Sissie Wong

Resveratrol, an extract of red wine, inhibits lipopolysaccharide induced airway neutrophilia and inflammatory mediators through an NF-κB-independent mechanism

Consumption of a naturally occurring polyphenol, resveratrol, in particular through drinking moderate amounts of red wine, has been suggested to be beneficial to health. A plethora of in vitro studies published demonstrate various anti‐inflammatory actions of resveratrol. The aim of this research was to determine whether any of these anti‐inflammatory effects translate in vivo in a rodent model of LPS induced airway inflammation. Resveratrol reduced lung tissue neutrophilia to a similar magnitude as that achieved by treatment with budesonide. This was associated with a reduction in pro‐inflammatory cytokines and prostanoid levels. Interestingly, the reduction did not appear to be due to an impact on NF‐κB activation or the expression of the respective genes as suggested by various in vitro publications. These results suggest that resveratrol may possess anti‐inflammatory properties via a novel mechanism. Elucidation of this mechanism may lead to potential new therapies for the treatment of chronic inflammation.

Impact of tobacco-smoke on key signaling pathways in the innate immune response in lung macrophages.

Many of the healthcare consequences of cigarette smoking could be due to its ability to compromise the immune system, and in respiratory diseases like chronic obstructive pulmonary disease (COPD), a constant low level of infection could be responsible for some of the symptoms/pathology. The aim was to assess the impact of cigarette smoke (CS) on the release of innate effector cytokines in THP‐1 cells and human lung macrophages, and to determine the molecular mechanism behind the altered response. Cells were exposed to CS with and without endotoxin stimulus, cytokines, glutathione, mitogen‐activated protein kinase (MAPK) phosphorylation, IκB kinase‐2 (IKK‐2) activity, nuclear factor kappa B (NF‐κB), and activator protein‐1 (AP‐1) pathway activation was measured. Attempts were made to mimic or block the effect of CS by using nicotine, nitric oxide donors/inhibitors, prostanoid inhibitors, and anti‐oxidants. Results showed that CS initially delayed the production of “innate” cytokines (e.g., IL‐1β and IL‐6) and reduced glutathione levels. This was associated with a reduction in NF‐κB pathway activation, which suggested a causative link. CS also increased the phosphorylation of MAPKs and the production of IL‐8 but interestingly only in stimulated cells. Exogenous glutathione treatment reversed both these effects of CS, which suggests that this molecule may play a central role. In conclusion, this data provides a novel mechanistic explanation for why smokers have increased prevalence/severity of respiratory infections. In addition, the suppression of the innate response is accompanied by an increase in the neutrophil chemoattractant, IL‐8, which may suggest a link to the pathogenesis of smoking‐related inflammatory disease. J. Cell. Physiol. 214:27–37, 2008.

P2X7 Receptor and Caspase 1 Activation Are Central to Airway Inflammation Observed after Exposure to Tobacco Smoke

Chronic Obstructive Pulmonary Disease (COPD) is a cigarette smoke (CS)-driven inflammatory airway disease with an increasing global prevalence. Currently there is no effective medication to stop the relentless progression of this disease. It has recently been shown that an activator of the P2X7/inflammasome pathway, ATP, and the resultant products (IL-1β/IL-18) are increased in COPD patients. The aim of this study was to determine whether activation of the P2X7/caspase 1 pathway has a functional role in CS-induced airway inflammation. Mice were exposed to CS twice a day to induce COPD-like inflammation and the role of the P2X7 receptor was investigated. We have demonstrated that CS-induced neutrophilia in a pre-clinical model is temporally associated with markers of inflammasome activation, (increased caspase 1 activity and release of IL-1β/IL-18) in the lungs. A selective P2X7 receptor antagonist and mice genetically modified so that the P2X7 receptors were non-functional attenuated caspase 1 activation, IL-1β release and airway neutrophilia. Furthermore, we demonstrated that the role of this pathway was not restricted to early stages of disease development by showing increased caspase 1 activation in lungs from a more chronic exposure to CS and from patients with COPD. This translational data suggests the P2X7/Inflammasome pathway plays an ongoing role in disease pathogenesis. These results advocate the critical role of the P2X7/caspase 1 axis in CS-induced inflammation, highlighting this as a possible therapeutic target in combating COPD.

