Researcher Profile and Settings

Affiliation

• Faculty of Advanced Life Science

Job Title

• Assistant Professor

Degree

• Doctor of Life Science（2020/03 Hokkaido University）

ORCID ID

•  https://orcid.org/0000-0002-8077-3200

J-Global ID

• 202001014870310894

Research Interests

• 炎症性腸疾患   生活習慣病   食と健康   腸内環境   エンテロイド   自然免疫   抗菌ペプチド   腸内細菌   幹細胞   腸管粘膜免疫   Paneth細胞   

Research Areas

• Life sciences / Cell biology
• Life sciences / Immunology

Academic & Professional Experience

• 2023/07 - Today 北海道大学大学院先端生命科学研究院 助教
• 2021/04 - 2023/06 北海道大学大学院先端生命科学研究院 特任助教
• 2020/04 - 2021/03 JSPS research fellow
• 2019/04 - 2020/03 JSPS research fellow

Education

• 2015/04 - 2020/03  Hokkaido University  Graduate School of Life Science
• 2010/04 - 2015/03  Hokkaido University  School of Science  Biological Sciences

Association Memberships

• Japanese Association for Laboratory Animal Science   The Intestinal Microbiology Society   Japanese Association for Food Immunology   Japanese Society for Immunology   The Molecular Biology Society of Japan   

Research Activities

Published Papers

• Effects of exposure to the neonicotinoid pesticide clothianidin on α-defensin secretion and gut microbiota in mice

  Sakura YONOICHI, Yukako HARA, Yuya ISHIDA, Asuka SHODA, Mako KIMURA, Midori MURATA, Sarika NUNOBIKI, Makiko ITO, Ayano YOSHIMOTO, Youhei MANTANI, Toshifumi YOKOYAMA, Tetsushi HIRANO, Yoshinori IKENAKA, Yuki YOKOI, Tokiyoshi AYABE, Kiminori NAKAMURA, Nobuhiko HOSHI

  Journal of Veterinary Medical Science 0916-7250 2024/01/23 [Refereed]
  
• A Basic Study of the Effects of Mulberry Leaf Administration to Healthy C57BL/6 Mice on Gut Microbiota and Metabolites

  Li Gan, Yuga Inamura, Yu Shimizu, Yuki Yokoi, Yuki Ohnishi, Zihao Song, Yasuhiro Kumaki, Takashi Kikukawa, Makoto Demura, Masaaki Ito, Tokiyoshi Ayabe, Kiminori Nakamura, Tomoyasu Aizawa

  Metabolites 13 (9) 1003 - 1003 2023/09/10 [Refereed]
   

  Mulberry leaves contain α-glucosidase inhibitors, which have hypoglycemic effects and are considered functional foods. However, few reports have covered the effects of mulberry leaf components on normal gut microbiota and gut metabolites. Herein, gut microbiota analysis and NMR-based metabolomics were performed on the feces of mulberry leaf powder (MLP)-treated mice to determine the effects of long-term MLP consumption. Gut microbiota in the mouse were analyzed using 16S-rRNA gene sequencing, and no significant differences were revealed in the diversity and community structure of the gut microbiota in the C57BL/6 mice with or without MLP supplementation. Thirty-nine metabolites were identified via 1H-NMR analysis, and carbohydrates and amino acids were significantly (p < 0.01–0.05) altered upon MLP treatment. In the MLP-treated group, there was a marked increase and decrease in maltose and glucose concentrations, respectively, possibly due to the degradation inhibitory activity of oligosaccharides. After 5 weeks, all amino acid concentrations decreased. Furthermore, despite clear fluctuations in fecal saccharide concentrations, short-chain fatty acid production via intestinal bacterial metabolism was not strongly affected. This study provides the knowledge that MLP administration can alter the gut metabolites without affecting the normal gut microbiota, which is useful for considering MLP as a healthy food source.
• Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

  Yi Wang, Yuchi Song, Shaonan Yan, Rina Hiramine, Yuki Ohnishi, Yuki Yokoi, Kiminori Nakamura, Takashi Kikukawa, Tokiyoshi Ayabe, Tomoyasu Aizawa

