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Toll-like receptors (TLRs) are germline-encoded pattern-recognition receptors (PRRs) to initiate innate immune responses by recognizing molecular structures shared by a wide range of pathogens, known as pathogen-associated molecular patterns (PAMPs). After tissue injury or cellular stress, TLRs can also detect endogenous ligands known as danger-associated molecular patterns (DAMPs). TLRs are expressed in various cell types in the central nervous system (CNS), including non-neuronal and neuronal cells, and contribute to both infectious and non-infectious disorders in the CNS. Following tissue insult and nerve injury, TLRs (such as TLR2, 3, and 4) induce the activation of microglia and astrocytes and the production of the proinflammatory cytokines in the spinal cord, leading to the development and maintenance of inflammatory pain and neuropathic pain. In particular, primary sensory neurons, such as nociceptors express TLRs (e.g., TLR4 and TLR7) to sense exogenous PAMPs and endogenous DAMPs released after tissue injury and cellular stress. These neuronal TLRs are new players in the processing of pain and itch by increasing the excitability of primary sensory neurons. Given the prevalence of chronic pain and itch and the suffering of the affected people, insights into TLR signaling in nervous system will open a new avenue for the management of clinical pain and itch.
Toll-like receptors (TLRs) are the first and best characterized germ line-encoded pattern-recognition receptors (PRRs) that initiate innate immune responses via recognition of molecular motifs of pathogens, known as pathogen-associated molecular patterns (PAMPs). Engagement of TLRs initiates intracellular signaling pathways, leading to the synthesis and secretion of various inflammatory cytokines and chemokines, typically by immune cells. TLR-induced innate immune responses are also a prerequisite for the generation of most adaptive immune responses. Therefore, TLRs represent the first line of host defense against pathogens and play a pivotal role in both innate and adaptive immunity.
The first-identified TLR family member was Drosophila Toll, which is essential not only for antifungal innate immune response but also for dorsoventral patterning during embryonic development [3,4]. Series of mammalian homologues of Toll were later identified. TLRs are evolutionarily conserved type I transmembrane proteins and comprise an ectodomain, which mediates the recognition of PAMPs, and characterized by the leucine-rich repeats, a transmembrane region, and cytosolic Toll-IL-1 receptor (TIR) domains that activate the downstream signaling pathways. To date, 10 (TLR1-10) and 12 (TLR1-9; TLR11-13) functional TLRs have been identified in human and mouse, respectively (Table 1). According to their subcellular localization, TLRs can be divided into surface-expressing TLRs (TLR1, 2, 4, 5, 6 and 10) and intracellular expressing TLRs (TLR3, 7/8 and 9) (Table 1). However, some TLRs (e.g., TLR3 and TLR7) could be localized both on membrane and in intracellular compartments, such as endosomes and the endoplasmic reticulum, depending on cell types and cell conditions . While most types of TLRs form homodimers between themselves, some TLRs can also form noncovalent dimmers, such as TLR1/TLR2 and TLR2/TLR6 heterodimers (Fig. 1) [7,8].
Each type of TLR detects distinct PAMPs derived from microorganisms, such as viruses, bacteria, mycobacteria, fungi, and parasites. For example, TLR1, TLR2, and TLR6 detect lipoproteins [9,10], TLR3 and TLR7/8 sense double-stranded (ds) and single-stranded (ss) RNAs, respectively[11–14], TLR4 responds to lipopolysaccharide (LPS) [15,16], TLR5 detects flagellin, TLR9 senses CpG DNA[18,19], and TLR11 sense profilin-like protein(Table 1). Importantly, TLRs can also recognize endogenous ligands[21–29] (Table 1), known as danger-associated molecular patterns termed (DAMPs) and induce sterile inflammatory responses in many pathological processes, which are known to release DAMPs as a consequence of cell necrosis and tissue remodeling. Thus, innate immune system is not only activated by TLR recognition of PAMPs but also by TLR recognition of DAMPs released after cell stress and injury.
