Details of a Researcher - Sato, Toshiro

Papers - Sato, Toshiro

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Transformation of intestinal stem cells into gastric stem cells on loss of transcription factor Cdx2

Simmini Salvatore, Bialecka Monika, Huch Meritxell, Kester Lennart, Van De Wetering Marc, Sato Toshiro, Beck Felix, Van Oudenaarden Alexander, Clevers Hans, Deschamps Jacqueline

Nature Communications 5 2014

Research paper (scientific journal), Joint Work, ISSN 2041-1723

The endodermal lining of the adult gastro-intestinal tract harbours stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation programme and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2 null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. We show that the intestinal genetic programme is critically dependent on the single transcription factor encoding gene Cdx2.

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Cross-talk between ROR_γt ⁺ innate lymphoid cells and intestinal macrophages induces mucosal IL-22 production In Crohn's disease

Mizuno Shinta, Mikami Yohei, Kamada Nobuhiko, Handa Tango, Hayashi Atsushi, Sato Toshiro, Matsuoka Katsuyoshi, Matano Mami, Ohta Yuki, Sugita Akira, Koganei Kazutaka, Sahara Rikisaburo, Takazoe Masakazu, Hisamatsu Tadakazu, Kanai Takanori

Inflammatory Bowel Diseases 20 ( 8 ) 1426 - 1434 2014

Research paper (scientific journal), Joint Work, ISSN 1078-0998

Background: Interleukin (IL)-22-producing ROR_γt ⁺ innate lymphoid cells (ILCs) play a pivotal role in intestinal immunity. Recent reports demonstrated that ILCs contribute to mucosal protection and intestinal inflammation in mice. In humans, numbers of ROR _γt
⁺ ILCs are significantly increased in the intestine of patients with Crohn's disease (CD), suggesting that ILCs may be associated with intestinal inflammation in CD. However, the mechanism by which ILCs are regulated in the intestine of patients with CD is poorly understood. This study aimed to determine the activation mechanism of intestinal ILCs in patients with CD. Methods: CD45+ lineage marker-ILCs were isolated from intestinal lamina propria of patients with CD. ILCs were then subdivided into 4 distinct populations based on the expression of CD56 and CD127. Purified ILC subsets were cocultured with intestinal CD14+ macrophages, and IL-22 production was evaluated. Results: CD127⁺CD56^- and CD127 ⁺CD56⁺ ILC, but not CD127^-CD56⁺ or CD127^-CD56^- ILC, subsets expressed ROR_γt and produced IL-22. IL-22 production by these ILC subsets was enhanced when ILCs were cocultured with intestinal macrophages. IL-23 or cell-to-cell contact was required for macrophage-mediated activation of ILCs. IL-22 production by ILCs was perturbed in inflamed mucosa compared with noninflamed mucosa. IL-22 induced the expression of Reg1_α and Claudin-1 in human intestinal epithelial organoids. Conclusions: ROR_γt
⁺ ILCs might enhance mucosal barrier function through the upregulation of Reg1 _α through production of IL-22. Although CD14⁺ macrophages augment intestinal inflammation in patients with CD, macrophages also promote a negative feedback pathway through the activation of IL-22 production by ROR_γt
⁺ ILCs.

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Culturing intestinal stem cells

Fujii Masayuki, Sato Toshiro

Frontiers in Genetics 5 ( JUN ) 2014

Research paper (scientific journal), Joint Work, ISSN 1664-8021

Recent advance of sequencing technology has revealed genetic alterations in colorectal cancer (CRC). The biological function of recurrently mutated genes has been intensively investigated through mouse genetic models and CRC cell lines. Although these experimental models may not fully reflect biological traits of human intestinal epithelium, they provided insights into the understanding of intestinal stem cell self-renewal, leading to the development of novel human intestinal organoid culture system. Intestinal organoid culture enabled to expand normal or tumor epithelial cells in vitro retaining their stem cell self-renewal and multiple differentiation. Gene manipulation of these cultured cells may provide an attractive tool for investigating genetic events involved in colorectal carcinogenesis.

