論文 - 木村　俊介

Protective effect of mesenchymal stromal cells in diabetic nephropathy: the In vitro and In vivo role of the M-Sec-tunneling nanotubes.

Federica Barutta, Beatrice Corbetta, Stefania Bellini, Roberto Gambino, Stefania Bruno, Shunsuke Kimura, Koji Hase, Hiroshi Ohno, Gabriella Gruden

Clinical science 138 （ 23 ） 1537 - 1559 2024年12月

研究論文（学術雑誌）, 査読有り

AP-1B regulates interactions of epithelial cells and intraepithelial lymphocytes in the intestine.

Matsumoto R, Ogata K, Takahashi D, Kinashi Y, Yamada T, Morita R, Tanaka K, Hattori K, Endo M, Fujimura Y, Sasaki N, Ohno H, Ishihama Y, Kimura S, Hase K

Cellular and molecular life sciences : CMLS （Springer Science and Business Media LLC）  81 （ 1 ） 425 - 425 2024年10月

責任著者, 査読有り,  ISSN  1420-682X

　概要を見る

Intraepithelial lymphocytes (IELs) reside in the epithelial layer and protect against foreign pathogens, maintaining the epithelial barrier function in the intestine. Interactions between IEL and epithelial cells are required for IELs to function effectively; however, the underlying molecular machinery remains to be elucidated. In this study, we found that intestinal epithelium-specific deficiency of the clathrin adaptor protein (AP)-1B, which regulates basolateral protein sorting, led to a massive reduction in IELs. Quantitative proteomics demonstrated that dozens of proteins, including known IEL-interacting proteins (E-cadherin, butyrophilin-like 2, and plexin B2), were decreased in the basolateral membrane of AP-1B-deficient epithelial cells. Among these proteins, CD166 interacted with CD6 on the surface of induced IEL. CD166 knockdown, using shRNA in intestinal organoid cultures, significantly inhibited IEL recruitment to the epithelial layer. These findings highlight the essential role of AP-1B-mediated basolateral sorting in IEL maintenance and survival within the epithelial layer. This study reveals a novel function of AP-1B in the intestinal immune system.

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Intestinal epithelium dysfunctions cause IgA deposition in the kidney glomeruli of intestine-specific Ap1m2-deficient mice.

Yusuke Kinashi, Keisuke Tanaka, Shunsuke Kimura, Masato Hirota, Seiga Komiyama 1, Tomoko Shindo 3, Akinori Hashiguchi, Daisuke Takahashi , Shinsuke Shibata, Shin-Ichiro Karaki, Hiroshi Ohno, Koji Hase

EBioMedicine 106   105256 2024年07月

研究論文（学術雑誌）, 共著, 責任著者, 査読有り

Purified diet affects intestinal epithelial proliferation and barrier functions through gut microbial alterations.

Hiroaki Shiratori, Kisara M Hattori, Kazuaki Nakata, Takuma Okawa, Seiga Komiyama, Yusuke Kinashi, Yuma Kabumoto, Yuria Kaneko, Motoyoshi Nagai, Tomoko Shindo, Nobuko Moritoki, Yuki I Kawamura, Taeko Dohi, Daisuke Takahashi, Shunsuke Kimura, Koji Hase

International immunology 2024年01月

研究論文（学術雑誌）, 査読有り

　概要を見る

The gut microbiota plays a crucial role in maintaining epithelial barrier function. Although multiple studies have demonstrated the significance of dietary factors on gut microbiota and mucosal barrier function, the impact of a purified diet, which has long been used in various animal experiments, on intestinal homeostasis remains to be elucidated. Here, we compared the impact of two different types of diets, a crude diet and an AIN-93G-formula purified diet, on epithelial integrity and the gut microbiota. Purified diet-fed mice exhibited shorter villi and crypt lengths and slower epithelial turnover, particularly in the ileum. In addition, antimicrobial products, including islet-derived protein 3γ (REG3γ), were substantially decreased in purified diet-fed mice. Purified diet feeding also suppressed α1,2-fucosylation on the epithelial surface. Furthermore, purified diet induced metabolic rewiring to fatty acid oxidation and ketogenesis. 16S ribosomal RNA gene sequencing of the ileal contents and mucus layer revealed distinct gut microbiota compositions between the purified and crude diet-fed mice. Purified diet feeding reduced the abundance of segmented filamentous bacteria (SFB), which potently upregulate REG3γ and fucosyltransferase 2 (Fut2) by stimulating group 3 innate lymphoid cells (ILC3) to produce IL-22. These observations illustrate that the intake of a crude diet secures epithelial barrier function by facilitating SFB colonization, whereas a purified diet insufficiently establishes the epithelial barrier, at least partly owing to the loss of SFB. Our data suggest that the influence of purified diets on the epithelial barrier integrity should be considered in experiments using purified diets.

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Dysfunction of Foxp3+ Regulatory T Cells Induces Dysbiosis of Gut Microbiota via Aberrant Binding of Immunoglobulins to Microbes in the Intestinal Lumen.

Kouhei Koshida, Mitsuki Ito, Kyosuke Yakabe, Yoshimitsu Takahashi, Yuki Tai, Ryouhei Akasako, Tatsuki Kimizuka, Shunsuke Takano, Natsumi Sakamoto, Kei Haniuda, Shuhei Ogawa, Shunsuke Kimura, Yun-Gi Kim, Koji Hase, Yohsuke Harada

International journal of molecular sciences 24 （ 10 ）  2023年05月

研究論文（学術雑誌）, 査読有り

　概要を見る

Foxp3+ regulatory T (Treg) cells prevent excessive immune responses against dietary antigens and commensal bacteria in the intestine. Moreover, Treg cells contribute to the establishment of a symbiotic relationship between the host and gut microbes, partly through immunoglobulin A. However, the mechanism by which Treg cell dysfunction disturbs the balanced intestinal microbiota remains unclear. In this study, we used Foxp3 conditional knockout mice to conditionally ablate the Foxp3 gene in adult mice and examine the relationship between Treg cells and intestinal bacterial communities. Deletion of Foxp3 reduced the relative abundance of Clostridia, suggesting that Treg cells have a role in maintaining Treg-inducing microbes. Additionally, the knockout increased the levels of fecal immunoglobulins and immunoglobulin-coated bacteria. This increase was due to immunoglobulin leakage into the gut lumen as a result of loss of mucosal integrity, which is dependent on the gut microbiota. Our findings suggest that Treg cell dysfunction leads to gut dysbiosis via aberrant antibody binding to the intestinal microbes.

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Protective effect of the tunneling nanotube-TNFAIP2/M-sec system on podocyte autophagy in diabetic nephropathy.

Barutta F, Bellini S, Kimura S, Hase K, Corbetta B, Corbelli A, Fiordaliso F, Bruno S, Biancone L, Barreca A, Papotti MG, Hirsh E, Martini M, Gambino R, Durazzo M, Ohno H, Gruden G

Autophagy 19 （ 2 ） 505 - 524 2023年02月

研究論文（学術雑誌）, 査読有り,  ISSN  1554-8627

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GP2-expressing cells: a new guardian with divergent functions in the intestine, eyes, and nose.

Toshihiko Iwanaga, Shunsuke Kimura

Biomedical research (Tokyo, Japan) 44 （ 6 ） 233 - 243 2023年

査読有り

　概要を見る

GP (glycoprotein)-2, originally identified as a predominant membranous component of pancreatic acinar cells, has attracted the interest of researchers in mucosal immunology for its role as a functional molecule specific for antigen-sampling cells in the intestinal Peyer's patches. GP2 is involved in the detection of pathological bacteria and is also histologically useful for the identification of the M cell lineage and their differentiation in lymphoid tissues. Subsequent immunohistochemistry for GP2 has revealed a broad distribution of M cells and related cells in the nasopharyngeal lymphoid tissues, conjunctiva, tear duct, and airway. Especially, GP2 cells in the paranasal sinuses and tear duct have been identified as novel types of epithelial cells. The systematic administration of RANKL can induce extra-M cells in conventional epithelia of body. The production and release of GP2 by conjunctival goblet cells and several mucous glands suggests leading roles for mucous cells in protection, including the entrapment of microorganisms for infections. The ocular surface and conjunctiva are connected to the lacrimal sac, nasolacrimal duct, and further nasal cavity, comprising another canal that passes through the body. The broad distribution of GP2-expressingcells may indicate its function as a new guardian in the intestine, eyes, and nose, all of which are exposed to external milieu.

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Spi-B alleviates food allergy by securing mucosal barrier and immune tolerance in the intestine.

Ishihara N, Nakamura Y, Yakabe K, Komiyama S, Fujimura Y, Kaisho T, Kimura S, Hase K

Frontiers in allergy （Frontiers Media SA）  3   996657 2022年10月

責任著者, 査読有り,  ISSN  2673-6101

　概要を見る

Food allergy is a type I allergic reaction induced by mast cells and is mainly activated by allergen-specific immunoglobulin (Ig)E. Spi-B is an E26-transformation-specific (Ets) family transcription factor essential for the differentiation and functional maturation of several immune cell subsets, including mast cells. However, the possible involvement of Spi-B in food allergy remains unclear. In this study, we found that Spi-B-deficient mice were highly susceptible to food allergy to ovalbumin (OVA), as indicated by the exacerbation of diarrhea and elevation of serum IgE levels. These pathological changes were associated with enhanced mast cell infiltration into the intestinal lamina propria. Activation of mast cells in the intestinal mucosa was observed in Spib^−/− mice, even under physiological conditions. Accordingly, Spi-B deficiency increased the translocation of fluorescently labeled dextran from the lumen to the serum, suggesting increased intestinal permeability in Spib^−/− mice. Moreover, Spib^−/− mice showed defects in oral tolerance induction to OVA. These data illustrate that Spi-B suppresses the development of food allergies by controlling the activation of intestinal mast cells and by inducing immune tolerance to food allergens.

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Inhibition of RANKL and Sema4D improves residual ridge resorption in mice.

