The diversity and extent of signaling functions of nitric oxide (NO) in cell physiology as well as its presence and influence as a common component of ambient air pollution and tobacco smoke are gaining increasing research attention relative to both health and disease. While cellular NO production is typically associated with inflammatory cells and processes, the airway epithelium particularly of the paranasal sinuses, has been documented to be a rich source of excreted NO. Inasmuch as excreted NO derives from both mucosal and inflammatory cell sources, distinguishing the individual contribution of these compartments to total excreted cellular NO is potentially problematic. We simulated an inflammatory mucosal environment by stimulating human nasal epithelial cultures with interleukin-13 (IL-13), a mediator produced by eosinophils in asthma, allergic rhinitis, and sinusitis. While a consistent baseline of NO excretion in control cultures was documented, widely variable individual responses to IL-13 exposure were observed in companion cultures maintained under identical conditions and tested at the same time. These studies suggest that cellular NO excretion by the healthy epithelial mucosa is subject to considerable individual variability and may be significantly elevated among some individuals in the presence of IL-13 stimulation.

Objective: Mucociliary clearance sustains a baseline functionality and an on demand capability to upregulate clearance upon irritant exposure involving mucus hypersecretion and accelerated ciliary beat frequency (CBF) modulated by nitric oxide (NO). This study characterized these elements as well as cellular and exogenous NO concentrations subsequent to a single exposure to tobacco smoke (TS) or e-cigarette vapor (EV) on cultured human airway epithelium.Materials and methods: Air-liquid interface (ALI) airway epithelial cultures per nonsmoking human subjects were subjected to single TS or EV exposures. Measures of ciliary function and secretion were performed and cellular and exogenous NO concentrations under control and experimental conditions were assessed.Results: Both TS and EV exposures resulted similar patterns of decline in CBF within 1min of the completion of exposure followed by a gradual return often exceeding baseline within 1h. Post-exposure examination of exposed cultures suggested morphologic differences in secretory function relative to controls. The relative NO concentrations of TS and EV chamber air were sharply different with EV NO being only slightly elevated relative to cellular NO production.Discussion and conclusions: Epithelial remodeling and mucociliary dysfunction have been clearly associated with TS exposure. However, information contrasting epithelial structure/function following a single acute TS or EV exposure is limited. This study demonstrates a similar pattern of epithelial response to acute TS or EV exposure. Inasmuch as NO may contribute to an inflammatory milieu and generation of toxic metabolites, it is plausible that recurrent exposures over time may be contributory to chronic pathologies.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.

The initial innate immune response to ozone (O3) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived signals affect Mac response to O3. Macs from the bronchoalveolar lavage (BAL) of healthy volunteers were cocultured with the human bronchial epithelial (16HBE) or alveolar (A549) epithelial cell lines. Cocultures, Mac monocultures, and epithelial cell monocultures were exposed to O3 or air, and Mac immunophenotype, phagocytosis, and cytotoxicity were assessed. Quantities of hyaluronic acid (HA) and IL-8 were compared across cultures and in BAL fluid from healthy volunteers exposed to O3 or air for in vivo confirmation. We show that Macs in coculture had increased markers of alternative activation, enhanced cytotoxicity, and reduced phagocytosis compared with Macs in monoculture that differed based on coculture with A549 or 16HBE. Production of HA by epithelial cell monocultures was not affected by O3, but quantities of HA in the in vitro coculture and BAL fluid from volunteers exposed in vivo were increased with O3 exposure, indicating that O3 exposure impairs Mac regulation of HA. Together, we show epithelial cell-Mac coculture models that have many similarities to the in vivo responses to O3, and demonstrate that epithelial cell-derived signals are important determinants of Mac immunophenotype and response to O3.

Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn2+) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn2+ toxicity is not fully understood. H2O2 and Zn2+ have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn2+ to cause cellular H2O2 production. To determine the role of Zn2+-induced H2O2 production in the human airway epithelial cell response to Zn2+ exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50µM Zn2+ for 5min in the presence of 1µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn2+ exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn2+-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn2+-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn2+ leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms.

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.

A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection.

We have previously shown that smokers have deficient innate immune response to influenza A virus (IAV) resulting in enhanced viral replication. Viral entry of IAV depends on proteolytic cleavage, and we have shown that the balance between proteases and antiproteases, such as secretory leukocyte protease inhibitor (SLPI) significantly affects viral replication. SLPI levels can be controlled transcriptionally and we have previously shown that the transcription factor Nrf2 positively regulates SLPI expression. In addition, the SLPI promoter contains ISRE regions, which may suppress transcription. We hypothesized that dysregulation of SLPI levels in epithelial cells from smokers enhances susceptibility to IAV. To test this hypothesis, apical washes and whole cell lysates from nasal epithelial cells from smokers and non‐smokers were analyzed for SLPI and STAT1 protein levels. At baseline, NEC from smokers had decreased SLPI and increased STAT1 levels. Moreover, apical washes from NEC of smokers resulted in enhanced cleavage and proteolytic activation of IAV, suggesting decreased SLPI activity. In addition, NEC treated with rhSLPI for 4h and infected with IAV for 24h, resulted in decreased HA gene expression and viral titers only in NEC from smokers. As such, dysregulation of SLPI at the levels of the epithelium may be key factor during IAV infection.

