Bronchoalveolar Lavage Neutrophils Research Articles

MiR-144/451 attenuates lipopolysaccharide-induced lung inflammation by downregulating Rac1 and STAT-3 in macrophages.

MicroRNAs have been shown to play a critical role in lung inflammatory diseases. Here, we report that knocking out miR-144/451 in mice exacerbates lipopolysaccharide (LPS)-induced lung inflammation. The lung inflammation in mice was induced by intratracheal instillation of LPS. Loss-of-function experiments demonstrated that miR-144/451 gene knockout (KO) increased LPS-induced lung inflammation and oxidant stress compared with wild-type (WT) mice, as manifested by increased total bronchoalveolar lavage fluid cells and neutrophil counts, elevated TNF-α and IL-6 levels in bronchoalveolar lavage fluid, enhanced myeloperoxidase activity, and reduced catalase and glutathione peroxidase activity in lung tissues. We also found that LPS significantly decreased miR-451 expression in lung tissues and macrophages; while miR-451 overexpression in LPS-induced RAW264.7 cells remarkably reduced TNF-α and IL-6 levels as well as reactive oxygen species (ROS) production, suggesting a feedback loop might exist in inflammatory cells. Rac1 mRNA and protein levels were downregulated in miR-451-overexpressed RAW264.7 cells. Ex vivo stimulation experiments, performed using alveolar macrophages isolated from miR-144/451 KO mice, confirmed that Rac1 inhibitor alleviated levels of TNF-α and ROS in response to LPS stimulation compared with WT controls. Luciferase reporter assay demonstrated that STAT-3 is a direct target of miR-451. STAT-3 protein levels were elevated in miR-144/451 KO macrophages. LPS treatment also resulted in higher phosphorylation levels of STAT-3 in macrophages from KO mice than in WT cells. Our study identified miR-144/451 as an anti-inflammatory factor in LPS-induced lung inflammation that acts by downregulating Rac1 and STAT-3.

Self-report underestimates the frequency of the acute respiratory exacerbations of COPD but is associated with BAL neutrophilia and lymphocytosis: an observational study

RationaleResearch studies typically quantify acute respiratory exacerbation episodes (AECOPD) among people with chronic obstructive pulmonary disease (COPD) based on self-report elicited by survey questionnaire. However, AECOPD quantification by self-report could be inaccurate, potentially rendering it an imprecise tool for identification of those with exacerbation tendency.ObjectiveDetermine the agreement between self-reported and health records-documented quantification of AECOPD and their association with airway inflammation.MethodsWe administered a questionnaire to elicit the incidence and severity of respiratory exacerbations in the three years preceding the survey among current or former heavy smokers with or without diagnosis of COPD. We then examined electronic health records (EHR) of those with COPD and those without (tobacco-exposed persons with preserved spirometry or TEPS) to determine whether the documentation of the three-year incidence of moderate to very severe respiratory exacerbations was consistent with self-report using Kappa Interrater statistic. A subgroup of participants also underwent bronchoalveolar lavage (BAL) to quantify their airway inflammatory cells. We further used multivariable regressions analysis to estimate the association between respiratory exacerbations and BAL inflammatory cell composition with adjustment for covariates including age, sex, height, weight, smoking status (current versus former) and burden (pack-years).ResultsOverall, a total of 511 participants completed the questionnaire, from whom 487 had EHR available for review. Among the 222 participants with COPD (70 ± 7 years-old; 96% male; 70 ± 38 pack-years smoking; 42% current smoking), 57 (26%) reported having any moderate to very severe AECOPD (m/s-AECOPD) while 66 (30%) had EHR documentation of m/s-AECOPD. However, 42% of those with EHR-identified m/s-AECOPD had none by self-report, and 33% of those who reported m/s-AECOPD had none by EHR, suggesting only moderate agreement (Cohen’s Kappa = 0.47 ± 0.07; P < 0.001). Nevertheless, self-reported and EHR-identified m/s-AECOPD events were both associated with higher BAL neutrophils (ß ± SEM: 3.0 ± 1.1 and 1.3 ± 0.5 per 10% neutrophil increase; P ≤ 0.018) and lymphocytes (0.9 ± 0.4 and 0.7 ± 0.3 per 10% lymphocyte increase; P ≤ 0.041). Exacerbation by either measure combined was associated with a larger estimated effect (3.7 ± 1.2 and 1.0 ± 0.5 per 10% increase in neutrophils and lymphocytes, respectively) but was not statistically significantly different compared to the self-report only approach. Among the 184 TEPS participants, there were fewer moderate to very severe respiratory exacerbations by self-report (n = 15 or 8%) or EHR-documentation (n = 9 or 5%), but a similar level of agreement as those with COPD was observed (Cohen’s Kappa = 0.38 ± 0.07; P < 0.001).DiscussionWhile there is modest agreement between self-reported and EHR-identified m/s-AECOPD, events are missed by relying on either method alone. However, m/s-AECOPD quantified by self-report or health records is associated with BAL neutrophilia and lymphocytosis.

Developing a mouse model of human coronavirus NL63 infection: comparison with rhinovirus-A1B and effects of prior rhinovirus infection.