Hisamoto M, Kimura S, Iwata K, Iwanaga T, Yokoyama A

Scientific reports 12 （ 1 ） 4094 - 4094 2022年03月

責任著者, 査読有り

　概要を見る

Residual ridge resorption (RRR) is a chronic and progressive bone resorption following tooth loss. It causes deterioration of the oral environments and leads to the pathogenesis of various systemic diseases. However, the molecular mechanisms and risk factors for RRR progression are still unclear and controversial. In this study, we developed a tooth extraction model using mice for analyzing long-term morphological and gene expression changes in the alveolar bone. We further applied ovariectomy to this model to elucidate the effects of osteoporosis on RRR progression. As a result, the alveolar bone loss was biphasic and consisted of rapid loss in the early stages and subsequently slow and sustained bone loss over a long period. Histological analysis indicated that ovariectomy prolonged the activation of osteoclasts in the alveolar bone. Furthermore, the expressions of Tnfsf11 and Sema4d kept increasing for a long time in OVX mice. Administration of neutralization antibodies for receptor activator of NF-κB ligand (RANKL) effectively suppressed RRR. Similarly, inhibition of Semaphorin 4D (Sema4D) also improved alveolar bone loss. This study demonstrated that reduced ovarian function may be a risk factor for RRR and that RANKL and Sema4D suppression are potential treatments.

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精巣マクロファージに発現するUSP2による精子運動機能の制御

北村 浩, 橋本 茉由子, 木村 俊介, 渡邊 敬文, 高橋 英機, 永野 昌志

日本病態生理学会雑誌 （日本病態生理学会）  30 （ 2 ） 34 - 34 2021年12月

Intestinal immunity: to be, or not to be, induced? That is the question.

Daisuke Takahashi, Shunsuke Kimura, Koji Hase

International immunology 33 （ 12 ） 755 - 759 2021年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0953-8178

　概要を見る

The intestinal immune system maintains intestinal homeostasis in collaboration with diverse immune cell subsets residing at the epithelial layer, lamina propria and gut-associated lymphoid tissue (GALT). Bacterial components and their metabolites are essential for the establishment of the gut immune system. In addition, nutritional signals contribute to maintaining the mucosal immune response. Specialized epithelial microfold (M) cells in GALT facilitate immune surveillance on the mucosal surface by actively taking up external antigens to transport them into the lymphoid follicles. Because hyperplasia of M cells causes an excessive immune response in GALT, there is a self-regulatory mechanism to control the development of M cells appropriately. In this review, we will discuss the molecular mechanisms of mucosal immune regulation and their biological importance.

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Symbiotic polyamine metabolism regulates epithelial proliferation and macrophage differentiation in the colon.

Atsuo Nakamura, Shin Kurihara, Daisuke Takahashi, Wakana Ohashi, Yutaka Nakamura, Shunsuke Kimura, Masayoshi Onuki, Aiko Kume, Yukiko Sasazawa, Yukihiro Furusawa, Yuuki Obata, Shinji Fukuda, Shinji Saiki, Mitsuharu Matsumoto, Koji Hase

Nature communications 12 （ 1 ） 2105 - 2105 2021年04月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Intestinal microbiota-derived metabolites have biological importance for the host. Polyamines, such as putrescine and spermidine, are produced by the intestinal microbiota and regulate multiple biological processes. Increased colonic luminal polyamines promote longevity in mice. However, no direct evidence has shown that microbial polyamines are incorporated into host cells to regulate cellular responses. Here, we show that microbial polyamines reinforce colonic epithelial proliferation and regulate macrophage differentiation. Colonisation by wild-type, but not polyamine biosynthesis-deficient, Escherichia coli in germ-free mice raises intracellular polyamine levels in colonocytes, accelerating epithelial renewal. Commensal bacterium-derived putrescine increases the abundance of anti-inflammatory macrophages in the colon. The bacterial polyamines ameliorate symptoms of dextran sulfate sodium-induced colitis in mice. These effects mainly result from enhanced hypusination of eukaryotic initiation translation factor. We conclude that bacterial putrescine functions as a substrate for symbiotic metabolism and is further absorbed and metabolised by the host, thus helping maintain mucosal homoeostasis in the intestine.

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Protective Role of the M-Sec-Tunneling Nanotube System in Podocytes.

Federica Barutta, Shunsuke Kimura, Koji Hase, Stefania Bellini, Beatrice Corbetta, Alessandro Corbelli, Fabio Fiordaliso, Antonella Barreca, Mauro Giulio Papotti, Gian Marco Ghiggeri, Gennaro Salvidio, Dario Roccatello, Valentina Audrito, Silvia Deaglio, Roberto Gambino, Stefania Bruno, Giovanni Camussi, Miriam Martini, Emilio Hirsch, Marilena Durazzo, Hiroshi Ohno, Gabriella Gruden

Journal of the American Society of Nephrology : JASN 32 （ 5 ） 1114 - 1130 2021年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1046-6673

　概要を見る

BACKGROUND: Podocyte dysfunction and loss are major determinants in the development of proteinuria. FSGS is one of the most common causes of proteinuria, but the mechanisms leading to podocyte injury or conferring protection against FSGS remain poorly understood. The cytosolic protein M-Sec has been involved in the formation of tunneling nanotubes (TNTs), membrane channels that transiently connect cells and allow intercellular organelle transfer. Whether podocytes express M-Sec is unknown and the potential relevance of the M-Sec-TNT system in FSGS has not been explored. METHODS: We studied the role of the M-Sec-TNT system in cultured podocytes exposed to Adriamycin and in BALB/c M-Sec knockout mice. We also assessed M-Sec expression in both kidney biopsies from patients with FSGS and in experimental FSGS (Adriamycin-induced nephropathy). RESULTS: Podocytes can form TNTs in a M-Sec-dependent manner. Consistent with the notion that the M-Sec-TNT system is cytoprotective, podocytes overexpressed M-Sec in both human and experimental FSGS. Moreover, M-Sec deletion resulted in podocyte injury, with mitochondrial abnormalities and development of progressive FSGS. In vitro, M-Sec deletion abolished TNT-mediated mitochondria transfer between podocytes and altered mitochondrial bioenergetics. Re-expression of M-Sec reestablishes TNT formation and mitochondria exchange, rescued mitochondrial function, and partially reverted podocyte injury. CONCLUSIONS: These findings indicate that the M-Sec-TNT system plays an important protective role in the glomeruli by rescuing podocytes via mitochondrial horizontal transfer. M-Sec may represent a promising therapeutic target in FSGS, and evidence that podocytes can be rescued via TNT-mediated horizontal transfer may open new avenues of research.

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Involvement of BMP and Wnt Signals Leadingto Epithelial-Mesenchymal Transition in Colon Adenocarcinoma with Heterotopic Ossification.

Naho Katono, Masumi Tsuda, Jun Suzuka, Yoshitaka Oda, Lei Wang, Zen-Ichi Tanei, Mishie Tanino, Takanobu Ohata, Eisuke Nagabuchi, Yusuke Ishida, Shunsuke Kimura, Toshihiko Iwanaga, Shinya Tanaka

Annals of clinical and laboratory science 51 （ 2 ） 271 - 276 2021年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0091-7370

　概要を見る

Here we present the case of a 73-year-old male with rectal adenocarcinoma with heterotopic ossification (HO). Cancer-associated HO in the digestive system is rare. Thus, the precise mechanism and clinicopathological significance of HO have not yet been defined. To clarify the molecular mechanisms of HO, we analyzed the expression levels of signaling molecules related to epithelial-mesenchymal transition (EMT) that lead to ossification in the tumor cells discriminating the ossified area (HO-area) and non-ossified area (non-HO area). Expression levels of BMP4 were elevated in both areas, whereas BMP2 was specifically increased in the HO-area by qPCR. EMT-related molecules such as Snail and Slug were especially higher in the HO-area. By immunohistochemistry, the expression of Smad4, nuclear staining of β-catenin, and the phosphorylated form of GSK-3β were detectable in both areas, and GSK-3β was highly phosphorylated in the HO-area. The tumor growth rate was extremely high, with the Ki-67 labeling index at 90%. In the HO-area, osteoblasts with alkaline phosphatase expression were distributed surrounding the tumor cells. This is the first demonstration of the involvement of EMT in HO of colon cancer through BMP/SMAD and WNT/β-catenin signaling pathways, which are especially prominent in the HO-area leading to the osteogenic property.

Protective Role of the M-Sec–Tunneling Nanotube System in Podocytes

F Barutta, S Kimura, K Hase, S Bellini, B Corbetta, A Corbelli, F Fiordaliso, ...

Journal of the American Society of Nephrology 32 (5), 1114-1130 2021年

Characterization of M Cells in Tear Duct-Associated Lymphoid Tissue of Mice: A Potential Role in Immunosurveillance on the Ocular Surface.

Yuki Oya, Shunsuke Kimura, Yutaka Nakamura, Narumi Ishihara, Shunsuke Takano, Ryo Morita, Mayumi Endo, Koji Hase

Frontiers in immunology （Frontiers Media SA）  12   779709 - 779709 2021年

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

The ocular mucosal tissues are exposed to potentially harmful foreign antigens in the air and tear fluid. The tear duct-associated lymphoid tissue (TALT) may contribute to immune surveillance in the eye region. Follicle-associated epithelium (FAE) of TALTs is classified as stratified squamous epithelium and consists of squamous epithelial cells arranged in layers on the basement membrane. In contrast, most mucosa-associated lymphoid tissue is covered by a monolayer of epithelium containing microfold (M) cells. Therefore, antigen uptake and the presence of M cells in TALT are not fully understood. The present study found that a small population of FAE cells in the TALT expressed intestinal M-cell markers, namely Sox8, Tnfaip2, GP2, and OPG. This cell population was identified as functional M cells because of their uptake capacity of luminal nanoparticles. In addition, RANKL, which is essential for M-cell differentiation, was expressed by stroma-like cells at the subepithelial region and its receptor RANK by the FAE in the TALT. The administration of RANKL markedly increased the number of Sox8+ M cells. In contrast, deficiency in OPG, an endogenous inhibitor of RANKL, increased the number of M cells in the TALT. These data demonstrate that the RANKL-RANK axis is essential for M-cell differentiation in the TALT. Furthermore, immunization via eye drops elicited the production of antigen-specific antibodies in tears, which was enhanced by RANKL administration. Thus, TALT M cells play an important role in the immunosurveillance of the eye region.

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Macrophage ubiquitin-specific protease 2 contributes to motility, hyperactivation, capacitation, and in vitro fertilization activity of mouse sperm.

