Kristof Raemdonck

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.

A role for sensory nerves in the late asthmatic response

Background In allergic asthma, exposure to relevant antigens leads to an early asthmatic response (EAR) followed, in certain subjects, by a late asthmatic response (LAR). Although many subjects with asthma consider LAR to be one of the defining symptoms of their disease, and despite its widespread use in the clinical assessment of new therapeutic entities, the mechanism underlying the LAR remains unclear. Method A study was undertaken using ovalbumin-sensitised and challenged Brown Norway rat and C57BL/6J mouse models which recapitulate phenotypic features of allergic asthma including the LAR and its susceptibility to clinically effective agents. Results In conscious animals an EAR was followed by a LAR. The LAR was subjectively evidenced by audible (wheeze) and visual signs of respiratory distress associated with quantifiable changes in non-invasive lung function assessment. Treatments that attenuated the EAR failed to impact on the LAR and, while anaesthesia did not impact on EAR, it abolished LAR. A key role for airway sensory neuronal reflexes in the LAR was therefore hypothesised, which was confirmed by the blockade observed after administration of ruthenium red (non-selective cation channel blocker), HC-030031 (TRPA1 inhibitor) and tiotropium bromide (anticholinergic) but not JNJ-17203212 (TRPV1 inhibitor). Conclusion These results suggest that LAR involves the following processes: allergen challenge triggering airway sensory nerves via the activation of TRPA1 channels which initiates a central reflex event leading to a parasympathetic cholinergic constrictor response. These data are supported by recent clinical trials suggesting that an anticholinergic agent improved symptoms and lung function in patients with asthma.

Tiotropium modulates transient receptor potential V1 (TRPV1) in airway sensory nerves: A beneficial off-target effect?

Background Recent studies have suggested that the long-acting muscarinic receptor antagonist tiotropium, a drug widely prescribed for its bronchodilator activity in patients with chronic obstructive pulmonary disease and asthma, improves symptoms and attenuates cough in preclinical and clinical tussive agent challenge studies. The mechanism by which tiotropium modifies tussive responses is not clear, but an inhibition of vagal tone and a consequent reduction in mucus production from submucosal glands and bronchodilation have been proposed. Objective The aim of this study was to investigate whether tiotropium can directly modulate airway sensory nerve activity and thereby the cough reflex. Methods We used a conscious cough model in guinea pigs, isolated vagal sensory nerve and isolated airway neuron tissue– and cell-based assays, and in vivo single-fiber recording electrophysiologic techniques. Results Inhaled tiotropium blocked cough and single C-fiber firing in the guinea pig to the transient receptor potential (TRP) V1 agonist capsaicin, a clinically relevant tussive stimulant. Tiotropium and ipratropium, a structurally similar muscarinic antagonist, inhibited capsaicin responses in isolated guinea pig vagal tissue, but glycopyrrolate and atropine did not. Tiotropium failed to modulate other TRP channel–mediated responses. Complementary data were generated in airway-specific primary ganglion neurons, demonstrating that tiotropium inhibited capsaicin-induced, but not TRPA1-induced, calcium movement and voltage changes. Conclusion For the first time, we have shown that tiotropium inhibits neuronal TRPV1-mediated effects through a mechanism unrelated to its anticholinergic activity. We speculate that some of the clinical benefit associated with taking tiotropium (eg, in symptom control) could be explained through this proposed mechanism of action.

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.

Selectivity profiling of the novel EP2 receptor antagonist, PF-04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

Understanding the role of the EP2 receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP2 receptor antagonist, PF‐04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF‐04418948 for the EP2 receptor over other EP receptors using a range of isolated tissue systems.

Respiratory infections cause the release of extracellular vesicles: implications in exacerbation of asthma/COPD.

Background Infection-related exacerbations of respiratory diseases are a major health concern; thus understanding the mechanisms driving them is of paramount importance. Despite distinct inflammatory profiles and pathological differences, asthma and COPD share a common clinical facet: raised airway ATP levels. Furthermore, evidence is growing to suggest that infective agents can cause the release of extracellular vesicle (EVs) in vitro and in bodily fluids. ATP can evoke the P2X7/caspase 1 dependent release of IL-1β/IL-18 from EVs; these cytokines are associated with neutrophilia and are increased during exacerbations. Thus we hypothesized that respiratory infections causes the release of EVs in the airway and that the raised ATP levels, present in respiratory disease, triggers the release of IL-1β/IL-18, neutrophilia and subsequent disease exacerbations. Methods To begin to test this hypothesis we utilised human cell-based assays, ex vivo murine BALF, in vivo pre-clinical models and human samples to test this hypothesis. Results Data showed that in a murine model of COPD, known to have increased airway ATP levels, infective challenge causes exacerbated inflammation. Using cell-based systems, murine models and samples collected from challenged healthy subjects, we showed that infection can trigger the release of EVs. When exposed to ATP the EVs release IL-1β/IL-18 via a P2X7/caspase-dependent mechanism. Furthermore ATP challenge can cause a P2X7 dependent increase in LPS-driven neutrophilia. Conclusions This preliminary data suggests a possible mechanism for how infections could exacerbate respiratory diseases and may highlight a possible signalling pathway for drug discovery efforts in this area.

