Irritant stimuli evoke ATP and inflammatory mediator release affecting P2X3 responses.

The cough reflex is activated through stimulation of vagal sensory nerves which may become hyperresponsive causing troublesome bouts of coughing. ATP-gated P2X3 receptors are believed to play a role in neuronal sensitization linked to chronic cough. This project aimed to examine the effect of irritant airway stimuli on ATP and cytokine release from primary bronchial epithelial cells (PBECs) and to subsequently examine the effect of these cytokines on P2X3 responses in a stem cell derived sensory neuronal model. PBECs were obtained from individuals undergoing bronchoscopy and expanded with submerged culture techniques. ATP release from PBECs treated with LPS, LTA, poly I:C or cigarette smoke extract (CSE) was examined using a luciferase assay. Cytokine release from PBECs treated with LPS, LTA and poly I:C was evaluated using an inflammatory microarray. To study neuronal P2X3 responses, human dental pulp stem cells were differentiated to peripheral neuronal equivalents (PNEs) in neurogenic media. P2X3 responses in PNEs were determined by FLIPR assay with the P2X3 agonist ATP [10 µM & 400 µM] and antagonist AF-353. The effect of IL-4, IP-10 & IL-16 on PNE ATP responses were evaluated by FLIPR assay. ATP release from PBECs was increased by 324% or 466% with poly I:C and CSE respectively(P<0.001). The expression of 19 cytokines (including IL-4, IP-10 & IL-16) was increased(>2 fold) with LPS, LTA or poly I:C treatment of PBECs. AF-353 inhibited 10 µM ATP(P<0.001) & 400 µM ATP(P<0.05) responses in PNEs. A leftward shift of ATP dose response curve in PNEs was observed with IL-4 pretreatment. Irritant airway stimuli induce the release of ATP and inflammatory cytokines from PBECs which can sensitise ATP responses in the PNE model