  Antibiotics 12 (6) 1047 - 1047 2023/06/14 [Refereed]
   

  Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.
• Application of Benchtop NMR for Metabolomics Study Using Feces of Mice with DSS-Induced Colitis

  Zihao Song, Yuki Ohnishi, Seiji Osada, Li Gan, Jiaxi Jiang, Zhiyan Hu, Hiroyuki Kumeta, Yasuhiro Kumaki, Yuki Yokoi, Kiminori Nakamura, Tokiyoshi Ayabe, Kazuo Yamauchi, Tomoyasu Aizawa

  Metabolites 13 (5) 611 - 611 2023/04/28 [Refereed][Not invited]
   

  Nuclear magnetic resonance (NMR)-based metabolomics, which comprehensively measures metabolites in biological systems and investigates their response to various perturbations, is widely used in research to identify biomarkers and investigate the pathogenesis of underlying diseases. However, further applications of high-field superconducting NMR for medical purposes and field research are restricted by its high cost and low accessibility. In this study, we applied a low-field, benchtop NMR spectrometer (60 MHz) employing a permanent magnet to characterize the alterations in the metabolic profile of fecal extracts obtained from dextran sodium sulfate (DSS)-induced ulcerative colitis model mice and compared them with the data acquired from high-field NMR (800 MHz). Nineteen metabolites were assigned to the 60 MHz 1H NMR spectra. Non-targeted multivariate analysis successfully discriminated the DSS-induced group from the healthy control group and showed high comparability with high-field NMR. In addition, the concentration of acetate, identified as a metabolite with characteristic behavior, could be accurately quantified using a generalized Lorentzian curve fitting method based on the 60 MHz NMR spectra.
• Shorter sleep time relates to lower human defensin 5 secretion and compositional disturbance of the intestinal microbiota accompanied by decreased short-chain fatty acid production

  Yu Shimizu, Ryodai Yamamura, Yuki Yokoi, Tokiyoshi Ayabe, Shigekazu Ukawa, Koshi Nakamura, Emiko Okada, Akihiro Imae, Takafumi Nakagawa, Akiko Tamakoshi, Kiminori Nakamura

  Gut Microbes 15 (1) 1949-0976 2023/03/21 [Refereed]
  
• Decreased Paneth cell α-defensins promote fibrosis in a choline-deficient L-amino acid-defined high-fat diet-induced mouse model of nonalcoholic steatohepatitis via disrupting intestinal microbiota

  Shunta Nakamura, Kiminori Nakamura, Yuki Yokoi, Yu Shimizu, Shuya Ohira, Mizu Hagiwara, Zihao Song, Li Gan, Tomoyasu Aizawa, Daigo Hashimoto, Takanori Teshima, Andre J. Ouellette, Tokiyoshi Ayabe

  Scientific Reports 13 (1) 2023/03/09 [Refereed]
   

  Abstract Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fibrosis that develops from fatty liver. Disruption of intestinal microbiota homeostasis, dysbiosis, is associated with fibrosis development in NASH. An antimicrobial peptide α-defensin secreted by Paneth cells in the small intestine is known to regulate composition of the intestinal microbiota. However, involvement of α-defensin in NASH remains unknown. Here, we show that in diet-induced NASH model mice, decrease of fecal α-defensin along with dysbiosis occurs before NASH onset. When α-defensin levels in the intestinal lumen are restored by intravenous administration of R-Spondin1 to induce Paneth cell regeneration or by oral administration of α-defensins, liver fibrosis is ameliorated with dissolving dysbiosis. Furthermore, R-Spondin1 and α-defensin improved liver pathologies together with different features in the intestinal microbiota. These results indicate that decreased α-defensin secretion induces liver fibrosis through dysbiosis, further suggesting Paneth cell α-defensin as a potential therapeutic target for NASH.
• Efficient and simple genetic engineering of enteroids using mouse isolated crypts for investigating intestinal functions

