SUMMARY Mechanical allodynia, induced by normally innocuous low-threshold mechanical stimulation, represents a cardinal feature of neuropathic pain. Blockade or ablation of high-threshold small-diameter unmyelinated C-fibers has limited effects on mechanical allodynia1–4. While large myelinated A-fibers, in particular Aβ-fibers, have previously been implicated in mechanical allodynia5–7, an A-fiber-selective pharmacological blocker is still lacking. Here we report a new method for targeted silencing of A-fibers in neuropathic pain. We found that Toll-like receptor 5 (TLR5) is co-expressed with neurofilament-200 in large-diameter A-fiber neurons in the dorsal root ganglion (DRG). Activation of TLR5 with its ligand flagellin results in neuronal entry of the membrane impermeable lidocaine derivative QX-314, leading to TLR5-dependent blockade of sodium currents predominantly in A-fiber neurons of mouse DRGs. Intraplantar co-application of flagellin and QX-314 (flagellin/QX-314) dose-dependently suppressed mechanical allodynia following chemotherapy, nerve injury, and diabetic neuropathy, but this blockade is abrogated in Tlr5-deficient mice. In vivo electrophysiology demonstrated that flagellin/QX-314 co-application selectively suppressed Aβ-fiber conduction in naive and chemotherapy-treated mice. TLR5-mediated Aβ blockade but not capsaicin-mediated C-fiber blockade also reduced chemotherapy-induced ongoing pain without impairing motor function. Finally, flagellin/QX-314 co-application suppressed sodium currents in large-diameter human DRG neurons. Thus, our findings provide a new tool for targeted silencing of Aβ-fibers and neuropathic pain treatment.

The mechanisms of pain induction by inflammation have been extensively studied. However, the mechanisms of pain resolution are not fully understood. Here, we report that GPR37, expressed by macrophages (MΦs) but not microglia, contributes to the resolution of inflammatory pain. Neuroprotectin D1 (NPD1) and prosaptide TX14 increase intracellular Ca2+ (iCa2+) levels in GPR37-transfected HEK293 cells. NPD1 and TX14 also bind to GPR37 and cause GPR37-dependent iCa2+ increases in peritoneal MΦs. Activation of GPR37 by NPD1 and TX14 triggers MΦ phagocytosis of zymosan particles via calcium signaling. Hind paw injection of pH-sensitive zymosan particles not only induces inflammatory pain and infiltration of neutrophils and MΦs, but also causes GPR37 upregulation in MΦs, phagocytosis of zymosan particles and neutrophils by MΦs in inflamed paws, and resolution of inflammatory pain in WT mice. Mice lacking Gpr37 display deficits in MΦ phagocytic activity and delayed resolution of inflammatory pain. Gpr37-deficient MΦs also show dysregulations of proinflammatory and antiinflammatory cytokines. MΦ depletion delays the resolution of inflammatory pain. Adoptive transfer of WT but not Gpr37-deficient MΦs promotes the resolution of inflammatory pain. Our findings reveal a previously unrecognized role of GPR37 in regulating MΦ phagocytosis and inflammatory pain resolution.

BACKGROUND AND PURPOSETemperature-sensitive transient receptor potential ion channels (thermoTRPs) expressed in primary sensory neurons and skin keratinocytes play a crucial role as peripheral pain detectors. Many natural and synthetic ligands have been found to act on thermoTRPs, but little is known about endogenous compounds that inhibit these TRPs. Here, we asked whether resolvin D1 (RvD1), a naturally occurring anti-inflammatory and pro-resolving lipid molecule is able to affect the TRP channel activation. EXPERIMENTAL APPROACHWe examined the effect of RvD1 on the six thermoTRPs using Ca 2+ imaging and whole cell electrophysiology experiments using the HEK cell heterologous expression system, cultured sensory neurons and HaCaT keratinocytes. We also checked changes in agonist-specific acute licking/flicking or flinching behaviours and TRP-related mechanical and thermal pain behaviours using Hargreaves, Randall-Selitto and von Frey assay systems with or without inflammation. KEY RESULTSRvD1 inhibited the activities of TRPA1, TRPV3 and TRPV4 at nanomolar and micromolar levels. Consistent attenuations in agonist-specific acute pain behaviours by immediate peripheral administration with RvD1 were also observed. Furthermore, local pretreatment with RvD1 significantly reversed mechanical and thermal hypersensitivity in inflamed tissues. CONCLUSIONS AND IMPLICATIONSRvD1 was a novel endogenous inhibitor for several sensory TRPs. The results of our behavioural studies suggest that RvD1 has an analgesic potential via these TRP-related mechanisms.

