Luisa E. Brighton

When cultured at an air-liquid interface (ALI) in the appropriate medium, primary human airway epithelial cells form a polarized, pseudostratified epithelium composed of ciliated and mucus-secreting cells. This culture system provides a useful tool for the in vitro study of airway epithelial biology and differentiation. We have performed microarray analysis on ALI cultures of human bronchial epithelial cells (HBECs) grown over a 28-d period to identify genes involved in mucociliary differentiation. We identified over 2,000 genes that displayed statistically significant 2-fold or greater changes in expression during the time course. Of the genes showing the largest increases, many are involved in processes associated with airway epithelial biology, such as cell adhesion, immunity, transport, and cilia formation; however, many novel genes were also identified. We compared our results with data from proteomic analyses of the ciliary axoneme and identified candidate genes that may have roles in cilia formation or function. Gene networks were generated using Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA) to identify signaling pathways involved in mucociliary cell differentiation or function. Networks containing genes involved in TGF-beta, WNT/beta-catenin, and epidermal growth factor receptor (EGFR) pathways were identified, suggesting potential roles for these families in airway epithelia. Microarray results were validated by real-time RT-PCR for a number of representative genes. This work has provided extensive information about gene expression changes during differentiation of airway epithelial cells, and will be a useful resource for researchers interested in respiratory function, pathology, and toxicology.

Several factors, such as age and nutritional status, can affect the susceptibility to influenza infections. Moreover, exposure to air pollutants, such as diesel exhaust (DE), has been shown to affect respiratory virus infections in rodent models. Influenza virus primarily infects and replicates in respiratory epithelial cells, which are also a major targets for inhaled DE. Using in vitro models of human respiratory epithelial cells, we determined the effects of an aqueous-trapped solution of DE (DE(as)) on influenza infections. Differentiated human nasal and bronchial epithelial cells, as well as A549 cells, were exposed to DE(as) and infected with influenza A/Bangkok/1/79. DE(as) enhanced the susceptibility to influenza virus infection in all cell models and increased the number of influenza-infected cells within 24 h post-infection. This was not caused by suppressing antiviral mediator production, since interferon (IFN) beta levels, IFN-dependent signaling, and IFN-stimulated gene expression were also enhanced by exposure to DE(as). Many of the adverse effects induced by DE exposure are mediated by oxidative stress. Exposure to DE(as) used in these studies generated oxidative stress in respiratory epithelial cells, and addition of the antioxidant glutathione-ethylester (GSH-ET) reversed the effects of DE(as) on influenza infections. Furthermore, DE(as) increased influenza virus attachment to respiratory epithelial cells within 2 h post-infection. Taken together, the results presented here suggest that in human respiratory epithelial cells oxidative stress generated by DE(as) increases the susceptibility to influenza infection and that exposure to DE(as) increases the ability of the virus to attach to and enter respiratory epithelial cells.

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

The ciliated epithelium of the respiratory airways is one of the first vital systemic surfaces in contact with the ambient air. Ex vivo nasal epithelial ciliary beat frequency (CBF) at room temperature is on the order of 7-8 Hz but may be stimulated by irritant exposure. The upregulation of CBF in response to acute irritant exposure is generally considered to be a transient event with eventual return to baseline. However, studies of CBF dynamics in response to typical lifestyle exposures are limited. This study assessed nasal epithelial CBF among human subjects as a function of quantifiable lifestyle tobacco smoke exposure. Nasal epithelial biopsies were obtained from human subjects with well documented histories of tobacco smoke exposure. CBF was determined using a digital photometric technique and concurrent assays of nasal nitric oxide and urine cotinine and creatinine were performed. Mean CBF among active smokers and non-smokers exposed to environmental tobacco smoke (ETS) was elevated over non-smokers. Although there were dramatic differences in relative levels of tobacco smoke exposure, CBF values among tobacco smoke-exposed groups were comparable. Parallel in vitro studies of cultured nasal epithelium exposed to cigarette smoke condensate further supported these observations. These studies suggest that persistent elevation in nasal epithelial CBF is an early, subtle, physiologic effect associated with lifestyle tobacco smoke exposure. The molecular mechanisms that upregulate CBF may also create a cell molecular milieu capable of provoking the eventual emergence of more overt adverse health effects and the pathogenesis of chronic airway disease.

Smokers are more susceptible to respiratory viral infections, including influenza virus, but the mechanisms mediating this effect are unknown. To determine how epithelial cells contribute to the enhanced susceptibility seen in smokers, we established an in vitro model of differentiated nasal epithelial cells (NECs) from smokers, which showed enhanced mucin expression. The NECs from smokers responded to influenza infection with greater cytotoxicity, release of interleukin-6, and viral shedding than NECs from nonsmokers. Focusing on type I interferon (IFN) expression, we observed that influenza-infected NECs from smokers produced significantly less IFN-alpha than NECs from nonsmokers. Similarly, the expression of IRF7, a key transcription factor controlling the expression of IFN-alpha, was significantly decreased in influenza-infected and IFN-beta-stimulated NECs from smokers. Furthermore, our data indicate that the DNA methylation of the IRF7 gene and expression of the DNA (cytosine-5-)-methyltransferase 1 was enhanced in NECs from smokers. To confirm these findings in vivo, we initiated a study in which smoking and nonsmoking healthy volunteers were inoculated nasally with the live-attenuated influenza virus (LAIV) vaccine, and nasal biopsies were obtained before and after the administration of LAIV. The LAIV-induced expression of IRF7 was lower in the nasal epithelium from smokers, supporting our in vitro observations. These data demonstrate that infection with influenza results in the reduced expression of transcription factor IRF7 in NECs from smokers, and that these effects may be mediated by an epigenetic modification of the IRF7 gene, thus providing a potential mechanism rendering smokers more susceptible to respiratory virus infections.