Regulation mechanisms of itch signal transduction by sensory neuronal toll-like receptor 4

Abstract

Itch, formally known as pruritus, has been defined as an unpleasant skin sensation that elicits the desire or reflex to scratch. The normal itch occurs in order to protect our body from external harmful stimuli however, excessive pruritus worsen the life quality of patient. The therapeutic strategies are limited to symptomatic treatment due to short of understanding to itch mechanism. Recent studies have indicated that Toll-like receptor 4 (TLR4) is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. In this study, I suggest that TLR4 expressed on sensory neuron regulates itch sensation by modulate TRPV1 activity. In chapter 1, data show that TLR4 on sensory neurons enhances histamine/chloroquine-induced itch signal transduction. I confirmed that TLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine/chloroquine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Chloroquine receptor expression was decreased in TLR4 deficient DRG sensory neuron, while expression of histamine receptors were comparable to wild type. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in the TLR4-deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated that TLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents. Otherwise, TLR4 regulates chloroquine-induced itch sensation by enhanced expression of MrgprA3 which is known as chloroquine receptor. In chapter 2, I revealed that direct association between TRPV1 and TLR4 through TIR domain decrease TRPV1 desensitization by dysregulation of channel expression on cell surface. I confirmed that TLR4 interact with TRPV1 through TIR domain. HEK 293T cells, transiently transfected with TIR-truncated TLR4 mutant and TRPV1, showed reduced capsaicin induced calcium signaling compared with HEK 293T cells expressing full length TLR4 and TRPV1. Although interaction between TLR4 and TRPV1 did not alter serine residue phosphorylation of TRPV1, the interaction decreased downregulation of TRPV1 after capsaicin stimuli. In consequence of increased TRPV1 expression on cell surface, capsaicin-induced desensitization was enhanced in TLR4 KO sensory neuron. In conclusion, TLR4 on sensory neurons enhances chloroquine/histamine-induced itch signal transduction by distinct mechanisms. TLR4 on sensory neurons enhances chloroquine-induced itch sensation by decreasing chloroquine receptor expression. However, TLR4 increases histamine-induced itch signal transduction by potentiating TRPV1 activity. The direct association between TRPV1 and TLR4 through TIR domain blocks capsaicin induced desensitization and moreover, TLR4 activation mediates TRPV1 sensitization by regulate TRPV1 trafficking to membrane. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation.