Neutralization of IL-33 modifies the type 2 and type 3 inflammatory signature of viral induced asthma exacerbation.

BACKGROUND: Respiratory viral infections are one of the leading causes of need for emergency care and hospitalizations in asthmatic individuals, and airway-secreted cytokines are released within hours of viral infection to initiate these exacerbations. IL-33, specifically, contributes to these allergic exacerbations by amplifying type 2 inflammation. We hypothesized that blocking IL-33 in RSV-induced exacerbation would significantly reduce allergic inflammation. METHODS: Sensitized BALB/c mice were challenged with aerosolized ovalbumin (OVA) to establish allergic inflammation, followed by RSV-A2 infection to yield four treatment groups: saline only (Saline), RSV-infected alone (RSV), OVA alone (OVA), and OVA-treated with RSV infection (OVA-RSV). Lung outcomes included lung mRNA and protein markers of allergic inflammation, histology for mucus cell metaplasia and lung immune cell influx by cytospin and flow cytometry. RESULTS: While thymic stromal lymphopoietin (TSLP) and IL-33 were detected 6 h after RSV infection in the OVA-RSV mice, IL-23 protein was uniquely upregulated in RSV-infected mice alone. OVA-RSV animals varied from RSV- or OVA-treated mice as they had increased lung eosinophils, neutrophils, group 2 innate lymphoid cells (ILC2) and group 3 innate lymphoid cells (ILC3) detectable as early as 6 h after RSV infection. Neutralized IL-33 significantly reduced ILC2 and eosinophils, and the prototypical allergic proteins, IL-5, IL-13, CCL17 and CCL22 in OVA-RSV mice. Numbers of neutrophils and ILC3 were also reduced with anti-IL-33 treatment in both RSV and OVA-RSV treated animals as well. CONCLUSIONS: Taken together, our findings indicate a broad reduction in allergic-proinflammatory events mediated by IL-33 neutralization in RSV-induced asthma exacerbation.

MyD88 controls airway epithelial Muc5ac expression during TLR activation conditions from agricultural organic dust exposure.

Inflammation from airborne microbes can overwhelm compensatory mucociliary clearance mechanisms, leading to mucous cell metaplasia. Toll-like receptor (TLR) activation via myeloid differentiation factor 88 (MyD88) signaling is central to pathogen responses. We have previously shown that agricultural organic dust extract (ODE), with abundant microbial component diversity, activates TLR-induced airway inflammation. With the use of an established model, C57BL/6J wild-type (WT) and global MyD88 knockout (KO) mice were treated with intranasal inhalation of ODE or saline, daily for 1 wk. ODE primarily increased mucin (Muc)5ac levels relative to Muc5b. Compared with ODE-challenged WT mice, ODE-challenged, MyD88-deficient mice demonstrated significantly increased Muc5ac immunostaining, protein levels by immunoblot, and expression by quantitative PCR. The enhanced Muc5ac levels in MyD88-deficient mice were not explained by differences in the differentiation program of airway secretory cells in naïve mice. Increased Muc5ac levels in MyD88-deficient mice were also not explained by augmented inflammation, IL-17A, or neutrophil elastase levels. Furthermore, the enhanced airway mucins in the MyD88-deficient mice were not due to defective secretion, as the mucin secretory capacity of MyD88-KO mice remained intact. Finally, ODE-induced Muc5ac levels were enhanced in MyD88-deficient airway epithelial cells in vitro. In conclusion, MyD88 deficiency enhances airway mucous cell metaplasia under environments with high TLR activation.

Dimethylarginine dimethylaminohydrolase (DDAH) overexpression enhances wound repair in airway epithelial cells exposed to agricultural organic dust.

OBJECTIVE: Workers exposed to dusts from concentrated animal feeding operations have a high prevalence of pulmonary diseases. These exposures lead to chronic inflammation and aberrant airway remodeling. Previous work shows that activating cAMP-dependent protein kinase (PKA) enhances airway epithelial wound repair while activating protein kinase C (PKC) inhibits wound repair. Hog barn dust extracts slow cell migration and wound repair via a PKC-dependent mechanism. Further, blocking nitric oxide (NO) production in bronchial epithelial cells prevents PKA activation. We hypothesized that blocking an endogenous NO inhibitor, asymmetric dimethylarginine, by overexpressing dimethylarginine dimethylaminohydrolase mitigates the effects of hog dust extract on airway epithelial would repair. MATERIALS/METHODS: We cultured primary tracheal epithelial cells in monolayers from both wild-type (WT) and dimethylarginine dimethylaminohydrolase overexpressing C57Bl/6 (DDAH1 transgenic) mice and measured wound repair using the electric cell impedance sensing system. RESULTS: Wound closure in epithelial cells from WT mice occurred within 24 h in vitro. In contrast, treatment of the WT cell monolayers with 5% hog dust extract prevented significant NO-stimulated wound closure. In cells from DDAH1 transgenic mice, control wounds were repaired up to 8 h earlier than seen in WT mice. A significant enhancement of wound repair was observed in DDAH cells compared to WT cells treated with hog dust extract for 24 h. Likewise, cells from DDAH1 transgenic mice demonstrated increased NO and PKA activity and decreased hog dust extract-stimulated PKC. DISCUSSION/CONCLUSION: Preserving the NO signal through endogenous inhibition of asymmetric dimethylarginine enhances wound repair even in the presence of dust exposure.