CONTEXT:Investigations of cell/molecular level effects of in vivo exposure of airway mucosa of experimental animals to common irritant gases have demonstrated structural and physiological changes reflective of breaches in epithelial barrier function, presence of inflammatory cell infiltrate and compromised ciliary function. These experimental animal studies provided useful perspectives of plausible, but more subtle pathologic outcomes having relevance to lifestyle exposure to gaseous environmental irritants including tobacco smoke.METHODS:Freeze-fracture technology was applied to ultrastructural examination of large airway epithelium, with appropriate controls, from guinea pigs exposed to ozone and of nasal mucosa of human subjects exposed to ozone or sulfur dioxide, and nasal mucosa of active smokers.RESULTS:We documented substantive membrane structural changes to tight junctional complexes and cilia as well as an infiltrate of neutrophils into the surface mucosal layer in exposed animals. These patterns also were evident but not as pervasive among human subjects acutely exposed experimentally to irritant gases and those chronically exposed by their lifestyle to tobacco smoke.DISCUSSION:Our intent was to characterize respiratory tract mucosal membrane disorganization associated with high level acute irritant exposures in an experimental animal model and to evaluate evidence of similar but perhaps more subtle pathologic change associated with lower level experimental or lifestyle exposures. Our studies demonstrate continuity, albeit subtle, of pathologic change from high dosage experimental animal exposure to low dosage human exposures.CONCLUSIONS:This study represents the first report of ultrastructural airway epithelial membrane anomalies associated with lifestyle exposure to tobacco smoke irritants.

We previously demonstrated that, in nasal epithelial cells (NECs) from smokers, methylation of an antiviral gene was associated with impaired antiviral defense responses. To expand these findings and better understand biological mechanisms underlying cigarette smoke (CS)-induced modifications of host defense responses, we aimed to compare DNA methylation of genes that may play a role in antiviral response. We used a two-tiered analytical approach, where we first implemented a genome-wide strategy. NECs from smokers differed in the methylation levels of 390 genes, the majority (84%) of which showed decreased methylation in smokers. Secondly, we generated an a priori set of 161 antiviral response-related genes, of which five were differentially methylated in NEC from smokers (CCL2, FDPS, GSK3B, SOCS3, and ULBP3). Assessing these genes at the systems biology level revealed a protein interaction network associated with CS-induced epigenetic modifications involving SOCS3 and ULBP3 signaling, among others. Subsequent confirmation studies focused on SOCS3 and ULBP3, which were hypomethylated and hypermethylated, respectively. Expression of SOCS3 was increased, whereas ULBP3 expression was decreased in NECs from smokers. Addition of the demethylating agent 5-Aza-2-deoxycytidine enhanced ULBP3 expression in NECs from smokers. Furthermore, infection of differentiated NECs with influenza virus resulted in significantly lower levels of ULBP3 in cells from smokers. Taken together, our findings show that genomic DNA methylation profiles are altered in NECs from smokers and that these changes are associated with decreased antiviral host defense responses, indicating that epigenenic dysregulation of genes such as SOCS3 and ULBP3 likely impacts immune responses in the epithelium.

Background: The leading cause of asthma exacerbation is respiratory viral infection. Innate antiviral defense pathways are altered in the asthmatic epithelium, yet involvement of inflammasome signaling in virus-induced asthma exacerbation is not known.Objective: This study compared influenza-induced activation of inflammasome and innate immune signaling in human bronchial epithelial cells from volunteers with and without asthma and investigated the role of caspase-1 in epithelial cell antiviral defense.Methods: Differentiated primary human bronchial epithelial cells from volunteers with and without asthma were infected with influenza Avirus. An inflammasome-specific quantitative real-time polymerase chain reaction array was used to compare baseline and influenza-induced gene expression profiles. Cytokine secretion, innate immune gene expression, and viral replication were compared between human bronchial epithelial cells from volunteers with and without asthma. Immunofluorescence microscopy was used to evaluate caspase-1 and PYCARD colocalization. Tracheal epithelial cells from caspase-1-deficient or wild-type mice were infected with influenza and assessed for antiviral gene expression and viral replication.Results: Human bronchial epithelial cells from asthmatic volunteers had altered influenza-induced expression of inflammasome-related and innate immune signaling components, which correlated with enhanced production of IL-1 beta, IL-6, and TNF-alpha. Specifically, influenza-induced caspase-1 expression was enhanced and localization differed in human bronchial epithelial cells from asthmatic volunteers compared to volunteers without asthma. Influenza-infected tracheal epithelial cells from caspase-1-deficient mice had reduced expression of antiviral genes and viral replication.Conclusion: Caspase-1 plays an important role in the airway epithelial cell response to influenza infection, which is enhanced in asthmatic volunteers, and may contribute to the enhanced influenza-related pathogenesis observed in vivo. (J Allergy Clin Immunol 2012; 130: 958-67.)