PPAR‐γ agonists as therapy for diseases involving airway neutrophilia

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear hormone receptors belonging to the steroid receptor superfamily. Previously, the present authors have shown that PPAR‐γ agonists inhibit the release of inflammatory cell survival factors and induce apoptosis in vitro. The aim of this study was to determine the effect of two structurally different PPAR agonists in an in vivo model of lipopolysaccharide (LPS)-induced airway inflammation. Mice were treated with PPAR agonists, rosiglitazone or SB 219994, prior to exposure to aerosolised LPS, and the extent of airway inflammation was assessed 3 h later. In these experiments, the PPAR ligands inhibited LPS-induced airway neutrophilia and associated chemoattractants/survival factors (keratinocyte-derived chemokine and granulocyte-colony stimulating factor) in the mouse lung. The present authors postulate that if a peroxisome proliferator-activated receptor agonist has the same effect in man, and neutrophils are important in the progression of respiratory diseases, such as chronic obstructive pulmonary disease, then this class of compounds could be a potential therapy. Furthermore, several peroxisome proliferator-activated receptor‐γ agonists have been shown to be clinically effective for the treatment of type II diabetes, suggesting that any benefit of peroxisome proliferator-activated receptor‐γ ligands in the progression of respiratory diseases, which may involve airway neutrophilia, could be explored relatively quickly.

IκB kinase-2 independent and dependent inflammation in airway disease models: relevance of IKK- 2 inhibition to the clinic

Nuclear factor κB (NF-κB) is a transcription factor believed to be central in the expression of numerous inflammatory genes and the pathogenesis of many respiratory diseases. We have previously demonstrated increased NF-κB pathway activation in a steroid-sensitive animal model of lipopolysaccharide (LPS)-driven airway inflammation. It is noteworthy that this phenomenon was not observed in a steroid-insensitive model of elastase-induced inflammation in the rat. The aim of this study was to gather further evidence to suggest that these similar profiles of neutrophilic inflammation can be NF-κB-dependent or -independent by determining the impact of an IκB kinase-2 (IKK-2) inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1). In the LPS model, TPCA-1 blocked the increase in NF-κB DNA binding, a marker of NF-κB pathway activation. This inhibition was associated with a reduction in inflammatory mediator release [tumor necrosis factor α (TNFα)/interleukin-1β (IL-1β)/matrix metalloproteinase-9 (MMP-9)] and lung inflammatory cell burden (neutrophilia/eosinophilia). These data were paralleled with a steroid and in human cell based assays. In the elastase-driven inflammation model, in which our group has previously failed to measure an increase in NF-κB DNA binding, neither TPCA-1 nor the steroid, affected mediator release (IL-1β/MMP-9) or cellular burden (neutrophilia/lymphomononuclear cells). This is the first study to examine the effect of an IKK-2 inhibitor in well validated models that mimic aspects of the inflammatory lesion evident in diseases such as COPD. In conclusion, we have demonstrated that animal models with similar profiles of airway inflammation can be IKK-2 inhibitor/steroid-sensitive or -insensitive. If both profiles of inflammation exist in the clinic, then this finding is extremely exciting and may lead to greater understanding of disease pathology and the discovery of novel anti-inflammatory targets.

Novel role for the Liver X nuclear receptor in the suppression of lung inflammatory responses

The liver X receptors (LXRα/β) are part of the nuclear receptor family and are believed to regulate cholesterol and lipid homeostasis. It has also been suggested that LXR agonists possess anti-inflammatory properties. The aim of this work was to determine the effect of LXR agonists on the innate immune response in human primary lung macrophages and a pre-clinical rodent model of lung inflammation. Before profiling the impact of the agonist, we established that both the human macrophages and the rodent lungs expressed LXRα/β. We then used two structurally distinct LXR agonists to demonstrate that activation of this transcription factor reduces cytokine production in THP-1 cells and lung macrophages. Then, using the expression profile of ATP binding cassettes A1 (ABCA-1; a gene directly linked to LXR activation) as a biomarker for lung exposure of the compound, we demonstrated an LXR-dependent reduction in lung neutrophilia rodents in vivo. This inhibition was not associated with a suppression of c-Fos/c-Jun mRNA expression or NF-κB/AP-1 DNA binding, suggesting that any anti-inflammatory activity of LXR agonists is not via inhibition of NF-κB/AP-1 transcriptional activity. These data do not completely rule out an impact of these agonists on these two prominent transcription factors. In summary, this study is the first to demonstrate anti-inflammatory actions of LXRs in the lung. Chronic innate inflammatory responses observed in some airway diseases is thought to be central to disease pathogenesis. Therefore, data suggest that LXR ligands have utility in the treatment of lung diseases that involves chronic inflammation mediated by macrophages and neutrophils.

