Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

Ozone (O3) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O3 oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. By using an alkynyl-tagged form of SecoA and shotgun proteomics, we identified 135 proteins as being modified in bronchial epithelial cells. Among them was NLRP2 (NLR family pyrin domain-containing protein 2), which forms an alkynyl-tagged SecoA-protein adduct at lysine residue 1019 (K1019) in the terminal leucine-rich repeat region, a known regulatory region for NLR proteins. NLRP2 expression in airway epithelial cells was characterized, and CRISPR-Cas9 knockout (KO) and shRNA knockdown of NLRP2 were used to determine its function in O3-induced inflammation. No evidence for NLPR2 inflammasome formation or an NLRP2-dependent increase in caspase-1 activity in response to O3 was observed. O3-induced proinflammatory gene expression for CXCL2 and CXCL8/IL8 was further enhanced in NLRP2-KO cells, suggesting a negative regulatory role. Reconstitution of NLRP2-KO cells with the NLRP2 K1019 mutated to arginine partially blocked SecoA adduction and enhanced O3-induced IL-8 release as compared with wild-type NLRP2. Together, our findings uncover NLRP2 as a highly abundant, key component of proinflammatory signaling pathways in airway epithelial cells and as a novel mediator of O3-induced inflammation.

Electronic-Cigarette Use Alters Nasal Mucosal Immune Response to Live-attenuated Influenza Virus. A Clinical Trial.

Inhalation of tobacco smoke has been linked to increased risk of viral infection, such as influenza. Inhalation of electronic-cigarette (e-cigarette) aerosol has also recently been linked to immune suppression within the respiratory tract, specifically the nasal mucosa. We propose that changes in the nasal mucosal immune response modify antiviral host-defense responses in e-cigarette users. Nonsmokers, cigarette smokers, and e-cigarette users were inoculated with live-attenuated influenza virus (LAIV) to safely examine the innate immune response to influenza infection. Before and after LAIV inoculation, we collected nasal epithelial-lining fluid, nasal lavage fluid, nasal-scrape biopsy specimens, urine, and blood. Endpoints examined include cytokines and chemokines, influenza-specific IgA, immune-gene expression, and markers of viral load. Statistical analysis included primary comparisons of cigarette and e-cigarette groups with nonsmokers, as well as secondary analysis of demographic factors as potential modifiers. Markers of viral load did not differ among the three groups. Nasal-lavage-fluid anti-LAIV IgA levels increased in nonsmokers after LAIV inoculation but did not increase in e-cigarette users and cigarette smokers. LAIV-induced gene-expression changes in nasal biopsy specimens differed in cigarette smokers and e-cigarette users as compared with nonsmokers, with a greater number of genes changed in e-cigarette users, mostly resulting in decreased expression. The top downregulated genes in cigarette smokers were SMPD3, NOS2A, and KLRB1, and the top downregulated genes in e-cigarette users were MR1, NT5E, and HRAS. Similarly, LAIV-induced cytokine levels in nasal epithelial-lining fluid differed among the three groups, including decreased antiviral host-defense mediators (IFNγ, IL6, and IL12p40). We also detected that sex interacted with tobacco-product exposure to modify LAIV-induced immune-gene expression. Our results demonstrate that e-cigarette use altered nasal LAIV-induced immune responses, including gene expression, cytokine and chemokine release, and LAIV-specific IgA levels. Together, these data suggest that e-cigarette use induces changes in the nasal mucosa that are consistent with the potential for altered respiratory antiviral host-defense function.Clinical trial registered with www.clinicaltrials.gov (NCT02019745).

Wood Smoke Exposure Alters Human Inflammatory Responses to Viral Infection in a Sex-Specific Manner. A Randomized, Placebo-controlled Study.

