Human immunodeficiency virus-1 (HIV-1) infection is associated with numerous effects on the nervous system, including pain and peripheral neuropathies. We now demonstrate that cultured rat dorsal root ganglion (DRG) neurons express a wide variety of chemokine receptors, including those that are thought to act as receptors for the HIV-1 coat protein glycoprotein120 (gp120). Chemokines that activate all of the known chemokine receptors increased [Ca 2ϩ ] i in subsets of cultured DRG cells. Many neurons responded to multiple chemokines and also to bradykinin, ATP, and capsaicin. Immunohistochemical studies demonstrated the expression of the CXCR4 and CCR4 chemokine receptors on populations of DRG neurons that also expressed substance P and the VR1 vanilloid receptor.RT-PCR analysis confirmed the expression of CXCR4, CX3CR1, CCR4, and CCR5 mRNAs in DRG neurons. Chemokines and gp120 produced excitatory effects on DRG neurons and also stimulated the release of substance P. Chemokines and gp120 also produced allodynia after injection into the rat paw. Thus these results provide evidence that chemokines and gp120 may produce painful effects via direct actions on chemokine receptors expressed by nociceptive neurons. Chemokine receptor antagonists may be important therapeutic interventions in the pain that is associated with HIV-1 infection and inflammation.
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 peripheral terminals of primary sensory neurons detect histamine and non-histamine itch-provoking ligands through molecularly distinct transduction mechanisms. It remains unclear, however, whether these distinct pruritogens activate the same or different afferent fibers. We utilized a strategy of reversibly silencing specific subsets of murine pruritogen-sensitive sensory axons by targeted delivery of a charged sodium-channel blocker and found that functional blockade of histamine itch did not affect the itch evoked by chloroquine or SLIGRL-NH2, and vice versa. Notably, blocking itch-generating fibers did not reduce pain-associated behavior. However, silencing TRPV1+ or TRPA1+ neurons allowed AITC or capsaicin respectively to evoke itch, implying that certain peripheral afferents may normally indirectly inhibit algogens from eliciting itch. These findings support the presence of functionally distinct sets of itch-generating neurons and suggest that targeted silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach for histaminergic and non-histaminergic pruritus.
Viral infection is one of the leading causes of brain encephalitis and meningitis. Recently, it was reported that Toll-like receptor-3 (TLR3) induces a double-stranded RNA (dsRNA)-mediated inflammatory signal in the cells of the innate immune system, and studies suggested that dsRNA may induce inflammation in the central nervous system (CNS) by activating the CNS-resident glial cells. To explore further the connection between dsRNA and inflammation in the CNS, we have studied the effects of dsRNA stimulation in astrocytes. Our results show that the injection of polyinosinic-polycytidylic acid (poly(I:C)), a synthetic dsRNA, into the striatum of the mouse brain induces the activation of astrocytes and the expression of TNF-alpha, IFN-beta, and IP-10. Stimulation with poly(I:C) also induces the expression of these proinflammatory genes in primary astrocytes and in CRT-MG, a human astrocyte cell line. Furthermore, our studies on the intracellular signaling pathways reveal that poly(I:C) stimulation activates IkappaB kinase (IKK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) in CRT-MG. Pharmacological inhibitors of nuclear factor-kappaB (NF-kappaB), JNK, ERK, glycogen synthase kinase-3beta (GSK-3beta), and dsRNA-activated protein kinase (PKR) inhibit the expression of IL-8 and IP-10 in astrocytes, indicating that the activation of these signaling molecules is required for the TLR3-mediated chemokine gene induction. Interestingly, the inhibition of PI3 kinase suppressed the expression of IP-10, but upregulated the expression of IL-8, suggesting differential roles for PI3 kinase, depending on the target genes. These data suggest that the TLR3 expressed on astrocytes may initiate an inflammatory response upon viral infection in the CNS.
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