Sexually dimorphic effects of pexidartinib on nerve injury-induced neuropathic pain in mice.

It is well-established that spinal microglia and peripheral macrophages play critical roles in the etiology of neuropathic pain; however, growing evidence suggests sex differences in pain hypersensitivity owing to microglia and macrophages. Therefore, it is crucial to understand sex- and androgen-dependent characteristics of pain-related myeloid cells in mice with nerve injury-induced neuropathic pain. To deplete microglia and macrophages, pexidartinib (PLX3397), an inhibitor of the colony-stimulating factor 1 receptor, was orally administered, and mice were subjected to partial sciatic nerve ligation (PSL). Following PSL induction, healthy male and female mice and male gonadectomized (GDX) mice exhibited similar levels of spinal microglial activation, peripheral macrophage accumulation, and mechanical allodynia. Treatment with PLX3397 significantly suppressed mechanical allodynia in normal males; this was not observed in female and GDX male mice. Sex- and androgen-dependent differences in the PLX3397-mediated preventive effects were observed on spinal microglia and dorsal root ganglia (DRG) macrophages, as well as in expression patterns of pain-related inflammatory mediators in these cells. Conversely, no sex- or androgen-dependent differences were detected in sciatic nerve macrophages, and inhibition of peripheral CC-chemokine receptor 5 prevented neuropathic pain in both sexes. Collectively, these findings demonstrate the presence of considerable sex- and androgen-dependent differences in the etiology of neuropathic pain in spinal microglia and DRG macrophages but not in sciatic nerve macrophages. Given that the mechanisms of neuropathic pain may differ among experimental models and clinical conditions, accumulating several lines of evidence is crucial to comprehensively clarifying the sex-dependent regulatory mechanisms of pain.

Characterization of spinal microglial activation in a mouse model of imiquimod-induced psoriasis.

Although microglia are associated with chronic pain, the role of spinal microglia in the regulation of itch remains unclear. In this study, we characterized spinal microglial activation in a mouse model of imiquimod (IMQ)-induced psoriasis. Hypertrophic (activated) microglia were observed throughout the spinal cord after the topical application of IMQ. Furthermore, the mRNA expression of microglial markers and inflammatory mediators was upregulated. Ablation of itch-related sensory neurons using resiniferatoxin decreased itch-related scratching behavior and the number of hypertrophic microglia in the spinal dorsal horn. Conclusively, sensory neuron input may partially contribute to spinal microglial activation after IMQ application.

Tas2R signaling enhances mouse neutrophil migration via a ROCK-dependent pathway.

Type-2 bitter taste receptors (Tas2Rs) are a large family of G protein-coupled receptors that are expressed in the oral cavity and serve to detect substances with bitter tastes in foods and medicines. Recent evidence suggests that Tas2Rs are also expressed extraorally, including in immune cells. However, the role of Tas2Rs in immune cells remains controversial. Here, we demonstrate that Tas2R126, Tas2R135, and Tas2R143 are expressed in mouse neutrophils, but not in other immune cells such as macrophages or T and B lymphocytes. Treatment of bone marrow-derived neutrophils from wild-type mice with the Tas2R126/143 agonists arbutin and d-salicin led to enhanced C-X-C motif chemokine ligand 2 (CXCL2)-stimulated migration in vitro, but this response was not observed in neutrophils from Tas2r126/135/143-deficient mice. Enhancement of CXCL2-stimulated migration by Tas2R agonists was accompanied by increased phosphorylation of myosin light chain 2 (MLC2) and was blocked by pretreatment of neutrophils with inhibitors of Rho-associated coiled-coil-containing protein kinase (ROCK), but not by inhibitors of the small GTPase RhoA. Taken together, these results demonstrate that mouse neutrophils express functional Tas2R126/143 and suggest a role for Tas2R126/143-ROCK-MLC2-dependent signaling in the regulation of neutrophil migration.

Chemogenetic activation of central gastrin-releasing peptide-expressing neurons elicits itch-related scratching behavior in male and female mice.

