Airway Lining Cells Research Articles

Cigarette smoking and microbiome composition; lessons from both epidemiology and experimental models

S15: Interaction between environmental chemicals and toxicants and the human microbiome, Room 217, Floor 2, August 28, 2019, 10:30 AM - 12:00 PM Lung resident bacteria reside in close proximity to the apical side of the airway lining cells, thus forming an intimate interface between cellular regulators of tissue homeostasis and the external environment. The lung microbiota has therefore been suggested to function as gate-keeper for pulmonary health. However, information on the structure and functionality of the human lung microbiota in healthy individuals is limited and it is even less clear how it responds to environmental challenges. We therefore investigated bacterial dynamics in the respiratory tract of healthy individuals as well as in mice in response to cigarette smoke. Within a small monocentric, prospective, controlled study (NCT03562442) the bacterial communities of the upper (nose and oropharynx) and lower respiratory tract (bronchoalveolar lavage) were examined in healthy, current or ex-smokers and never-smokers. Samples were analysed with Illumina MiSeq 16S rRNA gene amplicon sequencing. A microbial community structure dominated by Prevotella, Streptococcus, Veillonella and Actinomyces was found consistently in nasal and oropharyngeal swabs and lavage samples. Further, distinct genera and species of Proteobacteria were mainly found in BAL. In smokers, the oropharyngeal microbiota showed significantly more anaerobic Actinobacteria, as compared to aerobic β-proteobacteria and Fusobacteria. Nasal communities changed with smoking intensity, revealing a higher abundance of Staphylococcus epidermidis in heavy smokers. Although the current smoking status did not influence bronchoalveolar lavage microbial community significantly, changes associated with intense long-term smoking were visible. In particular, the abundance of β- and γ-proteobacteria in bronchoalveolar lavage, e.g. the opportunistic pathogens Stenotrophomonas maltophilia and Serratia marescens, increased while the abundance of Prevotella sp., that has been associated with lung health, decreased. Moreover, these changes persisted partly in ex-smokers. Thus, long-term smoking changes the pulmonary microbiome, leading to an increase of opportunistic pathogens that are known for their resistance to several antibiotics and for causing pneumonia in immunosuppressed patients.

Achaete-scute complex homolog-1 promotes DNA repair in the lung carcinogenesis through matrix metalloproteinase-7 and O(6)-methylguanine-DNA methyltransferase.

Lung cancer is the leading cause of cancer-related deaths in the world. Achaete-scute complex homolog-1 (Ascl1) is a member of the basic helix-loop-helix (bHLH) transcription factor family that has multiple functions in the normal and neoplastic lung such as the regulation of neuroendocrine differentiation, prevention of apoptosis and promotion of tumor–initiating cells. We now show that Ascl1 directly regulates matrix metalloproteinase-7 (MMP-7) and O(6)-methylguanine-DNA methyltransferase (MGMT). Loss- and gain-of-function experiments in human bronchial epithelial and lung carcinoma cell lines revealed that Ascl1, MMP-7 and MGMT are able to protect cells from the tobacco-specific nitrosamine NNK-induced DNA damage and the alkylating agent cisplatin-induced apoptosis. We also examined the role of Ascl1 in NNK-induced lung tumorigenesis in vivo. Using transgenic mice which constitutively expressed human Ascl1 in airway lining cells, we found that there was a delay in lung tumorigenesis. We conclude that Ascl1 potentially enhances DNA repair through activation of MMP-7 and MGMT which may impact lung carcinogenesis and chemoresistance. The study has uncovered a novel and unexpected function of Ascl1 which will contribute to better understanding of lung carcinogenesis and the broad implications of transcription factors in tobacco-related carcinogenesis.