Role of the ion channel, transient receptor potential cation channel subfamily V member 1 (TRPV1), in allergic asthma

BackgroundAsthma prevalence has increased world-wide especially in children; thus there is a need to develop new therapies that are safe and effective especially for patients with severe/refractory asthma. CD4+ T cells are thought to play a central role in disease pathogenesis and associated symptoms. Recently, TRPV1 has been demonstrated to regulate the activation and inflammatory properties of CD4+ cells. The aim of these experiments was to demonstrate the importance of CD4+ T cells and the role of TRPV1 in an asthma model using a clinically ready TRPV1 inhibitor (XEN-D0501) and genetically modified (GM) animals.MethodsMice (wild type, CD4 −/− or TRPV1 −/−) and rats were sensitised with antigen (HDM or OVA) and subsequently topically challenged with the same antigen. Key features associated with an allergic asthma type phenotype were measured: lung function (airway hyperreactivity [AHR] and late asthmatic response [LAR]), allergic status (IgE levels) and airway inflammation.ResultsCD4+ T cells play a central role in both disease model systems with all the asthma-like features attenuated. Targeting TRPV1 using either GM mice or a pharmacological inhibitor tended to decrease IgE levels, airway inflammation and lung function changes.ConclusionOur data suggests the involvement of TRPV1 in allergic asthma and thus we feel this target merits further investigation.

CD4(+) and CD8(+) T cells play a central role in a HDM driven model of allergic asthma.

BackgroundThe incidence of asthma is increasing at an alarming rate and while the current available therapies are effective in the majority of patients they fail to adequately control symptoms at the more severe end of the disease spectrum. In the search to understand disease pathogenesis and find effective therapies animal models are often employed. As exposure to house dust mite (HDM) has a causative link, it is thought of as the allergen of choice for modelling asthma.The objective was to develop a HDM driven model of asthmatic sensitisation and characterise the role of key allergic effector cells/mediators.MethodsMice were sensitised with low doses of HDM and then subsequently challenged. Cellular inflammation, IgE and airway responsiveness (AHR) was assessed in wild type mice or CD4+/CD8+ T cells, B cells or IgE knock out mice.ResultsOnly those mice sensitised with HDM responded to subsequent low dose topical challenge. Similar to the classical ovalbumin model, there was no requirement for systemic alum sensitisation. Characterisation of the role of effector cells demonstrated that the allergic cellular inflammation and AHR was dependent on CD4+ and CD8+ T cells but not B cells or IgE. Finally, we show that this model, unlike the classic OVA model, appears to be resistant to developing tolerance.ConclusionsThis CD4+/CD8+ T cell dependent, HDM driven model of allergic asthma exhibits key features of asthma. Furthermore, we suggest that the ability to repeat challenge with HDM means this model is amenable to studies exploring the effect of therapeutic dosing in chronic, established disease.

Characterisation of a murine model of the late asthmatic response

BackgroundThe incidence of asthma is increasing at an alarming rate. While the current available therapies are effective, there are associated side effects and they fail to adequately control symptoms in all patient subsets. In the search to understand disease pathogenesis and find effective therapies hypotheses are often tested in animal models before progressing into clinical studies. However, current dogma is that animal model data is often not predictive of clinical outcome. One possible reason for this is the end points measured such as antigen-challenge induced late asthmatic response (LAR) is often used in early clinical development, but seldom in animal model systems. As the mouse is typically selected as preferred species for pre-clinical models, we wanted to characterise and probe the validity of a murine model exhibiting an allergen induced LAR.MethodsC57BL/6 mice were sensitised with antigen and subsequently topically challenged with the same antigen. The role of AlumTM adjuvant, glucocorticoid, long acting muscarinic receptor antagonist (LAMA), TRPA1, CD4+ and CD8+ T cells, B cells, Mast cells and IgE were determined in the LAR using genetically modified mice and a range of pharmacological tools.ResultsOur data showed that unlike other features of asthma (e.g. cellular inflammation, elevated IgE levels and airway hyper-reactivity (AHR) the LAR required AlumTMadjuvant. Furthermore, the LAR appeared to be sensitive to glucocorticoid and required CD4+ T cells. Unlike in other species studied, the LAR was not sensitive to LAMA treatment nor required the TRPA1 ion channel, suggesting that airway sensory nerves are not involved in the LAR in this species. Furthermore, the data suggested that CD8+ T cells and the mast cell—B-cell - IgE axis appear to be protective in this murine model.ConclusionTogether we can conclude that this model does feature steroid sensitive, CD4+ T cell dependent, allergen induced LAR. However, collectively our data questions the validity of using the murine pre-clinical model of LAR in the assessment of future asthma therapies.

The role of CRAC channel in asthma

Asthma is increasing globally and current treatments only manage a proportion of patients. There is an urgent need to develop new therapies. Lymphocytes are thought to play a central role in the pathophysiology of asthma through the production of inflammatory mediators. This is thought to be via the transcription factor NFAT which in turn can be activated through Ca(2+) release-activated Ca(2+) (CRAC) channels. The aim of this work was to investigate the role of CRAC in clinical and pre-clinical models of allergic asthma. Initial data demonstrated that the NFAT pathway is increased in stimulated lymphocytes from asthmatics. To confirm a role for the channel we showed that a selective inhibitor, Synta 66, blocked mediator production from lymphocytes. Synta 66 inhibited CD2/3/28 induced IL-2, IL-7, IL-13 & IFNΥ in a concentration-dependent manner in healthy and severe asthma donors, with over 60% inhibition observed for all cytokines. NFAT pathway was also increased in a pre-clinical asthma model. In this model we have demonstrated that CRAC played a central role in the airway inflammation and late asthmatic response (LAR). In conclusion, our data provides evidence that suggests targeting CRAC channels could be of therapeutic benefit for asthma sufferers.