Malondialdehyde-Acetaldehyde (MAA) Protein Adducts Are Found Exclusively in the Lungs of Smokers with Alcohol Use Disorders and Are Associated with Systemic Anti-MAA Antibodies.

BACKGROUND: Malondialdehyde (MDA) and acetaldehyde (AA) exist following ethanol metabolism and tobacco pyrolysis. As such, lungs of individuals with alcohol use disorders (AUDs) are a target for the effects of combined alcohol and cigarette smoke metabolites. MDA and AA form a stable protein adduct, malondialdehyde-acetaldehyde (MAA) adduct, known to be immunogenic, profibrotic, and proinflammatory. MAA adduct is the dominant epitope in anti-MAA antibody formation. We hypothesized that MAA-adducted protein forms in lungs of those who both abuse alcohol and smoke cigarettes, and that this would be associated with systemically elevated anti-MAA antibodies. METHODS: Four groups were established: AUD subjects who smoked cigarettes (+AUD/+smoke), smokers without AUD (-AUD/+smoke), AUD without smoke (+AUD/-smoke), and non-AUD/nonsmokers (-AUD/-smoke). RESULTS: We observed a significant increase in MAA adducts in lung cells of +AUD/+smoke versus -AUD/-smoke. No significant increase in MAA adducts was observed in -AUD/+smoke or in +AUD/-smoke compared to -AUD/-smoke. Serum from +AUD/+smoke had significantly increased levels of circulating anti-MAA IgA antibodies. After 1 week of alcohol that MAA-adducted protein is formed in the lungs of those who smoke cigarettes and abuse alcohol, leading to a subsequent increase in serum IgA antibodies. CONCLUSIONS: MAA-adducted proteins could play a role in pneumonia and other diseases of the lung in the setting of AUD and smoking.

An association between MMP-9 and impaired T cell migration in ethanol-fed BALB/c mice infected with respiratory syncytial virus-2A.

Matrix metalloproteinases are important for proper airway matrix structure and wound healing. These enzymes are also implicated in many airway diseases. Previously, chronic ethanol consumption was shown to prolong inflammation and delay viral clearance in respiratory syncytial virus (RSV)-infected mice. We hypothesize that alcohol alters anti-viral immunity by disrupting immune cell chemotaxis in the lung. BALB/c mice were randomly selected to consume 18% alcohol ad libitum for 8 weeks prior to infection with RSV-2A. Bronchoalveolar lavage (BAL) cell populations were measured by flow cytometry, and chemokines were detected by Western blot or ELISA. MMP-9 levels were determined by polymerase chain reaction (PCR) in mouse lungs and in BAL fluid by ELISA. T cells were acquired from the spleens of water-fed, non-infected control mice (CTRL); alcohol-fed, non-infected (ETOH); water-fed, RSV-infected (RSV); or ethanol-fed, RSV-infected (ETOH-RSV) 4 days after RSV infection. T cells were placed in a transmigration system where chemokines had been treated with and without activated MMP-9. Lymphocyte recruitment was significantly reduced in the BAL 4 days after RSV infection in ETOH-RSV mice, whereas chemokine levels were the highest in this group at all experimental time points examined in comparison to RSV (p < 0.05). MMP-9 mRNA and protein were detected at high levels in ETOH-RSV mice compared to RSV. Using ex vivo transmigration to CCL2 and CXCL10, T cell migration was not impaired between any of the treatment groups, yet when CCL2 and CXCL10 were treated with activated MMP-9, significantly fewer T cells migrated across collagen-coated 5-µm membranes (p < 0.05). Immune cell recruitment is necessary for viral clearance. We show that immune cells are decreased in the lungs of ETOH-RSV mice. In contrast to decreased cell recruitment, key inflammatory chemokines were elevated in the lungs of ETOH-RSV mice. These proteins may be prematurely degraded by MMP-9 in the lung, leading to defective immunity and reduced viral clearance.

Alcohol potentiates RSV-mediated injury to ciliated airway epithelium.

