Single-cell resolution of human airway epithelial cells exposed to bronchiolitis obliterans-associated chemicals.

Bronchiolitis obliterans (BO) is a fibrotic lung disease characterized by progressive luminal narrowing and obliteration of the small airways. In the nontransplant population, inhalation exposure to certain chemicals is associated with BO; however, the mechanisms contributing to disease induction remain poorly understood. This study's objective was to use single-cell RNA sequencing for the identification of transcriptomic signatures common to primary human airway epithelial cells after chemical exposure to BO-associated chemicals-diacetyl or nitrogen mustard-to help explain BO induction. Primary airway epithelial cells were cultured at air-liquid interface and exposed to diacetyl, nitrogen mustard, or control vapors. Cultures were dissociated and sequenced for single-cell RNA. Differential gene expression and functional pathway analyses were compared across exposures. In total, 75,663 single cells were captured and sequenced from all exposure conditions. Unbiased clustering identified 11 discrete phenotypes, including 5 basal, 2 ciliated, and 2 secretory cell clusters. With chemical exposure, the proportion of cells assigned to keratin 5+ basal cells decreased, whereas the proportion of cells aligned to secretory cell clusters increased compared with control exposures. Functional pathway analysis identified interferon signaling and antigen processing/presentation as pathways commonly upregulated after diacetyl or nitrogen mustard exposure in a ciliated cell cluster. Conversely, the response of airway basal cells differed significantly with upregulation of the unfolded protein response in diacetyl-exposed basal cells, not seen in nitrogen mustard-exposed cultures. These new insights provide early identification of airway epithelial signatures common to BO-associated chemical exposures.NEW & NOTEWORTHY Bronchiolitis obliterans (BO) is a devastating fibrotic lung disease of the small airways, or bronchioles. This original manuscript uses single-cell RNA sequencing for identifying common signatures of chemically exposed airway epithelial cells in BO induction. Chemical exposure reduced the proportion of keratin 5+ basal cells while increasing the proportion of keratin 4+ suprabasal cells. Functional pathways contributory to these shifts differed significantly across exposures. These new results highlight similarities and differences in BO induction across exposures.

IL-17A neutralization fails to attenuate airway remodeling and potentiates a proinflammatory lung microenvironment in diacetyl-exposed rats.

Bronchiolitis obliterans (BO) is a devastating lung disease that can develop following inhalation exposure to certain chemicals. Diacetyl (DA) is one chemical commonly associated with BO development when inhaled at occupational levels. Previous studies in rats have shown that repetitive DA vapor exposures increased lung CD4+CD25+ T cells and bronchoalveolar (BAL) interleukin-17A (IL-17A) concentrations concurrent with the development of airway remodeling. We hypothesized that IL-17A neutralization would attenuate the severity of airway remodeling after repetitive DA vapor exposures. Sprague-Dawley rats were exposed to 200 parts-per-million DA vapor or filtered air (RA) for 6 h/day × 5 days and monitored for 2 wk postexposure. Treatment with IL-17A neutralization (αIL-17A) or IgG (control) began immediately following exposures and continued twice weekly until study's end. Lungs were harvested for histology, flow cytometry, and BAL analyses. Survival, oxygen saturations, and percent weight change decreased significantly in DA-exposed versus RA-exposed rats, but did not differ significantly between DA + αIL-17A versus DA + IgG. Similarly, the number nor severity of airway lesions did not differ significantly between DA + αIL-17A versus DA + IgG rats despite the percentage of lung regulatory T cells increasing with decreased BAL IL-17A concentrations. Ashcroft scoring of the distal lung parenchyma suggested worse parenchymal remodeling in DA + αIL-17A versus DA + IgG rats with increased expression of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and nuclear factor-kappa B (NF-κB). Collectively, IL-17A neutralization in DA-exposed rats failed to attenuate airway remodeling with increased expression of pro-inflammatory cytokines TNF-α, IL-1ß, and NF-κB.NEW & NOTEWORTHY Interleukin-17A (IL-17A) neutralization has shown benefit previously in preclinical models of transplant-associated bronchiolitis obliterans (BO), yet it remains unknown whether IL-17A neutralization has similar benefit for other forms of BO. Here, IL-17A neutralization fails to prevent severe airway remodeling in rats exposed repetitively to the flavoring chemical diacetyl, and instead, promotes a proinflammatory microenvironment with increased expression of TNF-α, IL-1ß, and NF-κB within the lung.

[Two cases of airway dysfunction related to diacetyl exposure].