Most TLRs (but not TLR3) signal through the adaptor protein MyD88 (Figure 1). TLR2 and TLR4 also signal through the adaptor protein TIRAP  (Table 1). After recognition of PAMPs by TLRs, MyD88 recruits the IL-1R associated kinases (IRAKs). IRAK activation results in an interaction with tumor necrosis factor receptor associated factor 6 (TRAF6) and recruitment of additional proteins, leading to the phosphorylation of the inhibitor of NFκB (IκB)–kinase complex (IKK complex). Phosphorylation of IκB causes the degradation of IκB, allowing the translocation of NFκB to the nucleus and subsequent gene transcription. Simultaneously, TRAF6 also activates the mitogen-activated protein kinase (MAPK) signaling pathways, such as the extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), leading to gene transcription and protein synthesis. Of note, activation of MAPK pathways also causes a rapid release (within 15 min) of inflammatory mediators such as chemokines. Activation of TLR signaling cascades has been shown to produce a wide array of pro-inflammatory mediators, including cytokines and chemokines, such as TNF-α, IL-1β, IL-6, IL-12, IL-8 and MIP2, as well as reactive oxygen/nitrogen intermediates such as NO (Figure 1).
TLRs 3 and 4 also utilize TRIF-dependent pathway, which culminates in the activation of NFκB and interferon regulatory factor 3 (IRF3) (Figure 1). TRIF also recruits TRAF6 for NFκB activation similar to those of the MyD88-dependent pathway. Additionally, TRIF recruits a signaling complex which leads to the phosphorylation of IRF3 and its nuclear translocation. Activation of IRF3 leads to the transcription of interferon-β (IFN-β), an anti-inflammatory mediator (Figure 1). Balanced production of inflammatory cytokines and type I interferon might have a role in controlling tumor growth and autoimmune diseases. The negative regulation of TLR-induced responses is also critical for suppressing inflammation and deleterious immune responses. TLR activation results in elimination of invaded pathogens via recruitment of neutrophils and activation of macrophages, as well as IFN-stimulated genes expression. Moreover, activation of TLR signaling also leads to maturation of dendrite cells (DCs), which is critical for the induction of adaptive immune responses .
TLRs are also expressed by many cell types in the central nerve system (CNS) and peripheral nerve system (PNS), including non-neuronal cells (e.g., microglia, astrocytes, oligodendrocytes, and Schwann cells) and neurons [35–37]. Activation of TLRs is known to produce various inflammatory mediators including cytokines (e.g., TNF-α), chemokines (e.g. MCP-1), and enzymes (e.g. COX-2 and MMP-9), as well as other inflammatory mediators (e.g., prostaglandins)[37–40]. Thus, TLRs play important roles in pathogenesis of multiple CNS disorders, including infectious diseases such as CNS viral infection  and non-infectious disorders, such as stroke, Alzheimer’s disease (AD), and multiple sclerosis (MS),
Increasing evidence indicates that TLRs and their associated signaling components contribute to pain hypersensitivity, and blockade of TLR signaling has been shown to reduce pathological pain[40,45–49]. Spinal application of TLR agonists, such as TLR4 agonist LPS or TLR3 agonist PolyI:C, are sufficient to induce pain-like behaviors in rodents[49–52]. Conversely, blockade of TLR signaling by different strategies can attenuate pain in animal models. For example, intrathecal injection of TLR4 antagonist, lipopolysaccharide Rhodobacter sphaeroides (LPS-RS) attenuated arthritic pain . Spinal targeting TLR4 with specific antisense oligonucleotides (ODN) or specific siRNA reduced neuropathic pain  and bone cancer pain . Intrathecal injection of green tea-derived epigallocatechin gallate (EGCG) reduced neuropathic pain through inhibition TLR4 signaling in rats. Intrathecal injection of ketamine also depresses neuropathic pain, at least in part, by inhibiting TLR3-induced p38 mitogen-activated protein kinase (MAPK) pathway in microglia. Additionally, application of TLR9 antagonist blocked tumor-induced thermal pain hypersensitivity. Although TLR activation in immune cells must play an important role in pain and itch, this review focuses on the role of TLRs in glial cells (microglia and astrocytes) and primary sensory neurons in processing pain and itch.
Molecular and cellular mechanisms underlying the development and maintenance of chronic pain remain unclear. It is generally believed that chronic pain results from neural plasticity including peripheral sensitization[58–60] (sensitization in primary sensory neurons) and central sensitization[39,61–65] (sensitization in spinal cord and other CNS neurons). Although pain is processed in neural networks, the interaction between neurons and glial cells (e.g., microglia and astrocytes) is also critical for the initiation and maintenance of chronic pain. Increasing evidence suggests that in chronic pain conditions glial cells are strongly activated in the DRG, spinal cord[68–74], as well as in the brain stem. Activation of glial cells contributes to the pathogenesis of chronic pain via neuron-glial interactions [71,75–78].