Novel Method for Isolation of Murine Clara Cell Secretory Protein-Expressing Cells with Traces of Stemness

Clara cells are non-ciliated, secretory bronchiolar epithelial cells that serve to detoxify harmful inhaled substances. Clara cells also function as stem/progenitor cells for repair in the bronchioles. Clara cell secretory protein (CCSP) is specifically expressed in pulmonary Clara cells and is widely used as a Clara cell marker. In addition CCSP promoter is commonly used to direct gene expression into the lung in transgenic models. The discovery of CCSP immunoreactivity in plasma membranes of airway lining cells prompted us to explore the possibility of enriching Clara cells by flow cytometry. We established a novel and simple method for the isolation of CCSP-expressing cell Clara cells using a combination of mechanical and enzymatic dissociation followed by flow cytometry sorting technology. We showed that ∼25% of dissociated cells from whole lung expressed CCSP. In the resulting preparation, up to 98% of cells expressed CCSP. Notably, we found that several common stem cell markers including CD44, CD133, Sca-1 and Sox2 were expressed in CCSP+ cells. Moreover, CCSP+ cells were able to form spheroid colonies in vitro with 0.97‰ efficiency. Parallel studies in vivo confirmed that a small population of CCSP−expressing cells in mouse airways also demonstrates stem cell-like properties such as label retention and harboring rare bronchioalveolar stem cells (BASCs) in terminal bronchioles (TBs). We conclude that CCSP+ cells exhibit a number of stem cell-like features including stem cell marker expression, bronchosphere colony formation and self-renewal ability. Clara cell isolation by flow cytometry sorting is a useful method for investigating the function of primary Clara cells in stem cell research and mouse models.

Achaete-Scute Homologue-1 Tapers Neuroendocrine Cell Differentiation in Lungs after Exposure to Naphthalene

The basic helix-loop-helix transcription factor achaete-scute homologue-1 (ASH1) plays a critical role in regulating the neuroendocrine (NE) phenotype in normal and neoplastic lung. Transgenic (TG) mice that constitutively express human ASH1 (hASH1) under control of the Clara cell 10-kDa protein (CC10) promoter in non-NE airway lining cells display progressive epithelial hyperplasia and bronchiolar metaplasia or bronchiolization of the alveoli (BOA). However, little is known about the involvement of hASH1 in regeneration of the conducting airway. In this study, we investigated the impact of hASH1 on airway cell injury and repair in the TG mice following an intraperitoneal injection of naphthalene, which specifically ablates bronchiolar Clara cells and induces pulmonary NE cell hyperplasia. We discovered an overall attenuation of NE maturation coupled with increased proliferation in TG mice during post-naphthalene repair. In addition, BOA lesions revealed enhanced epithelial cell proliferation while preserving Clara cell markers CC10 and the principal naphthalene-metabolizing enzyme cytochrome P4502F2. These data suggest that ASH1 may play an important role in maintaining a progenitor phenotype that promotes renewal of both NE and epithelial cells. Moreover, ASH1 may propagate a stem cell microenvironment in BOA where epithelium becomes resistant to naphthalene toxicity.

Engineered Common Cold Virus Helps Cultured Cystic Fibrosis Tissues Clear Mucus

Engineered Common Cold Virus Helps Cultured Cystic Fibrosis Tissues Clear Mucus

CD11b+ Myeloid Cells Are the Key Mediators of Th2 Cell Homing into the Airway in Allergic Inflammation

STAT6-mediated chemokine production in the lung is required for Th2 lymphocyte and eosinophil homing into the airways in allergic pulmonary inflammation, and thus is a potential therapeutic target in asthma. However, the critical cellular source of STAT6-mediated chemokine production has not been defined. In this study, we demonstrate that STAT6 in bone marrow-derived myeloid cells was sufficient for the production of CCL17, CCL22, CCL11, and CCL24 and for Th2 lymphocyte and eosinophil recruitment into the allergic airway. In contrast, STAT6 in airway-lining cells did not mediate chemokine production or support cellular recruitment. Selective depletion of CD11b(+) myeloid cells in the lung identified these cells as the critical cellular source for the chemokines CCL17 and CCL22. These data reveal that CD11b(+) myeloid cells in the lung help orchestrate the adaptive immune response in asthma, in part, through the production of STAT6-inducible chemokines and the recruitment of Th2 lymphocytes into the airway.