Human coronavirus (HCoV)-NL63 causes respiratory tract infections in humans and uses angiotensin-converting enzyme 2 (ACE2) as a receptor. We sought to establish a mouse model of HCoV-NL63 and determine whether prior rhinovirus (RV)-A1B infection affected HCoV-NL63 replication. HCoV-NL63 was propagated in LLC-MK2 cells expressing human ACE2. RV-A1B was grown in HeLa-H1 cells. C57BL6/J or transgenic mice expressing human ACE2 were infected intranasally with sham LLC-MK2 cell supernatant or 1 × 105 tissue culture infectious dose (TCID50) units HCoV-NL63. Wild-type mice were infected with 1 × 106 plaque-forming units (PFU) RV-A1B. Lungs were assessed for vRNA, bronchoalveolar lavage (BAL) cells, histology, HCoV-NL63 nonstructural protein 3 (nsp3), and host gene expression by next-generation sequencing and qPCR. To evaluate sequential infections, mice were infected with RV-A1B followed by HCoV-NL63 infection 4 days later. We report that hACE2 mice infected with HCoV-NL63 showed evidence of replicative infection with increased levels of vRNA, BAL neutrophils and lymphocytes, peribronchial and perivascular infiltrates, and expression of nsp3. Viral replication peaked 3 days after infection and inflammation persisted 6 days after infection. HCoV-NL63-infected hACE2 mice showed increased mRNA expression of IFNs, IFN-stimulated proteins, and proinflammatory cytokines. Infection with RV-A1B 4 days before HCoV-NL63 significantly decreased both HCoV-NL63 vRNA levels and airway inflammation. Mice infected with RV-A1B prior to HCoV-NL63 showed increased expression of antiviral proteins compared with sham-treated mice. In conclusion, we established a mouse model of HCoV-NL63 replicative infection characterized by relatively persistent viral replication and inflammation. Prior infection with RV-A1B reduced HCoV-NL63 replication and airway inflammation, indicative of viral interference.NEW & NOTEWORTHY We describe a mouse model of human coronavirus (HCoV) infection. Infection of transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) with HCoV-NL63 produced a replicative infection with peribronchial inflammation and nonstructural protein 3 expression. Mice infected with RV-A1B 4 days before HCoV-NL63 showed decreased HCoV-NL63 replication and airway inflammation and increased expression of antiviral proteins compared with sham-treated mice. This research may shed light on human coronavirus infections, viral interference, and viral-induced asthma exacerbations.

Pseudomonas aeruginosa infection correlates with high MFI donor-specific antibody development following lung transplantation with consequential graft loss and shortened CLAD-free survival

BackgroundDonor-specific antibodies (DSAs) are common following lung transplantation (LuTx), yet their role in graft damage is inconclusive. Mean fluorescent intensity (MFI) is the main read-out of DSA diagnostics; however its value is often disregarded when analyzing unwanted post-transplant outcomes such as graft loss or chronic lung allograft dysfunction (CLAD). Here we aim to evaluate an MFI stratification method in these outcomes.MethodsA cohort of 87 LuTx recipients has been analyzed, in which a cutoff of 8000 MFI has been determined for high MFI based on clinically relevant data. Accordingly, recipients were divided into DSA-negative, DSA-low and DSA-high subgroups. Both graft survival and CLAD-free survival were evaluated. Among factors that may contribute to DSA development we analyzed Pseudomonas aeruginosa (P. aeruginosa) infection in bronchoalveolar lavage (BAL) specimens.ResultsHigh MFI DSAs contributed to clinical antibody-mediated rejection (AMR) and were associated with significantly worse graft (HR: 5.77, p < 0.0001) and CLAD-free survival (HR: 6.47, p = 0.019) compared to low or negative MFI DSA levels. Analysis of BAL specimens revealed a strong correlation between DSA status, P. aeruginosa infection and BAL neutrophilia. DSA-high status and clinical AMR were both independent prognosticators for decreased graft and CLAD-free survival in our multivariate Cox-regression models, whereas BAL neutrophilia was associated with worse graft survival.ConclusionsP. aeruginosa infection rates are elevated in recipients with a strong DSA response. Our results indicate that the simultaneous interpretation of MFI values and BAL neutrophilia is a feasible approach for risk evaluation and may help clinicians when to initiate DSA desensitization therapy, as early intervention could improve prognosis.

The efficacy of N-acetylcysteine in decreasing airway inflammation and mucus accumulation in horses with 18 hours of head confinement.

During transportation many horses develop post-transportation infection, which can be life-threatening and end their sport career. Preventing mucus accumulation and inflammation during transportation is vital, emphasizing the need for effective strategies to enhance overall horse health welfare. Assess the impact of N-acetylcysteine (NAC) on mucus accumulation and inflammation in horses subjected to 18 hours of head confinement. Six healthy crossbred horses, 5.3 ± 2.1 years of age and weighing 387 ± 30 kg. Prospective placebo-controlled cross-over design study. The horses' heads were restrained in their stalls for a period of 18 hours. They were studied under 4 conditions: Not confined (NC): before head confinement, placebo (P), and confined head (CH): 18 hours of head confinement without treatment, and N-Acetylcysteine (NAC): 18 hours of head confinement treated with NAC before confinement (15 mg/kg/day NAC PO for 3 days). Bronchoalveolar lavage (BAL) was performed in each condition. Mucus accumulation along the trachea was evaluated by endoscopy. Endoscopic scores were significantly different between CH and other conditions, whereas no significant differences were found among NC, P, and NAC. The BAL cell count (34 291 ± 2624 cells/μL), neutrophil and lymphocyte count (18 601 ± 3193 cells/μL and 3337.4 ± 593 cells/μL, respectively) in CH were significantly higher compared to NAC. Neutrophil percentage was significantly higher in CH (53.8 ± 8%) compared to horses that received NAC (20.08 ± 8%). Conversely, in comparison to NAC (66.33 ± 9%), the percentage of macrophages was significantly lower in CH (35.7 ± 10%). N-acetylcysteine was found to significantly decrease mucus accumulation and inflammatory cell counts in horses with head confinement.