  Shuya Ohira, Yuki Yokoi, Tokiyoshi Ayabe, Kiminori Nakamura

  Biochemical and Biophysical Research Communications 637 153 - 160 0006-291X 2022/12/31 [Refereed]
  
• Hemodynamic response to intestinal pH stimulation measured with spectroscopic video imaging

  Tsukasa Funane, Yuki Yokoi, Masashi Kiguchi, Ryuga Shinozaki, Tokiyoshi Ayabe, Hirokazu Atsumori, Ayako Nishimura, Kiminori Nakamura, Akihiko Kandori

  Biomedical Physics & Engineering Express 2022/11/11 [Refereed]
   

  Abstract To investigate the relationship between the gut and skin (gut-skin axis), head skin hemodynamic responses to gut stimulation including the injection of acetic acid in nude mice were measured by spectroscopic video imaging, which was calculated using a modified Beer-Lambert formula. The relationship with blood proteins was also analyzed. The blood volume changes in three mice injected with acetic acid were highly reproducible in the mathematical model equation. Four proteins correlated with blood volume changes were all related to immunity. These results suggest that intestinal pH can alter the blood volume in the skin and induce immune-related responses.
• Intestinal commensal microbiota and cytokines regulate Fut2+ Paneth cells for gut defense

  Mariko Kamioka, Yoshiyuki Goto, Kiminori Nakamura, Yuki Yokoi, Rina Sugimoto, Shuya Ohira, Yosuke Kurashima, Shingo Umemoto, Shintaro Sato, Jun Kunisawa, Yu Takahashi, Steven E. Domino, Jean-Christophe Renauld, Susumu Nakae, Yoichiro Iwakura, Peter B. Ernst, Tokiyoshi Ayabe, Hiroshi Kiyono

  Proceedings of the National Academy of Sciences 119 (3) e2115230119 - e2115230119 0027-8424 2022/01/18 [Refereed]
   

  Paneth cells are intestinal epithelial cells that release antimicrobial peptides, such as α-defensin as part of host defense. Together with mesenchymal cells, Paneth cells provide niche factors for epithelial stem cell homeostasis. Here, we report two subtypes of murine Paneth cells, differentiated by their production and utilization of fucosyltransferase 2 (Fut2), which regulates α(1,2)fucosylation to create cohabitation niches for commensal bacteria and prevent invasion of the intestine by pathogenic bacteria. The majority of Fut2⁻ Paneth cells were localized in the duodenum, whereas the majority of Fut2⁺ Paneth cells were in the ileum. Fut2⁺ Paneth cells showed higher granularity and structural complexity than did Fut2⁻ Paneth cells, suggesting that Fut2⁺ Paneth cells are involved in host defense. Signaling by the commensal bacteria, together with interleukin 22 (IL-22), induced the development of Fut2⁺ Paneth cells. IL-22 was found to affect the α-defensin secretion system via modulation of Fut2 expression, and IL-17a was found to increase the production of α-defensin in the intestinal tract. Thus, these intestinal cytokines regulate the development and function of Fut2⁺ Paneth cells as part of gut defense.
• Decrease of α-defensin impairs intestinal metabolite homeostasis via dysbiosis in mouse chronic social defeat stress model

  Kosuke Suzuki, Kiminori Nakamura, Yu Shimizu, Yuki Yokoi, Shuya Ohira, Mizu Hagiwara, Yi Wang, Yuchi Song, Tomoyasu Aizawa, Tokiyoshi Ayabe

  Scientific Reports 11 (1) 9915 - 9915 2021/05 [Refereed]
   

  Psychological stress has been reported to relate to dysbiosis, imbalance of the intestinal microbiota composition, and contribute to the onset and exacerbation of depression, though, underlying mechanisms of psychological stress-related dysbiosis have been unknown. It has been previously established that α-defensins, which are effector peptides of innate enteric immunity produced by Paneth cells in the small intestine, play an important role in regulation of the intestinal microbiota. However, the relationship between disruption of intestinal ecosystem and α-defensin under psychological stress is yet to be determined. Here we show using chronic social defeat stress (CSDS), a mouse depression model that (1) the exposure to CSDS significantly reduces α-defensin secretion by Paneth cells and (2) induces dysbiosis and significant composition changes in the intestinal metabolites. Furthermore, (3) they are recovered by administration of α-defensin. These results indicate that α-defensin plays an important role in maintaining homeostasis of the intestinal ecosystem under psychological stress, providing novel insights into the onset mechanism of stress-induced depression, and may further contribute to discovery of treatment targets for depression.
• Simultaneous real-time analysis of Paneth cell and intestinal stem cell response to interferon-γ by a novel stem cell niche tracking method