Ozone (O3) causes significant adverse health effects worldwide. Nasal epithelial cells (NECs) are among the first sites within the respiratory system to be exposed to inhaled air pollutants. They recruit, activate, and interact with immune cells via soluble mediators and direct cell-cell contacts. Based on our recent observation demonstrating the presence of natural killer (NK) cells in nasal lavages, the goal of this study was to establish a coculture model of NECs and NK cells and examine how exposure to O3 modifies this interaction. Flow cytometry analysis was used to assess immunophenotypes of NK cells cocultured with either air- or O3-exposed NECs. Our data show that coculturing NK cells with O3-exposed NECs decreased intracellular interferon-γ (IFN-γ), enhanced, albeit not statistically significant, IL-4, and increased CD16 expression on NK cells compared with air controls. Additionally, the cytotoxicity potential of NK cells was reduced after coculturing with O3-exposed NECs. To determine whether soluble mediators released by O3-exposed NECs caused this shift, apical and basolateral supernatants of air- and O3-exposed NECs were used to stimulate NK cells. While the conditioned media of O3-exposed NECs alone did not reduce intracellular IFN-γ, O3 enhanced the expression of NK cell ligands ULBP3 and MICA/B on NECs. Blocking ULBP3 and MICA/B reversed the effects of O3-exposed NECs on IFN-γ production in NK cells. Taken together, these data showed that interactions between NECs and NK cells in the context of O3 exposure changes NK cell activity via direct cell-cell interactions and is dependent on ULBP3/MICA/B expressed on NECs.

Smokers are more susceptible to respiratory infections, including influenza. To explore the effect of smoking on influenza-induced responses within the nasal mucosa, we have developed a protocol using inoculation with live attenuated influenza virus (LAIV) vaccine followed by sampling of the nasal mucosa. Mucosal cell populations were harvested through superficial biopsy of the nasal inferior turbinate pre and post LAIV inoculation and analyzed using flow cytometry. The majority of nasal biopsy CD45+ immune cells at baseline were CD3+ T lymphocytes. Following LAIV, these lymphocytes increased in nonsmokers but not in smokers. A subset of individuals was negative for helper T cell marker CD4 and cytotoxic T cell marker CD8 but positive for the γδ T cell receptor (TCR). Increases in γδ TCR+ cells were greater in nonsmokers, than in smokers. Thus, LAIV-induced changes in CD3 T as well as γδ T lymphocyte percentages are suppressed in smokers compared to nonsmokers.

Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza-induced immune response, we established a model using differentiated nasal epithelial cells (NECs) from nonsmokers and smokers, co-cultured with peripheral blood monocyte-derived dendritic cells (mono-DCs) from nonsmokers. NEC/mono-DC co-cultures were infected with influenza A virus and analyzed for influenza-induced immune responses 24 hours after infection. We observed that NECs from smokers, as well as mono-DCs co-cultured with NECs from smokers, exhibited suppressed influenza-induced, interferon-related proteins interferon regulatory factor-7, Toll-like receptor-3, and retinoic acid inducible gene-1, likely because of the suppressed production of IFNα from the NECs of smokers. Furthermore, NEC/mono-DC co-cultures using NECs from smokers exhibited suppressed concentrations of T-cell/natural killer cell chemokine interferon gamma-induced protein 10 (IP-10) after infection with influenza, indicating that NECs from smokers may skew early influenza-induced Th1 responses. In contrast, NEC/mono-DC co-cultures using NEC from smokers contained increased influenza-induced concentrations of the Th2 chemokine thymic stromal lymphopoeitin (TSLP). In addition, NECs from smokers cultured alone had increased influenza-induced concentrations of the Th2 chemokine thymus and activation-regulated chemokine (TARC). Using this model, we demonstrated that in the context of infection with influenza, NECs obtained from smokers create an overall cytokine microenvironment that suppresses the interferon-mediated Th1 response and enhances the TSLP-TARC-mediated Th2 response, with the potential to modify the responses of DCs. Smoking-induced alterations in the Th1/Th2 balance may play a role in developing underlying susceptibilities to respiratory viral infections, and may also promote the likelihood of acquiring Th2 proallergic diseases.