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Mucosal healing with oral tacrolimus is associated with favorable medium- and long-term prognosis in steroid-refractory/dependent ulcerative colitis patients

Miyoshi Jun, Matsuoka Katsuyoshi, Inoue Nagamu, Hisamatsu Tadakazu, Ichikawa Riko, Yajima Tomoharu, Okamoto Susumu, Naganuma Makoto, Sato Toshiro, Kanai Takanori, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Journal of Crohn's and Colitis 7 ( 12 ) 2013.12

Research paper (scientific journal), Joint Work, ISSN 1873-9946

Background: Oral administration of tacrolimus is an effective remission induction therapy for steroid-refractory/dependent ulcerative colitis (UC). Aim: This study aimed to evaluate the short- as well as medium- and long-term effectiveness of tacrolimus therapy. Methods: The medical records of 51 patients treated with tacrolimus for UC at our hospital between July 2009 and December 2011 were reviewed retrospectively. Clinical remission and improvement were defined as a Lichtiger score of 4 or less and as a Lichtiger score of ≤. 10 and a reduction in the score of ≥. 3 compared with the baseline score, respectively. Endoscopic findings were evaluated based on the endoscopic activity index and Mayo endoscopic score. Results: The clinical effectiveness combining clinical remission and improvement was observed in 62.7% of the patients at 3. months. Thirty-six patients underwent colonoscopy at 3. months, and 12 (33.3%) and 10 patients (27.8%) showed Mayo endoscopic scores of 0 and 1, respectively. On Kaplan-Meier analysis, the overall percentage of event-free survivors, who did not require colectomy nor switching to other induction therapy such as infliximab, was 73.0% at 6. months, 49.9% at 1. year, and 37.8% at 2. years. Patients with a Mayo endoscopic score of 0-1 at 3. months showed significantly better medium- and long-term prognosis than those with a score of 2-3 (p<. 0.01). All adverse events, including infections in 2 patients, were reversible. Conclusions: Tacrolimus therapy was effective for inducing clinical and endoscopic remission of steroid-refractory/dependent UC. Endoscopic improvement was associated with favorable medium- and long-term prognosis.

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Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis

Huch Meritxell, Bonfanti Paola, Boj Sylvia F., Sato Toshiro, Loomans Cindy J M, Van De Wetering Marc, Sojoodi Mozhdeh, Li Vivian S W, Schuijers Jurian, Gracanin Ana, Ringnalda Femke, Begthel Harry, Hamer Karien, Mulder Joyce, Van Es Johan H., De Koning Eelco, Vries Robert G J, Heimberg Harry, Clevers Hans

EMBO Journal 32 ( 20 ) 2708 - 2721 2013.10

Research paper (scientific journal), Joint Work, ISSN 0261-4189

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5 + stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.

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[Intestinal stem cells].

Sato Toshiro

Seikagaku. The Journal of Japanese Biochemical Society (The Japanese Biochemical Society) 85 ( 9 ) 743 - 748 2013.09

Research paper (scientific journal), Accepted, ISSN 0037-1017

Establishment of novel prediction system of intestinal absorption in humans using human intestinal tissues

Miyake Masateru, Toguchi Hajime, Nishibayashi Toru, Higaki Kazutaka, Sugita Akira, Koganei Kazutaka, Kamada Nobuhiko, Kitazume Mina T., Hisamatsu Tadakazu, Sato Toshiro, Okamoto Susumu, Kanai Takanori, Hibi Toshifumi

Journal of Pharmaceutical Sciences 102 ( 8 ) 2564 - 2571 2013.08

Research paper (scientific journal), Joint Work, ISSN 0022-3549

The objective of this study was to establish a novel prediction system of drug absorption in humans by utilizing human intestinal tissues. Based on the transport index (TI), a newly defined parameter, calculated by taking account of the change in drug concentrations because of precipitation on the apical side and the amounts accumulated in the tissue and transported to the basal side, the absorbability of drugs in rank order as well as the fraction of dose absorbed (Fa) in humans were estimated. Human intestinal tissues taken from ulcerative colitis or Crohn's disease patients were mounted in a mini-Ussing chamber and transport studies were performed to evaluate the permeation of drugs, including FD-4, a very low permeable marker, atenolol, a low permeable marker, and metoprolol, a high permeable marker. Although apparent permeability coefficients calculated by the conventional equation did not reflect human Fa values for FD-4, atenolol, and metoprolol, TI values were well correlated with Fa values, which are described by 100 · [1 - e ^{- f · (TI - α)}]. Based on this equation, Fa values in humans for other test drugs were predicted successfully, indicating that our new system utilizing human intestinal tissues would be valuable for predicting oral drug absorption in humans.