  Yuki Yokoi, Takahiro Adachi, Rina Sugimoto, Mani Kikuchi, Tokiyoshi Ayabe, Kiminori Nakamura

  Biochemical and Biophysical Research Communications 545 14 - 19 0006-291X 2021/01/30 [Refereed]
   

  Paneth cells and Lgr5+ intestinal stem cells (Lgr5+ ISCs) constitute the stem cell niche and maintain small intestinal epithelial integrity by recognizing various niche factors derived from subepithelial cells and external antigens. Although it has been known that interferon-γ (IFN-γ), a Th1 cytokine, is associated with intestinal epithelial disruption during inflammation as a niche factor, dynamics of Paneth cells and Lgr5+ ISCs in response to IFN-γ remain to be understood. Here we show that CAG-tdTomato;Lgr5-EGFP (CT-LE) mice generated in this study enable to identify Paneth cells and Lgr5+ ISCs separately by fluorescence signals. Lgr5+ ISCs underwent cell death a little earlier than Paneth cells in response to IFN-γ by simultaneous tracking using CT-LE mice. In addition, the timing of cell death in most Paneth cells overlapped with Lgr5+ ISCs, suggesting that Paneth cell depletion is induced directly by IFN-γ. Taken together, we established a novel simultaneous stem cell niche tracking method and clarified the involvement of both Paneth cells and Lgr5+ ISCs in stem cell niche damage induced by IFN-γ, further contribute to understanding the mechanism for maintaining intestinal homeostasis by stem cell niche.
• Expression and Localization of Paneth Cells and Their α-Defensins in the Small Intestine of Adult Mouse

  Kiminori Nakamura, Yuki Yokoi, Rie Fukaya, Shuya Ohira, Ryuga Shinozaki, Takuto Nishida, Mani Kikuchi, Tokiyoshi Ayabe

  Frontiers in Immunology 11 2020/10/13 [Refereed]
   

  © Copyright © 2020 Nakamura, Yokoi, Fukaya, Ohira, Shinozaki, Nishida, Kikuchi and Ayabe. Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 103 to 1 x 106 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity.
• Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure.

  Hikaru Hanyu, Yuki Yokoi, Kiminori Nakamura, Tokiyoshi Ayabe, Keisuke Tanaka, Kinuko Uno, Katsuhiro Miyajima, Yuki Saito, Ken Iwatsuki, Makoto Shimizu, Miki Tadaishi, Kazuo Kobayashi-Hattori

  Toxins 12 (10) 2020/09/24 [Refereed]
   

  The different effects of deoxynivalenol (DON) on intestinal barrier and stem cells by its route of exposure remain less known. We explored the toxic effects of DON on intestinal barrier functions and stem cells after DON microinjection (luminal exposure) or addition to a culture medium (basolateral exposure) using three-dimensional mouse intestinal organoids (enteroids). The influx test using fluorescein-labeled dextran showed that basolateral DON exposure (1 micromolar (µM) disrupted intestinal barrier functions in enteroids compared with luminal DON exposure at the same concentration. Moreover, an immunofluorescence experiment of intestinal epithelial proteins, such as E-cadherin, claudin, zonula occludens-1 (ZO-1), and occludin, exhibited that only basolateral DON exposure broke down intestinal epithelial integrity. A time-lapse analysis using enteroids from leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescence protein (EGFP) transgenic mice and 5-ethynyl-2-deoxyuridine (EdU) assay indicated that only the basolateral DON exposure, but not luminal DON exposure, suppressed Lgr5+ stem cell count and proliferative cell ratio, respectively. These results revealed that basolateral DON exposure has larger impacts on intestinal barrier function and stem cells than luminal DON exposure. This is the first report that DON had different impacts on intestinal stem cells depending on the administration route. In addition, RNA sequencing analysis showed different expression of genes among enteroids after basolateral and luminal DON exposure.
• Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn’s disease model mice

