PI3Kγδ inhibition suppresses key disease features in a rat model of asthma.

BACKGROUND: Two isoforms of Phosphoinositide 3-kinase (PI3K), p110γ and p110δ, are predominantly expressed in leukocytes and represent attractive therapeutic targets for the treatment of allergic asthma. The study aim was to assess the impact of administration of an inhaled PI3Kγδ inhibitor (AZD8154) in a rat model of asthma. METHODS: Firstly, we checked that the tool compound, AZD8154, inhibited rat PI3K γ & δ kinases using rat cell-based assays. Subsequently, a time-course study was conducted in a rat model of asthma to assess PI3K activity in the lung and how it is temporally associated with other key transcription pathways and asthma like features of the model. Finally, the impact on lung dosed AZD8154 on target engagement, pathway specificity, airway inflammation and lung function changes was assessed. RESULTS: Data showed that AZD8154 could inhibit rat PI3K γ & δ isoforms and, in a rat model of allergic asthma the PI3K pathway was activated in the lung. Intratracheal administration of AZD8154 caused a dose related suppression PI3K pathway activation (reduction in pAkt) and unlike after budesonide treatment, STAT and NF-κB pathways were not affected by AZD8154. The suppression of the PI3K pathway led to a marked inhibition of airway inflammation and reduction in changes in lung function. CONCLUSION: These data show that a dual PI3Kγδ inhibitor suppress key features of disease in a rat model of asthma to a similar degree as budesonide and indicate that dual PI3Kγδ inhibition may be an effective treatment for people suffering from allergic asthma.

A microfluidic chip-based capillary zone electrophoresis-mass spectrometry method for measuring adenosine 5'-Triphosphate and its similar nucleotide analogues.

BACKGROUND: Extracellular ATP is involved in disorders that cause inflammation of the airways and cough, thus limiting its release has therapeutic benefits. Standard luminescence-based ATP assays measure levels indirectly through enzyme degradation and do not provide a simultaneous readout for other nucleotide analogues. Conversely, mass spectrometry can provide direct ATP measurements, however, common RPLC and HILIC methods face issues because these molecules are unstable, metal-sensitive analytes which are often poorly retained. These difficulties have traditionally been overcome using passivation or ion-pairing chromatography, but these approaches can be problematic for LC systems. As a result, more effective analytical methods are needed. RESULTS: Here, we introduce a new application that uses microfluidic chip-based capillary zone electrophoresis-mass spectrometry (µCZE-MS) to measure ATP and its analogues simultaneously in biofluids. The commercially available ZipChip Interface and a High-Resolution Bare-glass microchip (ZipChip, HRB, 908 Devices Inc.) coupled to a Thermo Scientific Tribrid Orbitrap, were successfully used to separate and detect various nucleotide standards, as well as ATP, ADP, AMP, and adenosine in plasma and BALF obtained from naïve Brown Norway rats. The findings demonstrate that this approach can rapidly and directly detect ATP and its related nucleotide analogues, while also highlighting the need to preserve these molecules in biofluids with chelators like EDTA. In addition, we demonstrate that this µCZE-MS method is also suitable for detecting a variety of metabolites, revealing additional potential future applications. SIGNIFICANCE: This innovative µCZE-MS approach provides a robust new tool to directly measure ATP and other nucleotide analogues in biofluids. This can enable the study of eATP in human disease and potentially contribute to the creation of ATP-targeting therapies for airway illnesses.

Novel airway smooth muscle-mast cell interactions and a role for the TRPV4-ATP axis in non-atopic asthma.

Mast cell-airway smooth muscle (ASM) interactions play a major role in the immunoglobulin (Ig)E- dependent bronchoconstriction seen in asthma but less is known about IgE-independent mechanisms of mast cell activation. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) activation causes contraction of human ASM via the release of cysteinyl leukotrienes (cysLTs) but the mechanism is unknown. The objective of the present study was to investigate a role for IgE-independent, mast cell-ASM interaction in TRPV4-induced bronchospasm.Bronchoconstriction was measured in anaesthetised guinea pigs and contraction of human and guinea-pig airway tissue assessed using isometric tension measurements. Increases in intracellular [Ca2+] were imaged using the Ca2+-sensitive dye FURA2, and time-lapse ptychography was utilised as a surrogate for contraction of ASM cells.The TRPV4 agonist GSK1016790A caused contraction in vivo in the guinea pig, and in human and guinea-pig tracheal tissue, which was inhibited by the TRPV4 antagonist GSK2193874. GSK1016790A increased [Ca2+]i and released ATP in human ASM cells without causing contraction. TRPV4 and ATP evoked contraction in isolated tracheal tissue but co-culture experiments indicated a requirement for human lung mast cells. Expression profiling and pharmacological studies demonstrated that mast cell activation was dependent upon ATP activating the P2X4 receptor. Trypsin was shown to evoke contraction of tracheal tissue via activation of PAR-2-TRPV4-ATP-cysLT axis indicating the potential disease relevance of this signalling pathway.TRPV4 activation increases [Ca2+]i and releases ATP from ASM cells triggering P2X4-dependent release of cysLTs from mast cells resulting in ASM contraction. This study delineates a novel mast cell-ASM interaction and TRPV4 as a driver of IgE-independent mast cell-dependent bronchospasm.

Mechanistic link between diesel exhaust particles and respiratory reflexes.

BACKGROUND: Diesel exhaust particles (DEPs) are a major component of particulate matter in Europe's largest cities, and epidemiologic evidence links exposure with respiratory symptoms and asthma exacerbations. Respiratory reflexes are responsible for symptoms and are regulated by vagal afferent nerves, which innervate the airway. It is not known how DEP exposure activates airway afferents to elicit symptoms, such as cough and bronchospasm. OBJECTIVE: We sought to identify the mechanisms involved in activation of airway sensory afferents by DEPs. METHODS: In this study we use in vitro and in vivo electrophysiologic techniques, including a unique model that assesses depolarization (a marker of sensory nerve activation) of human vagus. RESULTS: We demonstrate a direct interaction between DEP and airway C-fiber afferents. In anesthetized guinea pigs intratracheal administration of DEPs activated airway C-fibers. The organic extract (DEP-OE) and not the cleaned particles evoked depolarization of guinea pig and human vagus, and this was inhibited by a transient receptor potential ankyrin-1 antagonist and the antioxidant N-acetyl cysteine. Polycyclic aromatic hydrocarbons, major constituents of DEPs, were implicated in this process through activation of the aryl hydrocarbon receptor and subsequent mitochondrial reactive oxygen species production, which is known to activate transient receptor potential ankyrin-1 on nociceptive C-fibers. CONCLUSIONS: This study provides the first mechanistic insights into how exposure to urban air pollution leads to activation of guinea pig and human sensory nerves, which are responsible for respiratory symptoms. Mechanistic information will enable the development of appropriate therapeutic interventions and mitigation strategies for those susceptible subjects who are most at risk.

Transient receptor potential cation channel, subfamily V, member 4 and airway sensory afferent activation: Role of adenosine triphosphate.

BACKGROUND: Sensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored. OBJECTIVE: We hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung. METHODS: We used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues. RESULTS: Here we show TRPV4-induced activation of guinea pig airway-specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough. CONCLUSION: This study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP-mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.