Differential cell analysis in ovine bronchoalveolar lavage fluid: Effect of different sampling methods and seasons.

Bronchoalveolar lavage (BAL) is a method for collecting the cellular and fluid components of the airway surface in the lungs. The assessment of differential cell profiles is potentially valuable in the diagnosis of pulmonary diseases, but there is no information about the normal BAL profiles in the Gezel breed. This study aimed to characterize the normal cryptologic findings of BAL with Gezel sheep. Twenty healthy sheep (15 females: 5 males, bodyweight: 55-65 kg) were sedated with xylazine (0.02-0.04 mg/kg IV). Two methods; the transtracheal bronchoalveolar lavage technique and the bronchoscopic bronchoalveolar lavage using a scope were evaluated. Sampling was performed in the summer and winter seasons. Normal value (Mean ± SEM) for total cell, macrophage, lymphocyte, neutrophil, epithelial, and bronchial cells in BAL sampled in summer were (343.75 ± 30.23), ) 24.50 ± 2.62), (2.81 ± 0.51), (1.43 ± 0.88), and (3.12 ± 0.32), respectively. The normal values for the total cell, macrophage, lymphocyte, neutrophil, epithelial, and bronchial cells in BAL sampled in winter were (355.55 ± 37.67), (59.11 ± 4.30), (21.33 ± 3.10), (3.88 ± 1.07), (8.88 ± 3.78), and (6.33 ± 1.44), respectively. No significant change in the percentage of neutrophils was detected between seasons, although the percentages of bronchial and epithelial cells in winter were significantly high (p < 0.05). Except for the mentioned cases, neither the total cell number nor the percentage differential cell populations of BAL changed significantly (p < 0.05) in different sampling methods and seasons. Normal BAL profiles in the Gezel breed were determined and could be used in result interpretations. Also, both sampling methods can be used without significantly affecting the results.

Prognostic factors associated with mortality in acute exacerbations of idiopathic pulmonary fibrosis: A systematic review and meta-analysis

BackgroundAcute exacerbations of idiopathic pulmonary fibrosis (AE-IPF) increases mortality risk, but which factors increase mortality is unknown. We aimed to perform a prognostic review of factors associated with mortality in patients with IPF. Study designand methods: We searched MEDLINE, EMBASE, and CINAHL for studies that reported on the association between any prognostic factor and AE-IPF. We assessed risk of bias using the QUIPS tool. We conduced pairwise meta-analyses using REML heterogeneity estimator, and GRADE approach to assess the certainty of the evidence. ResultsWe included 35 studies in our analysis. We found that long-term supplemental oxygen at baseline (aHR 2.52 [95 % CI 1.68 to 3.80]; moderate certainty) and a diagnosis of IPF compared to non-IPF ILD (aHR 2.19 [95 % CI 1.22 to 3.92]; moderate certainty) is associated with a higher risk of death in patients with AE-IPF. A diffuse pattern on high resolution computed tomography (HRCT) compared to a non-diffuse pattern (aHR 2.61 [95 % CI 1.32 to 2.90]; moderate certainty) is associated with a higher risk of death in patients with AE-IPF. We found that using corticosteroids prior to hospital admission (aHR 2.19 [95 % CI 1.26 to 3.82]; moderate certainty) and those with increased neutrophils (by % increase) in bronchoalveolar lavage (BAL) during the exacerbation is associated with a higher risk of death (aHR 1.02 [1.01 to 1.04]; moderate certainty). InterpretationOur results have implications for healthcare providers in making treatment decisions and prognosticating the clinical trajectory of patients, for researchers to design future interventions to improve patient trajectory, and for guideline developers in making decisions about resource allocation.

2599. Impact of Alveolar Neutrophilia on Outcomes in Patients with Suspected Ventilated Hospital-Acquired Bacterial Pneumonia

Abstract Background Ventilated hospital-acquired bacterial pneumonia (vHABP) is a major source of morbidity &amp; mortality in hospitalized patients. Clinical risk scores and systemic biomarkers have limited clinical &amp; prognostic utility in vHABP. Alveolar biomarkers—specifically alveolar neutrophilia (AN), the percentage of polymorphonuclear cells (PMN%) present in bronchoalveolar lavage (BAL) fluid—gauge the host inflammatory response directly at the site of infection, offering an attractive but untested predictive risk biomarker in vHABP. We aimed to generate preliminary data exploring AN as a vHABP biomarker. Methods We performed a retrospective cohort study of intensive care unit (ICU) patients with vHABP from 2010-2023. vHABP was defined as development of acute hypoxic respiratory failure requiring invasive mechanical ventilation, clinical suspicion of pneumonia by treating teams prompting performance of BAL respiratory culture, and BAL PMN% &amp;gt; 50%. Patient demographic characteristics, comorbidities and acute severity of illness metrics were abstracted via chart review. PMN% values were dichotomized using median split rounded to the nearest PMN% decile ( &amp;gt; or &amp;lt; 80% PMN% in BAL fluid). Study outcomes included ventilator-free days, ICU length of stay and vHABP-directed antibiotic treatment duration within 30 days of vHABP. Results 44 patients met study inclusion criteria. 30 (68%) had a BAL neutrophil of &amp;gt; 80%. Patients with BAL PMN &amp;gt; 80% had a numerically higher use of tracheostomy and trend towards lower P/F ratio at the time of vHABP diagnosis [Table 1]. BAL PMN &amp;gt; 80% was associated with a 15% decrease in ventilator-free days (p &amp;lt; 0.01) and a 25% longer ICU length of stay (p &amp;lt; 0.01) than those with BAL PMN &amp;lt; 80% [Figure 1]. There was no significant difference in post-BAL vHABP-directed antibiotic duration in patients with and without BAL PMN &amp;gt; 80%. Conclusion Though limited by low sample size, this exploratory study suggests AN may be a potentially useful predictive risk biomarker in vHABP. Larger-scale, prospective studies of alveolar biomarkers—including AN—in vHABP are warranted. Disclosures Owen Albin, MD, Charles River Laboratories: Advisor/Consultant|Shionogi: Advisor/Consultant

Disease progression in patients with usual interstitial pneumonia and probable UIP patterns on computed tomography with various underlying etiologies: a retrospective cohort study.

