The respiratory system is a complex network of many cell types, including subsets of macrophages and dendritic cells that work together to maintain steady-state respiration. Due to limitations in acquiring cells from healthy human lung, these subsets remain poorly characterized transcriptionally and phenotypically. We set out to systematically identify these subsets in human airways by developing a schema of isolating large numbers of cells by whole lung bronchoalveolar lavage. Six subsets of phagocytic antigen presenting (HLA-DR+) cells were consistently observed. Aside from alveolar macrophages, subsets of Langerin+, BDCA1− CD14+, BDCA1+ CD14+, BDCA1+ CD14−, and BDCA1− CD14− cells were identified. These subsets varied in their ability to internalize Escherichia coli, Staphylococcus aureus, and Bacillus anthracis particles. All subsets were more efficient at internalizing S. aureus and B. anthracis compared to E. coli. Alveolar macrophages and CD14+ cells were overall more efficient at particle internalization compared to the four other populations. Subsets were further separated into two groups based on their inherent capacities to upregulate surface CD83, CD86, and CCR7 expression levels. Whole genome transcriptional profiling revealed a clade of “true dendritic cells” consisting of Langerin+, BDCA1+ CD14+, and BDCA1+ CD14− cells. The dendritic cell clade was distinct from a macrophage/monocyte clade, as supported by higher mRNA expression levels of several dendritic cell-associated genes, including CD1, FLT3, CX3CR1, and CCR6. Each clade, and each member of both clades, were discerned by specific upregulated genes, which can serve as markers for future studies in healthy and diseased states.

BackgroundGene expression changes resulting from conditions such as disease, environmental stimuli, and drug use, can be monitored in the blood. However, a less invasive method of sample collection is of interest because of the discomfort and specialized personnel necessary for blood sampling especially if multiple samples are being collected. Buccal mucosa cells are easily collected and may be an alternative sample material for biomarker testing. A limited number of studies, primarily in the smoker/oral cancer literature, address this tissue's efficacy as an RNA source for expression analysis. The current study was undertaken to determine if total RNA isolated from buccal mucosa could be used as an alternative tissue source to assay relative gene expression.MethodsTotal RNA was isolated from swabs, reverse transcribed and amplified. The amplified cDNA was used in RT-qPCR and microarray analyses to evaluate gene expression in buccal cells. Initially, RT-qPCR was used to assess relative transcript levels of four genes from whole blood and buccal cells collected from the same seven individuals, concurrently. Second, buccal cell RNA was used for microarray-based differential gene expression studies by comparing gene expression between a group of female smokers and nonsmokers.ResultsAn amplification protocol allowed use of less buccal cell total RNA (50 ng) than had been reported previously with human microarrays. Total RNA isolated from buccal cells was degraded but was of sufficient quality to be used with RT-qPCR to detect expression of specific genes. We report here the finding of a small number of statistically significant differentially expressed genes between smokers and nonsmokers, using buccal cells as starting material. Gene Set Enrichment Analysis confirmed that these genes had a similar expression pattern to results from another study.ConclusionsOur results suggest that despite a high degree of degradation, RNA from buccal cells from cheek mucosa could be used to detect differential gene expression between smokers and nonsmokers. However the RNA degradation, increase in sample variability and microarray failure rate show that buccal samples should be used with caution as source material in expression studies.

BackgroundCigarette smoking (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and most COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of the increased risk and severity of infections in smokers is likely multifactorial, but certainly includes changes in immunologic host defenses.MethodsWe investigated retinoic acid-inducible protein I (RIG-I) and interferon (IFN) induction by influenza A virus (IAV) in human bronchial epithelial cells (HBEC) isolated from smokers or nonsmokers. Subcultured HBEC cells were infected with A/Puerto Rico/8/1934 (PR8) IAV at an MOI of 1. After 24 h of infection, cells and supernatants were collected for qRT-PCR, immunoblot or ELISA to determine RIG-I, Toll-like receptor3 (TLR3) and IFN expression levels.ResultsIAV exposure induced a vigorous IFN-β, IFN-λ 1 and IFN-λ 2/3 antiviral response in HBEC from nonsmokers and significant induction of RIG-I and TLR3. In cells from smokers, viral RIG-I and TLR3 mRNA induction was reduced 87 and 79 % compared to the response from nonsmokers. CS exposure history was associated with inhibition of viral induction of the IFN-β, IFN-λ1 and IFN-λ 2/3 mRNA response by 85, 96 and 95 %, respectively, from that seen in HBEC from nonsmokers. The demethylating agent 5-Aza-2-deoxycytidine reversed the immunosuppressive effects of CS exposure in HBEC since viral induction of all three IFNs was restored. IFN-β induction of RIG-I and TLR3 was also suppressed in the cells from smokers.ConclusionOur results suggest that active smoking reduces expression of antiviral cytokines in primary HBEC cells. This effect likely occurs via downregulation of RIG-I and TLR3 due to smoke-induced epigenetic modifications. Reduction in lung epithelial cell RIG-I and TLR3 responses may be a major mechanism contributing to the increased risk and severity of viral respiratory infections in smokers and to viral-mediated acute exacerbations of COPD.