Mayuko Hashimoto, Shunsuke Kimura, Chihiro Kanno, Yojiro Yanagawa, Takafumi Watanabe, Jun Okabe, Eiki Takahashi, Masashi Nagano, Hiroshi Kitamura

Cellular and molecular life sciences : CMLS 78 （ 6 ） 2929 - 2948 2020年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1420-682X

　概要を見る

Macrophages are innate immune cells that contribute to classical immune functions and tissue homeostasis. Ubiquitin-specific protease 2 (USP2) controls cytokine production in macrophages, but its organ-specific roles are still unknown. In this study, we generated myeloid-selective Usp2 knockout (msUsp2KO) mice and specifically explored the roles of testicular macrophage-derived USP2 in reproduction. The msUsp2KO mice exhibited normal macrophage characteristics in various tissues. In the testis, macrophage Usp2 deficiency negligibly affected testicular macrophage subpopulations, spermatogenesis, and testicular organogenesis. However, frozen-thawed sperm derived from msUsp2KO mice exhibited reduced motility, capacitation, and hyperactivation. In addition, macrophage Usp2 ablation led to a decrease in the sperm population exhibiting high intracellular pH, calcium influx, and mitochondrial membrane potential. Interrupted pronuclei formation in eggs was observed when using frozen-thawed sperm from msUsp2KO mice for in vitro fertilization. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF), whose expression was decreased in testicular macrophages derived from msUsp2KO mice, restored mitochondrial membrane potential and total sperm motility. Our observations demonstrate a distinct role of the deubiquitinating enzyme in organ-specific macrophages that directly affect sperm function.

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Osteoprotegerin-dependent M cell self-regulation balances gut infection and immunity.

Shunsuke Kimura, Yutaka Nakamura, Nobuhide Kobayashi, Katsuyuki Shiroguchi, Eiryo Kawakami, Mami Mutoh, Hiromi Takahashi-Iwanaga, Takahiro Yamada, Meri Hisamoto, Midori Nakamura, Nobuyuki Udagawa, Shintaro Sato, Tsuneyasu Kaisho, Toshihiko Iwanaga, Koji Hase

Nature communications 11 （ 1 ） 234 - 234 2020年01月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.

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The Roles of Peyer's Patches and Microfold Cells in the Gut Immune System: Relevance to Autoimmune Diseases.

Kobayashi N, Takahashi D, Takano S, Kimura S, Hase K

Frontiers in immunology 10   2345 2019年10月

研究論文（学術雑誌）, 共著, 査読有り

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Mast cells play role in wound healing through the ZnT2/GPR39/IL-6 axis.

Nishida K, Hasegawa A, Yamasaki S, Uchida R, Ohashi W, Kurashima Y, Kunisawa J, Kimura S, Iwanaga T, Watarai H, Hase K, Ogura H, Nakayama M, Kashiwakura JI, Okayama Y, Kubo M, Ohara O, Kiyono H, Koseki H, Murakami M, Hirano T

Scientific reports 9 （ 1 ） 10842 - 10842 2019年07月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency and skin disorders. Various cells including mast cells release Zn-containing granules when activated; however, the biological role of the released Zn is currently unclear. Here we report our findings that Zn transporter ZnT2 is required for the release of Zn from mast cells. In addition, we found that Zn and mast cells induce IL-6 production from inflammatory cells such as skin fibroblasts and promote wound healing, a process that involves inflammation. Zn induces the production of a variety of pro-inflammatory cytokines including IL-6 through signaling pathways mediated by the Zn receptor GPR39. Consistent with these findings, wound healing was impaired in mice lacking IL-6 or GPR39. Thus, our results show that Zn and mast cells play a critical role in wound healing through activation of the GPR39/IL-6 signaling axis.

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Airway M cells arise in the lower airway due to RANKL signaling and reside in the bronchiolar epithelium associated with iBALT in murine models of respiratory disease

Shunsuke Kimura*, Mami Mutoh, Meri Hisamoto, Hikaru Saito, Shun Takahashi, Takanori Asakura, Makoto Ishii, Yutaka Nakamura, Junichiro Iida, Koji Hase and Toshihiko Iwanaga

Frontiers in Immunology 10   1323 - 1323 2019年06月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Microfold (M) cells residing in the follicle-associated epithelium of mucosa-associated lymphoid tissues are specialized for sampling luminal antigens to initiate mucosal immune responses. In the past decade, glycoprotein 2 (GP2) and Tnfaip2 were identified as reliable markers for M cells in the Peyer's patches of the intestine. Furthermore, RANKL-RANK signaling, as well as the canonical and non-canonical NFκB pathways downstream, is essential for M-cell differentiation from the intestinal stem cells. However, the molecular characterization and differentiation mechanisms of M cells in the lower respiratory tract, where organized lymphoid tissues exist rarely, remain to be fully elucidated. Therefore, this study aimed to explore M cells in the lower respiratory tract in terms of their specific molecular markers, differentiation mechanism, and functions. Immunofluorescence analysis revealed a small number of M cells expressing GP2, Tnfaip2, and RANK is present in the lower respiratory tract of healthy mice. The intraperitoneal administration of RANKL in mice effectively induced M cells, which have a high capacity to take up luminal substrates, in the lower respiratory epithelium. The airway M cells associated with lymphoid follicles were frequently detected in the pathologically induced bronchus-associated lymphoid tissue (iBALT) in the murine models of autoimmune disease as well as pulmonary emphysema. These findings demonstrate that RANKL is a common inducer of M cells in the airway and digestive tracts and that M cells are associated with the respiratory disease. We also established a two-dimensional culture method for airway M cells from the tracheal epithelium in the presence of RANKL successfully. This model may be useful for functional studies of M cells in the sampling of antigens at airway mucosal surfaces.

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Sox8 is essential for M cell maturation to accelerate IgA response at the early stage after weaning in mice.

Kimura S, Kobayashi N, Nakamura Y, Kanaya T, Takahashi D, Fujiki R, Mutoh M, Obata Y, Iwanaga T, Nakagawa T, Kato N, Sato S, Kaisho T, Ohno H, Hase K

The Journal of experimental medicine 216 （ 4 ） 831 - 846 2019年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-1007

　概要を見る

Microfold (M) cells residing in the follicle-associated epithelium (FAE) of the gut-associated lymphoid tissue are specialized for antigen uptake to initiate mucosal immune responses. The molecular machinery and biological significance of M cell differentiation, however, remain to be fully elucidated. Here, we demonstrate that Sox8, a member of the SRY-related HMG box transcription factor family, is specifically expressed by M cells in the intestinal epithelium. The expression of Sox8 requires activation of RANKL-RelB signaling. Chromatin immunoprecipitation and luciferase assays revealed that Sox8 directly binds the promoter region of Gp2 to increase Gp2 expression, which is the hallmark of functionally mature M cells. Furthermore, genetic deletion of Sox8 causes a marked decrease in the number of mature M cells, resulting in reduced antigen uptake in Peyer's patches. Consequently, juvenile Sox8-deficient mice showed attenuated germinal center reactions and antigen-specific IgA responses. These findings indicate that Sox8 plays an essential role in the development of M cells to establish mucosal immune responses.

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Osteoprotegerin-dependent M cell self-regulation balances the gut immunity and infection

Y Nakamura, S Kimura, N Kobayashi, K Hase

EUROPEAN JOURNAL OF IMMUNOLOGY 49, 1379-1379 2019年

Intestinal Lymphatic Endothelial Cells Produce R-Spondin3.

Ogasawara R, Hashimoto D, Kimura S, Hayase E, Ara T, Takahashi S, Ohigashi H, Yoshioka K, Tateno T, Yokoyama E, Ebata K, Kondo T, Sugita J, Onozawa M, Iwanaga T, Teshima T

Scientific reports 8 （ 1 ） 10719 - 10719 2018年07月

共著, 査読有り

　概要を見る

The R-Spondin (R-Spo) family regulates WNT signaling and stimulates the proliferation and differentiation of intestinal stem cells (ISCs). R-Spo plays a critical role in maintaining intestinal homeostasis, but endogenous producers of R-Spo in the intestine remain to be investigated. We found that R-Spo3 was the major R-Spo family member produced in the intestine and it was predominantly produced by CD45-CD90+CD31+ lymphatic endothelial cells (LECs) in the lamina propria of the intestinal mucosa. Transcriptome analysis demonstrated that LECs highly expressed R-Spo receptor, Lgr5, suggesting an autocrine stimulatory loop in LECs. LECs were significantly reduced in number, and their R-Spo3 production was impaired in intestinal graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. The impaired production of R-Spo3 in the intestine may be a novel mechanism of delayed tissue repair and defective mucosal defense in intestinal GVHD. We demonstrate a novel role of intestinal LECs in producing R-Spondin3 to maintain intestinal homeostasis.

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Histochemical characteristics of regressing vessels in the hyaloid vascular system of neonatal mice: Novel implication for vascular atrophy.

Kishimoto A, Kimura S, Nio-Kobayashi J, Takahashi-Iwanaga H, Park AM, Iwanaga T

Experimental eye research （Academic Press）  172   1 - 9 2018年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0014-4835

　概要を見る

The hyaloid vasculature constitutes a transitory system nourishing the internal structures of the developing eye, but the mechanism of vascular regression and its cell biological characteristics are not fully understood. The present study aimed to reveal the specificity of the hyaloid vessels by a systematic immunohistochemical approach for marker substances of myeloid cells and the extracellular matrix (ECM) in neonatal mice. Macrophages immunoreactive for F4/80, cathepsin D, and LYVE-1 gathered around the vasa hyaloidea propria (VHP), while small round cells in vascular lumen of VHP were selectively immunoreactive for galectin-3
their segmented nuclei and immunoreactivities for Ly-6G, CD11b, and myeloperoxidase indicated their neutrophilic origin. VHP possessed thick ECM and a dense pericyte envelope as demonstrated by immunostaining for laminin, type IV collagen, integrin β1, and NG2. The galectin-3+ cells loosely aggregated with numerous erythrocytes in the lumen of hyaloid vessels in a manner reminiscent of vascular congestion. Galectin-3 is known to polymerize and form a complex with ECM and NG2 as well as recruit leukocytes on the endothelium. Observation of galectin-3 KO mice implicated the involvement of galectin-3 in the regression of hyaloid vasculature. Since macrophages may play central roles including blocking of the blood flow and the induction of apoptosis in the regression, galectin-3+ neutrophils may play a supportive role in the macrophage-mediated involution of the hyaloid vascular system.