Occupational exposure to diacetyl can lead to bronchiolitis obliterans. In this paper, two patients with severe obstructive ventilation disorder who were exposed to diacetyl at a fragrance and flavours factory were analyzed. The clinical manifestations were cough and shortness of breath. One of them showed Mosaic shadows and uneven perfusion in both lungs on CT, while the other was normal. Field investigation found that 4 of the 8 workers in the factory were found to have obstructive ventilation disorder, and 2 had small airway dysfunction. This paper summarizes the diagnostic process of patients in order to improve the understanding of airway dysfunction caused by occupational exposure to diacetyl and promote the development of relevant standards.

p14ARF ameliorates inflammation and airway remodeling in nitric acid aerosol inhalation-induced bronchiolitis obliterans.

BACKGROUND: To investigate the protective effect of p14ARF in a nitric acid (NA) aerosol inhalation-induced bronchiolitis obliterans (BO) mouse model and its potential regulatory mechanism. METHODS: A BO mouse model was established by NA aerosol inhalation. The expressions of p14ARF, phosphatidylinositol-3-kinase (PI3K), and protein kinase B (AKT) were detected by quantitative reverse transcription PCR (qRT-PCR) and western blot (WB). Hematoxylin (HE) staining, Masson staining, and periodic acid-Schiff (PAS) staining observed pulmonary histological changes. TdT-mediated dUTP nick end labeling (TUNEL) staining detected pulmonary cell apoptosis, and enzyme-linked immunosorbent assay (ELISA) measured matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), interleukon-6 (IL-6), and transforminh growth factor-ß (TGF-ß) levels in lung tissue and bronchoalveolar lavage fluid (BALF). RESULTS: The expressions of p14ARF, PI3K, and AKT showed a time gradient change, with a decrease trend (*P < 0.05 and **P < 0.01). Severe inflammatory infiltration and tracheal fibrosis were found in lung tissue in the modeling group (BO group) compared with the control group (Con group). The pH, PaO2, and PaO2/FiO2 values significantly reduced, while the PaCO2 value and the number of TUNEL-positive cells increased in BO group (P < 0.05). In addition, MMP-2, MMP-9, IL-6, and TGF-ß levels remarkably increased, with an increase in the number of white blood cells, neutrophils, and lymphocytes in BO group (P < 0.05). Furthermore, p14ARF up-regulation reversed the trend of the aforementioned indexes in BO mice. CONCLUSIONS: p14ARF ameliorated the inflammatory response and airway remodeling in a BO mouse model via the PI3K/AKT pathway.

Inhalational Constrictive Bronchiolitis: The Evolution of our Understanding of this Disease.

The case definition of inhalational constrictive bronchiolitis (CB) has changed over the generations. We identify changes in the description of this illness over time associated with different exposures and present the natural history of CB in a case attributed to military burn pit exposure. The initial descriptions of this disease began with nitric acid spills and silage exposures. In these events, there was an acute exposure, typically a short-term resolution of the adverse respiratory events, and then a progression, leading to disability or a respiratory death. The life-saving role of corticosteroid therapy in this situation was recognized. War gas exposures of World War I and then Saddam Hussein's use of sulfur mustard gas in the Iran-Iraq War followed. More recently the findings associated with diacetyl exposure in commercial popcorn workers remained consistent with previously described presentations, but then the clinical presentation in troops returning from deployment to Southwest Asia was very different, yet with the same histologic findings. We recognize unreconciled disparities in the clinical, physiologic, and imaging presentation in those with inhalational bronchiolitis and acknowledge this as perhaps one of the difficult diagnoses in respiratory medicine.

Case Report: Flavoring-Related Lung Disease in a Coffee Roasting and Packaging Facility Worker With Unique Lung Histopathology Compared With Previously Described Cases of Obliterative Bronchiolitis.

Occupational exposure to diacetyl, a butter flavor chemical, can result in obliterative bronchiolitis. Obliterative bronchiolitis is characterized by exertional dyspnea, fixed airflow obstruction, and histopathologic constrictive bronchiolitis, with bronchiolar wall fibrosis leading to luminal narrowing and obliteration. We describe a case of advanced lung disease with histopathology distinct from obliterative bronchiolitis in a 37-year-old male coffee worker following prolonged exposure to high levels of diacetyl and the related compound 2,3-pentanedione, who had no other medical, avocational, or occupational history that could account for his illness. He began working at a coffee facility in the flavoring room and grinding area in 2009. Four years later he moved to the packaging area but continued to flavor and grind coffee at least 1 full day per week. He reported chest tightness and mucous membrane irritation when working in the flavoring room and grinding area in 2010. Beginning in 2014, he developed dyspnea, intermittent cough, and a reduced sense of smell without a work-related pattern. In 2016, spirometry revealed a moderate mixed pattern that did not improve with bronchodilator. Thoracoscopic lung biopsy results demonstrated focal mild cellular bronchiolitis and pleuritis, and focal peribronchiolar giant cells/granulomas, but no evidence of constrictive bronchiolitis. Full-shift personal air-samples collected in the flavoring and grinding areas during 2016 measured diacetyl concentrations up to 84-fold higher than the recommended exposure limit. Medical evaluations indicate this worker developed work-related, airway-centric lung disease, most likely attributable to inhalational exposure to flavorings, with biopsy findings not usual for obliterative bronchiolitis. Clinicians should be aware that lung pathology could vary considerably in workers with suspected flavoring-related lung disease.