Anti-inflammatory effects of PGE2 in the lung: role of the EP4 receptor subtype

Background Asthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the airway. Current treatment options (long acting β-adrenoceptor agonists and glucocorticosteroids) are not optimal as they are only effective in certain patient groups and safety concerns exist regarding both compound classes. Therefore, novel bronchodilator and anti-inflammatory strategies are being pursued. Prostaglandin E2 (PGE2) is an arachidonic acid-derived eicosanoid produced by the lung which acts on four different G-protein coupled receptors (EP1–4) to cause an array of beneficial and deleterious effects. The aim of this study was to identify the EP receptor mediating the anti-inflammatory actions of PGE2 in the lung using a range of cell-based assays and in vivo models. Methods and results It was demonstrated in three distinct model systems (innate stimulus, lipopolysaccharide (LPS); allergic response, ovalbumin (OVA); inhaled pollutant, cigarette smoke) that mice missing functional EP4 (Ptger4−/−) receptors had higher levels of airway inflammation, suggesting that endogenous PGE2 was suppressing inflammation via EP4 receptor activation. Cell-based assay systems (murine and human monocytes/alveolar macrophages) demonstrated that PGE2 inhibited cytokine release from LPS-stimulated cells and that this was mimicked by an EP4 (but not EP1–3) receptor agonist and inhibited by an EP4 receptor antagonist. The anti-inflammatory effect occurred at the transcriptional level and was via the adenylyl cyclase/cAMP/ cAMP-dependent protein kinase (PKA) axis. Conclusion This study demonstrates that EP4 receptor activation is responsible for the anti-inflammatory activity of PGE2 in a range of disease relevant models and, as such, could represent a novel therapeutic target for chronic airway inflammatory conditions.

House dust mite induces direct airway inflammation in vivo: implications for future disease therapy?

House dust mite (HDM) is the major source of allergen in house dust and is strongly associated with the development of asthma. HDM can evoke a direct, nonallergic inflammatory reaction in vitro. We aimed to determine whether this apparent nonallergic, inflammatory response can be observed in a more complex in vivo setting. Vehicle, AlumTM or HDM (Dermatophagoides pteronyssinus 5 μg, i.p. with Alum) sensitised Brown-Norway rats were challenged intratracheally with vehicle (saline), HDM (Der p 10 μg) or heat-inactivated HDM on day 21. Lung function changes and the associated inflammatory response were evaluated. Tissue and bronchoalveolar lavage from AlumTM sensitised Der p challenged animals exhibited strong eosinophilia and neutrophilia associated with an early release of pro-inflammatory cytokines (interleukin-13 and 1β, eotaxin and thymus and activation-regulated chemokine). This response was not attenuated by removal of HDM-associated protease activity. Interestingly, the vehicle sensitised group (no AlumTM) lacked this inflammatory response. HDM allergen evokes nonallergic airways inflammation with an inflammatory profile similar to that of the asthmatic airway. This response, independent of the protease activity of the HDM extract, appeared to be linked to prior administration of the adjuvant AlumTM and the subsequent increase in total immunoglobulin E. This finding could have important implications in the development of future asthma therapies.

Differences in the Phenotype, Cytokine Gene Expression Profiles, and In Vivo Alloreactivity of T Cells Mobilized with Plerixafor Compared with G-CSF

Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34+ cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4+ T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8+ T cells and altered expression levels of 16 cytokine-associated genes in CD3+ T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility–mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell–mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.

Role of Matrix Metalloproteinases in the Inflammatory Response in Human Airway Cell-Based Assays and in Rodent Models of Airway Disease

Since the discovery of the first matrix metalloproteinase (MMP), this ever-growing family of proteinases has been the subject of intense research. Although it was initially believed that MMPs were solely involved in matrix turnover and degradation, there are now data suggesting MMPs are actively involved in the inflammatory process. In previous studies, we have demonstrated an increase in MMP expression in human cell-based assays and in preclinical rat models of airway inflammation. Therefore, the aim of this study was to characterize the role of MMPs in these models by profiling the impact of a broad-spectrum MMP inhibitor. In lipopolysaccharide (LPS)-stimulated THP-1 cells and primary human lung tissue macrophages, the MMP inhibitor had no significant effect on the release of tumor necrosis factor-α, interleukin (IL)-8, IL-1β, growth-regulated oncogene-α, macrophage inflammatory protein-1α, or IL-6 whereas dexamethasone has a significant impact on all cytokines from both cell types. Similarly, in the more biologically complex LPS-driven rat model of airway inflammation, the MMP inhibitor did not have an impact on mediator release and cellular burden. The compound did, however, significantly reduce levels of lung MMP-9. Furthermore, in a “disease” model, the compound did not affect cellular inflammation but did significantly reduce elastase-induced experimental emphysema. In summary, these data demonstrate for the first time that MMPs do not play a role in the increase in inflammatory mediators or cellular burden observed in these preclinical models. However, they do appear to be involved in the elastase-driven breakdown of airway structure, which is not due to a direct effect of the stimulus.