Shinichi Kato

Background: Transient receptor potential melastatin 8 (TRPM8) is a cold‐sensitive Ca ²⁺ ‐permeable ion channel that regulates intestinal inflammation. This study was aimed to elucidate the role of TRPM8 in enteric macrophages and spinal afferent sensory neurons during dextran sulfate sodium (DSS)–induced colitis. Methods: The role of TRPM8 in the progression of colonic inflammation was examined in 2% DSS‐induced colitis by immunohistochemistry, reverse transcription‐polymerase chain reaction, visceral sensing assessment, and flow cytometry. The role of TRPM8 in sensory neurons and macrophages was investigated using primary cultured dorsal root ganglion (DRG) neurons and bone marrow‐derived macrophages (BMDMs). Key Results: DSS‐induced colitis was significantly aggravated in TRPM8‐deficient (KO) mice compared with that in wild‐type (WT) mice. In the colitis model, KO mice showed lower IL‐10 mRNA levels than WT mice, but IL‐6, IL‐1β, and TNF‐α levels were not affected. TRPM8 expression was upregulated in the distal colon and DRG following DSS administration. In the distal colon, TRPM8‐immunoreactive cells and nerve fibers colocalized with F4/80 and substance P/calcitonin gene‐related peptide (CGRP), respectively. In the DRG, TRPM8 neuron subtypes were labeled by neurofilament 200, substance P, CGRP, and tyrosine kinase receptor A. In cultured DRG neurons, TRPM8 deficiency attenuated lipopolysaccharide (LPS)–elicited CGRP release but did not affect substance P release. In BMDMs, TRPM8 deficiency significantly decreased LPS‐ and LPS/CGRP‐elicited upregulation of IL‐10 mRNA compared with that in WT animals. Conclusions & Inferences: TRPM8 protects against DSS‐induced experimental colitis via regulating CGRP release from sensory neurons and IL‐10 production in enteric macrophages during intestinal inflammation.

This study is the first to reveal the local role of TRPV6 in the intestine. TRPV6KO exacerbated dextran sulfate sodium-induced colitis and increased intestinal permeability compared with wild-type mice. Furthermore, intercellular junction protein expression was lower in TRPV6KO mice. TRPV6 protects against colitis by maintaining epithelial barrier function by regulating intercellular junction protein expression. Thus, TRPV6 may be a novel therapeutic candidate for treating inflammatory bowel disease.

Mast cell stabilizers including disodium cromoglycate (DSCG) were found to have potential as the agonists of an orphan G protein-coupled receptor, GPR35, although it remains to be determined whether GPR35 is expressed in mast cells and involved in suppression of mast cell degranulation. Our purpose in this study is to verify the expression of GPR35 in mast cells, and to clarify how GPR35 modulates the degranulation. We explored the roles of GPR35 using an expression system, a mast cell line constitutively expressing rat GPR35, peritoneal mast cells, and bone marrow-derived cultured mast cells. Immediate allergic responses were assessed using the IgE-mediated passive cutaneous anaphylaxis (PCA) model. Various known GPR35 agonists including DSCG and newly designed compounds suppressed IgE-mediated degranulation. GPR35 was expressed in mature mast cells, but not in immature bone marrow-derived cultured mast cells and the rat mast cell line. Degranulation induced by antigens was significantly down-modulated in the mast cell line stably expressing GPR35. A GPR35 agonist, zaprinast, induced a transient activation of RhoA and a transient decrease in the amount of filamentous actin. GPR35 agonists suppressed the PCA responses in the wild-type mice, but not in the GPR35^-/- mice. These findings suggest that GPR35 should prevent mast cells from undergoing degranulation induced by IgE-mediated antigen stimulation and be the primary target of mast cell stabilizers. Significance Statement The agonists of an orphan G protein-coupled receptor, GPR35, including disodium cromoglycate, were found to suppress degranulation of rat and mouse mature mast cells, and their anti-allergic effects were abrogated in the GPR35^-/- mice, indicating that the primary target of mast cell stabilizers should be GPR35.

Leukotriene B4 receptor type 1 (BLT1), a high‐affinity receptor for leukotriene B4 (LTB4), plays an important role in inflammatory responses, including allergic airway inflammation. In this study, we examined the effect of genetic BLT1 deletion (BLT1KO) on ovalbumin (OVA)‐induced allergic enteritis in mice to determine the pathogenic role of LTB4/BLT1 in allergic enteritis, a gastrointestinal form of food allergy. Repeated oral OVA challenges after sensitization with OVA and aluminium potassium sulphate induced allergic enteritis, characterized by systemic allergic symptoms (scratching, immobility and swelling), diarrhoea, colonic oedema and colonic goblet cell hyperplasia, accompanied by increased colonic peroxidase activity, colonic inflammatory cytokine expression and increased serum OVA‐specific IgE levels. The severity of enteritis was significantly attenuated in BLT1KO mice compared with wild‐type (WT) mice, without an increase in serum OVA‐specific IgE levels. The accumulation of neutrophils, eosinophils, M2‐macrophages, dendritic cells, CD4+ T cells and mast cells was observed in the colonic mucosa of allergic enteritis, and such accumulation was significantly lower in BLT1KO mice than in WT mice. BLT1 expression was upregulated and colocalized mostly in neutrophils and partly in eosinophils and dendritic cells in the colonic mucosa of allergic enteritis. These findings indicate that BLT1 deficiency ameliorates OVA‐induced allergic enteritis in mice and that LTB4/BLT1 contributes to neutrophil and eosinophil accumulation in the allergic colonic mucosa. Therefore, BLT1 is a promising drug target for treating food allergies.

