Prior cigarette smoke exposure does not affect acute post-stroke outcomes in mice

Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death globally and is characterized by airflow limitation that is progressive and not fully reversible. Cigarette smoking is the major cause of COPD. Fifty percent of deaths in the COPD population are due to a cardiovascular event and it is now recognised that COPD is a risk factor for stroke. Whether COPD increases stroke severity has not been explored. The aim of this study was to investigate whether functional and histological endpoints of stroke outcomes in mice after transient middle cerebral artery occlusion (tMCAo) were more severe in mice exposed to cigarette smoke (CS). 7-week-old male C57BL/6 mice were exposed to room air or CS generated from 9 cigarettes/day, 5 days/week for 2, 8 and 12 weeks. Following air or CS exposure, mice underwent tMCAO surgery with an ischaemic period of 30–40 min or sham surgery. Mice were euthanised 24 h following the induction of ischaemia and bronchoalveolar lavage fluid (BALF), lungs and brains collected. Mice exposed to CS for 2 weeks and subjected to a stroke had similar BALF macrophages to air-exposed and stroke mice. However, CS plus stroke mice had significantly more BALF total cells, neutrophils and lymphocytes than air plus stroke mice. Mice exposed to CS for 8 and 12 weeks had significantly greater BALF total cells, macrophages, neutrophils and lymphocytes than air-exposed mice, but stroke did not affect CS-induced BALF cellularity. Prior CS exposure did not worsen stroke-induced neurological deficit scores, reduced foregrip strength, infarct and oedema volumes. Collectively, we found that although CS exposure caused significant BALF inflammation, it did not worsen acute post-stroke outcomes in mice. This data suggests that while patients with COPD are at increased risk of stroke, it may not translate to COPD patients having more severe stroke outcomes

Cigarette smoke extract exacerbates hyperpermeability of cerebral endothelial cells after oxygen glucose deprivation and reoxygenation

Cigarette smoking is a risk factor for stroke and is linked to stroke severity. Previous studies have shown that cigarette smoke extract (CSE) triggers endothelial dysfunction in vitro by initiating oxidative stress and/or an inflammatory response. In addition, cerebral endothelial dysfunction (particularly at the level of the blood-brain barrier [BBB]) contributes to stroke pathogenesis. Therefore, we hypothesized that cigarette smoking may influence stroke, at least in part, by exacerbating ischaemia-induced BBB disruption. To test this, we examined the effect of CSE on the permeability of cerebral endothelial cells exposed to oxygen glucose deprivation and reoxygenation (OGD + RO). We found that the loss of BBB integrity following ischaemic/reperfusion-like conditions was significantly worsened by CSE. Despite this being associated with increased mRNA expression of Nox catalytic subunits, reactive oxygen species (ROS) levels were however markedly lower. Furthermore, this occurred in association with elevated expression of antioxidant enzymes (SOD1, SOD2, and Gpx-1), suggesting an antioxidant defence response. Lastly, we found that CSE significantly upregulated mRNA expression of cytokines (IL-6 and TGF-β). Collectively, these results show that acute exposure to CSE worsens BBB disruption caused by OGD + RO, however, this is not linked to elevated ROS levels but may involve inflammatory mechanisms

Akt in the pathogenesis of COPD

In this review we consider the therapeutic potential of targeting Akt for the treatment of COPD. Akt is a serine/threonine protein kinase that functions as a signaling intermediate linked to multiple signaling programs involved in survival, inflammation, and growth. Akt is closely associated with key membrane-bound receptors and represents a convergent integration point for multiple stimuli implicated in COPD pathogenesis. Persistent activation of Akt secondary to somatic mutations in regulatory oncogenes, such as PTEN, may explain why inflammation in COPD does not resolve when smoking is ceased. Akt is also implicated in the systemic manifestations of COPD such as skeletal muscle wasting and metabolic disturbances. Furthermore, targeting Akt may provide a useful means of limiting the severity and duration of disease exacerbations in COPD. As such, Akt represents a particularly attractive therapeutic target for the treatment of COPD. Interestingly, current knowledge suggests that both inhibitors and activators of Akt may be useful for treating different clinical subpopulations of COPD patients

Glutathione Peroxidase-1 Primes Pro-Inflammatory Cytokine Production after LPS Challenge In Vivo

