As key pathogen-responsive receptors, TLRs recognize pathogen-associated molecular patterns from foreign pathogens to mediate innate immunity. TLRs are typically expressed by immune cells, and activation of TLRs results in upregulation of proinflammatory cytokines via activation of the transcription factors NF-κB and interferon regulatory factor-3 (4
). TLR2–TLR4 are also expressed by glial cells in the CNS and contribute to neuropathic pain development through glial activation and induction of proinflammatory cytokines/chemokines in the spinal cord (7
). In this study, we demonstrated that TLR3 is also expressed by a subset of primary sensory neurons, namely TRPV1-expressing nociceptors. Importantly, the TLR3 agonist PIC is sufficient to induce inward currents and action potentials in DRG neurons and further elicit scratching in WT mice but not Tlr3–/–
mice. Further, the extracted total RNAs induced inward currents in DRG neurons via TLR3. At the spinal cord level, PIC also modulated the excitatory synaptic transmission by increasing sEPSC frequency. It appears that TLR3 synthesized in DRG cell bodies could be transported to the spinal central terminals to modulate synaptic transmission (30
). Thus, primary sensory neurons express TLR3 to detect foreign pathogens and endogenous ligands (e.g., dsRNAs), leading to rapid protective behaviors such as scratching.
Although itch and pain share many similarities (1
), increasing evidence points to distinct molecular mechanisms of itch (19
). Primary sensory neurons located in the trigeminal ganglion and DRG transduce itch stimuli to the CNS (1
). As the best-characterized itch mediator, histamine is released from mast cells and binds H1/H4 receptors on skin nerve terminals to elicit itch (37
) via activation of PLCβ3 and TRPV1 (29
). CQ (an agonist of sensory neuron-specific GPCR MrgprA3) and BAM8-22 (an endogenous agonist of MrgprC11) produce histamine-independent itch via TRPA1 activation (19
). Both histamine-dependent itch and histamine-independent itch (e.g., itch induced by proteases, serotonin, and endothelin) require the TRPV1-expressing nociceptors (29
). Loss of vesicular glutamate transporter-2 in nociceptors results in reduced pain but enhanced itch, indicating that glutamate release from these nociceptors is dispensable for itch in the absence of pain (41
). At the spinal cord level, the peptides GRP and SP, released from TRPV1-expressing nociceptors, activate GRP receptor and NK1 to elicit pruritus (28
). In this study, we further revealed TLR3 as a new player in itch control, to our knowledge, via its peripheral and central actions.
Of the most striking findings of this study are the marked deficits in histamine-dependent and -independent pruritus in Tlr3–/– mice: there was a profound reduction of scratching behaviors elicited by all the 9 pruritogens that we tested (Supplemental Figure 1K). Of note Tlr3–/– mice showed (a) no gross anatomical defects, (b) no neuronal loss in DRGs and spinal cords, (c) no deficits in the skin and spinal cord nerve innervations, (d) no impairment in the ascending itch pathway, and (e) no changes in skin morphology. Hence, it is unlikely that the itch phenotypes that we observed are results of developmental defects in Tlr3–/– mice. Consistently, knockdown of TLR3 in DRGs using AS-ODNs or siRNA and spinal inhibition of TLR3/TRIF signaling also reduced the compound 48/80– and CQ-induced itch in adult WT mice. Furthermore, direct activation of TLR3 by intradermal PIC was sufficient to elicit TLR3-dependent scratching.
Although TLR7 was implicated in pruritus in our previous study (13
), the present study has clearly demonstrated distinct roles of different TLRs in itch. Compared with a dramatic reduction in both histamine-dependent and -independent itch in Tlr3–/–
mice only showed a partial reduction in histamine-independent itch. Our findings also showed the limited role of TLRs in pain using the cheek model (25
): the wiping behaviors induced by compound 48/80 and CQ were unchanged after deletion of Tlr3
. However, there are at least 12 members in the mouse TLR family; the involvement of other TLRs in pain and itch needs further investigation.