Usual interstitial pneumonia (UIP) is a pattern of interstitial pneumonia that is caused by different etiologies. This study aimed to investigate the transplant-free survival (TFS) and the decline in forced vital capacity (FVC) of the patients with UIP and probable UIP patterns on CT caused by various underlying conditions. A retrospective cohort study was conducted, enrolling patients with interstitial lung disease exhibiting a CT pattern consistent with UIP or probable UIP. Clinical and prognostic data of patients categorized by the etiology were compared. A total of 591 patients were included and classified into the following groups: idiopathic pulmonary fibrosis (IPF) (n = 320), connective tissue disease (CTD)-UIP (n = 229), asbestosis-UIP (n = 28), and hypersensitivity pneumonitis (HP)-UIP (n = 14). Advanced age, elevated levels of serum cytokeratin fraction 21-1 and percentage of neutrophils in bronchoalveolar lavage were observed in all groups. IPF patients showed a more rapid decline in FVC (133.9 mL/year) compared to CTD-UIP (24.5 mL/year, p = 0.001) and asbestosis-UIP (61.0 mL/year, p = 0.008) respectively. Sub-analysis of CTD-UIP revealed that patients with rheumatoid arthritis (RA)-UIP (88.1 mL/year) or antineutrophil cytoplasmic antibody-associated vasculitis (AAV)-UIP (72.9 mL/year) experienced a faster deterioration in FVC compared to those with primary Sjögren's syndrome (pSS)-UIP (25.9 mL/year, p < 0.05). Kaplan-Meier curves showed that IPF had the poorest TFS (median 55.9 months), followed by HP-UIP (57.5 months), CTD-UIP (66.7 months), and asbestosis-UIP (TFS not reached). RA-UIP or AAV-UIP did not exhibit any prognostic advantages compared to IPF, while asbestosis-UIP and pSS-UIP showed better survival rates. Patients with UIP caused by different underlying conditions share certain common features, but the trajectories of disease progression and survival outcomes differ.

Clinical Utility of Bronchoalveolar Lavage Neutrophilia and Biomarkers for Evaluating Severity of Chronic Fibrosing Interstitial Lung Diseases.

Introduction It is hypothesized that bronchoalveolar lavage (BAL) neutrophilia, Krebs von den Lungen-6 (KL-6), and C-reactive protein (CRP) predict the severity of chronic fibrosing interstitial lung diseases (CF-ILDs). Methods This cross-sectional study enrolled 30 CF-ILD patients. Using Pearson's correlation analysis, BAL neutrophils, KL-6, and CRP were correlated with forced vital capacity (FVC), diffusing lung capacity for carbon monoxide (DLCO), six-minute walk distance (6MWD), partial pressure of oxygen (PaO2), computed tomography fibrosis score (CTFS), and pulmonary artery systolic pressure (PASP). Using the receiver operator characteristic (ROC) curve, BALKL-6and CRP were evaluated against FVC% and DLCO% in isolation and combination with BAL neutrophilia for predicting the severity of CF-ILDs. Results BAL neutrophilia significantly correlated only with FVC% (r = -0.38, P = 0.04) and DLCO% (r = -0.43, P = 0.03). BAL KL-6 showed a good correlation with FVC% (r = -0.44, P < 0.05) and DLCO% (r = -0.50, P = 0.02), while BAL CRP poorly correlated with all parameters (r = 0.0-0.2). Subset analysis of BAL CRP in patients with CTFS ≤ 15 showed a better association with FVC% (r = -0.28, P = 0.05)and DLCO% (r = -0.36, P = 0.04). BAL KL-6 cut-off ≥ 72.32 U/ml and BAL CRP ≥ 14.55 mg/L predicted severe disease with area under the curve (AUC) values of 0.77 and 0.71, respectively. The combination of BAL neutrophilia, KL-6, and CRP predicted severity with an AUC value of 0.89. Conclusion The combination of BAL neutrophilia, KL-6, and CRP facilitates the severity stratification of CF-ILDs complementing existing severity parameters.

Bronchoscopy for refractory/unexplained cough with mucus

IntroductionRefractory/unexplained cough (RUCC) is typically associated with throat symptoms and a dry cough. Some patients attending specialist cough clinics however, report sputum production (>1 tablespoon daily) and atypical sensations (urge-to-cough in chest). Bronchoscopy findings in this specific cohort have rarely been described. AimsWe aimed to evaluate bronchoscopy, bronchoalveolar lavage (BAL) cell differential and microbiology findings in RUCC with mucus production. MethodsWe retrospectively reviewed case notes, procedure results and treatment of patients undergoing bronchoscopy for RUCC with more than a tablespoon of sputum daily. ResultsData were included from 54 patients with RUCC, normal or trivial findings on CT (Computerised Tomography) imaging and no response to guideline-directed treatment of their cough. Most (84%) patients had BAL neutrophilia and excessive dynamic airway collapse (EDAC) was seen in 31%. Treatment strategies in these patients differed to those adopted in typical RUCC associated with a dry cough. Management was influenced or changed in 48/54 (89%) of the patients undergoing bronchoscopy. Conclusions: Bronchoscopy provides high diagnostic value in RUCC with mucus production (>1 tbsp daily), identifying specific treatable traits including neutrophilic airway inflammation and EDAC.