  Yu Shimizu, Kiminori Nakamura, Aki Yoshii, Yuki Yokoi, Mani Kikuchi, Ryuga Shinozaki, Shunta Nakamura, Shuya Ohira, Rina Sugimoto, Tokiyoshi Ayabe

  Life Science Alliance 3 (6) e201900592 - e201900592 2020/06 [Refereed]
   

  Crohn’s disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.
• Butyric Acid and Leucine Induce α-Defensin Secretion from Small Intestinal Paneth Cells.

  Akiko Takakuwa, Kiminori Nakamura, Mani Kikuchi, Rina Sugimoto, Shuya Ohira, Yuki Yokoi, Tokiyoshi Ayabe

  Nutrients 11 (11) 2019/11/18 [Refereed]
   

  The intestine not only plays a role in fundamental processes in digestion and nutrient absorption, but it also has a role in eliminating ingested pathogenic bacteria and viruses. Paneth cells, which reside at the base of small intestinal crypts, secrete α-defensins and contribute to enteric innate immunity through potent microbicidal activities. However, the relationship between food factors and the innate immune functions of Paneth cells remains unknown. Here, we examined whether short-chain fatty acids and amino acids induce α-defensin secretion from Paneth cells in the isolated crypts of small intestine. Butyric acid and leucine elicit α-defensin secretion by Paneth cells, which kills Salmonella typhimurium. We further measured Paneth cell secretion in response to butyric acid and leucine using enteroids, a three-dimensional ex vivo culture system of small intestinal epithelial cells. Paneth cells expressed short-chain fatty acid receptors, Gpr41, Gpr43, and Gpr109a mRNAs for butyric acid, and amino acid transporter Slc7a8 mRNA for leucine. Antagonists of Gpr41 and Slc7a8 inhibited granule secretion by Paneth cells, indicating that these receptor and transporter on Paneth cells induce granule secretion. Our findings suggest that Paneth cells may contribute to intestinal homeostasis by secreting α-defensins in response to certain nutrients or metabolites.
• Paneth cell granule dynamics on secretory responses to bacterial stimuli in enteroids.

  Yuki Yokoi, Kiminori Nakamura, Tsukasa Yoneda, Mani Kikuchi, Rina Sugimoto, Yu Shimizu, Tokiyoshi Ayabe

  Scientific reports 9 (1) 2710 - 2710 2019/02/25 [Refereed]
   

  Paneth cells at the base of small intestinal crypts secrete granules containing α-defensins in response to bacteria and maintain the intestinal environment by clearing enteric pathogens and regulating the composition of the intestinal microbiota. However, Paneth cell secretory responses remain debatable and the mechanisms that regulate the secretion are not well understood. Although enteroids, three-dimensional cultures of small intestinal epithelial cells, have proven useful for analyzing intestinal epithelial cell functions including ion transport, their closed structures have imposed limitations to investigating interactions between Paneth cells and the intestinal microbiota. Here, we report that microinjection of bacteria or lipopolysaccharide (LPS) into the enteroid lumen provides an ex vivo system for studying Paneth cell secretion in real-time. The results show that Paneth cells released granules immediately when the apical surfaces of enteroid epithelial cells were exposed to LPS or live bacteria by microinjection. However, Paneth cells did not respond to LPS delivered in culture media to enteroid exterior basolateral surface, although they responded to basolateral carbamyl choline. In addition, Paneth cells replenished their granules after secretion, enabling responses to second stimulation. These findings provide new insight for apically-induced Paneth cell secretory responses in regulating the intestinal environment.
• Essential role of IFN-γ in T cell-associated intestinal inflammation.