Several lines of evidence have clarified that the key transmission pathways of itching sensation travel from the periphery to the central nervous system (CNS). Despite the functional significance of gastrin-releasing peptide (GRP) and its cognate receptor in the itch processing mechanism in the spinal dorsal horn (SDH), the roles of GRP-expressing (GRP+ ) neurons in different regions remain unclear. This study aimed to determine whether GRP+ neurons in the CNS directly modulated itch processing. To specifically activate spinal and supraspinal GRP neurons by the designer receptors exclusively activated by designer drugs (DREADDs) system, CAG-LSL-Gq-DREADD mice were crossed with GRP-Cre mice, resulting in the development of GRP-hM3Dq mice. Immunohistochemistry showed that hM3Dq was highly expressed in the SDH and brainstem closely related to sensory processing. The intraperitoneal, intrathecal, or intracerebroventricular administration of clozapine-N-oxide, an agonist of hM3Dq, strongly elicited dermatome-dependent itch-related scratching behavior, but did not change pain sensitivity. Importantly, GRP-Gq-DREADD-mediated scratching behavior in GRP-hM3Dq mice was not affected by the ablation of transient receptor potential vanilloid 1+ sensory C-fibers, and it was also observed to a similar degree under chronic itch conditions. Furthermore, there were no significant sex differences in the scratching behavior elicited by GRP-Gq-DREADD, suggesting that itch-dominant roles of central GRP+ neurons might be common in both sexes, at least under normal physiological conditions. These novel findings not only contribute to understanding the functional roles of central GRP+ neurons further, but also propose the development of future effective therapeutics for intractable itching.

Chemogenetic Activation of CX3CR1-Expressing Spinal Microglia Using Gq-DREADD Elicits Mechanical Allodynia in Male Mice.

It is important to investigate the sex-dependent roles of microglia in pain hypersensitivity as reactive microglia within the spinal dorsal horn (DH) have been reported to be pivotal in neuropathic pain induction in male rodents upon nerve injury. Here, we aimed at determining the role of sex differences in the behavioral and functional outcomes of the chemogenetic activation of spinal microglia using Gq-designer receptors exclusively activated by designer drugs (Gq-DREADD) driven by the microglia-specific Cx3cr1 promoter. CAG-LSL-human Gq-coupled M3 muscarinic receptors (hM3Dq)-DREADD mice were crossed with CX3C chemokine receptor 1 (CX3CR1)-Cre mice, and immunohistochemistry images revealed that hM3Dq was selectively expressed on Iba1+ microglia, but not on astrocytes and neurons. Intrathecal (i.t.) administration of clozapine-N-oxide (CNO) elicited mechanical allodynia exclusively in male mice. Furthermore, the reactive microglia-dominant molecules that contributed to pain hypersensitivity in CX3CR1-hM3Dq were upregulated in mice of both sexes. The degree of upregulation was greater in male than in female mice. Depletion of spinal microglia using pexidartinib (PLX3397), a colony stimulating factor-1 receptor inhibitor, alleviated the male CX3CR1-hM3Dq mice from pain hypersensitivity and compromised the expression of inflammatory molecules. Thus, the chemogenetic activation of spinal microglia resulted in pain hypersensitivity in male mice, suggesting the sex-dependent molecular aspects of spinal microglia in the regulation of pain.

Chemogenetic Regulation of CX3CR1-Expressing Microglia Using Gi-DREADD Exerts Sex-Dependent Anti-Allodynic Effects in Mouse Models of Neuropathic Pain.

Despite growing evidence suggesting that spinal microglia play an important role in the molecular mechanism underlying experimental neuropathic pain (NP) in male rodents, evidence regarding the sex-dependent role of these microglia in NP is insufficient. In this study, we evaluated the effects of microglial regulation on NP using Gi-designer receptors exclusively activated by designer drugs (Gi-DREADD) driven by the microglia-specific Cx3cr1 promoter. For the Cre-dependent expression of human Gi-coupled M4 muscarinic receptors (hM4Di) in CX3C chemokine receptor 1-expressing (CX3CR1+) cells, R26-LSL-hM4Di-DREADD mice were crossed with CX3CR1-Cre mice. Mouse models of NP were generated by partial sciatic nerve ligation (PSL) and treatment with anti-cancer agent paclitaxel (PTX) or oxaliplatin (OXA), and mechanical allodynia was evaluated using the von Frey test. Immunohistochemistry revealed that hM4Di was specifically expressed on Iba1+ microglia, but not on astrocytes or neurons in the spinal dorsal horn of CX3CR1-hM4Di mice. PSL-induced mechanical allodynia was significantly attenuated by systemic (intraperitoneal, i.p.) administration of 10 mg/kg of clozapine N-oxide (CNO), a hM4Di-selective ligand, in male CX3CR1-hM4Di mice. The mechanical threshold in naive CX3CR1-hM4Di mice was not altered by i.p. administration of CNO. Consistently, local (intrathecal, i.t.) administration of CNO (20 nmol) significantly relieved PSL-induced mechanical allodynia in male CX3CR1-hM4Di mice. However, neither i.p. nor i.t. administration of CNO affected PSL-induced mechanical allodynia in female CX3CR1-hM4Di mice. Both i.p. and i.t. administration of CNO relieved PTX-induced mechanical allodynia in male CX3CR1-hM4Di mice, and a limited effect of i.p. CNO was observed in female CX3CR1-hM4Di mice. Unlike PTX-induced allodynia, OXA-induced mechanical allodynia was slightly improved, but not significantly relieved, by i.p. administration of CNO in both male and female CX3CR1-hM4Di mice. These results suggest that spinal microglia can be regulated by Gi-DREADD and support the notion that CX3CR1+ spinal microglia play sex-dependent roles in nerve injury-induced NP; however, their roles may vary among different models of NP.

