Self-resolving inflammatory exudates and lipid mediator metabolomics recently uncovered a new family of potent anti-inflammatory and proresolving mediators biosynthesized by macrophages (MΦs), denoted maresins. Here we determined that maresin 1 (MaR1) produced by human MΦs from endogenous docosahexaenoic acid (DHA) matched synthetic 7R,14S-dihydroxydocosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid. The MaR1 alcohol groups and Z/E geometry of conjugated double bonds were matched using isomers prepared by total organic synthesis. MaR1's potent defining actions were confirmed with synthetic MaR1, i.e., limiting polymorphonuclear neutrophil (PMN) infiltration in murine peritonitis (ng/mouse range) as well as enhancing human macrophage uptake of apoptotic PMNs. At 1 nM, MaR1 was slightly more potent than resolvin D1 in stimulating human MΦ efferocytosis, an action not shared by leukotriene B(4). MaR1 also accelerated surgical regeneration in planaria, increasing the rate of head reappearance. On injury of planaria, MaR1 was biosynthesized from deuterium-labeled (d(5))-DHA that was blocked with lipoxygenase (LOX) inhibitor. MaR1 dose-dependently inhibited TRPV1 currents in neurons, blocked capsaicin (100 nM)-induced inward currents (IC(50) 0.49±0.02 nM), and reduced both inflammation- and chemotherapy-induced neuropathic pain in mice. These results demonstrate the potent actions of MaR1 in regulating inflammation resolution, tissue regeneration, and pain resolution. These findings suggest that chemical signals are shared in resolution cellular trafficking, a key process in tissue regeneration. Moreover, immunoresolvents of the innate immune response, such as MaR1, offer new opportunities for assessing MΦs and their local DHA metabolome in the return to tissue homeostasis.

SUMMARY Intracellular microRNAs (miRNAs) are key regulators of gene expression. The role of extracellular miRNAs in neuronal activation and sensory behaviors are unknown. Here we report an unconventional role of extracellular miRNAs for rapid excitation of nociceptor neurons via toll-like receptor-7 (TLR7) and its coupling to TRPA1 ion channel. miRNA-let-7b induces rapid inward currents and action potentials in dorsal root ganglion (DRG) neurons. These responses require the GUUGUGU motif, only occur in neurons co-expressing TLR7 and TRPA1, and are abolished in mice lacking Tlr7 or Trpa1. Furthermore, let-7b induces TLR7/TRPA1-dependent single channel activities in DRG neurons and HEK293 cells over-expressing TLR7/TRPA1. Intraplantar injection of let-7b elicits rapid spontaneous pain via TLR7 and TRPA1. Finally, let-7b can be released from DRG neurons by neuronal activation, and let-7b inhibitor reduces formalin-induced TRPA1 currents and spontaneous pain. Thus, secreted extracellular miRNAs may serve as novelpain mediatorsvia activating TLR7 /TRPA1in nociceptor neurons.

Accumulating evidence suggests that spinal cord astrocytes play an important role in neuropathic pain sensitization by releasing astrocytic mediators (e.g. cytokines, chemokines and growth factors). However, it remains unclear how astrocytes control the release of astrocytic mediators and sustain late-phase neuropathic pain. Astrocytic connexin-43 (now known as GJ1) has been implicated in gap junction and hemichannel communication of cytosolic contents through the glial syncytia and to the extracellular space, respectively. Connexin-43 also plays an essential role in facilitating the development of neuropathic pain, yet the mechanism for this contribution remains unknown. In this study, we investigated whether nerve injury could upregulate connexin-43 to sustain late-phase neuropathic pain by releasing chemokine from spinal astrocytes. Chronic constriction injury elicited a persistent upregulation of connexin-43 in spinal astrocytes for >3 weeks. Spinal (intrathecal) injection of carbenoxolone (a non-selective hemichannel blocker) and selective connexin-43 blockers (connexin-43 mimetic peptides (43)Gap26 and (37,43)Gap27), as well as astroglial toxin but not microglial inhibitors, given 3 weeks after nerve injury, effectively reduced mechanical allodynia, a cardinal feature of late-phase neuropathic pain. In cultured astrocytes, TNF-α elicited marked release of the chemokine CXCL1, and the release was blocked by carbenoxolone, Gap26/Gap27, and connexin-43 small interfering RNA. TNF-α also increased connexin-43 expression and hemichannel activity, but not gap junction communication in astrocyte cultures prepared from cortices and spinal cords. Spinal injection of TNF-α-activated astrocytes was sufficient to induce persistent mechanical allodynia, and this allodynia was suppressed by CXCL1 neutralization, CXCL1 receptor (CXCR2) antagonist, and pretreatment of astrocytes with connexin-43 small interfering RNA. Furthermore, nerve injury persistently increased excitatory synaptic transmission (spontaneous excitatory postsynaptic currents) in spinal lamina IIo nociceptive synapses in the late phase, and this increase was suppressed by carbenoxolone and Gap27, and recapitulated by CXCL1. Together, our findings demonstrate a novel mechanism of astrocytic connexin-43 to enhance spinal cord synaptic transmission and maintain neuropathic pain in the late-phase via releasing chemokines.

