The role of the P2X7 receptor in cigarette smoke driven-inflammation associated with COPD

Chronic Obstructive Pulmonary Disease (COPD) is a cigarette smoke (CS)-driven inflammatory airway disease with an increasing global prevalence. Currently, there are no effective therapies to stop the relentless progression of this disease. In the inflammatory milieu present in the lungs of animal models of, and human patients with COPD are increased levels of cytokines (IL-1β/IL-18) linked to activation of the NLRP3-Inflammasome. It has been postulated that exposure to CS leads to the release of endogenous danger signals (e.g. ATP) activating the NLRP3-Inflammasome via the P2X7 receptors driving the maturation and release of IL-1β and IL-18. The literature suggests that these cytokines are central to the chronic inflammation in the airway which drives the pathological changes seen in COPD. My hypothesis is that blockade of the P2X7 - NLRP3-Inflammasome pathway will attenuate the inflammation present in CS-induced airway inflammation. I developed an acute (3-day) model of COPD-like inflammation to investigate the role of the P2X7 receptor in this pathway. I 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. I used genetically modified mice lacking functional P2X7 receptors to show attenuation in caspase-1 activity, IL-1β release and airway neutrophilia in response to acute CS exposure but not LPS-induced airway inflammation. These findings were validated using a specific P2X7 receptor antagonist. Furthermore, I confirmed that the role of this pathway was not restricted to early stages of disease development by showing increased caspase-1 activity in lungs from a more chronic exposure to CS (28-day) and patients with COPD. This translational data suggests the P2X7-Inflammasome pathway plays an on-going role in disease pathogenesis. These results advocate the crucial role of the P2X7 – caspase-1 axis in CS-induced inflammation, highlighting this as a possible therapeutic target in combating COPD

TLR4 activation induces IL-1ss release via an IPAF dependent but caspase 1/11/8 independent pathway in the lung

Background: The IL-1 family of cytokines is known to play an important role in inflammation therefore understanding the mechanism by which they are produced is paramount. Despite the recent plethora of publications dedicated to the study of these cytokines, the mechanism by which they are produced in the airway following endotoxin, Lipopolysaccharide (LPS), exposure is currently unclear. The aim was to determine the mechanism by which the IL-1 cytokines are produced after LPS inhaled challenge. Methods:Mice were challenged with aerosolised LPS, and lung tissue and bronchiolar lavage fluid (BALF) collected. Targets were measured at the mRNA and protein level; caspase activity was determined using specific assays. Results: BALF IL-1b/IL-18, but not IL-1a, was dependent on Ice Protease-Activating Factor (IPAF), and to a lesser extent Apoptosis-associated Speck-like protein containing a CARD (ASC). Interestingly, although we measured an increase in mRNA expression for caspase 1 and 11, we could not detect an increase in lung enzyme activity or a role for them in IL-1a/b production. Further investigations showed that whilst we could detect an increase in caspase 8 activity at later points in the time course (during resolution of inflammation), it appeared to play no role in the production of IL-1 cytokines in this model system. Conclusions: TLR4 activation increases levels of BALF IL-1b/IL-18 via an IPAF dependent and caspase 1/11/8 independent pathway. Furthermore, it would appear that the presence of IL-1a in the BALF is independent of these pathways. This novel data sheds light on innate signalling pathways in the lung that control the production of these key inflammatory cytokines

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

Role of the caspase 1 and 11 in CS-driven model.

<p>Caspase 1/11 or caspase 11 ^-/- mice were exposed to CS or room air (control) twice daily for 3 consecutive days alongside wild-type controls. BALF and lung tissue was collected 24 hours after the last exposure for measurement of neutrophilia (A – caspase 1/11 -/-, B – caspase 11-/-). Figures C to H depicts the levels of caspase 1 activity (C), IL-1β (D), IL-18 (E), IL-1α (F), ATP (G) and KC (H). Data are represented as mean ± S.E.M. for n = 8 animals in each group. Statistical significance was determined using Mann-Whitney U test. #  = P<0.05, denoting a significant difference between the smoke exposed and air exposed wild-type groups; *  = P<0.05, denoting a significant difference between the smoke exposed knock-outs and wild-types.</p

Role of the IL-1 family cytokines in CS-driven model.