  Yoshihiro Eriguchi, Kiminori Nakamura, Yuki Yokoi, Rina Sugimoto, Shuichiro Takahashi, Daigo Hashimoto, Takanori Teshima, Tokiyoshi Ayabe, Michael E Selsted, André J Ouellette

  JCI insight 3 (18) 2018/09/20 [Refereed]
   

  Paneth cells contribute to small intestinal homeostasis by secreting antimicrobial peptides and constituting the intestinal stem cell (ISC) niche. Certain T cell-mediated enteropathies are characterized by extensive Paneth cell depletion coincident with mucosal destruction and dysbiosis. In this study, mechanisms of intestinal crypt injury have been investigated by characterizing responses of mouse intestinal organoids (enteroids) in coculture with mouse T lymphocytes. Activated T cells induced enteroid damage, reduced Paneth cell and Lgr5+ ISC mRNA levels, and induced Paneth cell death through a caspase-3/7-dependent mechanism. IFN-γ mediated these effects, because IFN-γ receptor-null enteroids were unaffected by activated T cells. In mice, administration of IFN-γ induced enteropathy with crypt hyperplasia, villus shortening, Paneth cell depletion, and modified ISC marker expression. IFN-γ exacerbated radiation enteritis, which was ameliorated by treatment with a selective JAK1/2 inhibitor. Thus, IFN-γ induced Paneth cell death and impaired regeneration of small intestinal epithelium in vivo, suggesting that IFN-γ may be a useful target for treating defective mucosal regeneration in enteric inflammation.
• R-Spondin1 expands Paneth cells and prevents dysbiosis induced by graft-versus-host disease.

  Eiko Hayase, Daigo Hashimoto, Kiminori Nakamura, Clara Noizat, Reiki Ogasawara, Shuichiro Takahashi, Hiroyuki Ohigashi, Yuki Yokoi, Rina Sugimoto, Satomi Matsuoka, Takahide Ara, Emi Yokoyama, Tomohiro Yamakawa, Ko Ebata, Takeshi Kondo, Rina Hiramine, Tomoyasu Aizawa, Yoshitoshi Ogura, Tetsuya Hayashi, Hiroshi Mori, Ken Kurokawa, Kazuma Tomizuka, Tokiyoshi Ayabe, Takanori Teshima

  The Journal of experimental medicine 214 (12) 3507 - 3518 2017/12/04 [Refereed]
   

  The intestinal microbial ecosystem is actively regulated by Paneth cell-derived antimicrobial peptides such as α-defensins. Various disorders, including graft-versus-host disease (GVHD), disrupt Paneth cell functions, resulting in unfavorably altered intestinal microbiota (dysbiosis), which further accelerates the underlying diseases. Current strategies to restore the gut ecosystem are bacteriotherapy such as fecal microbiota transplantation and probiotics, and no physiological approach has been developed so far. In this study, we demonstrate a novel approach to restore gut microbial ecology by Wnt agonist R-Spondin1 (R-Spo1) or recombinant α-defensin in mice. R-Spo1 stimulates intestinal stem cells to differentiate to Paneth cells and enhances luminal secretion of α-defensins. Administration of R-Spo1 or recombinant α-defensin prevents GVHD-mediated dysbiosis, thus representing a novel and physiological approach at modifying the gut ecosystem to restore intestinal homeostasis and host-microbiota cross talk toward therapeutic benefits.

Conference Activities & Talks

• Selective Paneth cell granule secretion in response to pathogenic and commensal bacteria in enteric mucosal immunity  [Not invited]

  Yuki Yokoi, Shuya Ohira, Haruto Matsuoka, Shota Takemi, Tokiyoshi Ayabe, Kiminori Nakamura

  The 52nd Annual Meeting of the Japanese Society for Immunology  2024/01
• Establishment of a high-efficient method for generating transgenic enteroid revealing intestinal epithelial function  [Not invited]

  松下稜, 横井友樹, 大平修也, 松岡温音, 竹見祥大, 中村公則

  The 46th Annual Meeting of the Molecular Biology Society of Japan  2023/12
• 病原菌と共生菌に対する選択的なPaneth細胞顆粒分泌調節による自然免疫と共生のメカニズム  [Not invited]

