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.

Radiation-induced intestinal fibrosis (RIF) is a serious complication after abdominal radiotherapy for pelvic tumor or peritoneal metastasis. Herein, we show that RIF is mediated by eosinophil interactions with α-smooth muscle actin–positive (α-SMA⁺) stromal cells. Abdominal irradiation caused RIF especially in the submucosa (SM) of the small intestine, which was associated with the excessive accumulation of eosinophils in both human and mouse. Eosinophil-deficient mice showed markedly ameliorated RIF, suggesting the importance of eosinophils. After abdominal irradiation, chronic crypt cell death caused elevation of extracellular adenosine triphosphate, which in turn activated expression of C-C motif chemokine 11 (CCL11) by pericryptal α-SMA⁺ cells in the SM to attract eosinophils in mice. Inhibition of C-C chemokine receptor 3 (CCR3) by genetic deficiency or neutralizing antibody (Ab) treatment suppressed eosinophil accumulation in the SM after irradiation in mice, suggesting a critical role of the CCL11/CCR3 axis in the eosinophil recruitment. Activated α-SMA⁺ cells also expressed granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate eosinophils. Transforming growth factor-β1 from GM-CSF–stimulated eosinophils promoted collagen expression by α-SMA⁺ cells. In translational studies, treatment with a newly developed interleukin-5 receptor α–targeting Ab, analogous to the human agent benralizumab, depleted intestinal eosinophils and suppressed RIF in mice. Collectively, we identified eosinophils as a crucial factor in the pathogenesis of RIF and showed potential therapeutic strategies for RIF by targeting eosinophils.

Excess scar formation can occur after skin injurふy and lead to abnormal scar formation, such as keloids and hypertrophic scars, which are characterised by substantial deposition of extracellular matrix in the dermis. Periostin, an extracellular matrix protein that plays a crucial role in skin development and maintaining homeostasis, is also involved in skin disorders such as systemic/limited scleroderma, wound closure, and abnormal scar formation. However, the mechanism of periostin involvement in abnormal scar formation is not yet fully understood. In this study, we investigated the mechanism by which periostin is involved in abnormal scar formation. Treatment of human dermal fibroblasts (HDFs) with IL-4 and IL-13, which are cytokines of Th2 type immune responses that are up-regulated in abnormal scars, dramatically elevated the levels of periostin mRNA and protein, and also promoted the secretion of periostin by HDFs. Transforming growth factor-β1 (TGF-β1) had the same effect on HDFs as IL-4 and IL-13. Stimulation of HDFs with periostin promoted RhoA/ROCK pathway-mediated TGF-β1 secretion from HDFs. Our results suggest that IL-4 and IL-13 induce periostin expression and secretion, and in turn, secreted periostin induces RhoA/ROCK pathway-mediated TGF-β1 secretion. Secreted TGF-β1 then induces further periostin production and secretion, thereby promoting abnormal scar formation.

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.

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.

The chemokine receptor CCR7 contributes to various physiological and pathological processes including T cell maturation, T cell migration from the blood into secondary lymphoid tissues, and tumor cell metastasis to lymph nodes. Although a previous study suggested that the efficacy of CCR7 ligand-dependent T cell migration correlates with CCR7 homo- and heterodimer formation, the exact extent of contribution of the CCR7 dimerization remains unclear. Here, by inducing or disrupting CCR7 dimers, we demonstrated a direct contribution of CCR7 homodimerization to CCR7-dependent cell migration and signaling. Induction of stable CCR7 homodimerization resulted in enhanced CCR7-dependent cell migration and CCL19 binding, whereas induction of CXCR4/CCR7 heterodimerization did not. In contrast, dissociation of CCR7 homodimerization by a novel CCR7-derived synthetic peptide attenuated CCR7-dependent cell migration, ligand-dependent CCR7 internalization, ligand-induced actin rearrangement, and Akt and Erk signaling in CCR7-expressing cells. Our study indicates that CCR7 homodimerization critically regulates CCR7 ligand-dependent cell migration and intracellular signaling in multiple cell types.