Critical role of GRP receptor-expressing neurons in the spinal transmission of imiquimod-induced psoriatic itch.

AIM: Ample evidence indicates that gastrin-releasing peptide receptor (GRPR)-expressing neurons play a critical role in the transmission of acute itch. However, the pathophysiology of spinal mechanisms underlying intractable itch such as psoriasis remains unclear. In this study, we aimed to determine whether itch-responsive GRPR+ neurons contribute to the spinal transmission of imiquimod (IMQ)-induced psoriatic itch. METHODS: To generate a psoriasis model, C57BL/6J mice received a daily topical application of 5% IMQ cream on their shaved back skin for 7-10 consecutive days. GRP+ neurons were inhibited using Cre-dependent expression of Gi-designer receptors exclusively activated by designer drugs (DREADDs), while GRPR+ neurons were ablated by intrathecal administration of bombesin-saporin. RESULTS: Repeated topical application of IMQ elicited psoriasis-like dermatitis and scratching behaviors. The mRNA expression levels of GRP and GRPR were upregulated in the cervical spinal dorsal horn (SDH) on days 7 and 10 after IMQ application. Either chemogenetic silencing of GRP+ neurons by Gi-DREADD or ablation of GRPR+ neurons significantly attenuated IMQ-induced scratching behaviors. CONCLUSION: The GRP-GRPR system might be enhanced in the SDH, and itch-responsive GRPR+ neurons largely contribute to intractable itch in a mouse model of psoriasis.

GRP receptor and AMPA receptor cooperatively regulate itch-responsive neurons in the spinal dorsal horn.

Gastrin-releasing peptide (GRP) receptor-expressing (GRPR)+ neurons have a central role in the spinal transmission of itch. Because their fundamental regulatory mechanisms are not yet understood, it is important to determine how such neurons are excited and integrate itch sensation. In this study, we investigated the mechanisms for the activation of itch-responsive GRPR+ neurons in the spinal dorsal horn (SDH). GRPR+ neurons expressed the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) containing the GluR2 subunit. In mice, peripherally elicited histaminergic and non-histaminergic itch was prevented by intrathecal (i.t.) administration of the AMPAR antagonist NBQX, which was consistent with the fact that firing of GRPR+ neurons in SDH under histaminergic and non-histaminergic itch was completely blocked by NBQX, but not by the GRPR antagonist RC-3095. Because GRP+ neurons in SDH contain glutamate, we investigated the role of GRP+ (GRP+/Glu+) neurons in regulating itch. Chemogenetic inhibition of GRP+ neurons suppressed both histaminergic and non-histaminergic itch without affecting the mechanical pain threshold. In nonhuman primates, i.t. administration of NBQX also attenuated peripherally elicited itch without affecting the thermal pain threshold. In a mouse model of diphenylcyclopropenone (DCP)-induced contact dermatitis, GRP, GRPR, and AMPAR subunits were upregulated in SDH. DCP-induced itch was prevented by either silencing GRP+ neurons or ablation of GRPR+ neurons. Altogether, these findings demonstrate that GRP and glutamate cooperatively regulate GRPR+ AMPAR+ neurons in SDH, mediating itch sensation. GRP-GRPR and the glutamate-AMPAR system may play pivotal roles in the spinal transmission of itch in rodents and nonhuman primates.

Insufficient efferocytosis by M2-like macrophages as a possible mechanism of neuropathic pain induced by nerve injury.