Toll-like receptors (TLRs) are typically expressed in immune cells to regulate innate immunity. Here we report that functional TLR7 is expressed in C-fiber primary sensory neurons and important for inducing itch (pruritis) but not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Thus, we have uncovered TLR7 as a novel itch mediator and a potential therapeutic target for anti-itch treatment in skin disease conditions.TLRs play a critical role in triggering innate immune responses to pathogen-associated molecular patterns (PAMPs) in mammals. Except for TLR3, TLRs engage downstream signaling cascades via MyD88 to produce cytokines and chemokines and fight against pathogenic infection 1 . Mammalian TLR family comprises at least 13 members (TLR1 to TLR13). TLR7 recognizes single-stranded RNAs from RNA viruses 1 . As innate immunity is strongly implicated in abnormal pain hypersensitivity 2 , we first examined whether thermal and mechanical pain sensitivity or pathological pain is altered in Tlr7 knockout (Tlr7 −/− ) mice.Compared to wild type (WT) mice, Tlr7 −/− mice exhibited normal thermal pain sensitivity, assessed by Hargreave's test and tail-flick test ( Supplementary Fig. 1a, b), and normal mechanical pain sensitivity, assessed by graded von Frey filaments and Randall-Selitto test ( Supplementary Fig. 1c, d). Acute inflammatory pain elicited by intraplantar injection of capsaicin, mustard oil ( Supplementary Fig. 1e, f), or formalin in both the first and second phases (Fig. 1a), as well as carrageenan-induced persistent inflammatory pain (Fig. 1b) and spinal nerve ligation-induced neuropathic pain ( Supplementary Fig. 1g) was unaltered in Tlr7 −/− mice. Consistently, Tlr7 −/− mice did not show any developmental defects in the dorsal root ganglia (DRG) and spinal cord and the expression of neurochemical markers such as TRPV1, CGRP, and IB4 was normal (data not shown).Recent studies have revealed distinct molecular mechanisms underlying pain and itch 3-5 . We next evaluated whether TLR7 plays a role in itch sensation. We counted the number of scratches (bouts) by a hindpaw of mouse following intradermal injection of pruritogenic agents in the nape of the neck. Notably, scratches induced by histamine-dependent

Inflammatory pain such as arthritic pain is typically treated with opioids and COX-2 inhibitors with well-known side effects. Transient receptor potential subtype V1 (TRPV1) and A1 (TRPA1) contribute importantly to the genesis of inflammatory pain via both peripheral mechanisms (peripheral sensitization) and spinal cord mechanisms (central sensitization). Although these TRP channels have been intensively studied, little is known about their endogenous inhibitors. Recent studies have demonstrated that the endogenous lipid mediators resolvins (RvE1 and RvD1), derived from omega-3 unsaturated fatty acids, are potent inhibitors for inflammatory pain, without noticeable side effects. However, the molecular mechanisms underlying resolvins’ distinct analgesic actions in mice are unclear. Resolvin D2 (RvD2) is a novel family member of resolvins. Here we report that RvD2 is a remarkably potent inhibitor of TRPV1 (IC50=0.1 nM) and TRPA1 (IC50= 2 nM) in primary sensory neurons, whereas RvE1 and RvD1 selectively inhibited TRPV1 (IC50=1 nM) and TRPA1 (IC50=9 nM), respectively. Accordingly, RvD2, RvE1, and RvD1 differentially regulated TRPV1 and TRPA1 agonist-elicited acute pain and spinal cord synaptic plasticity (sEPSC frequency increase). RvD2 also abolished inflammation-induced sEPSC increases (frequency and amplitude), without affecting basal synaptic transmission. Intrathecal administration of RvD2 at very low doses (0.01-1 ng) prevented formalin-induced spontaneous pain. Intrathecal RvD2 also reversed adjuvant-induced inflammatory pain without altering baseline pain and motor function. Finally, intrathecal RvD2 reversed C-fiber stimulation-evoked long-term potentiation in the spinal cord. Our findings suggest distinct roles of resolvins in regulating TRP channels and identify RvD2 as a potent endogenous inhibitor for TRPV1/A1 and inflammatory pain.

