Validation of a human peripheral neuronal model for ATP-P2X3 mediated inflammatory pain signalling in the dental pulp.

INTRODUCTION: Extracellular adenosine triphosphate (ATP) is a key alarmin in inflammatory responses and amplifies pain signalling through the activation of P2X3 receptors. This study aimed to validate a peripheral neuronal model, differentiated from human pulp stem cells, for the study of neuronal immunomodulation with relevance to ATP-P2X3 signalling. METHODS: Human dental pulp stem cells (DPSCs) were enriched from dental pulp cells obtained by explant culture from the pulp tissue of extracted third molar teeth. DPSCs were differentiated to peripheral neuronal equivalents (PNEs) in neurogenic media for 14 days. Characterisation of the neuronal transcriptome of PNEs was carried out by single-cell sequencing (ScRNA-seq). Transcriptomic and secretome changes in PNEs treated with the synthetic ATP analogue αβ-methylene ATP (αβmeATP) were analysed using ScRNA-seq and antibody microarray respectively. ATP release was measured using the ATPlite luciferase assay (Perkin Elmer) following mechanical stimulation by stretch or lipopolysaccharide (LPS) treatment of PNEs. The functional expression of P2X3 receptors was assessed by calcium mobilisation assays. RESULTS: PNEs were shown to express a neuronal gene signature, without the expression of genes typical of odontogenic cells. Transcriptomic analysis of PNEs following αβmeATP treatment demonstrated an enriched expression of pain and neuropathy pathways (P<0.05). Antibody microarray results indicated that the increased expression of 9 cytokines with αβmeATP treatment was attenuated by a P2X3 antagonist. Mechanical stretch or LPS treatment of PNEs resulted in increased ATP release (P<0.05). Responses to ATP were inhibited by a specific P2X3 antagonist indicating functional expression of P2X3 receptors in PNEs (P<0.001). CONCLUSIONS: ScRNA-seq of the PNE model confirms a neuronal gene signature following differentiation, supporting its use as a relevant human model for studying neuronal immunomodulation and evaluating potential P2X3 receptor-targeted therapies for dental pain.