Peripheral nerve injury typically leads to chronic inflammation through recruitment of immune cells, which may induce neuropathic pain. We previously reported that M1-like macrophages at sites of peripheral nerve injury induced neuropathic pain; however, the involvement of other immune cells (e.g. M2-like macrophages) in the progression of neuropathic pain remains unclear. In addition, the immune responses that occur at sites of nerve injury have not been well characterized. In this study, we show that M2-like macrophages accumulate in injured nerves to participate in the clearance of dead or dying cells (i.e., efferocytosis). Because MerTK (a receptor of dead or dying cells) levels on the surface of macrophages are limited, it seems to induce the insufficient of efferocytosis, such that the levels of dead or dying cells cannot be controlled in injured nerves. Given that efferocytosis is pivotal for resolution of inflammation, our data suggest that insufficient efferocytosis is a contributing factor in the development of chronic inflammation in injured nerves.

Inflammatory Macrophages in the Sciatic Nerves Facilitate Neuropathic Pain Associated with Type 2 Diabetes Mellitus.

Despite the requirement for effective medication against neuropathic pain associated with type 2 diabetes mellitus (T2DM), mechanism-based pharmacotherapy has yet to be established. Given that long-lasting neuroinflammation, driven by inflammatory macrophages in the peripheral nerves, plays a pivotal role in intractable pain, it is important to determine whether inflammatory macrophages contribute to neuropathic pain associated with T2DM. To generate an experimental model of T2DM, C57BL/6J mice were fed a high-fat diet (HFD) ad libitum. Compared with control diet feeding, obesity and hyperglycemia were observed after HFD feeding, and the mechanical pain threshold evaluated using the von Frey test was found to be decreased, indicating the development of mechanical allodynia. The expression of mRNA markers for macrophages, inflammatory cytokines, and chemokines were significantly upregulated in the sciatic nerve (SCN) after HFD feeding. Perineural administration of saporin-conjugated anti-Mac1 antibody (Mac1-Sap) improved HFD-induced mechanical allodynia. Moreover, treatment of Mac1-Sap decreased the accumulation of F4/80+ macrophages and the upregulation of inflammatory mediators in the SCN after HFD feeding. Inoculation of lipopolysaccharide-activated peritoneal macrophages in tissue surrounding the SCN elicited mechanical allodynia. Furthermore, pharmacological inhibition of inflammatory macrophages by either perineural or systemic administration of TC-2559 [4-(5-ethoxy-3-pyridinyl)-N-methyl-(3E)-3-buten-1-amine difumarate], a α4ß2 nicotinic acetylcholine receptor-selective agonist, relieved HFD-induced mechanical allodynia. Taken together, inflammatory macrophages that accumulate in the SCN mediate the pathophysiology of neuropathic pain associated with T2DM. Inhibitory agents for macrophage-driven neuroinflammation could be potential candidates for novel pharmacotherapy against intractable neuropathic pain.

Chemokine CXCL1 is responsible for cocaine-induced reward in mice.

AIM: We have previously demonstrated that upregulation of CC chemokines through dopamine receptor signaling in the prefrontal cortex (PFC) underlies methamphetamine (Meth)-induced reward. Given the common pharmacological property of Meth and cocaine (Coca), which are highly addictive psychostimulants, we hypothesized that chemokines may also contribute to Coca-induced reward. The aim of this study was to identify a key chemokine-mediating Coca-induced reward in mice. METHODS: The mRNA expression levels of chemokines were measured by reverse transcription-quantitative polymerase chain reaction. Coca-induced reward was evaluated by conditioned place preference test. RESULTS: We found that mRNA expression levels of CC chemokine ligand 2 (CCL2), CCL7, and CXC chemokine ligand 1 (CXCL1) were upregulated in the PFC after a single administration of Coca (20 mg/kg, s.c.). Upregulation of CXCL1, but not CCL2 and CCL7, mRNA in the PFC was also observed after repeated administration of Coca. A single administration of dopamine D1 receptor agonist SKF 81297 (10 mg/kg, s.c.), but not D2 receptor agonist sumanirole, upregulated CXCL1 mRNA in the PFC. Coca-induced reward was attenuated by the pretreatment of SB 225002 (5 mg/kg, s.c.), a selective antagonist of CXC chemokine receptor 2 (CXCR2, cognate receptor for CXCL1). CONCLUSIONS: Collectively, we identified CXCL1 as a key regulator in Coca-induced reward and propose that pharmacological approach targeting CXCL1 could be a novel pharmacotherapy for Coca-induced reward.

