For a long time ago, people have believed that good medicine tastes bitter to the mouth; however, whether the bitter taste itself has therapeutic effects is less studied. Generally, bitter taste is recognized by Type-2 bitter-taste receptors (TAS2Rs) belonging to G-protein coupled receptors and TAS2Rs are localized on taste bud cells of the tongue. Growing evidence suggests that TAS2Rs are expressed not only in the taste bud cells but also in other cells including airway smooth muscle cells, intestinal tuft cells and immune cells. In this study, we show that bitter taste substance-TAS2R axis regulates neutrophil migration. By gene expression analysis, we found that neutrophils express TAS2R126, TAS2R135 and TAS2R143. Next, we observed the effect of TAS2R126/135/143 agonists on neutrophil migration. Although TAS2R135 agonists did not affect neutrophil migration, TAS2R126/143 agonists significantly enhanced CXCL2-induced neutrophil migration. The enhancing effects were not observed in a TAS2R126/143 deficient neutrophil-like cell line. In addition, TAS2R126/143 agonist also promotes neutrophil infiltration into zymosan-injected abdominal cavity. These results suggest that TAS2R126/143 signaling facilitates neutrophil-mediated immune responses and may be targets to promote host defense against infection.
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 α4β2 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.
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.In this study, we determined whether inhibition of inflammatory macrophages by administration of α4β2 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.Flow cytometry revealed that CD11b+ F4/80+ macrophages were accumulated in the injured sciatic nerve (SCN) after PSL. TC-2559, a full agonist for α4β2 nAChR, suppressed the upregulation of interleukin-1β (IL-1β) in the injured SCN after PSL and attenuated lipopolysaccharide-induced upregulation of IL-1β 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 α4β2 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-1β in the SDH after PSL.These findings support the notion that pharmacological inhibition of inflammatory macrophages using an α4β2 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.
Several lines of evidence indicate that spinal microglia exacerbate abnormal pain processing. Recent findings demonstrated there are significant functional differences of microglia in pain hypersensitivity between male and female animals, but underlying mechanisms are poorly understood. Here, we investigated whether androgens affect sex differences of microglia in neuropathic pain model mice. Peripheral nerve injury-induced mechanical allodynia was suppressed by the treatment of PLX3397, a microglial inhibitor, in male but not in female mice, and the effects of PLX3397 in the spinal dorsal horn of male mice was significantly greater than that of female mice. Gonadectomy (GDX) decreased in serum testosterone concentration and mechanical pain threshold in male mice. Susceptibility of spinal microglia for PLX3397 in GDX-treated male mice was similar to that of normal female mice. Moreover, intrathecal administration of colony-stimulating factor 1 (CSF1) elicited mechanical allodynia in male mice, but not GDX-treated male mice or normal female mice. Collectively, functional roles of spinal microglia contributing pain hypersensitivity are different between male and female, and sex-dependent characters of spinal microglia might be determined through androgen actions.
Nicotine (NIC) regulates various cellular functions acting on the nicotinic acetylcholine receptor (nAChR). And nAChR consists of ligand-gated cation channels with pentameric structure and composed of α and β subunits. In the central nervous system, α 4 β 2 and α 7 nAChRs are the most abundantly expressed as nAChR subtypes. There are several lines of evidence indicating that systemic administration of NIC elicits the release of endogenous opioids, such as, endorphins, enkephalins and dynorphins, in the brain. NIC exerts numerous acute effects, for example, antinociceptive effects and the activating effects of the hypothalamic-pituitary-adrenal (HPA) axis. In these effects, NIC-induced antinociception, but not HPA axis activation, was inhibited by opioid receptor antagonist, naloxone (NLX), and was also suppressed in morphine tolerated mice, indicating the participation of the endogenous opioid system in NIC-induced antinociception, but not HPA axis activation. Moreover, NIC-induced antinociception was antagonized by both α 4 β 2 and α 7 nAChR antagonists, while NIC-induced HPA axis activation was antagonized by α 4 β 2 nAChR antagonist, but not by α 7 nAChR antagonist. These results suggest that the endogenous opioid system may not be located on the downstream of α 4 β 2 nAChR. On the other hand, NIC has substantial physical dependence liability. NLX elicits NIC withdrawal after repeated NIC administration evaluated by corticosterone increase as a withdrawal sign, and NLX-precipitated NIC withdrawal is inhibited by concomitant administration of other opioid receptor antagonist, naltrexone, indicating the participation of endogenous opioid system in the development of physical dependence on NIC. NLX-precipitated NIC withdrawal was also inhibited by concomitant administration of an α 7 nAChR antagonist, but not an α 4 β 2 nAChR antagonist. Taken together, these findings suggest that the endogenous opioid system may be located on the downstream of α 7 nAChR and participates in the development of physical dependence on NIC.
Chronic neuroinflammation plays an important role in the molecular basis of neuropathic pain. It has been widely accepted that the various immune cells contribute to the development of neuropathic pain. Among immune cells, the role of macrophages and microglia has been well demonstrated. In this study, we evaluated the effects of macrophages/microglia on neuropathic pain using mice that can induce Gi-Designer Receptors Exclusively Activated by Designer Drugs（Gi-DREADD）driven by macrophages/microglia-specific cx3cr1 promoter (CX3CR1-hM4Di). Neuropathic pain model mice were generated by partial sciatic nerve ligation (PSL) or anti-cancer drugs, and mechanical allodynia was evaluated using von Frey test.
Because chronic vincristine (VCR) treatment causes neuropathic pain, as demonstrated by mechanical allodynia, effective therapeutic strategy is required. In this study, we investigated a suppressive effect of imipramine (IMI) on VCR-induced mechanical allodynia in mice. VCR (0.1 mg/kg, intraperitoneally (i.p.)) was administered once per day for 7 d in ICR male mice. Mechanical allodynia was evaluated by withdrawal response using von Frey filaments. In VCR-treated mice, mechanical allodynia was observed on day 3, 7, and 14. On day 14, morphine (3 mg/kg, subcutaneously) slightly but significantly suppressed VCR-induced mechanical allodynia. The percent inhibition by morphine of VCR-induced mechanical allodynia was less than that of the λ-carrageenan-induced inflammatory pain and was similar to that of nerve injury-induced neuropathic pain. Although single administration of IMI (30 mg/kg, i.p.) had no effect on VCR-induced mechanical allodynia, repeated administration of IMI (30 mg/kg, i.p.) for 7 d significantly suppressed VCR-induced mechanical allodynia. Suppressive effects by repeated IMI administration were observed in both early phase (day 0—6) and late phase (day 7—13) of VCR-induced mechanical allodynia. These results suggest that chronic VCR administration induces opioid analgesics-resistant mechanical allodynia, and repeated IMI administration may be an effective therapeutic approach for the treatment of VCR-induced mechanical allodynia.
Abstract Background 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. Methods The current study was performed using normal male and female mice, as well as gonadectomized (GDX) male mice. 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). Immunohistochemistry was performed to visualize microglia and macrophages, and PSL-induced mechanical allodynia was evaluated using the von Frey test. Results Following PSL induction, healthy male and female mice and male 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. Conclusion 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.
Abstract 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.
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