Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency.

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema.

Asthmatic airway neutrophilia after allergen challenge is associated with the glutathione S-transferase M1 genotype.

RATIONALE: Asthma is a heterogeneous lung disorder characterized by airway inflammation and airway dysfunction, manifesting as hyperresponsiveness and obstruction. Glutathione S-transferase M1 (GSTM1) is a multifunctional phase II enzyme and regulator of stress-activated cellular signaling relevant to asthma pathobiology. A common homozygous deletion polymorphism of the GSTM1 gene eliminates enzyme activity. OBJECTIVES: To determine the effect of GSTM1 on airway inflammation and reactivity in adults with established atopic asthma in vivo. METHODS: Nineteen GSTM1 wild-type and eighteen GSTM1-null individuals with mild atopic asthma underwent methacholine and inhaled allergen challenges, and endobronchial allergen provocations through a bronchoscope. MEASUREMENTS AND MAIN RESULTS: The influx of inflammatory cells, panels of cytokines and chemokines linked to asthmatic inflammation, F(2)-isoprostanes (markers of oxidative stress), and IgE were measured in bronchoalveolar lavage fluid at baseline and 24 hours after allergen instillation. Individuals with asthma with the GSTM1 wild-type genotype had greater baseline and allergen-provoked airway neutrophilia and concentrations of myeloperoxidase than GSTM1-null patients. In contrast, the eosinophilic inflammation was unaffected by GSTM1. The allergen-stimulated generation of acute-stress and proneutrophilic mediators, tumor necrosis factor-α, CXCL-8, IL-1ß, and IL-6, was also greater in the GSTM1 wild-type patients. Moreover, post-allergen airway concentrations of IgE and neutrophil-generated mediators, matrix metalloproteinase-9, B-cell activating factor, transforming growth factor-ß1, and elastase were higher in GSTM1 wild-type individuals with asthma. Total airway IgE correlated with B-cell activating factor concentrations. In contrast, levels of F(2)-isoprostane were comparable in both groups. Finally, GSTM1 wild-type individuals with asthma required lower threshold concentrations of allergen to produce bronchoconstriction. CONCLUSIONS: The functional GSTM1 genotype promotes neutrophilic airway inflammation in humans with atopic asthma in vivo.

Alveolar epithelial cells undergo epithelial-to-mesenchymal transition in response to endoplasmic reticulum stress.

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, ß-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

Peptidylarginine deiminase 2 suppresses inhibitory {kappa}B kinase activity in lipopolysaccharide-stimulated RAW 264.7 macrophages.

Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 µg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 µM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB ß/γ kinase (IKKß/γ) subunits, and PAD2. IKKß increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKß-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKß/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.

Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa.

Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA' that contains two genes for agmatine deiminases (agu2A and agu2A'). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A' contains a twin-arginine translocation signal at its N-terminus and site-directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA' promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA' provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA', specifically its secreted product Agu2A', reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA' operon in the biofilm development of P. aeruginosa.

IL-4 modulates transcriptional control of the mannose receptor in mouse FSDC dendritic cells.

The mannose receptor is a 175 kDa protein found on the surface of macrophages and dendritic cells whose functions include clearance of extracellular hydrolases, internalization of pathogens, and antigen capture. Receptor expression is closely linked to the functional state of these cells and is regulated by cytokines. Previous work has shown that treatment of macrophages and dendritic cells with interleukin-4 leads to increased mannose receptor expression. We have examined the mechanism of this IL-4-mediated up-regulation in the murine dendritic cell line FSDC. IL-4 increased mannose receptor activity, protein, and mRNA. The mannose receptor promoter was functional in FSDCs using transient transfection assays, and IL-4 treatment increased promoter activity 2.6-fold. The responsive region was localized to the proximal 228 bp. Electrophoretic mobility shift assays detected an IL-4-inducible protein that bound to the mannose receptor promoter at a site spanning the region between -147 and -108 bp. The sequence TTAC(N)4CACC (-135 and -124 bp) is similar to the IL-4 response region in the Fc receptor II. Mutation of the flanking TT and CC in this motif blocked IL-4 responsiveness and binding of the IL-4-induced mannose receptor binding protein. This protein does not appear to be STAT6 since neither an anti-STAT6 antibody nor a STAT6 consensus oligonucleotide altered factor binding.