molecule b (RGMb), in murine models of allergic asthma.MethodsIn models of allergic asthma using ovalbumin or cockroach allergen, mice were treated with anti-RGMb or control mAb and examined for airway inflammation and airway hyperreactivity (AHR), a cardinal feature of asthma. The mechanisms by which RGMb causes airways disease were also examined.ResultsWe found that blockade of RGMb by treatment with anti-RGMb mAb effectively blocked the development of airway inflammation and AHR. Importantly, blockade of RGMb completely blocked the development of airway inflammation and AHR, even if treatment occurred only during the challenge (effector) phase. IL-25 played an important role in these models of asthma because IL-25 receptor-deficient mice did not develop disease after sensitization and challenge with allergen. RGMb was expressed primarily by innate cells in the lungs, including bronchial epithelial cells (known producers of IL-25), activated eosinophils, and interstitial macrophages, which in the inflamed lung expressed the IL-25 receptor and produced IL-5 and IL-13. We also found that neogenin, the canonical receptor for RGMb, was expressed by interstitial macrophages and bronchial epithelial cells in the inflamed lung, suggesting that an innate RGMb-neogenin axis might modulate allergic asthma.ConclusionsThese results demonstrate an important role for a novel innate pathway in regulating type 2 inflammation in patients with allergic asthma involving RGMb and RGMb-expressing cells, such as interstitial macrophages and bronchial epithelial cells. Moreover, targeting this previously unappreciated innate pathway might provide an important treatment option for allergic asthma.

Previous studies have suggested a role for Tet1 in the pathogenesis of childhood asthma. However, how Tet1 contributes to asthma remains unknown. Here we used mice deficient for Tet1 in a well-established model of allergic airway inflammation and demonstrated that loss of Tet1 increased disease severity including airway hyperresponsiveness and lung eosinophilia. Increased expression of Muc5ac, Il13, Il33, Il17a, Egfr, and Tff2 were observed in HDM-challenged Tet1-deficient mice compared to Tet1+/+ littermates. Further, transcriptomic analysis of lung RNA followed by pathway and protein network analysis showed that the IFN signaling pathway was significantly upregulated and the aryl hydrocarbon receptor (AhR) pathway was significantly downregulated in HDM-challenged Tet1-/- mice. This transcriptional regulation of the IFN and AhR pathways by Tet1 was also present in human bronchial epithelial cells at base line and following HDM challenges. Genes in these pathways were further associated with changes in DNA methylation, predicted binding of transcriptional factors with relevant functions in their promoters, and the presence of histone marks generated by histone enzymes that are known to interact with Tet1. Collectively, our data suggest that Tet1 inhibits HDM-induced allergic airway inflammation by direct regulation of the IFN and AhR pathways.