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Molecular insights into the mechanisms of M-cell differentiation and transcytosis in the mucosa-associated lymphoid tissues.

Kimura S

Anatomical science international （Springer Tokyo）  93 （ 1 ） 23 - 34 2018年01月

単著, 査読有り,  ISSN  1447-6959

　概要を見る

Microfold cells (M cells), which are located in the follicle-associated epithelium (FAE) covering mucosal lymphoid follicles, are specialized epithelial cells that initiate mucosal immune responses. These cells take luminal antigens and transport them via transcytosis across the FAE to the antigen-presenting cells underneath. Several intestinal pathogens exploit M cells as their portal for entry to invade the host and cause disease conditions. Recent studies have revealed that the uptake of antigens by M cells is essential for efficient antigen-specific IgA production and that this process likely maintains the homeostasis of mucosal tissues. The present article reviews recent advances in understanding the molecular mechanism of M-cell differentiation and describes the molecules expressed by M cells that are associated with antigen uptake and/or the transcytosis process. Current efforts to augment M-cell-mediated uptake for use in the development of effective mucosal vaccines are also discussed.

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Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling.

Kanaya T, Sakakibara S, Jinnohara T, Hachisuka M, Tachibana N, Hidano S, Kobayashi T, Kimura S, Iwanaga T, Nakagawa T, Katsuno T, Kato N, Akiyama T, Sato T, Williams IR, Ohno H

The Journal of experimental medicine 215 （ 2 ） 501 - 519 2018年01月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-1007

　概要を見る

M cells are located in the follicle-associated epithelium (FAE) that covers Peyer's patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell-associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor-associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.

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M cell-dependent antigen uptake on follicle-associated epithelium for mucosal immune surveillance.

Nakamura Y, Kimura S, Hase K

Inflammation and regeneration 38   15 - 15 2018年

共著, 査読有り,  ISSN  1880-9693

　概要を見る

The follicle-associated epithelium (FAE) covering mucosa-associated lymphoid tissue is distinct from the villous epithelium in cellular composition and functions. Interleukin-22 binding protein (IL-22BP), provided by dendritic cells at the sub-epithelial dome region, inhibits the IL-22-mediated secretion of antimicrobial peptides by the FAE. The Notch signal from stromal cells underneath the FAE diminishes goblet cell differentiation. These events dampen the mucosal barrier functions to allow luminal microorganisms to readily gain access to the luminal surface of the FAE. Furthermore, receptor activator of nucleic factor-kappa B ligand (RANKL) from a certain stromal cell type induces differentiation into microfold (M) cells that specialize in antigen uptake in the mucosa. Microfold (M) cells play a key role in mucosal immune surveillance by actively transporting external antigens from the gut lumen to the lymphoid follicle. The molecular basis of antigen uptake by M cells has been gradually identified in the last decade. For example, GPI-anchored molecules (e.g., glycoprotein 2 (GP2) and cellular prion protein (PrPC)) and β1-integrin facilitate the transport of specific types of xenobiotics. The antigen transport by M cells initiates antigen-specific mucosal immune responses represented by the induction of secretory immunoglobulin A (S-IgA). Meanwhile, several invasive pathogens exploit M cells as a portal to establish a systemic infection. Recent findings have uncovered the molecular machinery of differentiation and functions of M cells.

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Enhanced gap junction intercellular communication inhibits catabolic and pro-inflammatory responses in tenocytes against heat stress.

Maeda E, Kimura S, Yamada M, Tashiro M, Ohashi T

Journal of cell communication and signaling （Springer Netherlands）  11 （ 4 ） 369 - 380 2017年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1873-9601

　概要を見る

Elevation of tendon core temperature during severe activity is well known. However, its effects on tenocyte function have not been studied in detail. The present study tested a hypothesis that heat stimulation upregulates tenocyte catabolism, which can be modulated by the inhibition or the enhancement of gap junction intercellular communication (GJIC). Tenocytes isolated from rabbit Achilles tendons were subjected to heat stimulation at 37 °C, 41 °C or 43 °C for 30 min, and changes in cell viability, gene expressions and GJIC were examined. It was found that GJIC exhibited no changes by the stimulation even at 43 °C, but cell viability was decreased and catabolic and proinflammatory gene expressions were upregulated. Inhibition of GJIC demonstrated further upregulated catabolic and proinflammatory gene expressions. In contrast, enhanced GJIC, resulting from forced upregulation of connexin 43 gene, counteracted the heat-induced upregulation of catabolic and proinflammatory genes. These findings suggest that the temperature rise in tendon core could upregulate catabolic and proinflammatory activities, potentially leading to the onset of tendinopathy, and such upregulations could be suppressed by the enhancement of GJIC. Therefore, to prevent tendon injury at an early stage from becoming chronic injury, tendon core temperature and GJIC could be targets for post-activity treatments.

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Intrarenal signaling mediated by CCK plays a role in salt intake-induced natriuresis.

Takahashi-Iwanaga H, Kimura S, Konno K, Watanabe M, Iwanaga T

American journal of physiology. Renal physiology （AMER PHYSIOLOGICAL SOC）  313 （ 1 ） ajprenal.00539.2016 - F29 2017年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1931-857X

　概要を見る

The natriuretic hormone CCK exhibits its gene transcripts in total kidney extracts. To test the possibility of CCK acting as an intrarenal mediator of sodium excretion, we examined mouse kidneys by 1) an in situ hybridization technique for CCK mRNA in animals fed a normal- or a high-sodium diet; 2) immuno-electron microscopy for the CCK peptide, 3) an in situ hybridization method and immunohistochemistry for the CCK-specific receptor CCKAR; 4) confocal image analysis of receptor-mediated Ca2+ responses in isolated renal tubules; and 5) metabolic cage experiments for the measurement of urinary sodium excretion in high-salt-fed mice either treated or untreated with the CCKAR antagonist lorglumide. Results showed the CCK gene to be expressed intensely in the inner medulla and moderately in the inner stripe of the outer medulla, with the expression in the latter being enhanced by high sodium intake. Immunoreactivity for the CCK peptide was localized to the rough endoplasmic reticulum of the medullary interstitial cells in corresponding renal regions, confirming it to be a secretory protein. Gene transcripts, protein products, and the functional activity for CCKAR were consistently localized to the late proximal tubule segments (S2 and S3) in the medullary rays, and the outer stripe of the outer medulla. Lorglumide significantly diminished natriuretic responses of mice to a dietary sodium load without altering the glomerular filtration rate. These findings suggest that the medullary interstitial cells respond to body fluid expansion by CCK release for feedback regulation of the late proximal tubular reabsorption.

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Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

Natsuko Saito, Shunsuke Kimura, Tomomi Miyamoto, Sanae Fukushima, Misato Amagasa, Yoshinori Shimamoto, Chieko Nishioka, Shiki Okamoto, Chitoku Toda, Kohei Washio, Atsushi Asano, Ichiro Miyoshi, Eiki Takahashi, Hiroshi Kitamura

Biochemistry and Biophysics Reports （Elsevier B.V.）  9   322 - 329 2017年03月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2405-5808

　概要を見る

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.

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Tunneling Nanotubes and Gap Junctions-Their Role in Long-Range Intercellular Communication during Development, Health, and Disease Conditions.

Ariazi J, Benowitz A, De Biasi V, Den Boer ML, Cherqui S, Cui H, Douillet N, Eugenin EA, Favre D, Goodman S, Gousset K, Hanein D, Israel DI, Kimura S, Kirkpatrick RB, Kuhn N, Jeong C, Lou E, Mailliard R, Maio S, Okafo G, Osswald M, Pasquier J, Polak R, Pradel G, de Rooij B, Schaeffer P, Skeberdis VA, Smith IF, Tanveer A, Volkmann N, Wu Z, Zurzolo C

Frontiers in molecular neuroscience （FRONTIERS MEDIA SA）  10   333 2017年

共著, 査読有り,  ISSN  1662-5099

　概要を見る

Cell-to-cell communication is essential for the organization, coordination, and development of cellular networks and multi-cellular systems. Intercellular communication is mediated by soluble factors (including growth factors, neurotransmitters, and cytokines/chemokines), gap junctions, exosomes and recently described tunneling nanotubes (TNTs). It is unknown whether a combination of these communication mechanisms such as TNTs and gap junctions may be important, but further research is required. TNTs are long cytoplasmic bridges that enable long-range, directed communication between connected cells. The proposed functions of TNTs are diverse and not well understood but have been shown to include the cell-to-cell transfer of vesicles, organelles, electrical stimuli and small molecules. However, the exact role of TNTs and gap junctions for intercellular communication and their impact on disease is still uncertain and thus, the subject of much debate. The combined data from numerous laboratories indicate that some TNT mediate a long-range gap junctional communication to coordinate metabolism and signaling, in relation to infectious, genetic, metabolic, cancer, and age-related diseases. This review aims to describe the current knowledge, challenges and future perspectives to characterize and explore this new intercellular communication system and to design TNT-based therapeutic strategies.

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Tunneling nanotubes and gap junctions–their role in long-range intercellular communication during development, health, and disease conditions

J Ariazi, A Benowitz, V De Biasi, ML Den Boer, S Cherqui, H Cui, ...

Frontiers in molecular neuroscience 10, 333 2017年

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress.

Ohashi W, Kimura S, Iwanaga T, Furusawa Y, Irié T, Izumi H, Watanabe T, Hijikata A, Hara T, Ohara O, Koseki H, Sato T, Robine S, Mori H, Hattori Y, Watarai H, Mishima K, Ohno H, Hase K, Fukada T

PLoS genetics 12 （ 10 ） e1006349 2016年10月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1553-7390

　概要を見る

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.

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Distinct Roles for the N- and C-terminal Regions of M-Sec in Plasma Membrane Deformation during Tunneling Nanotube Formation

Kimura S, Yamashita M, Yamakami-Kimura M, Sato Y, Yamagata A, Kobashigawa Y, Inagaki F, Amada T, Hase K, Iwanaga T, Ohno H and Fukai S

Scientific Reports （NATURE PUBLISHING GROUP）  6   33548 2016年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2045-2322

　概要を見る

The tunneling nanotube (TNT) is a structure used for intercellular communication, and is a thin membrane protrusion mediating transport of various signaling molecules and cellular components. M-Sec has potent membrane deformation ability and induces TNT formation in cooperation with the Ral/exocyst complex. Here, we show that the N-terminal polybasic region of M-Sec directly binds phosphatidylinositol (4,5)-bisphosphate for its localization to the plasma membrane during the initial stage of TNT formation. We further report a crystal structure of M-Sec, which consists of helix bundles arranged in a straight rod-like shape, similar to the membrane tethering complex subunits. A positively charged surface in the C-terminal domains is required for M-Sec interaction with active RalA to extend the plasma membrane protrusions. Our results suggest that the membrane-associated M-Sec recruits active RalA, which directs the exocyst complex to form TNTs.