Inhibition of peripheral macrophages by nicotinic acetylcholine receptor agonists suppresses spinal microglial activation and neuropathic pain in mice with peripheral nerve injury.

BACKGROUND: Neuro-immune interaction underlies chronic neuroinflammation and aberrant sensory processing resulting in neuropathic pain. Despite the pathological significance of both neuroinflammation-driven peripheral sensitization and spinal sensitization, the functional relationship between these two distinct events has not been understood. METHODS: In this study, we determined whether inhibition of inflammatory macrophages by administration of α4ß2 nicotinic acetylcholine receptor (nAChR) agonists improves neuropathic pain and affects microglial activation in the spinal dorsal horn (SDH) in mice following partial sciatic nerve ligation (PSL). Expression levels of neuroinflammatory molecules were evaluated by RT-qPCR and immunohistochemistry, and PSL-induced mechanical allodynia was defined by the von Frey test. RESULTS: Flow cytometry revealed that CD11b+ F4/80+ macrophages were accumulated in the injured sciatic nerve (SCN) after PSL. TC-2559, a full agonist for α4ß2 nAChR, suppressed the upregulation of interleukin-1ß (IL-1ß) in the injured SCN after PSL and attenuated lipopolysaccharide-induced upregulation of IL-1ß in cultured macrophages. Systemic (subcutaneous, s.c.) administration of TC-2559 during either the early (days 0-3) or middle/late (days 7-10) phase of PSL improved mechanical allodynia. Moreover, local (perineural, p.n.) administration of TC-2559 and sazetidine A, a partial agonist for α4ß2 nAChR, during either the early or middle phase of PSL improved mechanical allodynia. However, p.n. administration of sazetidine A during the late (days 21-24) phase did not show the attenuating effect, whereas p.n. administration of TC-2559 during this phase relieved mechanical allodynia. Most importantly, p.n. administration of TC-2559 significantly suppressed morphological activation of Iba1+ microglia and decreased the upregulation of inflammatory microglia-dominant molecules, such as CD68, interferon regulatory factor 5, and IL-1ß in the SDH after PSL. CONCLUSION: These findings support the notion that pharmacological inhibition of inflammatory macrophages using an α4ß2 nAChR agonist exhibit a wide therapeutic window on neuropathic pain after nerve injury, and it could be nominated as a novel pharmacotherapy to relieve intractable pain.

Upregulation of CCL7 and CCL2 in reward system mediated through dopamine D1 receptor signaling underlies methamphetamine-induced place preference in mice.

We previously showed that the CC-chemokine ligand 2 (CCL2)-CC-chemokine receptor 2 (CCR2) system is responsible for conditioned place preference (CPP) by methamphetamine (Meth). In this study, we investigated the roles for other chemokines mediating Meth-induced CPP and the upstream factors upregulating chemokines in mice. We found that CCL7 mRNA level was upregulated in the prefrontal cortex (PFC) after Meth administration (3mg/kg, subcutaneous), and increased CCL7 immunoreactivity was localized to the PFC NeuN-positive neurons. Meth-induced CPP was blocked by the dopamine D1 receptor antagonist SCH 23390 but not by the D2 receptor antagonists raclopride or haloperidol. The D1 receptor agonist SKF 81297 alone elicited CPP, suggesting a critical role of D1 receptor signaling in Meth-induced reward. Consistent with these results, the Meth-induced upregulation of CCL7 and CCL2 were attenuated by SCH 23390, and a single administration of SKF 81297 upregulated mRNA expression levels of CCL7 and CCL2 in the PFC. Furthermore, Meth-induced CPP was prevented by INCB 3284, a selective antagonist of CCR2, a receptor that binds both CCL7 and CCL2. Collectively, we identified two CC-chemokines (i.e., CCL7 and CCL2) as key regulatory factors in Meth-induced reward. Pharmacological inhibitors of these chemokines may warrant development as novel therapeutics for ameliorating Meth addiction.

Pharmacological Regulation of Neuropathic Pain Driven by Inflammatory Macrophages.