  横井友樹, 大平修也, 綾部時芳, 中村公則

  令和４年度北大細胞生物研究集会  2023/03
• Regulation of Paneth cell granule secretion by selective sensing of pathogenic and commensal bacteria in intestinal homeostasis  [Not invited]

  Yuki Yokoi, Shuya Ohira, Haruto Matsuoka, Yuito Kaibori, Ryuga Shinozaki, Tokiyoshi Ayabe, Kiminori Nakamura

  第45回日本分子生物学会年会  2022/12
• The mechanism of acetylcholine-induced Paneth cell secretory responses in innate enteric immunity  [Not invited]

  Yuki Yokoi, Shuya Ohira, Mani Kikuchi, Tokiyoshi Ayabe, Kiminori Nakamura

  The 50th Annual Meeting of the Japanese Society for Immunology  2021/12
• Paneth細胞顆粒分泌応答からみた食機能評価系の確立  [Not invited]

  横井友樹, 中村公則, 高桑明子, 菊池摩仁, 綾部時芳

  第17回日本食品免疫学会学術集会  2021/11
• Dynamics of Paneth Cell Granule Secretory Responses to Bacteria in Innate Enteric Immunity  [Not invited]

  Yuki Yokoi, Kiminori Nakamura, Shuya Ohira, Ryuga Shinozaki, Mani Kikuchi, Tokiyoshi Ayabe

  World Microbe Forum  2021/06
• Acetylcholine-induced Paneth cell granule secretion via muscarinic M3 receptor in mouse small intestine  [Not invited]

  Yokoi Y, Nakamura K, Sugimoto R, Ohira S, Kikuchi M, Ayabe T

  ASCB|EMBO 2019 meeting  2019/12
• Dynamics and mechanisms of Paneth cell granule secretory responses in enteroids  [Invited]

  Yokoi Y, Nakamura K, Sugimoto R, Ohira S, Kikuchi M, Ayabe T

  USC-HU Research Workshop “Gut-Organ Crosstalk”  2019/05
• Visualization of Paneth cell granule secretory responses to bacterial stimuli using enteroids

  Yokoi Y, Nakamura K, Yoneda T, Kikuchi M, Nakamura S, Ayabe T

  ASCB|EMBO 2018 meeting  2018/12
• A mechanism of acetylcholine-induced Paneth cell granule secretion

  Yokoi Y, Nakamura K, Ayabe T

  Clearing & Imaging Workshop in the Hokkaido University  2018/08
• Enteroidを用いたPaneth 細胞顆粒の分泌と再形成可視化

  横井 友樹, 中村 公則, 米田 司, 菊池 摩仁, 綾部 時芳

  第8回オルソオルガノジェネシス検討会  2018/07
• アセチルコリンによるPaneth細胞顆粒分泌反応の機序解明

  横井 友樹, 中村 公則, 綾部 時芳

  第7回オルソオルガノジェネシス検討会  2017/08
• Enteroidを用いたPaneth細胞顆粒分泌反応の可視化解析による機序解明  [Invited]

  横井 友樹

  平成29年度 北海道大学大学院生命科学院入学式「先輩からのメッセージ」  2017/04
• 腸内環境による嗜好の決定メカニズム－おいしく食べて健康になる－  [Invited]

  横井 友樹

  第2回COI2021会議  2017/03
• Enteroidを用いたPaneth細胞顆粒分泌反応の可視化解析による機序解明

  横井 友樹, 中村 公則, 櫻木 直也, 菊池 摩仁, 綾部 時芳

  第12回日本食品免疫学会学術大会  2016/11

Awards & Honors

• 2021/11 第17回日本食品免疫学会学術大会 ポスター賞
  
• 2016/11 第12回日本食品免疫学会学術大会 ポスター賞
  

Research Grants & Projects

• Paneth細胞分泌メカニズムから紐解く腸管粘膜免疫システムの解明

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists

  Date (from‐to) : 2021/04 -2024/03 

  Author : 横井 友樹

   