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Osteoprotegerinによる腸管上皮M細胞の分化抑制機構とその意義

木村 俊介, 武藤 麻未, 岩永 敏彦

Journal of Oral Biosciences Supplement （(一社)歯科基礎医学会）  2016   168 - 168 2016年09月

共著,  ISSN  2187-2333

Receptor activator of NFκB ligand(RANKL)によるマウス気管・気管支上皮からのM細胞分化誘導

武藤 麻未, 木村 俊介, 吉沢 早織, 岩永 敏彦, 飯田 順一郎

Journal of Oral Biosciences Supplement （(一社)歯科基礎医学会）  2016   277 - 277 2016年09月

共著,  ISSN  2187-2333

Neural FFA3 activation inversely regulates anion secretion evoked by nicotinic ACh receptor activation in rat proximal colon.

Kaji I, Akiba Y, Konno K, Watanabe M, Kimura S, Iwanaga T, Kuri A, Iwamoto K, Kuwahara A, Kaunitz JD

The Journal of physiology （WILEY-BLACKWELL）  594 （ 12 ） 3339 - 3352 2016年06月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-3751

　概要を見る

The proximal colonic mucosa is constantly exposed to high concentrations of microbially-produced short-chain fatty acids (SCFAs). Although luminal SCFAs evoke electrogenic anion secretion and smooth muscle contractility via neural and non-neural cholinergic pathways in the colon, the involvement of the SCFA receptor free fatty acid receptor (FFA)3, one of the free fatty acid receptor family members, has not been clarified. We investigated the contribution of FFA3 to cholinergic-mediated secretory responses in rat proximal colon. FFA3 was immunolocalized to enteroendocrine cells and to the enteric neural plexuses. Most FFA3-immunoreactive nerve fibres and nerve endings were cholinergic, colocalized with protein gene product (PGP)9.5, the vesicular ACh transporter, and the high-affinity choline transporter CHT1. In Ussing chambered mucosa-submucosa preparations (including the submucosal plexus) of rat proximal colon, carbachol (CCh)-induced Cl- secretion was decreased by TTX, hexamethonium, and the serosal FFA3 agonists acetate or propionate, although not by an inactive analogue 3-chloropropionate. Serosal application of a selective FFA3 agonist (N-[2-methylphenyl]-[4-furan-3-yl]-2-methyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxamide; MQC) dose-dependently suppressed the response to CCh but not to forskolin, with an IC50 of 13m. Pretreatment with MQC inhibited nicotine-evoked but not bethanechol-evoked secretion. The inhibitory effect of MQC was reversed by pretreatment with pertussis toxin, indicating that FFA3 acts via the G(i/o) pathway. Luminal propionate induced Cl- secretion via the cholinergic pathway, which was reduced by MQC, as well as by TTX, hexamethonium or removal of the submucosal plexus. These results suggest that the SCFA-FFA3 pathway has a novel anti-secretory function in that it inhibits cholinergic neural reflexes in the enteric nervous system.

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Non-neuronal, but atropine-sensitive ileal contractile responses to short-chain fatty acids: age-dependent desensitization and restoration under inflammatory conditions in mice.

Yajima M, Kimura S, Karaki S, Nio-Kobayashi J, Tsuruta T, Kuwahara A, Yajima T, Iwanaga T

Physiological reports （Wiley-Blackwell Publishing Ltd）  4 （ 7 ） 1 - 11 2016年04月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  2051-817X

　概要を見る

Intestinal epithelial cells sense short-chain fatty acids (SCFAs) to secrete non-neuronal acetylcholine (ACh). However, the roles of luminal SCFAs and epithelial ACh under normal and pathological conditions remain unknown. We examined ileal contractile responses to SCFAs at different ages and their mucosal cholinergic alterations under inflammatory conditions. Ileal contractile responses to SCFAs in 1-day-old pups to 7-week-old mice were compared using an isotonic transducer, and responses to an intraperitoneal injection of lipopolysaccharide (LPS) were analyzed in 7-week-old mice. The mRNA expression levels of a SCFA activate free fatty acid receptor, acetylcholinesterase (AChE), choline acetyltransferase (Chat), and choline transporter-like protein 4 (CTL4) were measured using real-time quantitative RT-PCR. AChE was analyzed by histochemical and optical enzymatic assays. Atropine-sensitive ileal contractile responses to SCFAs occurred in all 1-day-old pups, but were frequently desensitized after the weaning period. These contractile responses were not inhibited by tetrodotoxin and did not appear when the mucosal layer had been scraped off. Contractile desensitization in 7-week-old mice was abolished in the presence of the AChE inhibitor, eserine, which was consistent with increased AChE activity after weaning. Ileal contractions to SCFAs in adult mice were restored by LPS, which significantly increased the epithelial mRNA expression of Chat and CTL4. Atropine-sensitive ileal contractile responses to SCFAs constitutively occur in the newborn period, and are desensitized during developmental stages following the up-regulated expression of AChE in the villous mucosa, but are restored under inflammatory conditions possibly via the release of epithelial ACh.

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Potential Role of the Formation of Tunneling Nanotubes in HIV-1 Spread in Macrophages.

Hashimoto M, Bhuyan F, Hiyoshi M, Noyori O, Nasser H, Miyazaki M, Saito T, Kondoh Y, Osada H, Kimura S, Hase K, Ohno H, Suzu S

Journal of immunology (Baltimore, Md. : 1950) （AMER ASSOC IMMUNOLOGISTS）  196 （ 4 ） 1832 - 1841 2016年02月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0022-1767

　概要を見る

Tunneling nanotubes (TNTs), the long membrane extensions connecting distant cells, have emerged as a novel form of cell-to-cell communication. However, it is not fully understood how and to what extent TNTs contribute to intercellular spread of pathogens including HIV-1. In this study, we show that HIV-1 promotes TNT formation per se via its protein Nef and a cellular protein M-Sec, which appears to mediate approximately half of viral spread among monocyte-derived macrophages (MDMs). A small compound that inhibits M-Sec-induced TNT formation reduced HIV-1 production by almost half in MDMs. Such inhibition was not observed with Nef-deficient mutant HIV-1 that fails to promote TNT formation and replicates less efficiently than the wild-type HIV-1 in MDMs. The TNT inhibitor-sensitive/Nef-promoting viral production was also observed in a T cell line ectopically expressing M-Sec, but not in another M-Sec 2 T cell line. Our results suggest the importance of TNTs in HIV-1 spread among MDMs and might answer the long-standing question how Nef promotes HIV-1 production in a cell type-specific manner.

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Diversity of the intestinal microbiota differently affects non-neuronal and atropine-sensitive ileal contractile responses to short-chain fatty acids in mice.

Yajima M, Karaki SI, Tsuruta T, Kimura S, Nio-Kobayashi J, Kuwahara A, Yajima T

Biomedical research (Tokyo, Japan) （BIOMEDICAL RESEARCH PRESS LTD）  37 （ 5 ） 319 - 328 2016年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0388-6107

　概要を見る

Non-neuronal and atropine-sensitive ileal contractile responses to short chain fatty acids (SCFAs) are detected in the neonatal stage, and change with age or inflammatory conditions. However, the roles of luminal SCFAs in developmental changes have not yet been elucidated. We examined ileal contractile responses to SCFAs in mice colonized with different SCFA-producing intestinal microbiota under normal and inflammatory conditions. Using conventional (Conv), germ-free (GF), and gnotobiotic mice infected with Bifidobacterium (GB-bif), Propionibacterium (GB-prop), or Lactobacillus (GB-lact), ileal contractions were measured in 1-day-old neonates and 7-week-old mice using an isotonic transducer. Contractions occurred in all 1-day-old neonates, and were significantly desensitized in the adult stage in the Conv, GB-bif, and GB-prop groups, but not in the GF and GB-lact groups. An injection of lipopolysaccharide frequently restored desensitized contractions; however, the contraction rate did not change in the GF and GB-lact groups. The relative mRNA expression of a SCFA receptor (GPR43) or nicotinic acetylcholine receptor alpha 7 was weaker in the GF group (0.3-fold or 0.4-fold expression level, respectively) than in the Conv group. In conclusion, the luminal inhabitation of SCFA-producing bacteria may potentiate the regulation of non-neuronal and atropine-sensitive ileal contractile responses to SCFAs under healthy and inflammatory conditions.

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The selective distribution of LYVE-1-expressing endothelial cells and reticular cells in the reticulo-endothelial system (RES).

Zheng M, Kimura S, Nio-Kobayashi J, Iwanaga T

Biomedical research (Tokyo, Japan) （BIOMEDICAL RESEARCH PRESS LTD）  37 （ 3 ） 187 - 198 2016年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0388-6107

　概要を見る

LYVE-1, a receptor molecule for hyaluronan, is expressed in the lymphatic endothelium, blood sinus endothelium, and certain macrophage lineages. The present immunohistochemical study revealed a broader distribution of LYVE-1 in vascular endothelial cells of the murine lung, adrenal gland, and heart as well as the liver and spleen. In addition, sinus reticular cells-including sinuslining cells-in the medulla of the lymph node also intensely expressed LYVE-1. Ultrastructurally, immuno-gold particles for LYVE-1 were localized on the entire length of plasma membrane in all cell types. Most of these LYVE-1-expressing cells had previously been classified as the reticuloendothelial system (RES) specialized for eliminating foreign particles. An LPS stimulation decreased the LYVE-1 expression in macrophages but elevated the expression at mRNA and protein levels in the liver and lung, major organs for the elimination of blood-born waste substances. LYVE-1-expressing endothelial cells in these organs participated in the endocytosis of exogenous particles, and the uptake ability was conspicuously enhanced by the LPS challenge. Although the expression of the degrading enzyme, hyaluronidase, was generally low in the LYVE-1-expressing cells, they were topographically associated with a dense distribution of macrophages possessing hyaluronidase activities in each tissue. These findings suggest that the LYVE-1-expressing cells might be involved in the uptake of hyaluronan and other waste products as well as foreign particles circulating in the blood and lymph while participating in the subsequent degradation in relay with adjacent macrophage populations.