<p>IL-1β, IL-18 or IL-18R ^-/- mice were exposed to CS or room air (control) twice daily for 3 consecutive days alongside wild-type controls. BALF and lung tissue was collected 24 hours after the last exposure for measurement of ATP (A), caspase 1 activity (B), IL-1α (C), IL-1β (D), IL-18 (E), KC (F) and neutrophil (G) levels. Data are represented as mean ± S.E.M. for n = 8 animals in each group. Statistical significance was determined using Mann-Whitney U test. #  = P<0.05, denoting a significant difference between the smoke exposed and air exposed wild-type groups; *  = P<0.05, denoting a significant difference between the smoke exposed knock-outs and wild-types (one-way ANOVA).</p

Role of the IL-1β and IL-18 in CS-driven model.

<p>IL-1β, IL-18, IL-18R or caspase 1/11 ^-/- mice were exposed to CS or room air (control) twice daily for 3 consecutive days alongside wild-type controls. Lung tissue was collected 24 hours after the last exposure for measurement of E-selectin levels. Data are represented as mean ± S.E.M. for n = 8 animals in each group. Statistical significance was determined using Mann-Whitney U test. #  = P<0.05, denoting a significant difference between the smoke exposed and air exposed wild-type groups; *  = P<0.05, denoting a significant difference between the smoke exposed knock-outs and wild-types (one-way ANOVA).</p

Role of the inflammasome proteins in the CS-driven model.

<p>NALP3, IPAF or AIM2 ^-/- mice were exposed to CS or room air (control) twice daily for 3 consecutive days alongside wild-type controls. BALF was collected 24 hours after the last exposure for measurement of ATP (A), IL-18 (B) and neutrophil (C) levels. Data are represented as mean ± S.E.M. for n = 8 animals in each group. Statistical significance was determined using Mann-Whitney U test. #  = P<0.05, denoting a significant difference between the smoke exposed and air exposed wild-type groups; *  = P<0.05, denoting a significant difference between the smoke exposed knock-outs and wild-types (one-way ANOVA).</p

Role of the NALP3 inflammasome in CS-driven model.

<p>NALP3 or ASC ^-/- mice were exposed to CS or room air (control) twice daily for 3 consecutive days alongside wild-type controls. BALF and lung tissue was collected 24 hours after the last exposure for measurement of IL-1β (A), neutrophil (B), IL-1α (C), caspase 1 activity (D) and KC (E) levels. Data are represented as mean ± S.E.M. for n = 8 animals in each group. Statistical significance was determined using Mann-Whitney U test. #  = P<0.05, denoting a significant difference between the smoke exposed and air exposed wild-type groups; *  = P<0.05, denoting a significant difference between the smoke exposed knock-outs and wild-types (one-way ANOVA).</p

Schematic representation of the signalling cascade following CS-induced activation of the P2X₇ channel in the lung (Drawn by Dr E Dubuis).

<p>We hypothesise that CS exposure leads to the release of extracellular ATP which activates P2X₇ receptor, and this in turn triggers a signalling cascade involving the assembly of the NALP3/ASC inflammasome and recruitment of (pro)-caspase 1. Pro-Caspase 1 either auto-processes itself to the mature, active form or is cleaved by caspase 11. This functional inflammasome is essential for the release of mature, active IL-1β/IL-18, but not IL-1α which stimulates some of the production of proteins involved in the transmigration of neutrophils, like E-selectin. Another component of the P2X₇ receptor signalling cascade triggers the caspase 1 and/or caspase 11 dependent release of IL-1α. This cytokine also induces the production of proteins involved in the recruitment of a portion of the neutrophils observed in this model system. Interrupting steps involved in both signalling pathways i.e. at the P2X₇ receptor or via caspase 1/11 leads to a complete block of transmigration protein production and neutrophilia.</p