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.

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.

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.

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.

Non-malignant respiratory disease among workers in industries using styrene-A review of the evidence.

BACKGROUND: Asthma and obliterative bronchiolitis (OB) cases have occurred among styrene-exposed workers. We aimed to investigate styrene as a risk factor for non-malignant respiratory disease (NMRD). METHODS: From a literature review, we identified case reports and assessed cross-sectional and mortality studies for strength of evidence of positive association (i.e., strong, intermediate, suggestive, none) between styrene exposure and NMRD-related morbidity and mortality. RESULTS: We analyzed 55 articles and two unpublished case reports. Ten OB cases and eight asthma cases were identified. Six (75%) asthma cases had abnormal styrene inhalation challenges. Thirteen (87%) of 15 cross-sectional studies and 12 (50%) of 24 mortality studies provided at least suggestive evidence that styrene was associated with NMRD-related morbidity or mortality. Six (66%) of nine mortality studies assessing chronic obstructive pulmonary disease-related mortality indicated excess mortality. CONCLUSIONS: Available evidence suggests styrene exposure is a potential risk factor for NMRD. Additional studies of styrene-exposed workers are warranted. Am. J. Ind. Med. 60:163-180, 2017. © 2016 Wiley Periodicals, Inc.

Pathology of diacetyl and 2,3-pentanedione airway lesions in a rat model of obliterative bronchiolitis.

Inhalation of diacetyl vapors by workers has been associated with obliterative bronchiolitis (OB), a poorly understood fibroproliferative disease of the small airways. Significant insights into the pathogenesis of OB have been obtained through the use of a rat model. Inhalation exposure of rats to diacetyl or 2,3-pentanedione, a related flavoring agent, can cause severe injury to the airway epithelium and underlying basement membrane. Repeated exposure to diacetyl or 2,3-pentanedione leads to aberrant repair, fibroproliferation and partial to complete occlusion of the airway lumen. Fibroproliferative lesions in rat airways were found to include both intraluminal polyps and circumferential intramural lesions. Intraluminal polyps have been observed to form secondary attachments spanning the airway lumen causing increasing obstruction. These airway lesions in rats are accompanied by inflammation in the form of peribronchial and perivascular infiltrates of lymphocytes, eosinophils and neutrophils. Diacetyl-induced OB lesions in the rat are similar to OB lesions in humans and provide a good model for studying the pathogenesis of this disease.

An autopsy case of bronchiolitis obliterans as a previously unrecognized adverse event of afatinib treatment.

Interstitial lung disease is a well-known pulmonary adverse event that occurs during epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy and results in restrictive ventilatory dysfunction. However, obstructive changes such as those associated with bronchiolitis obliterans (BO) have never been reported as adverse events resulting from the use of any approved EGFR-TKI. This report documents an autopsy case of BO that developed during afatinib treatment for adenocarcinoma of the lung. Knowledge of the possibility of this fatal adverse event is important for adequate follow-up of patients with lung cancer undergoing afatinib treatment.

Long Term Follow-Up of Sulfur Mustard Related Bronchiolitis Obliterans Treatment.

Bronchiolitis obliterans (BO) is the most remarkable pulmonary sequels of war-related sulfur mustard inhalation. There is little if any data about long-term efficacy of associated BO treatment. Five years spirometric records of three groups of patients with obstructive pulmonary diseases (asthma, COPD, BO) and documented sulfur mustard inhalation were evaluated. The BO patients were treated with inhaled Seretide 125-250/25 (2 puffs BID), azithromycin (250 mg, three times/week) and N-acetylcysteine (1200-1800/day). Asthma and COPD patients were treated according to existing guidelines. Seventy-three (38 asthma, 16 COPD and 19 BO) patients completed the 5 years follow-up. Basal and final FEV1 in BO patients (2.69±0.81 and 2.39±0.65 respectively) were not significantly different from COPD patients (2.46±0.56 and 1.96±0.76 respectively). There was also no significant difference between the yearly FEV1 decline in BO patients compared to COPD patients (60±84 cc vs. 99±79 cc respectively, P=0.163). The non-significant difference of FEV1 decline in BO compared to COPD patients suggests the effectiveness of azithromycin, inhaled steroid and N-acetyl cysteine in BO patients. Considering safety and possible effectiveness, this treatment is recommended until more data is available from controlled clinical studies.

Gene expression in obliterative bronchiolitis-like lesions in 2,3-pentanedione-exposed rats.

Obliterative bronchiolitis (OB) is an irreversible lung disease characterized by progressive fibrosis in the small airways with eventual occlusion of the airway lumens. OB is most commonly associated with lung transplant rejection; however, OB has also been diagnosed in workers exposed to artificial butter flavoring (ABF) vapors. Research has been limited by the lack of an adequate animal model of OB, and as a result the mechanism(s) is unclear and there are no effective treatments for this condition. Exposure of rats to the ABF component, 2,3-pentanedione (PD) results in airway lesions that are histopathologically similar to those in human OB. We used this animal model to evaluate changes in gene expression in the distal bronchi of rats with PD-induced OB. Male Wistar Han rats were exposed to 200 ppm PD or air 6 h/d, 5 d/wk for 2-wks. Bronchial tissues were laser microdissected from serial sections of frozen lung. In exposed lungs, both fibrotic and non-fibrotic airways were collected. Following RNA extraction and microarray analysis, differential gene expression was evaluated. In non-fibrotic bronchi of exposed rats, 4683 genes were significantly altered relative to air-exposed controls with notable down-regulation of many inflammatory cytokines and chemokines. In contrast, in fibrotic bronchi, 3807 genes were significantly altered with a majority of genes being up-regulated in affected pathways. Tgf-ß2 and downstream genes implicated in fibrosis were significantly up-regulated in fibrotic lesions. Genes for collagens and extracellular matrix proteins were highly up-regulated. In addition, expression of genes for peptidases and peptidase inhibitors were significantly altered, indicative of the tissue remodeling that occurs during airway fibrosis. Our data provide new insights into the molecular mechanisms of OB. This new information is of potential significance with regard to future therapeutic targets for treatment.