Neuropathic pain can have a major effect on quality of life but current therapies are often inadequate. Growing evidence suggests that neuropathic pain induced by nerve damage is caused by chronic inflammation. Upon nerve injury, damaged cells secrete pro-inflammatory molecules that activate cells in the surrounding tissue and recruit circulating leukocytes to the site of injury. Among these, the most abundant cell type is macrophages, which produce several key molecules involved in pain enhancement, including cytokines and chemokines. Given their central role in the regulation of peripheral sensitization, macrophage-derived cytokines and chemokines could be useful targets for the development of novel therapeutics. Inhibition of key pro-inflammatory cytokines and chemokines prevents neuroinflammation and neuropathic pain; moreover, recent studies have demonstrated the effectiveness of pharmacological inhibition of inflammatory (M1) macrophages. Nicotinic acetylcholine receptor ligands and T helper type 2 cytokines that reduce M1 macrophages are able to relieve neuropathic pain. Future translational studies in non-human primates will be crucial for determining the regulatory mechanisms underlying neuroinflammation-associated neuropathic pain. In turn, this knowledge will assist in the development of novel pharmacotherapies targeting macrophage-driven neuroinflammation for the treatment of intractable neuropathic pain.

Peripheral administration of interleukin-13 reverses inflammatory macrophage and tactile allodynia in mice with partial sciatic nerve ligation.

Inflammatory macrophages play a fundamental role in neuropathic pain. In this study, we demonstrate the effects of peripheral interleukin-13 (IL-13) on neuropathic pain after partial sciatic nerve (SCN) ligation (PSL) in mice. IL-13 receptor α1 was upregulated in accumulating macrophages in the injured SCN after PSL. Treatment with IL-13 reduced inflammatory macrophage-dominant molecules and increased suppressive macrophage-dominant molecules in cultured lipopolysaccharide-stimulated peritoneal macrophages and ex vivo SCN subjected to PSL. Moreover, the perineural administration of IL-13 relieved tactile allodynia after PSL. These results suggest that IL-13 reverses inflammatory macrophage-dependent neuropathic pain via a phenotype shift toward suppressive macrophages.

[Exploration of novel therapeutic targets for neuropathic pain based on the regulation of immune cells].

The pathogenesis of neuropathic pain is quite complicated and diverse. Because pre-existing analgesics, such as opioid analgesics and nonsteroidal anti-inflammatory drugs, are not sufficient to treat it, it is a serious task to establish a strategy of remedy for neuropathic pain. Recently, increasing evidence suggests that immune cell-mediated neuroinflammation in the nervous system induces central and peripheral sensitization, resulting in chronic pain. Initially, the immune system plays an important role in host defense. Although intravital homeostasis is kept constant by innate and adaptive immunity, the immune system is activated excessively due to infection, stress and tissue injury. Activated immune cells produce and release several kinds of inflammatory mediators, which act directly on sensory neurons and promote a recruitment of immune cells, developing the feedback loop of inflammatory exacerbation. We've focused on the role of crosstalk between immune cells and neurons in peripheral neuroinflammation, and explored a novel candidate for a remedy of neuropathic pain. In this review, we will introduce recent reports and our research work that suggest the functional significance of neuroinflammation in neuropathic pain, and survey possibilities of new strategies for chronic pain from the point of view of basic research.

TC-2559, an α4ß2 nicotinic acetylcholine receptor agonist, suppresses the expression of CCL3 and IL-1ß through STAT3 inhibition in cultured murine macrophages.

The anti-inflammatory properties of TC-2559, an α4ß2 nicotinic acetylcholine receptor (nAChR) agonist, on cultured murine macrophages was investigated. TC-2559 suppressed the upregulation of CC-chemokine ligand 3 (CCL3) and interleukin-1ß (IL-1ß) following lipopolysaccharide (LPS) treatment in J774A.1 cells. TC-2559 inhibited the phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) but not nuclear factor-κB p65 after LPS. Blockade of pSTAT3 by AG490 inhibited the upregulation of CCL3 and IL-1ß after LPS. In conclusion, TC-2559-driven α4ß2 nAChR signaling suppressed the upregulation of CCL3 and IL-1ß by inhibiting pSTAT3 in inflammatory macrophages, resulting in the suppression of neuropathic pain.

Macrophage-T cell interactions mediate neuropathic pain through the glucocorticoid-induced tumor necrosis factor ligand system.

