Activation of olfactory receptors on mouse pulmonary macrophages promotes monocyte chemotactic protein-1 production

Background: Emerging evidence suggests that non-olfactory tissues and cells can express olfactory receptors (ORs), however, the exact function of ectopic OR expression remains unknown. We have previously shown in mouse models that a unique cooperation between interferon-γ (IFN-γ) and lipopolysaccharide (LPS) drives the activation of pulmonary macrophages and leads to the induction of pathogenic responses in the respiratory tract. Further, through gene array studies, we have shown that activation of macrophages by these molecules results in the selective expression of a number of ORs. In this study, we validated the expression of these ORs in mouse airway and pulmonary macrophages in response to IFN-γ and LPS (γ/LPS) stimulation, and further explored the effect of odorant stimulation on macrophage function. Methodology/Principal Findings: OR expression in airway and pulmonary macrophages in response to IFN-γ, LPS or γ/LPS treatments was assessed by microarray and validated by q-PCR. OR expression (e.g. OR622) on macrophages was confirmed by visualization in immunofluoresence assays. Functional responses to odorants were assessed by quantifying inflammatory cytokine and chemokine expression using q-PCR and cell migration was assessed by a modified Boyden chamber migration assay. Our results demonstrate that eight ORs are expressed at basal levels in both airway and pulmonary macrophages, and that γ/LPS stimulation cooperatively increased this expression. Pulmonary macrophages exposed to the combined treatment of γ/LPS+octanal (an odorant) exhibited a 3-fold increase in MCP-1 protein production, compared to cells treated with γ/LPS alone. Supernatants from γ/LPS+octanal exposed macrophages also increased macrophage migration in vitro. Conclusions/Significance: Eight different ORs are expressed at basal levels in pulmonary macrophages and expression is upregulated by the synergistic action of γ/LPS. Octanal stimulation further increased MCP-1 production and the motility of macrophages. Our results suggest that ORs may mediate macrophage function by regulating MCP-1 production and cell migration. © 2013 Li et al

Maturation of molybdoenzymes and its influence on the pathogenesis of non-typeable Haemophilus influenzae

© 2015 Dhouib, Pg Othman, Essilfie, Hansbro, Hanson, McEwan and Kappler. Mononuclear molybdenum enzymes of the dimethylsulfoxide (DMSO) reductase family occur exclusively in prokaryotes, and a loss of some these enzymes has been linked to a loss of bacterial virulence in several cases. The MobA protein catalyzes the final step in the synthesis of the molybdenum guanine dinucleotide (MGD) cofactor that is exclusive to enzymes of the DMSO reductase family. MobA has been proposed as a potential target for control of virulence since its inhibition would affect the activities of all molybdoenzymes dependent upon MGD. Here, we have studied the phenotype of a mobA mutant of the host-adapted human pathogen Haemophilus influenzae. H. influenzae causes and contributes to a variety of acute and chronic diseases of the respiratory tract, and several enzymes of the DMSO reductase family are conserved and highly expressed in this bacterium. The mobA mutation caused a significant decrease in the activities of all Mo-enzymes present, and also resulted in a small defect in anaerobic growth. However, we did not detect a defect in in vitro biofilm formation nor in invasion and adherence to human epithelial cells in tissue culture compared to the wild-type. In a murine in vivo model, the mobA mutant showed only a mild attenuation compared to the wild-type. In summary, our data show that MobA is essential for the activities of molybdenum enzymes, but does not appear to affect the fitness of H. influenzae. These results suggest that MobA is unlikely to be a useful target for antimicrobials, at least for the purpose of treating H. influenzae infections

Correction: Antagonism of miR-328 Increases the Antimicrobial Function of Macrophages and Neutrophils and Rapid Clearance of Non-typeable Haemophilus Influenzae (NTHi) from Infected Lung

[This corrects the article DOI: 10.1371/journal.ppat.1004549.]