Medical causes of poor performance and their associations with fitness in Standardbred racehorses.

Poor performance is a multifactorial syndrome of racehorses, commonly associated with subclinical disorders, which can be diagnosed by exercise testing. Describe the prevalence of medical causes of poor performance in Standardbreds unassociated with lameness, and evaluate their relationships with fitness variables measured by exercise treadmill test. Hospital population of 259 nonlame Standardbred trotters referred for poor performance. The horses' medical records were retrospectively reviewed. Horses underwent a diagnostic protocol including resting examination, plasma lactate concentration, treadmill test with continuous ECG and assessment of fitness variables, creatine kinase activity, treadmill endoscopy, postexercise tracheobronchoscopy, bronchoalveolar lavage (BAL), and gastroscopy. The prevalence of different disorders was evaluated, including cardiac arrhythmias, exertional myopathies, dynamic upper airway obstructions (DUAOs), exercise-induced pulmonary hemorrhage (EIPH), moderate equine asthma (MEA), and gastric ulcers (EGUS). The associations of these disorders with fitness variables were investigated individually and using multivariable models. Moderate equine asthma and EGUS were the most common disorders, followed by EIPH, DUAOs, cardiac arrhythmias, and exertional myopathies. Hemosiderin score was positively correlated with BAL neutrophils, eosinophils, and mast cells; increased creatine kinase activity was associated with BAL neutrophilia, DUAOs, premature complexes, and squamous gastric disease. Treadmill velocity at a plasma lactate concentration of 4 mmol/L and at heart rate of 200 beats per minute was negatively affected by BAL neutrophilia, multiple DUAOs, exertional myopathies, and squamous gastric disease. The multifactorial nature of poor performance was confirmed, with MEA, DUAOs, myopathies and EGUS representing the main diseases involved in fitness impairment.

Multiple Dosing and Preactivation of Mesenchymal Stromal Cells Enhance Efficacy in Established Pneumonia Induced by Antimicrobial-Resistant Klebsiella pneumoniae in Rodents.

Antimicrobial-resistant (AMR) bacteria, such as Klebsiella species, are an increasingly common cause of hospital-acquired pneumonia, resulting in high mortality and morbidity. Harnessing the host immune response to AMR bacterial infection using mesenchymal stem cells (MSCs) is a promising approach to bypass bacterial AMR mechanisms. The administration of single doses of naïve MSCs to ARDS clinical trial patient cohorts has been shown to be safe, although efficacy is unclear. The study tested whether repeated MSC dosing and/or preactivation, would attenuate AMR Klebsiella pneumonia-induced established pneumonia. Rat models of established K. pneumoniae-induced pneumonia were randomised to receive intravenous naïve or cytomix-preactivated umbilical cord MSCs as a single dose at 24 h post pneumonia induction with or without a subsequent dose at 48 h. Physiological indices, bronchoalveolar lavage (BAL), and tissues were obtained at 72 h post pneumonia induction. A single dose of naïve MSCs was largely ineffective, whereas two doses of MSCs were effective in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, while reducing bacteria and injury in the lung. Cytomix-preactivated MSCs were superior to naïve MSCs. BAL neutrophil counts and activation were reduced, and apoptosis increased. MSC therapy reduced cytotoxic BAL T cells, and increased CD4+/CD8+ ratios. Systemically, granulocytes, classical monocytes, and the CD4+/CD8+ ratio were reduced, and nonclassical monocytes were increased. Repeated doses of MSCs-particularly preactivated MSCs-enhance their therapeutic potential in a clinically relevant model of established AMR K. pneumoniae-induced pneumosepsis.

Rat bronchoalveolar lavage proteome changes following e-cigarette aerosol exposures.

E-cigarette liquids are complex mixtures of chemicals consisting of humectants, such as propylene glycol (PG) and vegetable glycerin (VG), with nicotine or flavorings added. Published literature emphasizes the toxicity of e-cigarette aerosols with flavorings whereas much less attention has been given to the biologic effects of humectants. The purpose of the current study was to provide a comprehensive view of the acute biologic effects of e-cigarette aerosols on rat bronchoalveolar lavage (BAL) using mass spectrometry-based global proteomics. Sprague-Dawley rats were exposed to e-cigarette aerosol for 3 h/day for three consecutive days. Groups included: PG/VG alone, PG/VG + 2.5% nicotine (N), or PG/VG + N + 3.3% vanillin (V). Right lung lobes were lavaged for BAL and supernatants prepared for proteomics. Extracellular BAL S100A9 concentrations and BAL cell staining for citrullinated histone H3 (citH3) were also performed. From global proteomics, ∼2,100 proteins were identified from rat BAL. The greatest change in number of BAL proteins occurred with PG/VG exposures alone compared with controls with biological pathways enriched for acute phase responses, extracellular trap formation, and coagulation. Extracellular BAL S100A9 concentrations and the number of citH3 + BAL cells also increased significantly in PG/VG and PG/VG + 2.5% N. In contrast to PG/VG or PG/VG + N, the addition of vanillin to PG/VG + N increased BAL neutrophilia and downregulated lipid transport proteins. In summary, global proteomics support e-cigarette aerosol exposures to PG/VG alone as having a significant biologic effect on the lung independent of nicotine or flavoring with increased markers of extracellular trap formation.