  小腸上皮細胞であるPaneth細胞は腸内の細菌刺激に応答して抗菌ペプチドα-defensinを豊富に含む細胞質顆粒を分泌することで腸管自然免疫に寄与している。本研究の目的は腸管内腔へ侵入した病原体のセンシングからPaneth細胞が顆粒を分泌するまでの一連のPaneth細胞を中心とした腸管自然免疫応答カスケードを明らかにすることである。令和3年度はまず、腸管内腔側からの細菌刺激が上皮細胞によってセンシングされ、Paneth細胞に伝わることで顆粒分泌に至る経路を明らかにするために、小腸上皮細胞の三次元培養系であるエンテロイドを用いたカルシウムイメージングを実施した。蛍光カルシウムセンサーであるGCaMP6ノックインマウスの小腸から作出したエンテロイドの内腔へマイクロインジェクション法により細菌を導入し、Paneth細胞を含む小腸上皮細胞の細胞内カルシウム動態を、共焦点レーザー顕微鏡を用いた三次元タイムラプスにより解析した。サルモネラ生菌をエンテロイド内腔へ導入すると、上皮細胞の細胞間カルシウムウェーブが起こった後、Paneth細胞が顆粒を分泌した。また、細胞間カルシウム伝播に関わる分子に対する阻害剤でエンテロイドを処理すると、細菌導入時の細胞間カルシウムウェーブが抑制され、Paneth細胞は顆粒を分泌しなかった。以上の結果より、Paneth細胞が上皮細胞間情報伝達によって腸内へ侵入した病原菌をセンシングし、顆粒分泌に至る小腸上皮細胞が連携した自然免疫応答を示すことを明らかにした。このことは、腸管粘膜免疫ネットワークの新たなメカニズム解明に進展すると考えられる。
• 細胞内カルシウム可視化エンテロイドによる食機能評価系の樹立

  ノーステック財団：札幌ライフサイエンス産業活性化事業

  Date (from‐to) : 2021/08 -2022/03
• 食と腸内細菌によるＰａｎｅｔｈ細胞分泌メカニズムからみた腸内環境ネットワーク

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  Date (from‐to) : 2019/04 -2021/03 

  Author : 横井 友樹

   

  小腸上皮細胞の一系統であるPaneth細胞は、抗菌ペプチドα-defensinを豊富に含む細胞質顆粒をコリン作動性刺激や細菌刺激に応答して腸管内腔へ分泌することで病原菌を排除し、腸内環境の恒常性維持に重要な役割を担っている。しかし、Paneth細胞の顆粒分泌応答メカニズムは未だ不明であった。本研究は腸内リガンドを感知し、顆粒分泌に至るまでのPaneth細胞分泌応答メカニズムを解明することを目的とする。令和2年度は上皮下の神経性アセチルコリン (ACh)が誘導するPaneth細胞顆粒分泌におけるM3受容体シグナル伝達経路を決定するために、単離Paneth細胞を材料としたnested PCRを行い、M3受容体下流の標的シグナル分子のmRNA発現を明らかにした。特にプロテインキナーゼC (PKC)について、Paneth細胞が発現する各アイソフォームに選択的な阻害剤を用いたex vivo顆粒分泌評価を行うことでPaneth細胞の分泌応答を制御するPKCアイソフォームを推定した。以上より、上皮下のコリン作動性神経からAChを介してPaneth細胞顆粒応答に至る分子メカニズムを明らかにすることで、Paneth細胞と上皮下細胞が連携した腸内環境ネットワークをはじめて示した。 本研究はPaneth細胞機能の新規ex vivo評価システムを確立し、Paneth細胞が腸内リガンドや上皮下細胞由来の細胞間シグナル因子を受容して、腸内環境を制御するメカニズムを明らかにした。さらに、これまで知られていなかったPaneth細胞を中心とした食、腸内細菌および種々の宿主細胞が形成する腸内環境ネットワークとその分子機構を示して目的を達成した。Paneth細胞機能に基づいた腸内環境の恒常性破綻が関与する様々な疾患の病態解明や新規治療法開発に大きく貢献することが期待される。