Inhibitors of bromodomain and extra‐terminal motif (BET) proteins are emerging epigenetic therapeutics that suppress gene expressions that drive cancer and inflammation. The present study examined anti‐inflammatory effects of a quinazoline‐based BET inhibitor, CN210, in a murine ileitis model. CN210 was given orally 30 min before and 24 h after a subcutaneous administration of indomethacin. Macroscopic and histological evidences of ileitis, mucosal myeloperoxidase (MPO) activity and cytokine expressions were evaluated 48 h after the indomethacin administration. To further characterize the anti‐inflammatory pathways modulated by CN210, its effects on RAW264 cells treated with lipopolysaccharide (LPS) were investigated. Competitive ligand binding and docking studies of CN210 to CREB‐binding protein (CBP) and p300 were also performed. Oral administration of CN210 significantly reduced the severity of ileitis, normalized both proinflammatory MPO activity and concomitant cytokine expressions induced by indomethacin administration. Furthermore, CN210 attenuated the expression of cytokines and reversed the activation of nuclear factor κB (NF‐κB) and mitogen‐activated protein kinases (MAPK) induced by LPS. Competitive ligand binding assays showed that CN210 bound to the bromodomains of two paralogous histone acetyltransferases, CBP and p300, in addition to the bromodomains of BET proteins. Docking studies of CN210 to the bromodomains of CBP and p300 showed a similarity to the binding mode of SGC‐CBP30, a specific CBP/p300 inhibitor. CN210 ameliorates indomethacin‐induced ileitis by inhibiting the expression of inflammatory cytokines through the attenuation of NF‐κB and MAPK pathways. CN210 thus represents a new mode of therapy for non‐steroidal anti‐inflammatory drug‐induced ileitis and inflammatory bowel disease.

Inhibitors of bromodomain and extra-terminal motif proteins (“BET inhibitors”) are emerging epigenetic therapeutics that exert their effects by suppressing the expression of genes that drive cancer and inflammation. The present study examined the anti-inflammatory effects of a quinazoline-based BET inhibitor, CN210, which also inhibits bromodomains of two paralogous histone acetyltransferases (HATs), such as cAMP-responsive element binding protein-binding protein (CBP) and p300. To assess its effectiveness against inflammatory bowel disease (IBD), CN210 was tested in an experimentally-induced murine Crohn’s disease (CD)-like ileitis model. Ileitis was induced in mice by subcutaneous administration of indomethacin and CN210 was given orally 30 min before and 24 h after the indomethacin administration. Whilst indomethacin produced severe ileitis accompanied by an increase in ileal mucosal myeloperoxidase (MPO) activity, administration of CN210 reduced the severity of ileitis in a dose-dependent manner and suppressed the MPO activity. Similarly, upregulation of inflammatory cytokines was observed in ileitis mucosa after indomethacin injection but this response was significantly attenuated by CN210 administration. To further characterize the effects of CN210 on inflammatory pathways, monocyte/macrophage-like cell line RAW264 treated with lipopolysaccharide (LPS) was exposed to CN210. CN210 significantly attenuated the expression of inflammatory cytokines and reversed the activation of NF-κB and MAP kinases induced by LPS. These findings suggest that CN210 ameliorates indomethacin-induced ileitis due at least in part to the inhibition of inflammatory cytokine expressionviaattenuation of NF-κB and MAP kinase pathways, thus representing a new mode of therapy for CD.

Motilin is a gastrointestinal peptide hormone that stimulates gastrointestinal motility. Motilin is produced primarily in the duodenum and jejunum. Motilin receptors (MTLRs) are G protein-coupled receptors that may represent a clinically useful pharmacological target as they can be activated by erythromycin. The functions of motilin are highly species-dependent and remain poorly understood. As a functional motilin system is absent in rodents such as rats and mice, these species are not commonly used for basic studies. In this study, we examine the usefulness of human MTLR-overexpressing transgenic (hMTLR-Tg) mice by identifying the mechanisms of the gastric motor response to human motilin and erythromycin. The distribution of hMTLR was examined immunohistochemically in male wild-type (WT) and hMTLR-Tg mice. The contractile response of gastric strips was measured isometrically in an organ bath, while gastric emptying was determined using phenol red. hMTLR expression was abundant in the gastric smooth muscle layer. Interestingly, higher levels of hMTLR expression were observed in the myenteric plexus of hMTLR-Tg mice but not WT mice. hMTLR was not co-localized with vesicular acetylcholine transporter, a marker of cholinergic neurons in the myenteric plexus. Treatment with human motilin and erythromycin caused concentration-dependent contraction of gastric strips obtained from hMTLR-Tg mice but not from WT mice. The contractile response to human motilin and erythromycin in hMTLR-Tg mice was affected by neither atropine nor tetrodotoxin and was totally absent in Ca²⁺-free conditions. Furthermore, intraperitoneal injection of erythromycin significantly promoted gastric emptying in hMTLR-Tg mice but not in WT mice. Human motilin and erythromycin stimulate gastric smooth muscle contraction in hMTLR-Tg mice. This action is mediated by direct contraction of smooth muscle via the influx of extracellular Ca²⁺. Thus, hMTLR-Tg mice may be useful for the evaluation of MTLR agonists as gastric prokinetic agents.