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The broad distribution of GP2 in mucous glands and secretory products.

Kimura S, Nio-Kobayashi J, Kishimoto A, Iwanaga T

Biomedical research (Tokyo, Japan) （BIOMEDICAL RESEARCH PRESS LTD）  37 （ 6 ） 351 - 358 2016年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0388-6107

　概要を見る

GP2, a GPI-anchored glycoprotein that is a useful marker for M cells of Peyer's patches, is functionally related to the uptake of pathogenic bacteria in the gut lumen. Our immunostaining throughout the whole body of mice detected a broader localization than previously found of GP2 in various mucous glands and secretory cells. In the oral cavity, the palatine gland and lingual gland intensely expressed GP2 with immunolabeling along the basolateral membrane of acini and in luminal secretions of ducts. Secretory portions of the duodenal gland as well as the pancreas were immunoreactive for GP2 in the digestive tract. Luminal contents in the small intestine contained aggregations of GP2-immunoreactive substances which mixed with bacteria. The bulbourethral gland of Cowper displayed the GP2 immunoreactivity among the male reproductive organs. The vaginal epithelium contained many GP2-immunoreactive goblet-like cells, the occurrence of which dramatically changed according to the estrous cycle. These findings show that GP2 is a popular secretory product released from mucous glands and secretory cells and may support defense mechanisms against pathogenic bacteria in the tubular organs open to the external milieu.

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Epithelium-Intrinsic MicroRNAs Contribute to Mucosal Immune Homeostasis by Promoting M-Cell Maturation.

Nakato G, Hase K, Sato T, Kimura S, Sakakibara S, Sugiyama M, Obata Y, Hanazato M, Iwanaga T, Ohno H

PloS one （PUBLIC LIBRARY SCIENCE）  11 （ 3 ） e0150379 2016年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1932-6203

　概要を見る

cells in the follicle-associated epithelium (FAE) of Peyer's patches (PPs) serve as a main portal for external antigens and function as a sentinel in mucosal immune responses. The scarcity of these cells has hampered identification of M cell-specific molecules. Recent efforts have begun to provide insight into antigen transcytosis and differentiation of M cells; however, the molecular mechanisms underlying these processes are not fully elucidated. Small non-coding RNAs including microRNA (miRNA) have been reported to regulate gene expression and control various biological processes such as cellular differentiation and function. To evaluate the expression of miRNAs in FAE, including M cells, we previously performed microarray analysis comparing intestinal villous epithelium (VE) and PP FAE. Here we confirmed FAE specific miRNA expression levels by quantitative PCR. To gain insight into miRNA function, we generated mice with intestinal epithelial cell-specific deletion of Dicer1 (Dicer(Delta IEC)) and analyzed intestinal phenotypes, including M-cell differentiation, morphology and function. Dicer(Delta IEC) mice had a marked decrease in M cells compared to control floxed Dicer mice, suggesting an essential role of miRNAs in maturation of these cells. Furthermore, transmission electron microscopic analysis revealed that depletion of miRNA caused the loss of endosomal structures in M cells. In addition, antigen uptake by M cells was impaired in Dicer(Delta IEC) mice. These results suggest that miRNAs play a significant role in M cell differentiation and help secure mucosal immune homeostasis.

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Non‐neuronal, but atropine‐sensitive ileal contractile responses to short‐chain fatty acids: age‐dependent desensitization and restoration under inflammatory conditions in mice

M Yajima, S Kimura, S Karaki, J Nio‐Kobayashi, T Tsuruta, A Kuwahara, ...

Physiological reports 4 (7), e12759 2016年

RANKL regulates differentiation of microfold cells in mouse nasopharynx-associated lymphoid tissue (NALT).

Mutoh M, Kimura S, Takahashi-Iwanaga H, Hisamoto M, Iwanaga T, Iida J

Cell and tissue research （SPRINGER）  364 （ 1 ） 175 - 84 2015年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0302-766X

　概要を見る

Murine nasopharynx-associated lymphoid tissue (NALT), located at the base of the nasal cavity, serves as a major site for the induction of mucosal immune responses against airway antigens. The follicle-associated epithelium (FAE) covering the luminal surface of NALT is characterized by the presence of microfold cells (M cells), which take up and transport luminal antigens to lymphocytes. Glycoprotein 2 (GP2) has recently been identified as a reliable marker for M cells in Peyer's patches of the intestine. However, the expression of GP2 and other functional molecules in the M cells of NALT has not yet been examined. We have immunohistochemically detected GP2-expressing cells in the FAE of NALT and the simultaneous expression of other intestinal M-cell markers, namely Tnfaip2, CCL9, and Spi-B. These cells have been further identified as M cells because of their higher uptake capacity of luminal microbeads. Electron microscopic observations have shown that GP2-expressing cells on the FAE display morphological features typical of M cells: they possess short microvilli and microfolds on the luminal surface and are closely associated with intraepithelial lymphocytes. We have also found that the receptor activator of nuclear factor kappa-B ligand (RANKL) is expressed by stromal cells underneath the FAE, which provides its receptor RANK. The administration of RANKL markedly increases the number of GP2(+)Tnfaip2(+) cells on the NALT FAE and that of intestinal M cells. These results suggest that GP2(+)Tnfaip2(+) cells in NALT are equivalent to intestinal M cells, and that RANKL-RANK signaling induces their differentiation.

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鼻咽頭粘膜組織における抗原取り込み細胞であるM細胞分化機構の解析

武藤 麻未, 木村 俊介, 吉沢 早織, 岩永 敏彦, 飯田 順一郎

日本矯正歯科学会大会プログラム・抄録集 （(公社)日本矯正歯科学会）  74回   191 - 191 2015年11月

共著

Regulated C-C motif ligand 2 (CCL2) in luteal cells contributes to macrophage infiltration into the human corpus luteum during luteolysis.

Nio-Kobayashi J, Kudo M, Sakuragi N, Kimura S, Iwanaga T, Duncan WC

Molecular human reproduction （OXFORD UNIV PRESS）  21 （ 8 ） 645 - 654 2015年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1360-9947

　概要を見る

Intense macrophage infiltration is observed during luteolysis in various animals including women; however, we still do not know how macrophage infiltration into the human corpus luteum (CL) during luteolysis is regulated. In this study, we examined the expression, localization and regulation of an important chemokine for the recruitment of monocyte/macrophage lineages, C-C motif ligand 2 (CCL2), in the human CL across the luteal phase and in cultured human luteinized granulosa cells (LGCs), with special reference to the number of infiltrating macrophages and luteal cell function. CCL2 mRNA increased in the non-functional regressing CL during menstruation (P < 0.01), corresponding to an elevated mRNA expression of a macrophage-derived cytokine, tumor necrosis factor (TNF), and an increased number of infiltrating macrophages positively stained with a macrophage marker, CD68. CCL2 protein was immunohistochemically localized to the cytoplasm of granulosa-lutein and theca-lutein cells, and CCL2 mRNA was significantly reduced by hCG both in vivo (P < 0.05) and in vitro (P < 0.01). CCL2 was also down-regulated by luteotrophic prostaglandin (PG) E (P < 0.0001), but up-regulated by luteolytic PGF (P < 0.05) in vitro. Administration of TNF significantly enhanced the CCL2 mRNA expression in cultured LGCs (P < 0.01). A greater abundance of infiltrating macrophages were found around granulosa-lutein cells lacking 3 beta-HSD or PGE synthase (PGES) immunostaining. CCL2 mRNA expression was negatively correlated with both HSD3B1 and PGES, suggesting that locally produced progesterone and PGE suppress macrophage infiltration into the CL. Taken together, the infiltration of macrophages in the human CL is regulated by endocrine and paracrine molecules via regulation of the CCL2 expression in luteal cells.

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Visualization of the entire differentiation process of murine M cells: suppression of their maturation in cecal patches.

Kimura S, Yamakami-Kimura M, Obata Y, Hase K, Kitamura H, Ohno H, Iwanaga T

Mucosal immunology （NATURE PUBLISHING GROUP）  8 （ 3 ） 650 - 660 2015年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1933-0219

　概要を見る

粘膜組織の各部位に存在するリンパ濾胞は管腔内の抗原に対するIgA抗体を産生し、粘膜免疫応答の主役となるリンパ性器官である。リンパ濾胞上皮に存在するM細胞は異物を取込むことで、粘膜免疫応答の開始に働く特殊な上皮細胞である。
我々は濾胞上皮のホールマウント染色法を開発し、成熟M細胞と未成熟M細胞の可視化に成功した。そして、転写因子RelBの活性がM細胞の分化に必須であったことから、RelB活性の制御がM細胞成熟に重要であることを見出した。

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GP2-expressing cells in the conjunctiva and tear ducts of mice: identification of a novel type of cells in the squamous stratified epithelium.

Kimura S, Kishimoto A, Mutoh M, Takahashi-Iwanaga H, Iwanaga T

Biomedical research (Tokyo, Japan) （BIOMEDICAL RESEARCH PRESS LTD）  36 （ 4 ） 263 - 272 2015年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0388-6107

　概要を見る

GP2 is a membrane-associated secretory protein originally identified in zymogen granules of pancreatic acinar cells. Recently, this glycoprotein has attracted attention as a marker substance of M cells of Peyer's patches and for its involvement in the selective uptake of pathological bacteria via M cells. When we stained the conjunctiva and tear ducts of mice using a GP2 antibody, all goblet cells in the squamous stratified epithelium of the conjunctiva were intensely immunolabeled, while goblet cells in the intestine and airway were devoid of the immunoreactivity, indicating that the conjunctiva contains a special type of goblet cell. Further immunostaining for GP-2 labeled dispersed cells of peculiar shapes within the stratified squamous epithelium in the lacrimal canaliculi, lacrimal sac, and nasolacrimal duct. The GP2-immunoreactive cells in the tear duct projected arched or branched processes toward the basement membrane. Electron-microscopically, immunogold particles for GP2 outlined the basolateral plasma membrane of both the conjuntival goblet cells and the peculiarly shaped cells in the tear duct. Intracellularly, GP2 products of the goblet cells were localized around secretory granules in the apical cytoplasm and those of the tear duct cells inside the vesicles. The luminal contents close to apical plasma membrane were heavily labeled with immunogold particles, suggesting an exocytosis-based targeting of GP2 to the plasma membrane and its release into the lumen. The possible function of GP2 in tear ducts is discussed in relation to a defense system against invasive microoranisms and antigens.