Microglial cells are resident innate immune cell origin from primitive myeloid precursors and comprise approximately 10% of the total cells within the CNS. Spinal cord microglia is strongly activated after nerve injury, surgical incision, and chronic opioid exposure[80,81]. Activated microglia not only exhibit increased expression of microglial markers CD 11b and Iba1, but also display elevated phosphorylation of p38 mitogen-activated protein kinase (MAPK) (Figure 2a). Activation of p38 in spinal microglia results in increased synthesis and release of the proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and brain-derived neurotrophic factor (BDNF) [52,69,82–84]. Microglial mediators (e.g., TNF-α, IL-1β, and BDNF) have been shown to powerfully regulate synaptic transmission by enhancing excitatory synaptic transmission and suppressing inhibitory synaptic transmission in spinal cord neurons[85–87]. TNF-α and BDNF also contribute importantly to the induction of spinal long-term potentation[83,88–90].
Microglia express most known members of the TLR family identified to date. Microglia constitutively express TLR2 and TLR3 [91,92] and respond to their agonists for the production of cytokines, including IFN-β, IL-1β and IL-6 (Figure 2b). TLRs are often up-regulated in microglia following exposure to their own ligands, although TLR3 does not appear to be up-regulated by its ligand poly(I:C) in microglia. TLR9 expressed by microglia was shown to respond to CpG DNA to produce pro-inflammatory mediators and regulate microglial-mediated neurotoxicity[93,94]. Microglia also express TLR7/8, which are highly homologous and important for the expression of microglial pro-inflammatory mediators and cross-talk with TLR9 [92,95]. LPS is one of the best known activator of microglia by activating TLR4 . LPS not only induces substantial expression and release of proinflammatory mediators but also cause dramatic morphological changes and proliferation of microglial cells [51,97,98].
Of note, nerve injury-induced microglia activation (e.g., up-regulation of microglial markers) and cytokine expression in the spinal cord are abrogated in TLR4 deficient mice and also in wild-type mice treated with TLR4 antisense-oligodeoxynucleotides . In addition, TLR2 and TLR3 are also required for nerve injury-induced microglial responses in the spinal cord. Antisense knockdown of spinal TLR3 reduced the nerve injury-induced phosphorylation of p38 MAPK in spinal microglia[49,52]. Importantly, nerve injury-induced neuropathic pain is impaired after deletion or inhibition of TLR2, TLR3, or TLR4 [47–49,99,100]. In addition to neuropathic pain, spinal TLR4 is important for microglial reaction after arthritis and the development of chronic arthritic pain. Intrathecal injection of TLR4 ligands is sufficient to induce spinal TNF-α production and persistent mechanical allodynia, a cardinal feature of chronic pain, which is a suppressed by minocycline, a non-selective microglial inhibitor. In particular, activation of p38 MAPK in microglia by TLR4 is critical for the release of TNF-α, IL-1β, BDNF, PGE2, and nitric oxide, leading to pain hypersensitivity [101,102] (Figure 2).
Astrocytes, derived from neuronal stem cells, are the most abundant glial cell type in the CNS and play various functional and structural roles, including formation of the blood brain barrier, regulation of cerebral blood flow, and participation in the tripartite synapse[103,104]. Astrocytes also play a critical role in chronic pain sensitization [70,105–108]. Following nerve injury, arthritis, and tumor growth, astrocyte reaction (e.g., up-regulation of GFAP and S100b) is more persistent than microglial reaction (e.g., up-regulation of CD11b and Iba1) (Figure 3a), and importantly, astrocyte activation is correlated with chronic pain behaviors [32,108–111]. Nerve injury and inflammation activate extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) pathways in spinal cord astrocytes (Figure 3b), leading to the synthesis and release of inflammatory mediators such as the proinflammatory cytokine interleukin 1β (IL-1β) and the proinflammatory chemokine CCL2 (also called MCP-1), CXCL1 (KC), and CXCL10 (IP-10) [71,112–114] (Figure 3b). Of note, IL-1β and CCL2/MCP-1 have been shown to induce central sensitization by increasing the activity of NMDA receptor in dorsal horn neurons[32,85,115,116].