TMC genes encode a broadly-conserved family of multipass integral membrane proteins in animals 1,2 . Human TMC1 and TMC2 are deafness genes required for hair cell mechantransduction; however, the molecular functions of these and other TMC proteins have not been determined [3][4][5][6] . We show here that the C. elegans TMC-1 gene encodes a sodium sensor that functions specifically in salt taste chemosensation. TMC-1 is expressed in the ASH polymodal avoidance neurons, where it is required for salt-evoked neuronal activity and behavioural avoidance of high concentrations of NaCl. However, tmc-1 has no effect on responses to other stimuli sensed by the ASH neurons including high osmolarity and chemical repellents, indicating a specific role in salt sensation. When expressed in mammalian cell culture, TMC-1 generates a predominantly cationic conductance activated by high extracellular sodium but not by other cations or uncharged small molecules. Thus, TMC-1 is both necessary for salt sensation in vivo and sufficient to generate a sodium-sensitive channel in vitro, identifying it as a likely candidate ionotropic sensory receptor.TMC1 is an important deafness gene in humans 1,3 , and mutant mice carrying semidominant (Beethoven) or recessive (deafness) TMC1 alleles are hearing-deficient 3,4 . TMC1 is expressed in cochlear hair cells, and is required for hair cell function 5 . Recently, knockout mice containing deletions of both TMC1 and a closely related gene, TMC2, were shown to lack hair cell mechanosensory potentials 6 . TMC1 and TMC2 are members of a larger family of putative multipass transmembrane proteins that includes eight proteins in mammals, one in Drosophila, and two in C. elegans 2,3 , one of which (tmc-2) is enriched mechanoreceptors 7 . However, it is not known whether TMC genes encode channel proteins, nor whether TMC1 and TMC2 are components of the hair cell mechanotransducer or are required indirectly for its activity. To learn more about the function of the C. elegans tmc-1 gene, we first investigated its expression pattern. In transgenic lines expressing a fluorescent reporter under the tmc-1 promoter, expression was observed in a small number of neurons, including the ASH neurons, which are important for sensing chemical repellents 8 , high osmolarity and nose touch 9 ( Figure 1A, B). Expression was also seen in other sensory neurons, including the ADFs, ASEs, ADLs and PHAs (Supplemental Figures 1-2). To investigate the subcellular distribution of TMC-1, we expressed a translational fusion between the tmc-1 cDNA and mCherry under the ASH promoter gpa-11 ( Figure 1C). This TMC-1::mCherry fusion was localised to the cell body as well as the sensory cilia, the site of sensory transduction. These results suggested a possible role for TMC-1 in ASH sensory function.To investigate the possible role of TMC-1 in sensory transduction, we assayed the effect of a tmc-1 deletion allele on on ASH-mediated behaviours. The ASH neurons are important for avoidance of diverse noxious stimuli, including nose...

