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

Cigarette Smoke Induced Airway Inflammation Is Independent of NF-κB Signalling

<div><h3>Rationale</h3><p>COPD is an inflammatory lung disease largely associated with exposure to cigarette smoke (CS). The mechanism by which CS leads to the pathogenesis of COPD is currently unclear; it is known however that many of the inflammatory mediators present in the COPD lung can be produced via the actions of the transcription factor Nuclear Factor-kappaB (NF-κB) and its upstream signalling kinase, Inhibitor of κB kinase-2 (IKK-2). Therefore the NF-κB/IKK-2 signalling pathway may represent a therapeutic target to attenuate the inflammation associated with COPD.</p> <h3>Aim</h3><p>To use a range of assays, genetically modified animals and pharmacological tools to determine the role of NF-κB in CS-induced airway inflammation.</p> <h3>Methods</h3><p>NF-κB pathway activation was measured in pre-clinical models of CS-induced airway inflammation and in human lung tissue from COPD patients. This data was complemented by employing mice missing a functional NF-κB pathway in specific cell types (epithelial and myeloid cells) and with systemic inhibitors of IKK-2.</p> <h3>Results</h3><p>We showed in an airway inflammation model known to be NF-κB-dependent that the NF-κB pathway activity assays and modulators were functional in the mouse lung. Then, using the same methods, we demonstrated that the NF-κB pathway appears not to play an important role in the inflammation observed after exposure to CS. Furthermore, assaying human lung tissue revealed that in the clinical samples there was also no increase in NF-κB pathway activation in the COPD lung, suggesting that our pre-clinical data is translational to human disease.</p> <h3>Conclusions</h3><p>In this study we present compelling evidence that the IKK-2/NF-κB signalling pathway does not play a prominent role in the inflammatory response to CS exposure and that this pathway may not be important in COPD pathogenesis.</p> </div

Profiling the impact of a clinically relevant glucocortoid, budesonide (3 mg/kg) and two structurally distinct IKK-2 inhibitors GSK 657311A (100 mg/kg) and TPCA-1 (30 mg/kg) in CS-induced airway inflammation.

<p>Vehicle or compound was orally dosed to the mice one hour prior to the morning CS challenge and one hour after the afternoon CS challenge. BALF samples were collected 20 hours after the last CS challenge. Figures A and C represent the levels of KC in the BALF and Figures B and D the BALF neutrophilia. Data are presented as mean ± s.e.m. of n = 6–8 observations. # indicates a statistically significant difference (p<0.05) from the air challenged control groups (Mann-Whitney test). * indicates statistical significance (p<0.05) from CS challenged vehicle dosed control groups by Kruskal-Wallis one-way-ANOVA with Dunn's multiple comparison post-hoc test. Bars with no annotation were not significantly different from their respective control group.</p

Characterisation of NF-κB pathway activation after LPS or 3 days of CS challenge.

<p>Mice were challenged with LPS (1 mg/ml) or endotoxin free saline for 30 minutes or challenged for 3 days with CS (500 ml/min, 1 hour, twice daily) or ambient air. Samples were collected 2 hours after the last challenge. NF-κB subunits were measured in the lung nuclear fractions. Data are presented as mean ± s.e.m. of n = 6–8 observations. * indicates statistical significance (p<0.05) against saline control group (Students t test).</p

Profile of IKK-2^ΔEpi mice in the 3 and 14 day CS-driven models.

<p>IKK-2^ΔEpi mice were challenged with 3 or 14 days CS (500 ml/min, 1 hour, twice daily) or ambient air. BALF samples were collected twenty-four hours after the final challenge. BALF neutrophilia after 3 days of CS challenge (A) BALF neutrophilia, macrophages and lymphocytes after 14 days of CS challenge (B, C and D, respectively). Data are presented as mean ± s.e.m. of n = 8–16 observations. # indicates a statistically significant difference (p<0.05) from air challenged control groups (Mann-Whitney test). Any changes in data collected from the GM control mice i.e. fl/fl, TET alone and CRE alone did not reach statistical significance when compared to the appropriate control group.</p

Temporal characterisation of the airway inflammation after LPS or 3 days of CS challenge.

<p>Mice were challenged with LPS (1 mg/ml) or endotoxin free saline for 30 minutes. Samples were collected at increasing time points after challenge. A) LPS-induced BALF neutrophilia. B) LPS-induced NF-κB(p65):DNA association in lung nuclear extract. Mice were challenged for 3 days with CS (500 ml/min, 1 hour, twice daily) or ambient air. Samples were collected at increasing time points after the final challenge. C) CS-induced BALF neutrophilia. D) CS-induced NF-κB (p65):DNA association in lung nuclear extract. Data are presented as mean ± s.e.m. of n = 6–8 observations.</p