Repetitive diacetyl vapor exposure promotes ubiquitin proteasome stress and precedes bronchiolitis obliterans pathology.

Bronchiolitis obliterans (BO) is a devastating lung disease seen commonly after lung transplant, following severe respiratory tract infection or chemical inhalation exposure. Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha-diketone known to cause BO when inhaled, however, the mechanisms of how inhalation exposure leads to BO development remains poorly understood. In the current work, we combined two clinically relevant models for studying the pathogenesis of DA-induced BO: (1) an in vivo rat model of repetitive DA vapor exposures with recovery and (2) an in vitro model of primary human airway epithelial cells exposed to pure DA vapors. Rats exposed to 5 consecutive days 200 parts-per-million DA 6 h per day had worsening survival, persistent hypoxemia, poor weight gain, and histologic evidence of BO 14 days after DA exposure cessation. At the end of exposure, increased expression of the ubiquitin stress protein ubiquitin-C accumulated within DA-exposed rat lung homogenates and localized primarily to the airway epithelium, the primary site of BO development. Lung proteasome activity increased concurrently with ubiquitin-C expression after DA exposure, supportive of significant proteasome stress. In primary human airway cultures, global proteomics identified 519 significantly modified proteins in DA-exposed samples relative to controls with common pathways of the ubiquitin proteasome system, endosomal reticulum transport, and response to unfolded protein pathways being upregulated and cell-cell adhesion and oxidation-reduction pathways being downregulated. Collectively, these two models suggest that diacetyl inhalation exposure causes abundant protein damage and subsequent ubiquitin proteasome stress prior to the development of chemical-induced BO pathology.

Bronchiolitis obliterans following toxic epidermal necrolysis: a case report.

BACKGROUND: Toxic epidermal necrolysis (TEN) is a rare, acute and potentially fatal skin condition usually induced by drugs. Although much attention is focused on the life threatening acute cutaneous and sight threatening ocular manifestations of this disease, chronic pulmonary complications like bronchiolitis obliterans are occasionally encountered. However, little is known about its incidence, pathogenesis, clinical course and outcome in children recovering from TEN. CASE PRESENTATION: We report a five-year-old boy who presented four months after the first manifestation of drug-induced TEN with cough and shortness of breath and was subsequently diagnosed with bronchiolitis obliterans. He was treated with supportive therapy that improved his hypercapnia allowing him to be discharged on domiciliary oxygen, chest physiotherapy and bronchodilators. CONCLUSIONS: This case highlights the need to be vigilant for adverse drug reactions and consider chronic pulmonary complications like Bronchiolitis Obliterans in children recovering from TEN.

[Advances in flavoring-related bronchiolitis obliterans].

Occupational exposure to the diacetyl flavoring may cause bronchiolitis obliterans (BO) in workers. The first case of flavoring-related bronchiolitis obliterans was reported in 2002. Since then, similar cases have been identified among workers in various production industries in some countries and regions. At present, there are no cases reported in China. In order to improve the awareness of the disease and promote the prevention work, this article reviews the research progress of case recognition, risk factors, clinical manifestation and possible pathogenesis.

SP-A and TLR4 localization in lung tissue of SM-exposed patients.