Temperature-sensitive transient receptor potential ion channels (thermoTRPs) expressed in epidermal keratinocytes and sensory afferents play an important role as peripheral pain detectors for our body. Many natural and synthetic compounds have been found to act on the thermoTRPs leading to altered nociception, but little is known about endogenous painful molecules activating TRPV3. Here, we show that farnesyl pyrophosphate (FPP), an intermediate metabolite in the mevalonate pathway, specifically activates TRPV3 among six thermoTRPs using Ca 2؉ imaging and electrophysiology with cultured keratinocytes and TRPV3-overexpressing cells. Agonistic potencies of related compounds in the FPP metabolism were ignorable. Voltage-dependence of TRPV3 was shifted by FPP, which appears to be the activation mechanism. An intraplantar injection of FPP acutely elicits nociceptive behaviors in inflamed animals, indicating that FPP is a novel endogenous pain-producing substance via TRPV3 activation. Co-culture experiments demonstrated that this FPP-evoked signal in the keratinocytes is transmitted to sensory neurons. In addition, FPP reduced TRPV3 heat threshold resulting in heightened behavioral sensitivity to noxious heat. Taken together, our data suggest that FPP is the firstly identified endogenous TRPV3 activator that causes nociception. Our results may provide useful chemical information to elucidate TRPV3 physiology and novel pain-related metabolisms.Skin keratinocytes and sensory neurons express sensor ion channels that detect changes in the external or internal environments. TRPV3 channel has been found to be expressed in the skin keratinocytes of mice and humans and in the sensory neurons of humans and is activated by temperatures exceeding 33°C (1-3). Studies using TRPV3-knock-out mice or TRPV3-overexpressing transgenic mice have demonstrated that TRPV3 is involved in behavioral responses to innocuous and noxious heat (4 -5).Searching for pharmacological tools to modulate TRPV3 activity seems active (see review, Ref. 6). Several phytosubstances and synthetic compounds, such as camphor, menthol, carvacrol, citral, incensole acetate, and 2-aminoethoxydiphenyl borate (2-APB) 2 are shown to activate TRPV3 (4, 7-13). It has been reported that some of endogenous molecules are able to modulate TRPV3 activation. For example, unsaturated fatty acids potentiated TRPV3 activation by 2-APB (14). S-Nitrosylation of the channel protein activates TRPV3, as well as other TRPVs (15). Ca 2ϩ and calmodulin suppressed the sensitization of TRPV3 to recurrent stimuli (16). However, little is known about endogenous compounds that are able to directly and specifically activate TRPV3.A variety of metabolites are formed in the human body as a result of the diverse biochemical processes and some of the metabolites are potentially involved in pain development (17). Farnesyl pyrophosphate (FPP) is an intermediate molecule in the cholesterol synthesis pathway. In the present study, we examined whether FPP is an activator for TRPV3. We performed Ca 2ϩ ...

BACKGROUND AND PURPOSETransient receptor potential ion channel vanilloid 3 (TRPV3) is expressed in skin keratinocytes and plays an important role in thermal and chemical nociceptions in the periphery. The presence of TRPV3 inhibitors would improve our understanding of TRPV3 function and help to develop receptor-specific analgesics. However, little is known about physiological substances that specifically inhibit TRPV3 activity. Here, we investigated whether 17(R)-resolvin D1 (17R-RvD1), a naturally occurring pro-resolving lipid specifically affects TRPV3 activity. EXPERIMENTAL APPROACHWe examined the effect of 17R-RvD1 on sensory TRP channels using Ca 2+ imaging and whole cell electrophysiology experiments in a HEK cell heterologous expression system, cultured sensory neurons and keratinocytes. We also examined changes in sensory TRP agonist-specific acute licking/flicking or flinching behaviours and mechanical and thermal pain behaviours using Hargreaves, Randall-Selitto and von Frey assay systems in the absence and presence of inflammation. KEY RESULTSWe showed that 17R-RvD1 specifically suppresses TRPV3-mediated activity at nanomolar and micromolar concentrations. The voltage-dependence of TRPV3 activation by camphor was shifted rightwards by 17R-RvD1, which indicates its inhibitory mechanism is as a result of a shift in voltage-dependence. Consistently, TRPV3-specific acute pain behaviours were attenuated by locally injected 17R-RvD1. Moreover, the administration of 17R-RvD1 significantly reversed the thermal hypersensitivity that occurs during an inflammatory response. Knockdown of epidermal TRPV3 blunted these antinociceptive effects of 17R-RvD1. CONCLUSIONS AND IMPLICATIONS17R-RvD1 is a novel natural inhibitory substance specific for TRPV3. The results of our behavioural studies suggest that 17R-RvD1 has acute analgesic potential via TRPV3-specific mechanisms.

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Authorship note: KW and YG share first authorship. RRJ and KW share senior authorship. Conflict of interest: RRJ is a consultant of Boston Scientific and received a research grant from the company. He also serves on the board of directors of Ascletis Pharma. RRJ and KW also filed a patent, Methods and kits for treating pain (16/612,909), in association with Duke University.