Sensory Neuron Expressed TRPC3 Mediates Acute and Chronic Itch

Abstract Background: Chronic pruritus is a prominent symptom of allergic contact dermatitis (ACD) and represent a huge unmet health problem. However, its underlying cellular and molecular mechanisms remain largely unexplored. TRPC3 is highly expressed in primary sensory neurons and has been implicated in peripheral sensitization induced by proinflammatory mediators. However, the role of TRPC3 in acute and chronic itch is still not well defined. Methods: RNAscope in situ hybridization and immunohistochemical staining were performed on mouse trigeminal ganglion (TG) neurons. Fura-2 calcium imaging was used to characterize the function of TRPC3 in dissociated TG neurons. In native mice, the TRPC3 agonist and pruritogens were subcutaneously injected to the cheek and nape of the neck of mice, respectively. Site directed scratching and/or wiping behaviors were video recorded. Contact hypersensitivity (CHS) model was induced in mouse ears by topical application of SADBE or DNCB. Spontaneous scratching behaviors were recorded by video monitoring. Global and conditional Trpc3 knockout mice were employed to determine the contribution of TRPC3 to acute and chronic itch. The mRNA expression levels of Trpc3 and proinflammatory cytokines were assayed by quantitative real-time PCR. H&E. staining was used for the evaluation of the thickness of mouse ears. Flow cytometry was performed to assess immune cell infiltration in mouse ear tissues. Results: Among mouse TG neurons, RNAscope assay revealed that Trpc3 mRNA was predominantly expressed in nonpeptidergic small diameter neurons. Moreover, Trpc3 mRNA signal was present in the majority of itch sensing neurons. TRPC3 agonism induced TG neuronal activation and acute nonhistaminergic itch- and pain-like behaviors in naïve mice. In addition, genetic deletion of Trpc3 attenuated acute itch evoked by certain common nonhistaminergic pruritogens, including endothelin-1 and SLIGRL-NH2. In a murine model of CHS, Trpc3 mRNA expression level and function were upregulated in the TG following CHS. Pharmacological inhibition and global knockout of Trpc3 significantly alleviated spontaneous scratching behaviors without affecting concurrent cutaneous inflammation in the CHS model. Furthermore, conditional deletion of Trpc3 in primary sensory neurons but not in keratinocytes produced similar antipruritic effects in this model. Conclusions: These findings suggest that TRPC3 expressed in primary sensory neurons may contribute to acute and chronic itch via a histamine independent mechanism and that targeting neuronal TRPC3 might benefit the treatment of chronic itch associated with ACD and other inflammatory skin disorders.