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

다국어 초록 (Multilingual 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.

목차 (Table of Contents)

• BACKGROUND 13
• 1. Ich sensation 13
• 1.1. Overview of Itch Sensation 13
• 1.2. Histamine induce Itch Sensation 15
• 2. Toll-Like Recetors 16
• 2.1. Overview of Toll-Like Receptor 16
• 2.2. TLRs Expression in the Nervous System 17
• 3. Transient receptor potential vanilloid 1 21
• PURPOSE 26
• CHAPTER 1 TLR4 enhances histamine-mediated pruritus by potentiating TRPV1 activity 27
• ABSTRACT 28
• INTRODUCTION 29
• MATERIALS AND METHODS 31
• Mice 31
• Behavior study 31
• Primary DRG neuron culture 32
• Cell culture and transfection 32
• Plasmid 33
• Single-cell RT-PCR 33
• Immunofluorescence 34
• Real-time PCR 36
• Calcium Assay 37
• Whole cell patch-clamp recording 38
• Western blot 38
• Statistical Analysis 39
• RESULTS 40
• TLR4 enhances histamine and chloroquine induced itch sensation 40
• TLR4 enhances both HRH1 and HRH4 activation induced itch sensation 40
• Sensory neurons express TLR4 41
• TLR4 expression detected in various type of sensory neuron 41
• Histamine- or chloroquine- induced calcium signals are reduced in TLR4 KO sensory neuron 42
• Histamine receptor expression was not affected by TLR4 expression 43
• Capsaicin induced calcium signal and inward current were reduced in TLR4 KO sensory neuron 43
• TRPA1 activity was not altered in TLR4 KO sensory neuron 44
• TLR4 expression enhances TRPV1 activity 44
• DISCUSSION 64
• CHAPTER 2 TLR4 enhances TRPV1 activity by direct association through TIR domain 68
• ABSTRACT 69
• INTRODUCTION 71
• MATERIALS AND METHODS 73
• Mice 73
• Behavior study 73
• Primary DRG neuron culture 73
• Cell culture and transfection 74
• Plasmid 75
• Immunofluorescence 75
• Calcium Assay 76
• Immunoprecipitation 77
• Western blot 78
• Cell-surface biotinylation assay 79
• Statistical Analysis 79
• RESULTS 81
• TRPV1 expression on TLR4 KO sensory neurons was similar to WT sensory neurons 81
• Direct association between TLR4 and TRPV1 protein 81
• TLR4 associates with TRPV1 through TIR domain 82
• Interaction between TLR4 and TRPV1 does not alter serine residues phosphorylation of TRPV 83
• TRPV1 on TLR4 deficient sensory neuron undergoes excessive desensitizatio 83
• TLR4 expression decreases capsaicin induced TRPV1 downregulation 84
• Capsaicin induced acute pain attenuated in TLR4 deficient mice 85
• DISCUSSION 103
• CONCLUSION 108
• REFERENCES 110
• ABSTRACT IN KOREAN 128