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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. [ABSTRACT FROM AUTHOR]

Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate (IRS)-2 in macrophages. We hypothesized that negative regulation of IRS-2 activity following IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (pY) and serine phosphorylation (pS) of IRS-2 following IL-4 stimulation. Inhibiting serine phosphatase activity increased pS-IRS-2 and decreased pY-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished pS-IRS-2 and prolonged pY-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both pY-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα and γC following IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 minutes of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be manipulated therapeutically to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation. [ABSTRACT FROM AUTHOR]

Alcohol-use disorders (AUD) persist in the United States and are heavily associated with an increased susceptibility to respiratory viral infections. Respiratory syncytial virus (RSV) in particular has received attention as a viral pathogen commonly detected in children and immune-compromised populations (elderly, asthmatics), yet more recently was recognized as an important viral pathogen in young adults. Our study evaluated the exacerbation of RSV-associated illness in mice that chronically consumed alcohol for 6 weeks prior to infection. Prior studies showed that lung viral titers remained elevated in these animals, leading to a hypothesis that T-cell activation and immune specificity were deficient in controlling viral spread and replication in the lungs. Herein, we confirm a reduction in RSV-specific IFNγ production by CD8 T cells and a depolarization of Th1 (CD4+IFNγ+) and Th2 (CD4+IL-4+) T cells at day 5 after RSV infection. Furthermore, over the course of viral infection (day 1 to day 7 after RSV infection), we detected a delayed influx of neutrophils, monocytes/macrophages, and lymphocytes into the lungs. Taken together, the data show that both the early and late adaptive immunity to RSV infection are altered by chronic ethanol consumption. Future studies will determine the interactions between the innate and adaptive immune systems to delineate therapeutic targets for individuals with AUD often hospitalized by respiratory infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]