INTRODUCTION: Long-term pulmonary complications are one of the major long-term consequences of sulfur mustard (SM) exposure. Toll-like receptor 4 (TLR4) involves in the pathogenesis of several pulmonary disorders. Surfactant protein-A (SP-A) regulates LPS-induced TLR4 localization and activation responses. However, the intensity and significance of TLR4 and SP-A expression by lung cells in SM-exposed patients is not clear. METHODS: The gene expression of TLR4 (through real-time PCR) and TLR4 and SP-A positive cells and alveolar type II cells, as SP-A producers, (using IHC) were assessed in formalin fixed paraffin embedded (FFPE) specimens from SM-exposed (n = 17), and non-SM exposed individuals (n = 12). RESULTS: TLR4 gene expression did not change between study groups. However, its cell surface presentation was significantly reduced in SM-exposed patients and particularly in which with constrictive bronchiolitis compared with the control group (P < 0.001 and P = 0.002, respectively). Frequency of alveolar type II cells was lower in the case group rather than the control group while the number of SP-A positive cells did not alter. CONCLUSIONS: These findings suggest that reduced TLR4 cell surface presentation may have anti-inflammatory function and SP-A may have a critical role in regulation of inflammatory responses in SM-exposed patients. Further investigation on other possible mechanisms involved in TLR4 internalization maybe help to illustrate the modulatory or inflammatory activity of TLR4 in these patients.

Flavorings-Related Lung Disease: A Brief Review and New Mechanistic Data.

Flavorings-related lung disease is a potentially disabling and sometimes fatal lung disease of workers making or using flavorings. First identified almost 20 years ago in microwave popcorn workers exposed to butter-flavoring vapors, flavorings-related lung disease remains a concern today. In some cases, workers develop bronchiolitis obliterans, a severe form of fixed airways disease. Affected workers have been reported in microwave popcorn, flavorings, and coffee production workplaces. Volatile α-dicarbonyl compounds, particularly diacetyl (2,3-butanedione) and 2,3-pentanedione, are implicated in the etiology. Published studies on diacetyl and 2,3-pentanedione document their ability to cause airway epithelial necrosis, damage biological molecules, and perturb protein homeostasis. With chronic exposure in rats, they produce airway fibrosis resembling bronchiolitis obliterans. To add to this knowledge, we recently evaluated airway toxicity of the 3-carbon α-dicarbonyl compound, methylglyoxal. Methylglyoxal inhalation causes epithelial necrosis at even lower concentrations than diacetyl. In addition, we investigated airway toxicity of mixtures of diacetyl, acetoin, and acetic acid, common volatiles in butter flavoring. At ratios comparable to workplace scenarios, the mixtures or diacetyl alone, but not acetic acid or acetoin, cause airway epithelial necrosis. These new findings add to existing data to implicate α-dicarbonyl compounds in airway injury and flavorings-related lung disease.

Pembrolizumab-induced obstructive bronchiolitis in a patient with stage IV non-small-cell lung cancer.

Objective: Immune checkpoint inhibitors are now a standard of care for the management of many metastatic cancers, including non-small-cell lung cancer. Pembrolizumab, a selective anti-PD-1 monoclonal antibody, augments the host antitumoural response. This hyperactivation of the immune system has side effects, the so-called immune-related adverse effects. The objective of this case report was to review and point out a new pattern of immune checkpoint inhibitor-associated pneumonitis. Case Description: A 69-year-old woman with stage iv non-small-cell lung cancer receiving pembrolizumab presented for increased dyspnea. Pembrolizumab-related obstructive bronchiolitis was diagnosed based on a new severe obstructive disorder, without bronchodilator reversibility, and mosaic attenuation on angiography, without other identifiable causes. Summary: To our knowledge, this is the first description of a case of pembrolizumab-induced obstructive bronchiolitis. Various patterns of immune checkpoint inhibitor-associated lung disease have been described, and bronchiolitis should be included in the differential diagnosis.

EGFR-Dependent IL8 Production by Airway Epithelial Cells After Exposure to the Food Flavoring Chemical 2,3-Butanedione.

2,3-Butanedione (DA), a component of artificial butter flavoring, is associated with the development of occupational bronchiolitis obliterans (BO), a disease of progressive airway fibrosis resulting in lung function decline. Neutrophilic airway inflammation is a consistent feature of BO across a range of clinical contexts and may contribute to disease pathogenesis. Therefore, we sought to determine the importance of the neutrophil chemotactic cytokine interleukin-8 (IL-8) in DA-induced lung disease using in vivo and in vitro model systems. First, we demonstrated that levels of Cinc-1, the rat homolog of IL-8, are increased in the lung fluid and tissue compartment in a rat model of DA-induced BO. Next, we demonstrated that DA increased IL-8 production by the pulmonary epithelial cell line NCI-H292 and by primary human airway epithelial cells grown under physiologically relevant conditions at an air-liquid interface. We then tested the hypothesis that DA-induced epithelial IL-8 protein occurs in an epidermal growth factor receptor (EGFR)-dependent manner. In these in vitro experiments we demonstrated that epithelial IL-8 protein is blocked by the EGFR tyrosine kinase inhibitor AG1478 and by inhibition of tumor necrosis factor-alpha converting enzyme using the small molecule inhibitor, TAPI-1. Finally, we demonstrated that DA-induced IL-8 is dependent upon ERK1/2 and Mitogen activated protein kinase kinase activation downstream of EGFR signaling using the small molecule inhibitors AG1478 and PD98059. Together these novel in vivo and in vitro observations support that EGFR-dependent IL-8 production occurs in DA-induced BO. Further studies are warranted to determine the importance of IL-8 in BO pathogenesis.