Reactive oxygen species produced during the innate immune response to LPS are important agents of anti-pathogen defence but may also cause oxidative lung damage. Glutathione peroxidase-1 (gpx-1) is an anti-oxidant enzyme that may protect lungs from such damage. We assessed the in vivo importance of gpx-1 in LPS-induced lung inflammation. Male wild-type (WT) or gpx-1 deficient (gpx-1−/−) mice were treated intranasally with PBS or 10 µg LPS and killed 3 and 24 h post LPS. Lungs were lavaged with PBS and then harvested for inflammatory marker expression. LPS caused an intense neutrophilia in WT BALF evident 3 and 24 h post challenge that was reduced in gpx-1−/− mice. In addition, LPS-treated gpx-1−/− mice had significantly fewer macrophages than LPS-treated WT mice. To understand the basis for this paradoxical reduction we assessed inflammatory cytokines and proteases at protein and transcript levels. MMP-9 expression and net gelatinase activity in BALF of gpx-1−/− mice treated with LPS for 3 and 24 h was no different to that found in LPS-treated WT mice. BALF from LPS-treated gpx-1−/− mice (3 h) had less TNF-α, MIP-2 and GM-CSF protein than LPS-treated WT mice. In contrast, LPS-induced increases in TNF-α, MIP-2 and GM-CSF mRNA expression in WT mice were similar to those observed in gpx-1−/− mice. These attenuated protein levels were unexpectedly not mirrored by reduced mRNA transcripts but were associated with increased 20S proteasome expression. Thus, these data suggest that gpx-1 primes pro-inflammatory cytokine production after LPS challenge in vivo

Resolving Viral-Induced Secondary Bacterial Infection in COPD: A Concise Review

Chronic obstructive pulmonary disease (COPD) is a leading cause of disability and death world-wide, where chronic inflammation accelerates lung function decline. Pathological inflammation is worsened by chronic bacterial lung infections and susceptibility to recurrent acute exacerbations of COPD (AECOPD), typically caused by viral and/or bacterial respiratory pathogens. Despite ongoing efforts to reduce AECOPD rates with inhaled corticosteroids, COPD patients remain at heightened risk of developing serious lung infections/AECOPD, frequently leading to hospitalization and infection-dependent delirium. Here, we review emerging mechanisms into why COPD patients are susceptible to chronic bacterial infections and highlight dysregulated inflammation and production of reactive oxygen species (ROS) as central causes. This underlying chronic infection leaves COPD patients particularly vulnerable to acute viral infections, which further destabilize host immunity to bacteria. The pathogeneses of bacterial and viral exacerbations are significant as clinical symptoms are more severe and there is a marked increase in neutrophilic inflammation and tissue damage. AECOPD triggered by a bacterial and viral co-infection increases circulating levels of the systemic inflammatory marker, serum amyloid A (SAA). SAA is a functional agonist for formyl peptide receptor 2 (FPR2/ALX), where it promotes chemotaxis and survival of neutrophils. Excessive levels of SAA can antagonize the protective actions of FPR2/ALX that involve engagement of specialized pro-resolving mediators, such as resolvin-D1. We propose that the anti-microbial and anti-inflammatory actions of specialized pro-resolving mediators, such as resolvin-D1 should be harnessed for the treatment of AECOPD that are complicated by the co-pathogenesis of viruses and bacteria

Urokinase-type plasminogen activator and arthritis progression: role in systemic disease with immune complex involvement

INTRODUCTION: Urokinase-type plasminogen activator (u-PA) has been implicated in fibrinolysis, cell migration, latent cytokine activation, cell activation, T-cell activation, and tissue remodeling, all of which are involved in the development of rheumatoid arthritis. Previously, u-PA has been reported to play a protective role in monoarticular arthritis models involving mBSA as the antigen, but a deleterious role in the systemic polyarticular collagen-induced arthritis (CIA) model. The aim of the current study is to determine how u-PA might be acting in systemic arthritis models. METHODS: The CIA model and bone marrow chimeras were used to determine the cellular source of u-PA required for the arthritis development. Gene expression of inflammatory and destructive mediators was measured in joint tissue by quantitiative PCR and protein levels by ELISA. The requirement for u-PA in the type II collagen mAb-induced arthritis (CAIA) and K/BxN serum transfer arthritis models was determined using u-PA(-/-) mice. Neutrophilia was induced in the peritoneal cavity using either ovalbumin/anti-ovalbumin or the complement component C5a. RESULTS: u-PA from a bone marrow-derived cell was required for the full development of CIA. The disease in u-PA(-/-) mice reconstituted with bone marrow from C57BL/6 mice was indistinguishable from that in C57BL/6 mice, in terms of clinical score, histologic features, and protein and gene expression of key mediators. u-PA(-/-) mice were resistant to both CAIA and K/BxN serum transfer arthritis development. u-PA(-/-) mice developed a reduced neutrophilia and chemokine production in the peritoneal cavity following ovalbumin/anti-ovalbumin injection; in contrast, the peritoneal neutrophilia in response to C5a was u-PA independent. CONCLUSIONS: u-PA is required for the full development of systemic arthritis models involving immune complex formation and deposition. The cellular source of u-PA required for CIA is bone marrow derived and likely to be of myeloid origin. For immune complex-mediated peritonitis, and perhaps some other inflammatory responses, it is suggested that the u-PA involvement may be upstream of C5a signaling