Antagonism of miR-328 Increases the Antimicrobial Function of Macrophages and Neutrophils and Rapid Clearance of Non-typeable Haemophilus Influenzae (NTHi) from Infected Lung

© 2015 Tay et al. Pathogenic bacterial infections of the lung are life threatening and underpin chronic lung diseases. Current treatments are often ineffective potentially due to increasing antibiotic resistance and impairment of innate immunity by disease processes and steroid therapy. Manipulation miRNA directly regulating anti-microbial machinery of the innate immune system may boost host defence responses. Here we demonstrate that miR-328 is a key element of the host response to pulmonary infection with non-typeable haemophilus influenzae and pharmacological inhibition in mouse and human macrophages augments phagocytosis, the production of reactive oxygen species, and microbicidal activity. Moreover, inhibition of miR-328 in respiratory models of infection, steroid-induced immunosuppression, and smoke-induced emphysema enhances bacterial clearance. Thus, miRNA pathways can be targeted in the lung to enhance host defence against a clinically relevant microbial infection and offer a potential new anti-microbial approach for the treatment of respiratory diseases

A novel, molybdenum-containing methionine sulfoxide reductase supports survival of Haemophilus influenzae in an in vivo model of infection

© 2016 Dhouib, Othman, Lin, Lai, Wijesinghe, Essilfie, Davis, Nasreen, Bernhardt, Hansbro, McEwan and Kappler. Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while ~3.6 × 103 CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia coli TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized clade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens

Nontypeable haemophilus influenzae induces sustained lung oxidative stress and protease expression

© 2015 King et al. Nontypeable Haemophilus influenzae (NTHi) is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1), oxidative stress and 2), protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS) production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT) in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps

Combined Haemophilus influenzae respiratory infection and allergic airways disease drives chronic infection and features of neutrophilic asthma

Background: 20-30% of patients with asthma have neutrophilic airway inflammation and reduced responsiveness to steroid therapy. They often have chronic airway bacterial colonisation and Haemophilus influenzae is one of the most commonly isolated bacteria. The relationship between chronic airway colonisation and the development of steroid-resistant neutrophilic asthma is unclear. Objectives: To investigate the relationship between H influenzae respiratory infection and neutrophilic asthma using mouse models of infection and ovalbumin (OVA)- induced allergic airways disease. Methods: BALB/c mice were intratracheally infected with H influenzae (day 10), intraperitoneally sensitised (day 0) and intranasally challenged (day 12-15) with OVA. Treatment groups were administered dexamethasone intranasally during OVA challenge. Infection, allergic airways disease, steroid sensitivity and immune responses were assessed (days 11, 16 and 21). Results: The combination of H influenzae infection and allergic airways disease resulted in chronic lung infection that was detected on days 11, 16 and 21 (21, 26 and 31 days after infection). Neutrophilic allergic airways disease and T helper 17 cell development were induced, which did not require active infection. Importantly, all features of neutrophilic allergic airways disease were steroid resistant. Toll-like receptor 4 expression and activation of phagocytes was reduced, but most significantly the influx and/or development of phagocytosing neutrophils and macrophages into the airways was inhibited. Conclusions: The combination of infection and allergic airways disease promotes bacterial persistence, leading to the development of a phenotype similar to steroid-resistant neutrophilic asthma and which may result from dysfunction in innate immune cells. This indicates that targeting bacterial infection in steroid-resistant asthma may have therapeutic benefit

Time-resolved proteomic profiling of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. METHODS: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. RESULTS: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. CONCLUSION: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies

Do Young Hepatocellular Carcinoma Patients Have Worse Prognosis? The Paradox of Age as a Prognostic Factor in the Survival of Hepatocellular Carcinoma Patients

Background/Aims: Our previous study showed that male hepatocellular carcinoma (HCC) patients below 40 years of age had the worst survival in the initial several years, but had the best prognosis thereafter. Thus, it seems that age has a paradoxical influence on the prognosis. To further clarify the issue of age on HCC prognosis, we initiated this study. Methods: A total of 11 312 HCC cases from seven medical centers from 1986 to 2002 were included. We analyzed the 1-year survival and survival after 1 year. Results: Male gender, age younger than 40 years old and hepatitis B virus (HBV) were associated with worse 1-year survival. In contrast, male gender, age younger than 40 years old and HBV were associated with better survival after 1 year. Higher percentage of the young HCC patients had a tumor size larger than 3 cm. 83.7% of HCC patients below 40 years of age were male and 89.8% of them were HBV carriers. Conclusions: If we encountered a young HCC patient, the patient will probably be a male HBV carrier. He would probably have larger tumor and is more likely to expire within 1 year than the older HCC patients. However, if the young HCC patient can survive for more than 1 year, he would probably have better survival in the following years than the older patients