Lung transplantation for pleuroparenchymal fibroelastosis after chemotherapy.

We report the youngest patient ever reported in the literature to exhibit pleuroparenchymal fibroelastosis (PPFE) as a late-onset pulmonary toxicity after treatment with anticancer chemotherapy. The patient was diagnosed with mature B-cell leukemia at age 14. He was successfully treated with intensive chemotherapy; however, 7 years later, he experienced recurrent pneumothoraces. He was clinically diagnosed with upper lobe pulmonary fibrosis. At age 28, he underwent single left lung transplantation. Histologic examination of the resected lung revealed PPFE in the upper lobe and constrictive bronchiolitis obliterans in the lower lobe, which implied a close relationship between PPFE and constrictive bronchiolitis obliterans.

[Obliterative bronchiolitis due to inhalation of toxic gases and fumes--case report]. / Zarostowe zapalenie oskrzelików w wyniku inhalacji toksycznych gazów i oparów--opis przypadku.

Obliterative bronchiolitis is a rare pulmonary disease, characterised by narrowing and eventual obliteration of bronchioles by peribronchial and submucosal fibrosis. One of the identified causes of bronchiolitis is acute injury due to inhalation of toxic gases and fumes. Physiological criteria, essential in preliminary diagnostics, include irreversible airflow limitation, forced expiratory volume in 1 second (FEV1) < 60%, and exclusion of other causes of airflow obstruction. Surgical lung biopsy with histologic examination confirms diagnosis definitely. Prognosis of obliterative bronchiolitis, irrespective of aetiology, is rather poor, and treatment is rarely efficacious. We present a young chemist exposed to inhalation of toxic gases and fumes due to lack of usage of any personal protective equipment. He was referred to our lung disease department because of shortness of breath on exertion and irreversible airflow limitation. Definitive diagnosis of obliterative bronchiolitis was established by histological examination of specimen from open lung biopsy.

The role of N-acetylcysteine in the management of acute and chronic pulmonary complications of sulfur mustard: a literature review.

CONTEXT: Sulfur mustard exposure, as the most widely used chemical weapon, can lead to acute and long-term pulmonary complications via various pathways, such as triggering an imbalance between the oxidant and antioxidant system. Currently, there is no validated antidote, chemoprophylaxis and curative modality for pulmonary toxicities secondary to sulfur mustard exposure. OBJECTIVE: The aim of this literature review is to collect available experimental and clinical data on the efficacy of N-acetylcysteine (NAC), as a prominent antioxidant agent, in the prevention and/or treatment of sulfur mustard-induced acute and chronic pulmonary toxicities. METHODS: A literature search was performed by the relevant keywords like "N-acetyl cysteine", "Sulfur mustard" and "Lung injury" in databases such as Scopus, Medline, Embase and ISI Web of Knowledge. No time limitation was considered. Nineteen articles were selected for review. RESULTS: A number of in vitro and experimental studies concluded that oral, intravenous, intraperitoneal and intra-tracheal administration of NAC is effective in the management of sulfur mustard-induced acute lung injury, in a time-dependent manner, via direct scavenging, inhibition of oxidative stress, inflammatory responses and apoptosis. In addition, oral NAC alone (1200 or 1800 mg/day for 4 months) or at a dose 600 mg/day for 6 months in combination with clarithromycin (500 mg/day) have led to improvements of clinical and paraclinical pulmonary parameters of patients with bronchiolitis obliterans due to sulfur mustard, through undetermined mechanisms. CONCLUSION: Despite limitations of relevant experimental and clinical studies, NAC can be considered as a candidate agent for prevention and/or treatment of sulfur mustard-induced acute lung injuries, as well as its long-term pulmonary toxicities, especially bronchiolitis obliterans.

Diacetyl induces amphiregulin shedding in pulmonary epithelial cells and in experimental bronchiolitis obliterans.

Diacetyl (DA), a component of artificial butter flavoring, has been linked to the development of bronchiolitis obliterans (BO), a disease of airway epithelial injury and airway fibrosis. The epidermal growth factor receptor ligand, amphiregulin (AREG), has been implicated in other types of epithelial injury and lung fibrosis. We investigated the effects of DA directly on the pulmonary epithelium, and we hypothesized that DA exposure would result in epithelial cell shedding of AREG. Consistent with this hypothesis, we demonstrate that DA increases AREG by the pulmonary epithelial cell line NCI-H292 and by multiple independent primary human airway epithelial donors grown under physiologically relevant conditions at the air-liquid interface. Furthermore, we demonstrate that AREG shedding occurs through a TNF-α-converting enzyme (TACE)-dependent mechanism via inhibition of TACE activity in epithelial cells using the small molecule inhibitor, TNF-α protease inhibitor-1, as well as TACE-specific small inhibitor RNA. Finally, we demonstrate supportive in vivo results showing increased AREG transcript and protein levels in the lungs of rodents with DA-induced BO. In summary, our novel in vitro and in vivo observations suggest that further study of AREG is warranted in the pathogenesis of DA-induced BO.