Although the previous reports indicated roles of TLR3 in suppressing axonal growth of DRG neurons (12
) and memory retention in the hippocampus (43
), our study is the first to our knowledge to demonstrate a critical role of TLR3 in synaptic transmission. Notably, the frequency of sEPSCs in spinal lamina II neurons was reduced in Tlr3–/–
mice, suggesting that TLR3 is required for glutamate release from the spinal presynaptic terminals. Consistently, PIC increased sEPSC frequency in the lamina II neurons via TLR3 activation. Despite a moderate reduction in spinal cord basal synaptic transmission, basal pain perception after thermal, mechanical, and chemical stimuli was intact in Tlr3–/–
mice (Figure , B–G). However, the central sensitization–induced pain hypersensitivity, i.e., the formalin-induced second-phase pain and the capsaicin-induced secondary mechanical hyperalgesia, was significantly impaired in Tlr3–/–
mice (Figure , H and I). In parallel, spinal cord synaptic plasticity underlying pain or itch hypersensitivity was abrogated in Tlr3–/–
mice. Importantly, TLR3 deficiency resulted in a failure in the induction of spinal LTP. Accordingly, the capsaicin-induced sEPSC frequency increase in dorsal horn neurons was substantially reduced in Tlr3–/–
mice (Figure ). In sharp contrast, Tlr7–/–
mice displayed normal spinal cord synaptic transmission and LTP induction (Figure ) and unaltered second-phase pain in the formalin test (13
), despite a reduction in nonhistaminergic pruritus (13
). Thus, impairment in spinal synaptic plasticity and central sensitization should contribute additionally to profound itch deficits in Tlr3–/–
We postulate that TLR3 expression on the DRG neuronal surface could be coupled to ion channels to induce inward currents and actions potentials, whereas TLR3 localization in intracellular compartments (endosomes) of DRG neurons (data not shown) could be involved in the trafficking and axonal transport of signaling molecules. A reduction in the axonal transport of TRPV1 and GRP, as observed in Tlr3–/–
mice (Supplemental Figure 8), should also contribute to deficits in central sensitization and itch. Of interest, a genome-scale functional screening reveals that the microtubule regulator stathmin is a potential ligand of TLR3 (44
). Whether stathmin plays a role in itch by interacting with TLR3 is of great interest.
Central sensitization has been implicated in itch hypersensitivity. For example, scratching is greatly potentiated after loss of Bhlhb5-expressing inhibitory interneurons in the dorsal horn (23
). Central sensitization appears to underlie the increasingly appreciated “population coding” hypothesis of itch (20
). According to this hypothesis, itch-responsive neurons are localized to a small population of C-fiber nociceptors expressing MrgprA3/MrgprC11/GRP/SP/TRPV1. While activation of this small population of neurons with pruritogens elicits itch, activation of the larger population of nociceptors induces pain to suppress itch. Of interest, TLR3 is expressed in a subset of TRPV1+
nociceptors that contain GRP (Figure F). This unique distribution pattern of TLR3 may explain why acute pain is largely intact, whereas itch is impaired, in Tlr3–/–
mice. A specific role of TLR3 in central sensitization is supported by (a) failure of spinal LTP induction in Tlr3–/–
mice and (b) the observation that intrathecal PIC- and capsaicin-induced licking behaviors were abolished in Tlr3–/–
Although our data support a neuronal mechanism of TLR3 for itch control, we should not exclude other mechanisms that are important in disease conditions. It is known that skin cells, such as mast cells and keratinocytes, play important roles in itch sensation in skin disease conditions (3
). TLR3 is expressed by mast cells and keratinocytes, and PIC activates mast cells and keratinocytes to release the proinflammatory cytokines (46
). Although we did not find obvious morphological and biochemical changes in the skin of Tlr3–/–
mice under the normal conditions, we observed marked TLR3 upregulation in the dry skin condition, and this upregulation may drive chronic itch via producing the proinflammatory cytokines (3
). In addition, TLR3-mediated upregulation of NGF in dry skin could also regulate chronic itch, since increased epidermal NGF expression was implied in the pathogenesis of pruritic contact dermatitis and psoriasis (48
In conclusion, we have identified TLR3 as a critical signaling molecule that is required for pruritus, regardless of histamine dependence. Functional TLR3 is expressed by primary sensory neurons that coexpress TRPV1 and GRP, and it is known that this subset of nociceptors is indispensable for itch sensation. The TLR3 agonist PIC was sufficient to induce inward currents and action potentials in DRG neurons, increase synaptic transmission in spinal dorsal horn neurons, and elicit scratching in WT mice. In particular, TLR3 deficiency resulted in impairment in central sensitization and spinal cord synaptic plasticity underlying itch hypersensitivity. TLR3 is further upregulated in dry skin and essential for the development of chronic itch. Given (a) the prevalence of chronic itch during skin diseases (e.g., atopic and contact dermatitis) as well as kidney, liver, and metabolic diseases and (b) the ineffectiveness of current antihistamine treatments in these chronic itchy conditions (2
), targeting TLR3 may provide a novel strategy for developing anti-itch therapies.