Extracellular Regulated Kinase/Mitogen Activated Protein Kinase Is Up-regulated in Pulmonary Emphysema and Mediates Matrix Metalloproteinase-1 Induction by Cigarette Smoke

The interstitial collagenase matrix metalloprotein-ase-1 (MMP-1) is up-regulated in the lung during pulmonary emphysema. The mechanisms underlying this aberrant expression are poorly understood. Although cigarette smoking is the predominant cause of emphysema, only 15-20% of smokers develop the disease. To define the signaling pathways activated by smoke and to identify molecules responsible for emphysema-associated MMP-1 expression, we performed several in vitro and in vivo experiments. In this study, we showed that cigarette smoke directly induced MMP-1 mRNA and protein expression and increased the collagenolytic activity of human airway cells. Treatment with various chemical kinase inhibitors revealed that this response was dependent on the extracellular regulated kinase-1/2 (ERK) mitogen activated protein kinase pathway. Cigarette smoke increased phosphorylation of residues Thr-202 and Tyr-204 of ERK in airway lining cells and alveolar macrophages in mice at 10 days and 6 months of exposure. Moreover, analysis of lung tissues from emphysema patients revealed significantly increased ERK activity compared with lungs of control subjects. This ERK activity was evident in airway lining and alveolar cells. The identification of active ERK in the lungs of emphysema patients and the finding that induction of MMP-1 by cigarette smoke in pulmonary epithelial cells is ERK-dependent reveal a molecular mechanism and potential therapeutic target for excessive matrix remodeling in smokers who develop emphysema.

SURFACTANT PROTEIN-B (SP-B) PROMOTER IN REPLICATION DEFICIENT ADENOVIRUS(rAd) DIRECTS PULMONARY EPITHELIAL CELL-SPECIFIC GENE EXPRESSION. • 1986

Gene therapy using rAd shows promise in treating genetic pulmonary diseases. Inherited deficiency of SP-B, a proteolipid critical to pulmonary surfactant, results in severe respiratory failure in term infants. SP-B is produced by alveolar type II cells and bronchiolar Clara cells and may be harmful within other cells due to its fusogenic properties. A SP-B promoter(SPB; -640/+319 relative to transcription start site) shows lung epithelial cell line specificity in transient transfection of H441 pulmonary adenocarcinoma cells with promoter strength similar to the promiscuous viral RSV promoter. To explore cell-type specific gene therapy for SP-B deficiency, we compared expression of the lacZ reporter gene driven by the RSV or SP-B promoters in rAd. Dose-response and time course experiments in H441 cells demonstrated efficient rAd.SPB.lacZ and rAd.RSV.lacZ infection with 24h of virus exposure at a multiplicity of infection (MOI) of≈1-10 with high-level β-galactosidase activity after 72h. X-gal staining showed specificity of rAd.SPB.lacZ for pulmonary derived cell lines (A549 and H441 versus HeLa) whereas all cell lines stained extensively with rAd.RSV.lacZ. This finding was confirmed by quantitative β-gal assay: the ratios of rAd.SPB.lacZ to rAd.RSV.lacZ β-gal activities(average ± SEM) were A549 = 1.96 ± 0.69; H441 = 0.66 ± 0.13; HeLa = 0.05 ± 0.01; p<0.05; n=3). Human fetal lung explants(24wk gestation) infected with rAd.SPB.lacZ had preferential X-gal staining of airway lining cells versus interstitial cells. Epithelia of explanted trachea showed minimal staining. All lung and trachea explant cell types stained extensively with rAd.RSV.lacZ. rAd.SPB.lacZ infection of mixed cells isolated from lung explants showed staining of most epithelial cells and rare fibroblasts. Primary fibroblasts infected with rAd.SPB.lacZ (MOI 1-300) had very low β-gal activities (7-94 A420/min/mg protein) whereas β-gal activities of fibroblasts infected with rAd.RSV.lacZ increased in a dose-dependent manner (480-55200 A420/min/mg protein). These results indicate that the SP-B promoter in rAd maintains cell line specificity and may be useful for limiting transgene expression to type II and Clara cells. (supported by 5 P30 HD28815)

Enhanced production of rat interleukin-8 by in vitro and in vivo infections with influenza A NWS virus

We investigated the interleukin-8 (IL-8)-producing activity of influenza A NWS virus in cultured rat kidney NRK-52E cells and a rat influenza model. The production of rat IL-8 increased significantly in the virus-infected cells but not in UV-inactivated virus- or split-product-treated cells. The increase in IL-8 production could be detected in the bronchoalveolar lavage of infected rats. These data suggest that infectious virus has the potential to accelerate the production of IL-8 in cultured cells and in vivo in airway-lining cells.