Bronchiolitis obliterans murine model induced by nitric acid aerosol inhalation: An economical and reproducible model.

PURPOSE: Bronchiolitis obliterans (BO) is a highly debilitative and fatal syndrome associated with a series of severe lower airway disorders. The pathogenesis of BO is complicated and not entirely understood. An appropriate animal model of BO may aid research into its pathogenesis. Here, we establish a mouse model of BO to provide insight into this disease. MATERIALS AND METHODS: 6-8 week old BABL/c mice were exposed to 5% nitric acid (NA) aerosol through a nebulizer for 3 hours, and controls were exposed to distilled water instead. Symptoms, airway resistance and pathological process were observed dynamically. The levels of matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), 8-isoprostane and myeloperoxidase (MPO) in lung tissue and bronchoalveolar lavage fluids (BLAF) were determined by ELISA on day 3, 7, 14, 28 and 56 after the aerosol nebulization. RESULTS: Typical BO lesions were observed in NA nebulized mice characterized histologically by initial necrotizing bronchiolitis and final airway fibrosis at day 28 after the aerosol nebulization. NA nebulized mice also exhibited labored breathing and significantly increased airway resistance. Expression of MMP-2, MMP-9, TIMP-1, 8-isoprostane and MPO were significantly elevated in NA nebulized mice in different time frame. CONCLUSION: A murine BO model was established by NA aerosol inhalation. It provides an easy, economic, and reproducible mice model for BO research.

Bronchiolitis Obliterans and Pulmonary Fibrosis after Sulfur Mustard Inhalation in Rats.

Inhalation of powerful chemical agents, such as sulfur mustard (SM), can have debilitating pulmonary consequences, such as bronchiolitis obliterans (BO) and parenchymal fibrosis (PF). The underlying pathogenesis of disorders after SM inhalation is not clearly understood, resulting in a paucity of effective therapies. In this study, we evaluated the role of profibrotic pathways involving transforming growth factor-ß (TGF-ß) and platelet-derived growth factor (PDGF) in the development of BO and PF after SM inhalation injury using a rat model. Adult Sprague-Dawley rats were intubated and exposed to SM (1.0 mg/kg), then monitored daily for respiratory distress, oxygen saturation changes, and weight loss. Rats were killed at 7, 14, 21, or 28 days, and markers of injury were determined by histopathology; pulmonary function testing; and assessment of TGF-ß, PDGF, and PAI-1 concentrations. Respiratory distress developed over time after SM inhalation, with progressive hypoxemia, respiratory distress, and weight loss. Histopathology confirmed the presence of both BO and PF, and both gradually worsened with time. Pulmonary function testing demonstrated a time-dependent increase in lung resistance, as well as a decrease in lung compliance. Concentrations of TGF-ß, PDGF, and PAI-1 were elevated at 28 days in lung, BAL fluid, and/or plasma. Time-dependent development of BO and PF occurs in lungs of rats exposed to SM inhalation, and the elevated concentrations of TGF-ß, PDGF, and PAI-1 suggest involvement of these profibrotic pathways in the aberrant remodeling after injury.

Constrictive Bronchiolitis Attributable to Inhalation of Toxic Agents: Considerations for a Case Definition.

OBJECTIVE: We identified cases of constrictive bronchiolitis (CB), an inflammatory injury obliterating the small airways, in adults caused by inhalational exposure to determine an appropriate case definition. METHODS: We performed a systematic review with meta-analysis for these cases from 1990 to 2017. Publications were included if there was 1) inhalational exposure; 2) respiratory symptoms/signs; 3) pulmonary function test results; and 4) computerized tomographic chest imaging. Many had a lung biopsy. RESULTS: Two hundred seventy-four articles were retrieved; 22 manuscripts comprising 102 cases were included. Diagnostic criteria from cases associated with military deployment to southwest Asia were statistically different from criteria of other etiologies. CONCLUSION: In three cases, the scan was consistent with CB, the biopsy nondiagnostic, yet the diagnosis was made. CB associated with military deployment presented with diagnostic features statistically different from features in the other cases.