RATIONALE: Exposure to particulates from burning biomass is an increasing global health issue. Burning biomass, including wood smoke, is associated with increased lower respiratory infections. OBJECTIVES: To determine whether acute exposure to wood smoke modifies nasal inflammatory responses to influenza. METHODS: Healthy young adults (n = 39) were randomized to a 2-hour controlled chamber exposure to wood smoke, where exposure levels were controlled to particulate number (wood smoke particles [WSP]; 500 µg/cm3) or filtered air, followed by nasal inoculation with a vaccine dose of live attenuated influenza virus (LAIV). Nasal lavage was performed before exposure (Day 0) and on Days 1 and 2 after exposure. Nasal lavage fluid cells were analyzed for inflammatory gene expression profiles, and cell-free fluid was assayed for cytokines. MEASUREMENTS AND MAIN RESULTS: Only IP-10 protein levels were affected, suppressed, by WSP exposure in aggregate analysis. Subsequent analysis indicated an exposure × sex interaction, prompting additional analyses of WSP- and LAIV-induced changes in males and females. Inflammation-related gene expression profiles differed between the sexes, at baseline (males greater than females), after LAIV inoculation (females greater than males), and after WSP exposure (increase in males and decrease in females), demonstrating that WSP- and LAIV-induced changes in antiviral defense responses in the nasal mucosa occur in a sex-specific manner. CONCLUSIONS: WSP exposure resulted in minimal modification of LAIV-induced responses in aggregate analysis. In contrast, analyzing WSP-induced modification of LAIV responses in the sexes separately unmasked sex-specific differences in response to exposure. These data highlight the need for additional studies to understand sex-specific pollutant-induced effects. Clinical trial registered with www.clinicaltrials.gov (NCT02183753).

Small Molecule Antipsychotic Aripiprazole Potentiates Ozone-Induced Inflammation in Airway Epithelium.

Inhaled ground level ozone (O3) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O3-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O3 reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O3-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O3-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O3-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O3-induced biological responses.

Macrophages from the upper and lower human respiratory tract are metabolically distinct.

The function and cell surface phenotype of lung macrophages vary within the respiratory tract. Alterations in the bioenergetic profile of macrophages may also be influenced by their location within the respiratory tract. This study sought to characterize the bioenergetic profile of macrophages sampled from different locations within the respiratory tract at baseline and in response to ex vivo xenobiotic challenge. Surface macrophages recovered from healthy volunteers by induced sputum and by bronchial and bronchoalveolar lavage were profiled using extracellular flux analyses. Oxygen consumption and extracellular acidification rates were measured at rest and after stimulation with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), or 1,2-naphthoquinone (1,2-NQ). Oxygen consumption and extracellular acidification rates were highly correlated for all macrophage samples. Induced sputum macrophages had relatively higher oxygen consumption and extracellular acidification rates and were largely reliant on glycolysis. In contrast, bronchial fraction and bronchoalveolar macrophages depended more heavily on mitochondrial respiration. Bronchoalveolar macrophages showed elevated LPS-induced cytokine responses. Unlike their autologous peripheral blood monocytes, lung macrophages from any source did not display bioenergetic changes following LPS stimulation. The protein kinase C activator PMA did not affect mitochondrial respiration, whereas the air pollutant 1,2-NQ induced marked mitochondrial dysfunction in bronchoalveolar and bronchial fraction macrophages. The bioenergetic characteristics of macrophages from healthy individuals are dependent on their location within the respiratory tract. These findings establish a regional bioenergetic profile for macrophages from healthy human airways that serves as a reference for changes that occur in disease.

Ozone-derived Oxysterols Affect Liver X Receptor (LXR) Signaling: A POTENTIAL ROLE FOR LIPID-PROTEIN ADDUCTS.

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -ß and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3 Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

Novel applications for a noninvasive sampling method of the nasal mucosa.

Reliable methods for sampling the nasal mucosa provide clinical researchers with key information regarding respiratory biomarkers of exposure and disease. For quick and noninvasive sampling of the nasal mucosa, nasal lavage (NL) collection has been widely used as a clinical tool; however, limitations including volume variability, sample dilution, and storage prevent NL collection from being used in nonlaboratory settings and analysis of low abundance biomarkers. In this study, we optimize and validate a novel methodology using absorbent Leukosorb paper cut to fit the nasal passage to extract epithelial lining fluid (ELF) from the nasal mucosa. The ELF sampling method limits the dilution of soluble mediators, allowing quantification of both high- and low-abundance soluble biomarkers such as IL-1ß, IL-8, IL-6, interferon gamma-induced protein 10 (IP-10), and neutrophil elastase. Additionally, we demonstrate that this method can successfully detect the presence of respiratory pathogens such as influenza virus and markers of antibiotic-resistant bacteria in the nasal mucosa. Efficacy of ELF collection by this method is not diminished in consecutive-day sampling, and percent recovery of both recombinant IL-8 and soluble mediators are not changed despite freezing or room temperature storage for 24 h. Our results indicate that ELF collection using Leukosorb paper sampling of ELF provides a sensitive, easy-to-use, and reproducible methodology to collect concentrated amounts of soluble biomarkers from the nasal mucosa. Moreover, the methodology described herein improves upon the standard NL collection method and provides researchers with a novel tool to assess changes in nasal mucosal host defense status.