Alcohol impairs resolution of respiratory viral infections. Numerous immune response pathways are altered in response to alcohol misuse, including alcohol-induced ciliary dysfunction in the lung. We hypothesized that mucociliary clearance-mediated innate immunity to respiratory syncytial virus (RSV) would be compromised by alcohol exposure. Cilia were assayed using Sisson-Ammons Video Analysis by quantitating the average number of motile points in multiple whole field measurements of mouse tracheal epithelial cells grown on an air-liquid interface. Pretreatment with ethanol alone (100 mM for 24 hours) had no effect on the number of motile cilia. A single dose (TCID50 1 × 105) of RSV resulted in a significant (p < 0.05) decrease in motile cilia after 2 days. Ethanol pretreatment significantly (p < 0.05) potentiated RSV-induced cilia loss by 2 days. Combined RSV and ethanol treatment led to a sustained activation-induced auto-downregulation of PKC epsilon (PKCε). Ethanol-induced enhancement of ciliated cell detachment was confirmed by dynein ELISA and LDH activity from the supernates. RSV-induced cilia loss was evident until 7 days, when RSV-only infected cells demonstrated no significant cilia loss vs. control cells. However, cells pretreated with ethanol showed significant cilia loss until 10 days post-RSV infection. To address the functional significance of ethanol-enhanced cilia detachment, mice fed alcohol ad libitum (20% for 12 weeks) were infected once with RSV, and clearance was measured by plaque-forming assay from lung homogenates for up to 7 days. After 3 days, RSV plaque formation was no longer detected from the lungs of control mice, while significant (p < 0.01) RSV plaque-forming units were detected at 7 days in alcohol-fed mice. Alcohol-fed mice demonstrated enhanced cilia loss and delayed cilia recovery from tracheal measurements in wild-type C57BL/6 mice, but not PKCε KO mice. These data suggest that alcohol worsens RSV-mediated injury to ciliated epithelium in a PKCε-dependent manner.

Autophagy regulates DUOX1 localization and superoxide production in airway epithelial cells during chronic IL-13 stimulation.

The airway epithelium is a broad interface with the environment, mandating well-orchestrated responses to properly modulate inflammation. Classically, autophagy is a homeostatic pathway triggered in response to external cellular stresses, and is elevated in chronic airway diseases. Recent findings highlight the additional role of autophagy in vesicle trafficking and protein secretion, implicating autophagy pathways in complex cellular responses in disease. Th2 cytokines, IL-13 and IL-4, are increased in asthma and other airway diseases contributing to chronic inflammation. Previously, we observed that IL-13 increases reactive oxygen species (ROS) in airway epithelial cells in an autophagy-dependent fashion. Here, we tested our hypothesis that autophagy is required for IL-13-mediated superoxide production via the NADPH oxidase DUOX1. Using a mouse model of Th2-mediated inflammation induced by OVA-allergen, we observed elevated lung amounts of IL-13 and IL-4 accompanied by increased autophagosome levels, determined by LC3BII protein levels and immunostaining. ROS levels were elevated and DUOX1 expression was increased 70-fold in OVA-challenged lungs. To address the role of autophagy and ROS in the airway epithelium, we treated primary human tracheobronchial epithelial cells with IL-13 or IL-4. Prolonged, 7-day treatment increased autophagosome formation and degradation, while brief activation had no effect. Under parallel culture conditions, IL-13 and IL-4 increased intracellular superoxide levels as determined by electron paramagnetic resonance (EPR) spectroscopy. Prolonged IL-13 activation increased DUOX1, localized at the apical membrane. Silencing DUOX1 by siRNA attenuated IL-13-mediated increases in superoxide, but did not reduce autophagy activities. Notably, depletion of autophagy regulatory protein ATG5 significantly reduced superoxide without diminishing total DUOX1 levels. Depletion of ATG5, however, diminished DUOX1 localization at the apical membrane. The findings suggest non-canonical autophagy activity regulates DUOX1-dependent localization required for intracellular superoxide production during Th2 inflammation. Thus, in chronic Th2 inflammatory airway disease, autophagy proteins may be responsible for persistent intracellular superoxide production.

Malondialdehyde-acetaldehyde (MAA) adducted proteins bind to scavenger receptor A in airway epithelial cells.

Co-exposure to cigarette smoke and ethanol generates malondialdehyde and acetaldehyde, which can subsequently lead to the formation of aldehyde-adducted proteins. We have previously shown that exposure of bronchial epithelial cells to malondialdehyde-acetaldehyde (MAA) adducted protein increases protein kinase C (PKC) activity and proinflammatory cytokine release. A specific ligand to scavenger receptor A (SRA), fucoidan, blocks this effect. We hypothesized that MAA-adducted protein binds to bronchial epithelial cells via SRA. Human bronchial epithelial cells (BEAS-2B) were exposed to MAA-adducted protein (either bovine serum albumin [BSA-MAA] or surfactant protein D [SPD-MAA]) and SRA examined using confocal microscopy, fluorescent activated cell sorting (FACS), and immunoprecipitation. Differentiated mouse tracheal epithelial cells (MTEC) cultured by air-liquid interface were assayed for MAA-stimulated PKC activity and keratinocyte-derived chemokine (KC) release. Specific cell surface membrane dye co-localized with upregulated SRA after exposure to MAA for 3-7 min and subsided by 20 min. Likewise, MAA-adducted protein co-localized to SRA from 3 to 7 min with a subsequent internalization of MAA by 10 min. These results were confirmed using FACS analysis and revealed a reduced mean fluorescence of SRA after 3 min. Furthermore, increased amounts of MAA-adducted protein could be detected by Western blot in immunoprecipitated SRA samples after 3 min treatment with MAA. MAA stimulated PKCε-mediated KC release in wild type, but not SRA knockout mice. These data demonstrate that aldehyde-adducted proteins in the lungs rapidly bind to SRA and internalize this receptor prior to the MAA-adducted protein stimulation of PKC-dependent inflammatory cytokine release in airway epithelium.