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Epithelial-stromal interaction via Notch signaling is essential for the full maturation of gut-associated lymphoid tissues.

Obata Y, Kimura S, Nakato G, Iizuka K, Miyagawa Y, Nakamura Y, Furusawa Y, Sugiyama M, Suzuki K, Ebisawa M, Fujimura Y, Yoshida H, Iwanaga T, Hase K, Ohno H

EMBO reports （WILEY-BLACKWELL）  15 （ 12 ） 1297 - 1304 2014年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1469-221X

　概要を見る

Intrinsic Notch signaling in intestinal epithelial cells restricts secretory cell differentiation. In gut-associated lymphoid tissue (GALT), stromal cells located beneath the follicle-associated epithelium (FAE) abundantly express the Notch ligand delta-like 1 (Dll1). Here, we show that mice lacking Rbpja gene encoding a transcription factor implicated in Notch signalingin intestinal epithelial cells have defective GALT maturation. This defect can be attributed to the expansion of goblet cells, which leads to the down-regulation of CCL20 in FAE. These data demonstrate that epithelial Notch signaling maintained by stromal cells contributes to the full maturation of GALT by restricting secretory cell differentiation in FAE.

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A novel type of cells expressing GP2 in the respiratory epithelium of the paranasal sinuses in mice.

Kimura S, Muto M, Hisamoto M, Zheng M, Iwanaga T

Biomedical research (Tokyo, Japan) （BIOMEDICAL RESEARCH PRESS LTD）  35 （ 5 ） 329 - 337 2014年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0388-6107

　概要を見る

GP2, a GPI-anchored glycoprotein, is a useful marker of M cells in Peyer's patches. Our immunostaining of the paranasal sinuses in mice detected a condensed distribution of GP2-immunoreactive cells within the epithelium, apart from lymphoid tissues. In the paranasal sinuses, the cells exhibited a unique morphology characterized by a slender neck portion and huge terminal bulb, quite different from M cells. Electron microscopically, the GP2 immunoreactivity centered on the luminal plasma membrane of the terminal bulb, being less intense in the baso-lateral plasma membrane and not visible at all in the cytoplasm. The cells frequently came in contact with nerve fibers containing small synaptic vesicles. These nerve fibers contained neither CGRP nor substance P indicators of sensory neurons; moreover, no signal molecules used for a sensory function were expressed in the GP2-immunoreactive cells, implying that these nerves are efferent in nature. A weak but significant stainability in PAS reaction and an intense GP2 immunoreactivity for typical goblet cells in the tunica conjunctiva suggest that the GP2-expressing cells in paranasal sinuses are in the lineage of goblet cells.

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Three types of macrophagic cells in the mesentery of mice with special reference to LYVE-1-immunoreactive cells.

Zheng M, Kimura S, Nio-Kobayashi J, Takahashi-Iwanaga H, Iwanaga T

Biomedical research (Tokyo, Japan) （BIOMEDICAL RESEARCH PRESS LTD）  35 （ 1 ） 37 - 45 2014年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0388-6107

　概要を見る

Immunohistochemistry using whole mount preparations of the murine mesentery revealed two types of LYVE-1-immunoreactive cells with dendritic morphology other than F4/80(+) typical macrophages. The two types of LYVE-1(+) cells were regularly distributed with constant intervals throughout the mesentery and appeared to possess their own territory. Both types of LYVE-1(+) cells were weakly or moderately immunopositive for F4/80 antibody, a marker of macrophages, while F4/80(+) round macrophages were absolutely free from the LYVE-1 immunoreactivity. Only macrophages could ingest latex particles of 20 nm in diameter 3 h after a peritoneal injection. Peritoneal administration of lipopolysaccharide (LPS) induced a rapid reduction of LYVE-1 immunoreactivity in the cells with dendritic morphology followed by an increased immunoreactivity to F4/80 antibody, and simultaneously by dynamic changes in their shape. Under nonnal conditions, F4/80(+) macrophages in various connective tissues expressed LYVE-1, in contrast to lack of LYVE-1 in F4/80(+) macrophages within the parenchyma of visceral organs and macrophages residing in hepatic sinusoids and pulmonary alveoli. LYVE-1 may play a role in cell adhesion and migration of macrophagic cells within connective tissues rich in hyaluronan, and loss of LYVE-1 becomes a reliable sign of activated conditions in inflammation.

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Epithelial–stromal interaction via N otch signaling is essential for the full maturation of gut‐associated lymphoid tissues

Y Obata, S Kimura, G Nakato, K Iizuka, Y Miyagawa, Y Nakamura, ...

EMBO reports 15 (12), 1297-1304 2014年

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation.

Kitamura H, Kimura S, Shimamoto Y, Okabe J, Ito M, Miyamoto T, Naoe Y, Kikuguchi C, Meek B, Toda C, Okamoto S, Kanehira K, Hase K, Watarai H, Ishizuka M, El-Osta A, Ohara O, Miyoshi I

FASEB journal : official publication of the Federation of American Societies for Experimental Biology （FEDERATION AMER SOC EXP BIOL）  27 （ 12 ） 4940 - 4953 2013年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0892-6638

　概要を見る

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/ FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (6-fold) and SAA3 (15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P&lt
0.01), which was consistent with a 40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (&gt
2.1-fold), methylation of histone H3 lysine 4 (&gt
4.5- fold), and acetylation of histone H4 (&gt
2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.-Kitamura, H., Kimura, S., Shimamoto, Y., Okabe, J., Ito, M., Miyamoto, T., Naoe, Y., Kikuguchi, C., Meek, B., Toda, C., Okamoto, S., Kanehira, K., Hase, K., Watarai, H., Ishizuka, M., El-Osta, A., Ohara, O., Miyoshi, I. Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation. © FASEB.

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Beneficial effects of Brazilian propolis on type 2 diabetes in ob/ob mice: Possible involvement of immune cells in mesenteric adipose tissue.

Kitamura H, Naoe Y, Kimura S, Miyamoto T, Okamoto S, Toda C, Shimamoto Y, Iwanaga T, Miyoshi I

Adipocyte 2 （ 4 ） 227 - 236 2013年10月

共著, 査読有り,  ISSN  2162-3945

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Ubiquitin‐specific protease 2‐69 in macrophages potentially modulates metainflammation

H Kitamura, S Kimura, Y Shimamoto, J Okabe, M Ito, T Miyamoto, Y Naoe, ...

The FASEB Journal 27 (12), 4940-4953 2013年

The molecular basis of induction and formation of tunneling nanotubes.

Kimura S, Hase K, Ohno H

Cell and tissue research （SPRINGER）  352 （ 1 ） 67 - 76 2012年11月

共著, 査読有り,  ISSN  0302-766X

　概要を見る

Tunneling nanotubes (TNTs) and associated structures are recently recognized structures for intercellular communication. They are F-actin-containing thin protrusions of the plasma membrane of a cell and allow a direct physical connection to the plasma membranes of remote cells. TNTs and associated structures serve as mediators for intercellular transfer of organelles as well as membrane components and cytoplasmic molecules. Moreover, several pathogens have been shown to exploit these structures to spread among cells. Because of their contribution to normal cellular functions and importance in pathological conditions, studies on TNTs and related structures have accelerated over the past few years. These studies have revealed key molecules for their induction and/or formation; HIV Nef and M-Sec can induce the formation of TNTs in coordination with the remodeling of the actin cytoskeleton and vesicle trafficking.

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Tunneling nanotubes: emerging view of their molecular components and formation mechanisms.

Kimura S, Hase K, Ohno H

Experimental cell research （14）  318 （ 14 ） 1699 - 1706 2012年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0014-4827

　概要を見る

Cell-to-cell communication is essential for the development and maintenance of multicellular organisms. The tunneling nanotube (TNT) is a recently recognized distinct type of intercellular communication device. This are thin protrusions of the plasma membrane and allow direct physical connections of the plasma membranes between remote cells. The proposed functions for TNTs include the cell-to-cell transfer of large cellular structures such as membrane vesicles and organelles, as well as signal transduction molecules in a wide variety of cell types. Moreover TNT and TNT-related structures are thought to facilitate the intercellular spreading of virus and/or pathogenic proteins. Despite their contribution to normal cellular functions and importance in pathological conditions, virtually nothing is known about the molecular basis for their formation. We have recently shown that M-Sec (also called TNFaip2) is a key molecule for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton and vesicle trafficking are involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels. (C) 2012 Elsevier Inc. All rights reserved.

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Roles of a macrophage molecule M-mod in type 2 diabetes

H Kitamura, M Ito, S Kimura, Y Shimamoto, T Miyamoto, H Watarai, ...

The FASEB Journal 26 (1_supplement), 686.5-686.5 （FEDERATION AMER SOC EXP BIOL）  26 2012年

共著, 査読有り,  ISSN  0892-6638

Roles of a macrophage molecule M‐mod in type 2 diabetes

H Kitamura, M Ito, S Kimura, Y Shimamoto, T Miyamoto, H Watarai, ...

The FASEB Journal 26, 686.5-686.5 2012年

The epithelia-specific membrane trafficking factor AP-1B controls gut immune homeostasis in mice.

