Matrix metalloproteinase-14 mediates a phenotypic shift in the airways to increase mucin production.

RATIONALE: Induced mainly by cigarette smoking, chronic obstructive pulmonary disease (COPD) is a global public health problem characterized by progressive difficulty in breathing and increased mucin production. Previously, we reported that acrolein levels found in COPD sputum could activate matrix metalloproteinase-9 (MMP9). OBJECTIVES: To determine whether acrolein increases expression and activity of MMP14, a critical membrane-bound endopeptidase that can initial a MMP-activation cascade. METHODS: MMP14 activity and adduct formation were measured following direct acrolein treatment. MMP14 expression and activity was measured in human airway epithelial cells. MMP14 immunohistochemistry was performed with COPD tissue, and in acrolein- or tobacco-exposed mice. MEASUREMENTS AND MAIN RESULTS: In a cell-free system, acrolein, in concentrations equal to those found in COPD sputum, directly adducted cysteine 319 in the MMP14 hemopexin-like domain and activated MMP14. In cells, acrolein increased MMP14 activity, which was inhibited by a proprotein convertase inhibitor, hexa-d-arginine. In the airway epithelium of COPD subjects, immunoreactive MMP14 protein increased. In mouse lung, acrolein or tobacco smoke increased lung MMP14 activity and protein. In cells, acrolein-induced MMP14 transcripts were inhibited by an epidermal growth factor receptor (EGFR) neutralizing antibody, EGFR kinase inhibitor, metalloproteinase inhibitor, or mitogen-activated protein kinase (MAPK) 3/2 or MAPK8 inhibitors, but not a MAPK14 inhibitor. Decreasing the MMP14 protein and activity in vitro by small interfering (si)RNA to MMP14 diminished the acrolein-induced MUC5AC transcripts. In acrolein-exposed mice or transgenic mice with lung-specific transforming growth factor-alpha (an EGFR ligand) expression, lung MMP14 and MUC5AC levels increased and these effects were inhibited by a EGFR inhibitor, erlotinib. CONCLUSIONS: Taken together, these findings implicate acrolein-induced MMP14 expression and activity in mucin production in COPD.

Critical role of NOD2 in regulating the immune response to Staphylococcus aureus.

NOD2 (the nucleotide-binding oligomerization domain containing protein 2) is known to be involved in host recognition of bacteria, although its role in the host response to Staphylococcus aureus infection is unknown. NOD2-deficient (Nod2(-/-)) mice and wild-type (WT) littermate controls were injected intraperitoneally with S. aureus suspension (10(7) bacteria/g of body weight), and their survival was monitored. Cultured bone marrow-derived neutrophils were harvested from Nod2(-/-) and WT mice and tested for cytokine production and phagocytosis. Compared to WT mice, Nod2(-/-) mice were significantly more susceptible to S. aureus infection (median survival of 1.5 days versus >5 days; P = 0.003) and had a significantly higher bacterial tissue burden. Cultured bone marrow-derived neutrophils from Nod2(-/-) and WT mice had similar levels of peritoneal neutrophil recruitment and intracellular killing, but bone marrow-derived neutrophils from Nod2(-/-) mice had significantly reduced ability to internalize fluorescein-labeled S. aureus. Nod2(-/-) mice had significantly higher levels of Th1-derived cytokines in serum (tumor necrosis factor alpha, gamma interferon, and interleukin-2 [IL-2]) compared to WT mice, whereas the levels of Th2-derived cytokines (IL-1beta, IL-4, IL-6, and IL-10) were similar in Nod2(-/-) and WT mice. Thus, mice deficient in NOD2 are more susceptible to S. aureus. Increased susceptibility is due in part to defective neutrophil phagocytosis, elevated serum levels of Th1 cytokines, and a higher bacterial tissue burden.

Acrolein-activated matrix metalloproteinase 9 contributes to persistent mucin production.