Summary Voltage-gated sodium (NaV) channels control the upstroke of the action potentials in excitable cells. Multiple studies have shown distinct roles of NaV channel subtypes in human physiology and diseases, but subtype-specific therapeutics are lacking and the current efforts have been limited to small molecules. Here we present a monoclonal antibody that targets the voltage-sensor paddle of NaV1.7, the subtype critical for pain sensation. This antibody not only inhibits NaV1.7 with high selectivity but also effectively suppresses inflammatory and neuropathic pain in mice. Interestingly, the antibody inhibits acute and chronic itch, despite well-documented differences in pain and itch modulation. Using this antibody, we discovered that NaV1.7 plays a key role in spinal cord nociceptive and pruriceptive synaptic transmission. Our studies reveal that NaV1.7 is a target for itch management and the antibody has therapeutic potential for suppressing pain and itch. Our antibody strategy may have broad applications for voltage-gated cation channels.

Temperature signaling can be initiated by members of transient receptor potential family (thermo-TRP) channels. Hot and cold substances applied to teeth usually elicit pain sensation. This study investigated the expression of thermo-TRP channels in dental primary afferent neurons of the rat identified by retrograde labeling with a fluorescent dye in maxillary molars. Single cell reverse transcription-PCR and immunohistochemistry revealed expression of TRPV1, TRPM8, and TRPA1 in subsets of such neurons. Capsaicin (a TRPV1 agonist), menthol (a TRPM8 agonist), and icilin (a TRPM8 and TRPA1 agonist) increased intracellular calcium and evoked cationic currents in subsets of neurons, as did the appropriate temperature changes (>43°, <25°, and <17°C, respectively). Some neurons expressed more than one TRP channel and responded to two or three corresponding stimuli (ligands or thermal stimuli). Immunohistochemistry and single cell reverse transcription-PCR following whole cell recordings provided direct evidence for the association between the responsiveness to thermo-TRP ligands and expression of thermo-TRP channels. The results suggest that activation of thermo-TRP channels expressed by dental afferent neurons contributes to tooth pain evoked by temperature stimuli. Accordingly, blockade of thermo-TRP channels will provide a novel therapeutic intervention for the treatment of tooth pain.Recent studies have demonstrated that subfamilies of TRP 2 channels play critical roles in the transduction of temperature and pain sensation (1). The temperature-activated TRP channels (thermo-TRP channels) include TRPV1, TRPM8, and TRPA1; they are activated by Ͼ43°, Ͻ25°, and Ͻ17°C, respectively (1). They can also be activated by the exogenous ligands, capsaicin (TRPV1), menthol (TRPM8), and icilin (TRPM8 and TRPA1) (2-5). Given that the thermo-TRPs are involved in converting thermal information into electrical signals within the sensory nervous system (1), it is possible that thermo-TRPs expressed by dental primary afferents play a crucial role for transduction process of tooth pain, especially caused by noxious thermal stimulation.The different forms of pain are produced by distinctive molecular and cellular mechanisms. It is now thought that elucidation of the main mechanism involved in a certain form of pain is key to the development of pain treatments, which specifically target underlying the cause rather than just symptoms (6). Tooth pain is commonly induced by the presence of hot or cold foods in the oral cavity (7-9). Tooth pain results from the exposure of dentin, which is caused by dissolution of the protective enamel covering the tooth crown by dental caries or gingival recession (7,8). Since the proposition of Brännström, arguing that the fluid movement in dentinal tubules induced by diverse stimuli, including thermal stimuli, elicits tooth nerve firing to produce tooth pain (referred to as the hydrodynamic hypothesis) (10, 11), much evidence has been reported to support the hypothesis (12). However, it is not fully under...

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