Astrocytes express relatively limited TLR repertoire, in part due to neuroectodermal origin of astrocytes. Astrocytes express TLR2 and proinflammatory stimuli lead to augmented TLR2 expression in astrocytes. Astrocytes also express TLR3, and TLR3 activation by poly(I:C) treatment contributes to a proinflammatory phenotype of human and murine astrocytes via production of the proinflammatory mediators such as IL-6[118–120]. Interestingly, astrocytic TLR3 activation also induced the expression of neurotrophic factors and cytokines that are involved in cellular growth, differentiation and migration . Low but constitutive expression of TLR4 is detected in astrocytes, and activation of astrocytic TLR4 induces proinflammatory reactions mediated by the NFκB, MAPK and Jak1/Stat1 signaling pathways [118,122]. A recent study has demonstrated that astrocyte TLR7 activation and cross-talk with TLR9 signaling can regulate production of proinflammatory mediators[117,118,123]. Astrocytes also express TLR1, TLR5, and TLR6, but their functions are still unclear .
TLR2, 3, and 4 have been shown to regulate spinal cord astrocyte activation following nerve injury[47–49], although it remains to be investigated whether astrocyte activation is secondary to microglial activation by these TLRs. However, stimulation of astrocytic TLR4 with LPS induced a strong JNK activation and CCL2 release, which required TRAF6. Interestingly, TRAF6 was up-reregulated in spinal cord astrocytes after nerve injury, and spinal inhibition of TRAF6 signaling reduced mechanical allodynia. Thus, activation of TLR4 and possible TLR3 in astrocytes also contributes to chronic pain via activating the JNK/chemokines cascade (Figure 3b). The detailed mechanisms of TLRs signaling in astrocyte activation in chronic pain conditions still need further investigation.
Apart from TLR expression in glial cells, increasing evidence has also demonstrated TLR expression in primary sensory neurons, such as DRG and trigeminal ganglion (TG) neurons, and expression of TLRs in these primary sensory neurons is involved in pain and itch sensation [40,57,126,127]. It is possible that primary sensory neurons can directly detect PAMPs (exogenous TLR ligands) or DAMPs (endogenous TLR ligands) to send warning signals to the brain. Early immunohistochemical analysis revealed that TLR4 and its co-receptor CD14 are expressed in transient receptor potential vanilloid subtype 1 (TRPV1)-expressing trigeminal neurons. Further studies showed that TLR4 agonist LPS could bind to trigeminal neurons, elicit intracellular calcium release and inward currents, and increase TRPV1 activity . In addition, TLR4 is colocalized with calciton gene-related peptide (CGRP) in sensory neurons, and LPS was able to enhance TRPV1-dependent release of CGRP. Recently, it has been shown that activation TLRs including TLR3, TLR7, and TLR9 in DRG neurons by their respective ligands resulted in the expression of proinflammatory and pronociceptive mediators such as PGE2, CGRP, and IL-1β. Of note, mouse DRG neurons require MD-1 and CD14 but not MD-2 for TLR4 signaling . The intracellular signaling triggered by neuronal TLR activation needs to be characterized. It is likely that TLR signaling in neurons is distinct from that revealed in immune cells.
Recent evidence also points to a role of TLRs expressed by primary sensory neurons for itch sensation. Itch or pruritus, is defined as an unpleasant sensation that elicits the desire or reflex to scratch. Although acute itch severs as a warning and self-protective mechanism, chronic itch is a common clinical problem associated with skin diseases[132,133], systemic diseases[134,135], and metabolism disorders. Primary sensory neurons located in DRG and TG are responsible for transducing peripheral itch signals to brain via spinal cord [131,137]. It is well known that TRPV1-containing C-fibers are required for both histamine-dependent and independent itch[138,139].
Recently, we identified that functional TLR7 is expressed in small-sized DRG neurons, especially in TRPV1-expressing nociceptors to mediate itch sensation (Figure 4a). Immunohistochemistry revealed that TLR7 is highly colocalized with gastrin-releasing peptide (GRP), a neuropeptide that is known to elicit itch via GRP receptor expressed by superficial dorsal horn neurons in the spinal cord [140,141]. Single-cell RT-PCR analysis, conducted selectively in small DRG neurons, also confirmed that TLR7 population is within GRP and TRPV1 populations (Figure 4b). Notably, the G protein–coupled receptor MrgprA3, which is known to mediate chloroquine-induced and histamine-independent itch5, is completely colocalized with TLR7 (Figure 4b).