Chronic obstructive pulmonary disease (COPD), a global public health problem, is characterized by progressive difficulty in breathing, with increased mucin production, especially in the small airways. Acrolein, a constituent of cigarette smoke and an endogenous mediator of oxidative stress, increases airway mucin 5, subtypes A and C (MUC5AC) production; however, the mechanism remains unclear. In this study, increased mMUC5AC transcripts and protein were associated with increased lung matrix metalloproteinase 9 (mMMP9) transcripts, protein, and activity in acrolein-exposed mice. Increased mMUC5AC transcripts and mucin protein were diminished in gene-targeted Mmp9 mice [Mmp9((-/-))] or in mice treated with an epidermal growth factor receptor (EGFR) inhibitor, erlotinib. Acrolein also decreased mTissue inhibitor of metalloproteinase protein 3 (an MMP9 inhibitor) transcript levels. In a cell-free system, acrolein increased pro-hMMP9 cleavage and activity in concentrations (100-300 nM) found in sputum from subjects with COPD. Acrolein increased hMMP9 transcripts in human airway cells, which was inhibited by an MMP inhibitor, EGFR-neutralizing antibody, or a mitogen-activated protein kinase (MAPK) 3/2 inhibitor. Together these findings indicate that acrolein can initiate cleavage of pro-hMMP9 and EGFR/MAPK signaling that leads to additional MMP9 formation. Augmentation of hMMP9 activity, in turn, could contribute to persistent excessive mucin production.

Genotypic characteristics of Staphylococcus aureus isolates from a multinational trial of complicated skin and skin structure infections.

The impact of bacterial genetic characteristics on the outcome of patients with Staphylococcus aureus infections is uncertain. This investigation evaluated potential associations between bacterial genotype and clinical outcome using isolates collected as part of an international phase 2 clinical trial (FAST II) evaluating telavancin for the treatment of complicated skin and skin structure infections (cSSSI). Ninety S. aureus isolates from microbiologically evaluable patients with cSSSI enrolled in the FAST II trial from 11 sites in the United States (56 isolates, or 62%) and 7 sites in South Africa (34 isolates, or 38%) were examined for staphylococcal cassette chromosome mec, agr, and the presence of 31 virulence genes and subjected to pulsed-field gel electrophoresis (PFGE). South African methicillin-susceptible S. aureus (MSSA) isolates were more likely to carry certain virulence genes, including sdrD (P = 0.01), sea (P < 0.01), and pvl (P = 0.01). All 44 (49%) methicillin-resistant S. aureus (MRSA) isolates were from the United States; 37 (84%) were strain USA 300 by PFGE. In the United States, MRSA isolates were more likely than MSSA isolates to carry genes for sdrC (P = 0.03), map/eap (P = 0.05), fnbB (P = 0.11), tst (P = 0.02), sea (P = 0.04), sed (P = 0.04), seg (P = 0.11), sej (P = 0.11), agr (P = 0.09), V8 (P = 0.06), sdrD, sdrE, eta, etb, and see (P < 0.01 for all). MRSA isolates were more often clonal than MSSA isolates by PFGE. Isolates from patients who were cured were significantly more likely to contain the pvl gene than isolates from patients that failed or had indeterminate outcomes (79/84 [94%] versus 3/6 [50%]; P = 0.01). S. aureus strains from different geographic regions have different distributions of virulence genes.

The microbiota regulates neutrophil homeostasis and host resistance to Escherichia coli K1 sepsis in neonatal mice.