Triple-tyrosine kinase inhibition by BIBF1000 attenuates airway and pulmonary arterial remodeling following chronic allergen challenges in mice

BackgroundAirway remodeling is an important pathological feature of chronic airway diseases, which leads to a progressive decline in lung function. The present study examined the anti-remodeling and anti- inflammatory effect of BIBF1000, a triple-tyrosine kinase inhibitor that targets VEGF, PDGF, and FGF receptor signaling in a mouse model of repeated ovalbumin (OVA) challenges.MethodsFemale Balb-c mice were immunized intraperitoneally on days 0 and 12 with 50 µg ovalbumin plus 1 mg of Al(OH)3 in 200 μl saline. Intranasal OVA challenges (20 µg/50 µl in PBS) were administered on days 26, 29, and 31, and were repeated twice a week for 3 months. Animals received vehicle or BIBF1000 (25 mg/kg, b.i.d.) through gavage from day 26 to the end of fourth month. On day 120, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immunohistological analysis.ResultsCompared to vehicle controls, treatment with BIBF1000 reduced the numbers of BAL eosinophils, macrophages, neutrophils, and lymphocytes by 70.0%, 57.9%, 47.5%, and 63.0%, respectively, and reduced IL-5 and IL-13 in BAL. Treatment with BIBF1000 reduced airway mucus secretion, peribronchial fibrosis, small airway, and pulmonary arterial wall thickness, compared to vehicle controls. Furthermore, treatment with BIBF1000 also reduced the expression of inflammatory mediators (TNF-α, IL-1β, IL-5, IL-13, MMP-2, MMP-9, COX-2, and iNOS) and inhibited ERK and AKT phosphorylation.ConclusionsThe protective effect afforded by triple-tyrosine kinase inhibition with BIBF1000 in reducing allergen-induced airway and arterial remodeling was associated with down-regulation of inflammatory mediators, as well as inhibition of ERK and AKT signaling pathways.

Long-term clinical course and outcomes of patients with microscopic polyangiitis-associated interstitial lung disease.

Background: Interstitial lung disease (ILD) is a significant complication associated with microscopic polyangiitis (MPA) that has a poor prognosis. However, the long-term clinical course, outcomes, and prognostic factors of MPA-ILD are not well defined. Hence, this study aimed to investigate the long-term clinical course, outcomes, and prognostic factors in patients with MPA-ILD. Methods: Clinical data of 39 patients with MPA-ILD (biopsy proven cases, n = 6) were retrospectively analyzed. High resolution computed tomography (HRCT) patterns were assessed based on the 2018 idiopathic pulmonary fibrosis diagnostic criteria. Acute exacerbation (AE) was defined as the worsening of dyspnea within 30days, with new bilateral lung infiltration that is not fully explained by heart failure or fluid overload and that does not have identified extra-parenchymal causes (pneumothorax, pleural effusion, or pulmonary embolism). Results: The median follow-up period was 72.0months (interquartile range: 44-117months). The mean age of the patients was 62.7years and 59.0% were male. Usual interstitial pneumonia (UIP) and probable usual interstitial pneumonia patterns on high resolution computed tomography were identified in 61.5 and 17.9% of the patients, respectively. During the follow-up, 51.3% of patients died, and the 5- and 10-year overall survival rates were 73.5% and 42.0%, respectively. Acute exacerbation occurred in 17.9% of the patients. The non-survivors had higher neutrophil counts in bronchoalveolar lavage (BAL) fluid and more frequent acute exacerbation than the survivors. In the multivariable Cox analysis, older age (hazard ratio [HR], 1.07; 95% confidence interval [CI], 1.01-1.14; p = 0.028) and higher BAL counts (HR, 1.09; 95% CI, 1.01-1.17; p = 0.015) were found to be the independent prognostic factors associated with mortality in patients with MPA-ILD. Conclusion: During the 6years-follow-up, about half of patients with MPA-ILD died and approximately one-fifth experienced acute exacerbation. Our results suggest that older age and higher BAL neutrophil counts mean poor prognosis in patients with MPA-ILD.

Gastrointestinal factors associated with risk of bronchiectasis in children.

To evaluate gastrointestinal (GI) risk factors for bronchiectasis in children. We hypothesized that upper GI tract dysmotility would be associated with increased risk of bronchiectasis. Subjects in this retrospective cohort study included those evaluated for persistent pulmonary symptoms in the Aerodigestive Center at Boston Children's Hospital who underwent chest computed tomography (CT) between 2002 and 2019. To determine gastrointestinal predictors of bronchiectasis, baseline characteristics, comorbidities, enteral tube status, medications received, gastroesophageal reflux burden, adequacy of swallow function, esophageal dysmotility, gastric dysmotility, and neutrophil count on bronchoalveolar lavage (BAL) were compared between patients with and without bronchiectasis. Proportions were compared with Fisher's exact test and binary logistic regression with stepwise selection was used for multivariate analysis. ROC analyses were utilized to compare BAL neutrophils and bronchiectasis. Of 192 subjects, 24% were found to have evidence of bronchiectasis on chest CT at age 7.9 ± 0.5 years. Enteral tubes (OR 5.77, 95% CI 2.25-14.83, p < 0.001) and increased BAL neutrophil count (OR 5.79, 95% CI 1.87-17.94, p = 0.002) were associated with increased risk while neurologic comorbidities were associated with decreased risk (OR 0.24, 95% CI 0.09-0.66, p = 0.006). Gastroesophageal reflux was not found to be a significant risk factor. Neutrophil counts >10% had 72% sensitivity and 60% specificity for identifying bronchiectasis. Enteral tubes were associated with significantly increased risk of bronchiectasis but gastroesophageal reflux was not. Providers should consider obtaining chest CT to evaluate for bronchiectasis in children found to have unexplained elevated BAL neutrophil count.