TLR7 recognizes imidazoquinoline derivatives, such as imiquimod and resiquimod (R848), and guanine analogs, such as loxoribine . Intradermal injection of imiquimod, R848, and loxoribine induced itch-indicative scratching behavior in wild-type mice. Importantly, the scratches induced by imiquimod, R848 and loxoribine are reduced in Tlr7−/− mice, suggesting these responses are TLR7-dependent. However, imiquimod also elicited TLR7-indendent itch[130,143], which may attribute to its off-target effects, since imiquimod has been shown to act on adenosine receptors or IP3R[143–145]. Furthermore, we found that TRPV1-containing C fibers, but not TRPV1 per se, are required for imiquimod-elicited itch. Strikingly, application of TLR7 ligands to dissociated DRG neurons elicited very rapid inward currents and action potentials (Figure 4c). By contrast, these ligands failed to induce inward currents and action potentials in TLR7 knockout mice (Figure 4c). Thus, activation of TLR7 leads to an immediate increase in neuronal excitability. This non-genomic action of TLR7 suggests a possible coupling of TLRs with ion channels in primary sensory neurons that can trigger immediate pain and/or itch sensation.
Compared with wild-type mice, Tlr7−/− mice exhibited normal thermal and mechanical pain and unaltered inflammatory and neuropathic pain. Of interest, Tlr7−/− mice showed a significant reduction in scratching behaviors in response to nonhistaminergic pruritogens, including chloroquine, endothlin-1, and SLIGRL-NH2, an agonist of protease-activated receptor 2 (PAR2). Thus, TLR7 is required for histamine-dependent itch but dispensable for pain sensation.
Despite recent progress and growing interest in understanding the crucial roles of TLR signaling in regulating pain and itch, many questions remain unanswered. First, what are the endogenous ligands for TLRs that are released following cell stress, tissue insult, or nerve injury? And what are the specific contributions of these endogenous TLR ligands to glial and neuronal activation in persistent pain and itch conditions? Second, is intracellular signaling of neuronal TLRs distinct from that of immune and glial TLR signaling? Third, what is the role of different co-receptors of TLRs that can mediate the interaction between endogenous ligands and TLRs in the processing of pain and itch? Fourth, what is the molecular mechanisms underlying the excitatory effects of TLR ligands on sensory neurons? Fourth, in addition to glial cells and sensory neurons, TLRs are expressed in different types of cells in skin tissue, including keratinocytes, Langerhans cells, monocytes macrophages, dendritic cells, T and B cells, and mast cells [146–148], which is implicated in the pathogenesis of several types of pruritic skin diseases, such as psoriasis, atopic dermatitis, allergic contact dermatitis, and skin infections. Thus, the precise role of TLR signaling in different types of cells (e.g., neurons, glia, and keratinocytes) for pain and itch sensation require the generation of conditional knockout mice with specific deletion of TLRs in different cell types. Fifth, compared with the well known roles of TLRs in pain control, much less is known about the roles of TLRs in the regulation of itch. It remains to be tested whether TLR2, TLR3, and TLR4, the most studied TLR family members that have been implicated in neuropathic pain sensitization, play a role in acute and chronic itch.
Chronic pain, such as tissue injury-induced inflammatory pain and nerve injury-induced neuropathic pain affects 1.5 billion people all over the world, and current treatments for chronic pain is insufficient [40,149]. Chronic itch is also a common clinical problem associated with skin diseases[132,133], systemic diseases[134,135], and metabolism disorders. Chronic pain and itch substantially reduces the quality of life that is affected . Clinically, the current treatments for both chronic pain and chronic itch are far from sufficient . Given the important role of TLRs in pain and itch, targeting TLRs may offer new treatment for treating debilitating pain and itch-related problems.
Finally, it is important to point out that TLR activation is a double-edged sword, producing both beneficial and detrimental effects. While persistent activation of TLRs could cause chronic inflammation and pathological changes of various diseases, limited activation of TLRs may be beneficial for resolving acute inflammation and restoring the homoeostatic balance. Thus, the challenge for developing new therapies is to block the detrimental effects of TLRs, meanwhile leaving the beneficial effects of TLRs intact.
The work was supported by US National Institutes of Health grants R01-DE17794, R01-NS54362 and R01-NS67686 to RRJ.
Conflict of interest
The authors state no conflict of interest