Comparison of permeable cell culture inserts for use in culture of a human in vitro air-liquid interface model system.

In this study, we compared 12 mm cell culture inserts with permeable polyester membranes (0.4 µm pores) from two different manufacturers: CELLTREAT® and Corning®. Physical dimensions and masses of the inserts were found to be very similar between the two brands, with CELLTREAT® inserts having a slightly smaller diameter and growth area (11.91 mm; 1.11 cm2 ) compared to Corning® Transwells® (12 mm; 1.13 cm2 ). We compared cell differentiation outcomes of human nasal epithelial cells (HNECs) at air-liquid interface grown on inserts from the two different manufacturers, including trans-epithelial electrical resistance, ciliary beat frequency, ciliated area, and gene expression. HNECs from three male donors were used for all endpoints. No statistically significant differences were observed between paired cultures grown on different brands of insert. In conclusion, these inserts are comparable for use with airway epithelial cell model systems and likely do not impact cellular differentiation or cell culture quality.

Benchmark Dose Modeling Approaches for Volatile Organic Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System.

Inhalation is the most relevant route of volatile organic chemical (VOC) exposure; however, due to unique challenges posed by their chemical properties and poor solubility in aqueous solutions, in vitro chemical safety testing is predominantly performed using direct application dosing/submerged exposures. To address the difficulties in screening toxic effects of VOCs, our cell culture exposure system permits cells to be exposed to multiple concentrations at air-liquid interface (ALI) in a 24-well format. ALI exposure methods permit direct chemical-to-cell interaction with the test article at physiological conditions. In the present study, BEAS-2B and primary normal human bronchial epithelial cells (pHBEC) are used to assess gene expression, cytotoxicity, and cell viability responses to a variety of volatile chemicals including acrolein, formaldehyde, 1,3-butadiene, acetaldehyde, 1-bromopropane, carbon tetrachloride, dichloromethane, and trichloroethylene. BEAS-2B cells were exposed to all the test agents, whereas pHBECs were only exposed to the latter 4 listed above. The VOC concentrations tested elicited only slight cell viability changes in both cell types. Gene expression changes were analyzed using benchmark dose (BMD) modeling. The BMD for the most sensitive gene set was within one order of magnitude of the threshold-limit value reported by the American Conference of Governmental Industrial Hygienists, and the most sensitive gene sets impacted by exposure correlate to known adverse health effects recorded in epidemiologic and in vivo exposure studies. Overall, our study outlines a novel in vitro approach for evaluating molecular-based points-of-departure in human airway epithelial cell exposure to volatile chemicals.

Protection of HepG2 cells against acrolein toxicity by 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide via glutathione-mediated mechanism.

Acrolein is an environmental toxicant, mainly found in smoke released from incomplete combustion of organic matter. Several studies showed that exposure to acrolein can lead to liver damage. The mechanisms involved in acrolein-induced hepatocellular toxicity, however, are not completely understood. This study examined the cytotoxic mechanisms of acrolein on HepG2 cells. Acrolein at pathophysiological concentrations was shown to cause apoptotic cell death and an increase in levels of protein carbonyl and thiobarbituric acid reactive acid substances. Acrolein also rapidly depleted intracellular glutathione (GSH), GSH-linked glutathione-S-transferases, and aldose reductase, three critical cellular defenses that detoxify reactive aldehydes. Results further showed that depletion of cellular GSH by acrolein preceded the loss of cell viability. To further determine the role of cellular GSH in acrolein-mediated cytotoxicity, buthionine sulfoximine (BSO) was used to inhibit cellular GSH biosynthesis. It was observed that depletion of cellular GSH by BSO led to a marked potentiation of acrolein-mediated cytotoxicity in HepG2 cells. To further assess the contribution of these events to acrolein-induced cytotoxicity, triterpenoid compound 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) was used for induction of GSH. Induction of GSH by CDDO-Im afforded cytoprotection against acrolein toxicity in HepG2 cells. Furthermore, BSO significantly inhibited CDDO-Im-mediated induction in cellular GSH levels and also reversed cytoprotective effects of CDDO-Im in HepG2 cells. These results suggest that GSH is a predominant mechanism underlying acrolein-induced cytotoxicity as well as CDDO-Im-mediated cytoprotection. This study may provide understanding on the molecular action of acrolein which may be important to develop novel strategies for the prevention of acrolein-mediated toxicity.