478. Lung Hybrid Neutrophils and Subsequent Extracellular Traps Are Protective In COVID-19-Associated Pulmonary Aspergillosis

Abstract Background COVID-19-associated pulmonary aspergillosis (CAPA) is a severe superinfection with the fungus Aspergillus frequently affecting critically ill COVID-19 patients. Pathophysiological insight, key to improve diagnostic and immunomodulatory therapeutic options, is lacking. Methods We performed single-cell RNA sequencing (scRNA-seq) on 37 bronchoalveolar lavage (BAL) samples from 37 critically ill COVID-19 patients. Three groups were defined: patients who did not develop aspergillosis (COVID-19-only, n=22), CAPA patients with sampling &amp;lt; 5 days after CAPA diagnosis (early CAPA, n=6) and CAPA patients with sampling 5-11 days after CAPA diagnosis (late CAPA, n=9). All CAPA patients had probable/proven CAPA according to the 2020 ECMM/ISHAM consensus criteria. Additionally, we assessed neutrophil extracellular trap (NET) levels in a separate cohort of 33 biobanked COVID-19-only BAL samples and 24 early CAPA samples. Results A total of 69008 cells passed quality filtering. CAPA patients had significantly lower BAL neutrophil proportions than COVID-19-only patients, particularly in early CAPA (Fig. 1A). Pseudotime inference revealed two neutrophil trajectories: a regular maturation trajectory, and a trajectory giving rise to “hybrid” neutrophils which express genes encoding proteins with antigen-presenting functions (Fig. 1B). The latter trajectory was dominant in CAPA patients (Fig. 1C). NETosis analyses revealed significantly higher levels of citrullinated histone H3 DNA complexes (H3Cit-DNA) in CAPA patients (Fig. 2A). This explains the low CAPA BAL neutrophil proportions, as neutrophils that underwent NETosis are no longer detected via scRNA-seq. CAPA patients with the lowest H3Cit-DNA levels had significantly decreased survival rates (Fig. 2B). Figure 1:Bronchoalveolar lavage fluid (BALF) neutrophil proportions are significantly decreased in CAPA patients, accompanied by a shift to hybrid neutrophil formation. Panel (A): BALF neutrophil proportions as analyzed by single-cell RNA sequencing using the Seurat R package are significantly lower in CAPA patients compared to COVID-19-only patients. Patients with early CAPA have significantly lower BALF neutrophil proportions than patients with late CAPA. Macrophage/monocyte and epithelial cell proportions are reciprocally increased in CAPA patients compared to COVID-19-only patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Panel (B): Two trajectories are defined using pseudotime inference calculated using the Slingshot R package: a trajectory dominant in COVID-19-only patients with regular maturation of progenitor neutrophils, and a trajectory dominant in CAPA patients with maturation towards a ‘hybrid neutrophil’ state, with neutrophils expression genes encoding proteins with functions in antigen presentation. Subsequently, the hybrid neutrophil proportion is significantly higher in CAPA patients compared to COVID-19-only patients, and is significantly higher in patients with early CAPA than those with late CAPA. The mature neutrophil proportion is reciprocally reduced in CAPA patients. P-values shown for differences between the pooled CAPA patients and the COVID-19-only patients. P-values were calculated using a generalized linear model correcting for age, Charlson Comorbidity Index at hospital admission, and administration of corticosteroids (prednisone equivalent dose 20 mg or higher) within 48 hours of BALF sampling. Figure 2:Neutrophil extracellular trap (NET) levels are increased in early CAPA and are associated with increased survival in CAPA patients specifically. Panel (A): Myeloperoxidase (MPO) DNA levels were analyzed as measure for general NET-formation, while citrullinated histone H3 bound DNA (H3Cit-DNA) levels were analyzed as more specific PAD4-dependent NET-formation, in BALF samples from early CAPA and COVID-19-only patients. A trend towards higher MPO-DNA levels was found in early CAPA patients, while H3Cit-DNA levels were significantly higher in early CAPA compared to COVID-19-only patients. P-values calculated using Mann-Whitney U test. Panel (B): Kaplan-Meier analysis of patients with NETosis analyses, divided in early CAPA and COVID-19-only patients and subdivided according to H3Cit-DNA levels (cut-off at 20000 ng/mL for early CAPA and at 8000 ng/mL for COVID-19-only). Log-rank test was used to compare survival distributions. For the comparison early CAPA (low H3Cit-DNA) versus early CAPA (high H3Cit-DNA), the log-rank p-value was 0.033. Conclusion CAPA patients display extremely high levels of released NETs in the lower respiratory tract, associated with a shift from the normal neutrophil maturation process towards “hybrid neutrophil” formation, probably upon encountering the fungus. In contrast to high NETosis contributing to mortality in severe COVID-19, CAPA patients likely require these NETs to survive aspergillosis. BAL NET levels hold promise as a tool to guide diagnosis, prognosis and treatment in these patients. Disclosures Simon Feys, MD, Pfizer: Travel support Katrien Lagrou, PharmD, PhD, FUJIFILM Wako: Speaker fee|Gilead: Advisor/Consultant|Gilead: Speaker fee|MRM Health: Advisor/Consultant|MSD: Advisor/Consultant|Pfizer: Speaker fee|Thermo fisher Scientific: Service fee Joost Wauters, MD, PhD, Gilead: Grant/Research Support|Gilead: Speaker's fee, travel support, advisory board|MSD: Grant/Research Support|MSD: Speaker's fee, travel support, posaconazole for interventional trial|Pfizer: Grant/Research Support|Pfizer: Speaker's fee, travel support, advisory board.