Peripheral neuroinflammation caused by activated immune cells can provoke neuropathic pain. Herein, we investigate the actions of macrophages and T cells through glucocorticoid-induced tumor neurosis factor receptor ligand (GITRL) and its receptor (GITR) in neuropathic pain. After partial sciatic nerve ligation (PSL) in enhanced green fluorescent protein (eGFP) chimeric mice generated by the transplantation of eGFP(+) bone marrow cells, eGFP(+) macrophages, and T cells markedly migrated to the injured site after PSL. Administration of agents to deplete macrophages (liposome-clodronate and Clophosome-A(TM)) or T cells (anti-CD4 antibody and FTY720) could suppress PSL-induced thermal hyperalgesia and tactile allodynia. The expression levels of co-stimulatory molecules GITRL and GITR were increased on infiltrating macrophages and T cells, respectively. The perineural injection of a GITRL neutralizing antibody that could inhibit the function of the GITRL-GITR pathway attenuated PSL-induced neuropathic pain. Additionally, the induction of inflammatory cytokines and the accumulation of GITR(+) T cells in the injured SCN were abrogated after macrophage depletion by Clophosome-A(TM). In conclusion, GITRL expressed on macrophages drives cytokine release and T cell activation, resulting in neuropathic pain via GITR-dependent actions. The GITRL-GITR pathway might represent a novel target for the treatment of neuropathic pain.

Peripheral interleukin-4 ameliorates inflammatory macrophage-dependent neuropathic pain.

There is increasing evidence that inflammatory (M1-polarized) macrophages drive the nonresolving neuroinflammation that causes neuropathic pain after nerve injury. As interleukin-4 (IL-4) promotes the suppressive (M2-polarized) state in macrophages, we examined whether exploiting an IL-4-mediated pathway could ameliorate M1 macrophage-dependent neuropathic pain. The mRNA and protein expression of IL-4 receptor α chain (IL-4Rα) were upregulated in accumulating F4/80 macrophages in injured sciatic nerve (SCN). In mouse macrophage cell line J774A.1, IL-4 downregulated the mRNA expression of M1 macrophage-specific molecules (IL-1ß, CC chemokine ligand 3, and CD86) normally provoked by lipopolysaccharide, while increasing the mRNA expression of M2 macrophage-specific molecules (arginase-1, IL-10, and CD206) through a STAT6-mediated pathway. In ex vivo SCN culture, M1 molecules were highly expressed in the injured SCN on day 7 after partial SCN ligation (PSL) but were decreased by IL-4 treatment. In contrast, M2 molecules were upregulated by IL-4. IL-4 also increased phosphorylated STAT6 (pSTAT6) expression and shifted IL-1ß M1 macrophages toward a CD206 M2 phenotype. Perineural administration of IL-4 in mice subject to PSL ameliorated development and maintenance of tactile allodynia and thermal hyperalgesia. These effects of IL-4 were based on that IL-4 treatment increased the proportions of pSTAT6 and CD206 macrophages in injured SCN on day 14 after PSL. We found that neuropathic pain can be ameliorated by IL-4 treatment, which exerts its therapeutic effect on accumulating macrophages through a STAT6-dependent pathway. A shift in macrophage phenotype from the inflammatory to the suppressive phenotype, driven by IL-4R signaling, may have benefits in the treatment of neuropathic pain.

[The role of CC-chemokine ligand 2 in the development of psychic dependence on methamphetamine].

Addiction is described as a chronic neurological disorder associated with plasticity in the mesolimbic system. Recently, it has been suggested that neuroinflammation plays an important role in the induction of neuronal plasticity and the formation of pathogenesis in chronic neurological disorders. Therefore, we examined the role of CC-chemokine ligand 2 (CCL2), a proinflammatory chemokine, in the development of psychic dependence on methamphetamine. In mice treated with methamphetamine, CCL2 mRNA was significantly increased in prefrontal cortex and nucleus accumbens. Moreover, phosphorylated tyrosine hydroxylase serine40 (pTH Ser40) levels in the ventral tegmental area (VTA) were increased by methamphetamine. Similarly, pTH Ser40 levels in the VTA were also increased by the intracerebroventricular administration of recombinant CCL2. The increment of pTH Ser40 levels in the VTA by methamphetamine was attenuated by RS504393, a selective CC-chemokine receptor 2 (CCR2) antagonist, indicating that the increased CCL2 activates the brain reward system via CCR2 activation. In the conditioned place preference test, methamphetamine produced place preference in a dose-dependent manner, which was attenuated by RS504393. These results suggest that the activation of the brain reward system via CCL2-CCR2 pathway plays an important role in the development of psychic dependence on methamphetamine.