Inhibition of Nox2 Oxidase Activity Ameliorates Influenza A Virus-Induced Lung Inflammation

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2−/y mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1β) cytokines/chemokines, CD8+ T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2−/y mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1β were ∼3-fold higher in Nox2−/y mice. The numbers of influenza-specific CD8+DbNP366+ and DbPA224+ T cells in the BALF and spleen were comparable in WT and Nox2−/y mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner

Lipopolysaccharide does not alter small airway reactivity in mouse lung slices

The bacterial endotoxin, lipopolysaccharide (LPS) has been associated with occupational airway diseases with asthma-like symptoms and in acute exacerbations of COPD. The direct and indirect effects of LPS on small airway reactivity have not been fully elucidated. We tested the hypothesis that both in vitro and in vivo LPS treatment would increase contraction and impair relaxation of mouse small airways. Lung slices were prepared from naïve Balb/C mice and cultured in the absence or presence of LPS (10 µg/ml) for up to 48 h for measurement of TNFα levels in conditioned media. Alternatively, mice were challenged with PBS or LPS in vivo once a day for 4 days for preparation of lung slices or for harvest of lungs for Q-PCR analysis of gene expression of pro-inflammatory cytokines and receptors involved in airway contraction. Reactivity of small airways to contractile agonists, methacholine and serotonin, and bronchodilator agents, salbutamol, isoprenaline and rosiglitazone, were assessed using phase-contrast microscopy. In vitro LPS treatment of slices increased TNFα release 6-fold but did not alter contraction or relaxation to any agonists tested. In vivo LPS treatment increased lung gene expression of TNFα, IL-1β and ryanodine receptor isoform 2 more than 5-fold. However there were no changes in reactivity in lung slices from these mice, even when also incubated with LPS ex vivo. Despite evidence of LPS-induced inflammation, neither airway hyperresponsiveness or impaired dilator reactivity were evident. The increase in ryanodine receptor isoform 2, known to regulate calcium signaling in vascular smooth muscle, warrants investigation. Since LPS failed to elicit changes in small airway reactivity in mouse lung slices following in vitro or in vivo treatment, alternative approaches are required to define the potential contribution of this endotoxin to altered small airway reactivity in human lung diseases

Aspirin-triggered resolvin D1 reduces pneumococcal lung infection and inflammation in a viral and bacterial coinfection pneumonia model

Formyl peptide receptor 2/lipoxin A4 (LXA4) receptor (Fpr2/ALX) co-ordinates the transition from inflammation to resolution during acute infection by binding to distinct ligands including serum amyloid A (SAA) and Resolvin D1 (RvD1). Here, we evaluated the proresolving actions of aspirin-triggered RvD1 (AT-RvD1) in an acute coinfection pneumonia model. Coinfection with Streptococcus pneumoniae and influenza A virus (IAV) markedly increased pneumococcal lung load and neutrophilic inflammation during the resolution phase. Fpr2/ALX transcript levels were increased in the lungs of coinfected mice, and immunohistochemistry identified prominent Fpr2/ALX immunoreactivity in bronchial epithelial cells and macrophages. Levels of circulating and lung SAA were also highly increased in coinfected mice. Therapeutic treatment with exogenous AT-RvD1 during the acute phase of infection (day 4–6 post-pneumococcal inoculation) significantly reduced the pneumococcal load. AT-RvD1 also significantly reduced neutrophil elastase (NE) activity and restored total antimicrobial activity in bronchoalveolar lavage (BAL) fluid (BALF) of coinfected mice. Pneumonia severity, as measured by quantitating parenchymal inflammation or alveolitis was significantly reduced with AT-RvD1 treatment, which also reduced the number of infiltrating lung neutrophils and monocytes/macrophages as assessed by flow cytometry. The reduction in distal lung inflammation in AT-RvD1-treated mice was not associated with a significant reduction in inflammatory and chemokine mediators. In summary, we demonstrate that in the coinfection setting, SAA levels were persistently increased and exogenous AT-RvD1 facilitated more rapid clearance of pneumococci in the lungs, while concurrently reducing the severity of pneumonia by limiting excessive leukocyte chemotaxis from the infected bronchioles to distal areas of the lungs