S3.3d Influenza versus COVID-19-associated pulmonary aspergillosis: Profiling lower respiratory tract epithelial and myeloid innate immunity in patient samples

S3.3 Innate immune responses to pathogenic fungi, September 21, 2022, 4:45 PM - 6:15 PM ObjectivesUp to 20% and 15% of critically ill influenza and coronavirus disease 2019 (COVID-19) patients are affected by influenza- and COVID-19-associated pulmonary aspergillosis (IAPA and CAPA) respectively. These viral-fungal coinfections are difficult to diagnose and are associated with increased mortality. Mechanistic insights into the development of IAPA and CAPA are a prerequisite for the development of new biomarkers and novel immunomodulatory therapeutic targets. However, data on the pathophysiology are scarce. With this study, we aimed at expanding our knowledge of IAPA and CAPA pathophysiology in an explorative way, resorting to lower respiratory tract samples and focusing on the epithelial and myeloid innate immunity components of the antifungal host response.MethodsWe performed nCounter gene expression analyses of 755 genes linked to innate immunity, and determined protein levels of 47 cytokines, chemokines, growth factors, and other inflammatory mediators on bronchoalveolar lavage (BAL) fluid samples from 166 ICU-admitted influenza and COVID-19-patients with or without aspergillosis. Additionally, we performed spatial transcriptomics and RNAscope on in vivo tracheobronchial biopsies from four IAPA and CAPA patients.ResultsSeveral genes encoding proteins with important effector functions in antifungal immunity are downregulated in BAL fluid of IAPA and CAPA patients compared with influenza-only or COVID-19-only patients. Cellular deconvolution of the gene expression data reveals a significantly lower BAL neutrophil fraction in CAPA patients compared to COVID-19-only patients.IAPA and CAPA patients have high BAL fluid levels of pro-inflammatory cytokines, but these are not significantly different from the levels seen in influenza-only and COVID-19-only patients. By integrating the BAL fluid cytokine levels with their respective transcriptional responses, we show that IAPA patients, and to a lesser extent CAPA patients, have an aberrant transcriptional response to pro-inflammatory cytokines as well as type I and type II interferons, which may result in poor cellular effector functions (Fig. 1a). Interferon-gamma signaling is abrogated in both IAPA and CAPA patients when compared with influenza-only and COVID-19-only patients.We observe significantly higher levels of growth factors associated with lung fibrosis in both IAPA and CAPA BAL fluid, which may contribute to the higher mortality seen in these coinfections (Fig. 1b).Using spatial transcriptomics, we show that different epithelial defense mechanisms are at play in IAPA and CAPA (Fig. 2a).Finally, using RNAscope ultrasensitive single-molecule RNA in situ hybridization, we visualize fungal and viral co-localization in CAPA tracheobronchial tissue, proving that virus-induced epithelial barrier disruption paves the way for tissue-invasive aspergillosis (Fig. 2b).ConclusionUsing state-of-the-art techniques in lower respiratory tract samples obtained from a large representative patient cohort, we provide arguments for a three-level breach in antifungal immunity in IAPA and CAPA. A hampered ability to phagocytize and kill fungal spores enables Aspergillus germination and growth, leading to hyphae that are not contained because of restrained extracellular defense mechanisms. These hyphae may easily become tissue-invasive through an epithelium that is weakened by the viral infection, causing detrimental damage to the respiratory system. Functional studies will be necessary to further unravel the pathophysiology of IAPA and CAPA.

Smoking status impacts treatment efficacy in smoke-induced lung inflammation: A pre-clinical study.

Rationale: Smoking status and smoking history remain poorly accounted for as variables that could affect the efficacy of new drugs being tested in chronic obstructive pulmonary disease (COPD) patients. As a proof of concept, we used a pre-clinical model of cigarette smoke (CS) exposure to compare the impact of treatment during active CS exposure or during the cessation period on the anti-inflammatory effects IL-1α signaling blockade. Methods: Mice were exposed to CS for 2 weeks, followed by a 1-week cessation, then acutely re-exposed for 2 days. Mice were treated with an anti-IL-1α antibody either during CS exposure or during cessation and inflammatory outcomes were assessed. Results: We found that mice re-exposed to CS displayed reduced neutrophil counts and cytokine levels in the bronchoalveolar lavage (BAL) compared to mice exposed only acutely. Moreover, we found that treatment with an anti-IL-1α antibody during the initial CS exposure delayed inflammatory processes and interfered with pulmonary adaptation, leading to rebound pulmonary neutrophilia, increased BAL cytokine secretion (CCL2) and upregulated Mmp12 expression. Conversely, administration of anti-IL-1α during cessation had the opposite effect, improving BAL neutrophilia, decreasing CCL2 levels and reducing Mmp12 expression. Discussion: These results suggest that pulmonary adaptation to CS exposure dampens inflammation and blocking IL-1α signaling during CS exposure delays the inflammatory response. More importantly, the same treatment administered during cessation hastens the return to pulmonary inflammatory homeostasis, strongly suggesting that smoking status and treatment timing should be considered when testing new biologics in COPD.