Neonatal colonization by microbes, which begins immediately after birth, is influenced by gestational age and the mother's microbiota and is modified by exposure to antibiotics. In neonates, prolonged duration of antibiotic therapy is associated with increased risk of late-onset sepsis (LOS), a disorder controlled by neutrophils. A role for the microbiota in regulating neutrophil development and susceptibility to sepsis in the neonate remains unclear. We exposed pregnant mouse dams to antibiotics in drinking water to limit transfer of maternal microbes to the neonates. Antibiotic exposure of dams decreased the total number and composition of microbes in the intestine of the neonates. This was associated with decreased numbers of circulating and bone marrow neutrophils and granulocyte/macrophage-restricted progenitor cells in the bone marrow of antibiotic-treated and germ-free neonates. Antibiotic exposure of dams reduced the number of interleukin-17 (IL-17)-producing cells in the intestine and production of granulocyte colony-stimulating factor (G-CSF). Granulocytopenia was associated with impaired host defense and increased susceptibility to Escherichia coli K1 and Klebsiella pneumoniae sepsis in antibiotic-treated neonates, which could be partially reversed by administration of G-CSF. Transfer of a normal microbiota into antibiotic-treated neonates induced IL-17 production by group 3 innate lymphoid cells (ILCs) in the intestine, increasing plasma G-CSF levels and neutrophil numbers in a Toll-like receptor 4 (TLR4)- and myeloid differentiation factor 88 (MyD88)-dependent manner and restored IL-17-dependent resistance to sepsis. Specific depletion of ILCs prevented IL-17- and G-CSF-dependent granulocytosis and resistance to sepsis. These data support a role for the intestinal microbiota in regulation of granulocytosis, neutrophil homeostasis and host resistance to sepsis in neonates.

The role of metallothionein in the pathogenesis of acute lung injury.

Often fatal, acute lung injury has a complicated etiology. Previous studies from our laboratory in mice have demonstrated that survival during acute lung injury is a complex trait governed by multiple loci. We also found that the increase in metallothionein (MT) is one of the greatest noted in transcriptome-wide analyses of gene expression. To assess the role of MT in nickel-induced acute lung injury, the survival of Mt-transgenic, Mt1/2(+/+), and Mt1/2(-/-) mice was compared. Pulmonary inflammation and global gene expression were compared in Mt1/2(+/+) and Mt1/2(-/-) mice. Gene-targeted Mt1/2(-/-) mice were more susceptible than Mt1/2(+/+) mice to nickel-induced inflammation, surfactant-associated protein B transcript loss, and lethality. Similarly, Mt-transgenic mice exhibited increased survival. MAPPFinder analyses also noted significant decreases in genes involved in protein processing (e.g., ubiquitination, folding), which were greater in Mt1/2(-/-) mice as compared with Mt1/2(+/+) mice early in the progression of acute lung injury, possibly due to a zinc-mediated transcript destabilization. In contrast, transcript levels of genes associated with the inflammatory response, extracellular matrix regulation, and coagulation/fibrinolysis were increased more in Mt1/2(-/-) mice as compared with Mt1/2(+/+) mice late in the development of acute lung injury. Thus, MT ultimately improves survival in the progression of acute lung injury in mice. Transcriptome-wide analysis suggests that this survival may be mediated through changes in the destabilization of transcripts associated with protein processing, the subsequent augmentation of transcripts controlling inflammation, extracellular matrix regulation, coagulation/fibrinolysis, and disruption of surfactant homeostasis.

Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation.

Chronic obstructive pulmonary disease is marked by alveolar enlargement and excess production of airway mucus. Acrolein, a component of cigarette smoke, increases mucin 5AC (MUC5AC), a prevalent airway mucin in NCI-H292 cells by transcriptional activation, but the signal transduction pathways involved in acrolein-induced MUC5AC expression are unknown. Acrolein depleted cellular glutathione at doses of 10 muM or greater, higher than those sufficient (0.03 muM) to increase MUC5AC mRNA, suggesting that MUC5AC expression was independent of oxidative stress. In contrast, acrolein increased MUC5AC mRNA levels by phosphorylating epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase 3/2, or MAPK 3/2(ERK1/2). Pretreating the cells with an EGFR-neutralizing antibody, or a metalloproteinase inhibitor, decreased the acrolein-induced MUC5AC mRNA increase. Small, interfering RNA directed against ADAM17 or MMP9 inhibited the acrolein-induced MUC5AC mRNA increase. Acrolein increased the release and subsequent activation of pro-MMP9. Acrolein increased MMP9 and decreased tissue inhibitor of metalloproteinase 3 (TIMP3), an endogenous inhibitor of ADAM17, transcripts. Together, these data suggest that acrolein induces MUC5AC expression via an initial ligand-dependent activation of EGFR mediated by ADAM17 and MMP9. In addition, a prolonged effect of acrolein may be mediated by altering MMP9 and TIMP3 transcription.