Takahashi D, Hase K, Kimura S, Nakatsu F, Ohmae M, Mandai Y, Sato T, Date Y, Ebisawa M, Kato T, Obata Y, Fukuda S, Kawamura YI, Dohi T, Katsuno T, Yokosuka O, Waguri S, Ohno H

Gastroenterology （2）  141 （ 2 ） 621 - 632 2011年08月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0016-5085

　概要を見る

BACKGROUND & AIMS: Epithelial cells that cover the intestinal mucosal surface maintain immune homeostasis and tolerance in the gastrointestinal tract. However, little is known about the molecular mechanisms that regulate epithelial immune functions. Epithelial cells are distinct in that they are highly polarized; this polarity is, at least in part, established by the epithelium-specific polarized sorting factor adaptor protein (AP)-1B. We investigated the role of AP-1B-mediated protein sorting in the maintenance of gastrointestinal immune homeostasis. METHODS: The role of AP-1B in intestinal immunity was examined in AP-1B-deficient mice (Ap1m2(-/-)) by monitoring their phenotypes, intestinal morphology, and epithelial barrier functions. AP-1B-mediated protein sorting was examined in polarized epithelial cells from AP-1B knockdown and Ap1m2(-/-) mice. RESULTS: Ap1m2(-/-) mice developed spontaneous chronic colitis, characterized by accumulation of interleukin-17A-producing, T-helper 17 cells. Deficiency of AP-1B caused epithelial immune dysfunction, such as reduced expression of antimicrobial proteins and impaired secretion of immunoglobulin A. These defects promoted intestinal dysbiosis and increased bacterial translocation within the mucosa. Importantly, AP-1B deficiency led to mistargeting of a subset of basolateral cytokine receptors to the apical plasma membrane in a polarized epithelial cell line and in colonic epithelial cells from mice. AP1M2 expression was reduced significantly in colonic epithelium samples from patients with Crohn's disease. CONCLUSIONS: AP-1B is required for proper localization of a subset of cytokine receptors in polarized epithelial cells, which allows them to respond to cytokine signals from underlying lamina propria cells. The AP-1B-mediated protein sorting machinery is required for maintenance of immune homeostasis and prevention of excessive inflammation.

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M-Sec: Emerging secrets of tunneling nanotube formation.

Ohno H, Hase K, Kimura S

Communicative & integrative biology （3）  3 （ 3 ） 231 - 233 2010年05月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1942-0889

　概要を見る

Tunneling nanotubes (TNT) are the latest addition to the array of strategies used for intercellular signaling. TNTs are continuous conduits of the plasma membrane that allow direct physical connection of plasma membranes and cytosol among remote cells. They are important for intercellular communication by mediating exchange of cellular components as well as signal transduction molecules. Despite ample evidence suggesting the pathophysiological importance of TNTs, virtually nothing is known about the molecular basis for their formation. With the lack of specific TNT markers, their study has relied solely on morphological analyses, and the precise identity of TNT and TNTlike structures have been difficult to define. We have now shown that M-Sec is a TNT marker and a central factor for TNT formation. In cooperation with the RalA small GTPase and the exocyst complex, M-Sec can induce the formation of functional TNTs, indicating that the remodeling of the actin cytoskeleton is involved in M-Sec-mediated TNT formation. Discovery of the role of M-Sec will accelerate our understanding of TNTs, both at the molecular and physiological levels. © 2010 Landes Bioscience.

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Essential role of membrane trafficking factor AP-1B in gut immune homeostasis (90.4)

D Takahashi, K Hase, S Kimura, H Ohno

The Journal of Immunology 184 (1 Supplement), 90.4-90.4 2010年

M-Sec promotes membrane nanotube formation by interacting with Ral and the exocyst complex.

Hase K, Kimura S, Takatsu H, Ohmae M, Kawano S, Kitamura H, Ito M, Watarai H, Hazelett CC, Yeaman C, Ohno H

Nature cell biology （12）  11 （ 12 ） 1427 - 1432 2009年12月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1465-7392

　概要を見る

Cell-cell communication is essential for the development and homeostasis of multicellular organisms. Recently, a new type of cell-cell communication was discovered that is based on the formation of thin membranous nanotubes between remote cells(1,2). These long membrane tethers, termed tunneling nanotubes (TNTs), form an intercellular conduit and have been shown to enable the transport of various cellular components and signals. However, the molecular basis for TNT formation remains to be elucidated. Here we report that a mammalian protein, M-Sec, induces de novo formation of numerous membrane protrusions extending from the plasma membrane, some of which tether onto adjacent cells and subsequently form TNT-like structures. Depletion of M-Sec by RNA interference (RNAi) greatly reduced endogenous TNT formation as well as intercellular propagation of a calcium flux in a macrophage cell line. Furthermore, blockage of the interaction of M-Sec with Ral and the exocyst complex, which serves as a downstream effector of Ral, attenuated the formation of membrane nanotubes. Our results reveal that M-Sec functions as a key regulator of membrane nanotube formation through interaction with the Ral-exocyst pathway.

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Uptake through glycoprotein 2 of FimH(+) bacteria by M cells initiates mucosal immune response.

Hase K, Kawano K, Nochi T, Pontes GS, Fukuda S, Ebisawa M, Kadokura K, Tobe T, Fujimura Y, Kawano S, Yabashi A, Waguri S, Nakato G, Kimura S, Murakami T, Iimura M, Hamura K, Fukuoka S, Lowe AW, Itoh K, Kiyono H, Ohno H

Nature （7270）  462 （ 7270 ） 226 - 230 2009年11月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0028-0836

　概要を見る

The mucosal immune system forms the largest part of the entire immune system, containing about three-quarters of all lymphocytes and producing grams of secretory IgA daily to protect the mucosal surface from pathogens(1-3). To evoke the mucosal immune response, antigens on the mucosal surface must be transported across the epithelial barrier into organized lymphoid structures such as Peyer's patches(4). This function, called antigen transcytosis, is mediated by specialized epithelial M cells(5,6). The molecular mechanisms promoting this antigen uptake, however, are largely unknown. Here we report that glycoprotein 2 (GP2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for mucosal antigens. Recombinant GP2 protein selectively bound a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane. Consistently, these bacteria were colocalized with endogenous GP2 on the apical plasma membrane as well as in cytoplasmic vesicles in M cells. Moreover, deficiency of bacterial FimH or host GP2 led to defects in transcytosis of type-I-piliated bacteria through M cells, resulting in an attenuation of antigen-specific immune responses in Peyer's patches. GP2 is therefore a previously unrecognized transcytotic receptor on M cells for type-I-piliated bacteria and is a prerequisite for the mucosal immune response to these bacteria. Given that M cells are considered a promising target for oral vaccination against various infectious diseases(7,8), the GP2-dependent transcytotic pathway could provide a new target for the development of M-cell-targeted mucosal vaccines.

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Monitoring autophagy in mammalian cultured cells through the dynamics of LC3.

Kimura S, Fujita N, Noda T, Yoshimori T

Methods in enzymology 452 （ C ） 1 - 12 2009年

共著, 査読有り,  ISSN  0076-6879

　概要を見る

In this chapter, we introduce several methods that rely on the analysis of LC3, a versatile marker protein of autophagic structures in mammalian cultured cells. The appearance of LC3-positive puncta is indicative of the induction of autophagy, and it is observed either by immunofluorescence or by GFP-based microscopy. The maturation process by which autophagosomes are converted into autolysosomes can be monitored by the GFP and RFP tandemly tagged LC3 (tfLC3) method. Lysosomal turnover of LC3 is a good index of the proceeding of autophagy and can be assessed by Western blotting. These methods will provide a relatively easy assessment of autophagy, and the details of the procedure will be described along with possible pitfalls. © 2009 Elsevier Inc. All rights reserved.

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Uptake through glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response

K Hase, K Kawano, T Nochi, GS Pontes, S Fukuda, M Ebisawa, ...

Nature 462 (7270), 226-230 2009年

Dynein-dependent movement of autophagosomes mediates efficient encounters with lysosomes.

Kimura S, Noda T, Yoshimori T

Cell structure and function （1）  33 （ 1 ） 109 - 122 2008年

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0386-7196|1347-3700

　概要を見る

Autophagy is a membrane trafficking pathway that carries cytosolic components to the lysosome for degradation. During this process, the autophagosome, a double-membraned organelle, is generated de novo, sequesters cytoplasmic proteins and organelles, and delivers them to lysosomes. However, the mechanism by which autophagosomes are targeted to lysosomes has not been determined. Here, we observed the real-time behavior of microtubule-associated protein light chain 3 (LC3), which localizes to autophagosomes, and showed that autophagosomes move in a microtubule- and dynein-dynactin motor complex-dependent manner. After formation, autophagosomes show a rapid vectorial movement in the direction of the centrosome, where lysosomes are usually concentrated. Microinjection of antibodies against LC3 inhibited this movement; furthermore, using FRAP, we showed that anti-LC3 antibody injection caused a defect in targeting of autophagosomes to lysosomes. Collectively, our data demonstrate the functional significance of autophagosome movement that enables effective delivery from the cytosol to lysosomes.

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Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3.

Kimura S, Noda T, Yoshimori T

Autophagy （5）  3 （ 5 ） 452 - 460 2007年09月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  1554-8627

　概要を見る

During the process of autophogy, autophagosomes undergo a maturation process consisting of multiple fusions with enclosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.

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[Autophagic degradation of unfolding proteins].

Kimura S, Kamimoto T, Yoshimori T

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme （10 Suppl）  51 （ 10 Suppl ） 1494 - 1498 2006年08月

共著, 査読有り,  ISSN  0039-9450

Autophagic degradation of unfolding proteins

S Kimura, T Kamimoto, T Yoshimori

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 51 (10 Suppl … 2006年

The analysis of autophagosome movement

S Kimura, T Yoshimori

Autophagy 2 (4), 355-355 2006年

Mutations in the membrane component, chromosome 1, surface marker 1 (M1S1) gene in gelatinous drop-like corneal dystrophy.

Murakami A, Kimura S, Fujiki K, Fujimaki T, Kanai A

Japanese journal of ophthalmology （4）  48 （ 4 ） 317 - 320 2004年07月

研究論文（学術雑誌）, 共著, 査読有り,  ISSN  0021-5155

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Purpose: To report mutations in the membrane component, chromosome 1, surface marker 1 (M1S1) gene in two members of the same family who showed symptoms of gelatinous drop-like corneal dystrophy (GDLD).
Methods: DNA was extracted from leukocytes of peripheral blood of the two affected members of the family and from controls, and the coding region of M1S1 was amplified by polymerase chain reaction (PCR). The PCR products were analyzed by direct sequencing. Normal and mutant M1S1 expression vectors were constructed and transfected into CHO cells to identify the cellular location of the gene products.
Results: The affected members had compound heterozygous mutations consisting of a nonsense change at codon 84 (K84X) and a missense mutation resulting in a substitution of arginine for cysteine at codon 108 (C108R). Neither of these mutations was found in the 50 controls. Protein expression analysis showed that the C108R product was distributed diffusely in the cytoplasm, whereas the normal gene product accumulated at cell-to-cell adhesion borders.
Conclusion: These data indicate that the K84X and C108R mutations in M1S1 cause GDLD. (C) Japanese Ophthalmological Society 2004.

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