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A single strain of Clostridium butyricum induces intestinal IL-10-producing macrophages to suppress acute experimental colitis in mice

Hayashi Atsushi, Sato Toshiro, Kamada Nobuhiko, Mikami Yohei, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Hibi Toshifumi, Roers Axel, Yagita Hideo, Ohteki Toshiaki, Yoshimura Akihiko, Kanai Takanori

Cell Host and Microbe 13 ( 6 ) 711 - 722 2013.06

Research paper (scientific journal), Joint Work, ISSN 1931-3128

Imbalance in gut bacterial composition provokes host proinflammatory responses causing diseases such as colitis. Colonization with a mixture of Clostridium species from clusters IV and XIVa was shown to suppress colitis through the induction of IL-10-producing regulatory T (Treg) cells. We demonstrate that a distinct Clostridium strain from cluster I, Clostridium butyricum (CB), prevents acute experimental colitis in mice through induction of IL-10, an anti-inflammatory cytokine. However, while CB treatment had no effect on IL-10 production by T cells, IL-10-producing F4/80⁺CD11b ⁺CD11c^int macrophages accumulated in the inflamed mucosa after CB treatment. CB directly triggered IL-10 production by intestinal macrophages in inflamed mucosa via the TLR2/MyD88 pathway. The colitis-preventing effect of CB was negated in macrophage-specific IL-10-deficient mice, suggesting that induction of IL-10 by intestinal macrophages is crucial for the probiotic action of CB. Collectively, CB promotes IL-10 production by intestinal macrophages in inflamed mucosa, thereby preventing experimental colitis in mice.

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TGR5 signalling inhibits the production of pro-inflammatory cytokines by in vitro differentiated inflammatory and intestinal macrophages in Crohn's disease

Yoneno Kazuaki, Hisamatsu Tadakazu, Shimamura Katsuyoshi, Kamada Nobuhiko, Ichikawa Riko, Kitazume Mina T., Mori Maiko, Uo Michihide, Namikawa Yuka, Matsuoka Katsuyoshi, Sato Toshiro, Koganei Kazutaka, Sugita Akira, Kanai Takanori, Hibi Toshifumi

Immunology 139 ( 1 ) 19 - 29 2013.05

Research paper (scientific journal), Joint Work, ISSN 0019-2805

Bile acids (BAs) play important roles not only in lipid metabolism, but also in signal transduction. TGR5, a transmembrane receptor of BAs, is an immunomodulative factor, but its detailed mechanism remains unclear. Here, we aimed to delineate how BAs operate in immunological responses via the TGR5 pathway in human mononuclear cell lineages. We examined TGR5 expression in human peripheral blood monocytes, several types of in vitro differentiated macrophages (Mφ{symbol}s) and dendritic cells. Mφ{symbol}s differentiated with macrophage colony-stimulating factor and interferon-γ (Mγ-Mφ{symbol}s), which are similar to the human intestinal lamina propria CD14⁺ Mφ{symbol}s that contribute to Crohn's disease (CD) pathogenesis by production of pro-inflammatory cytokines, highly expressed TGR5 compared with any other type of differentiated Mφ{symbol} and dendritic cells. We also showed that a TGR5 agonist and two types of BAs, deoxycholic acid and lithocholic acid, could inhibit tumour necrosis factor-α production in Mγ-Mφ{symbol}s stimulated by commensal bacterial antigen or lipopolysaccharide. This inhibitory effect was mediated by the TGR5-cAMP pathway to induce phosphorylation of c-Fos that regulated nuclear factor-κB p65 activation. Next, we analysed TGR5 levels in lamina propria mononuclear cells (LPMCs) obtained from the intestinal mucosa of patients with CD. Compared with non-inflammatory bowel disease, inflamed CD LPMCs contained more TGR5 transcripts. Among LPMCs, isolated CD14⁺ intestinal Mφ{symbol}s from patients with CD expressed TGR5. In isolated intestinal CD14⁺ Mφ{symbol}s, a TGR5 agonist could inhibit tumour necrosis factor-α production. These results indicate that TGR5 signalling may have the potential to modulate immune responses in inflammatory bowel disease.

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IL-22-Producing RORγt-Dependent Innate Lymphoid Cells Play a Novel Protective Role in Murine Acute Hepatitis

Matsumoto Atsuhiro, Kanai Takanori, Mikami Yohei, Chu Po Sung, Nakamoto Nobuhiro, Ebinuma Hirotoshi, Saito Hidetsugu, Sato Toshiro, Yagita Hideo, Hibi Toshifumi

PLoS One 8 ( 4 ) 2013.04

Research paper (scientific journal), Joint Work, Accepted

<p>Retinoid-related orphan receptor (ROR) γt is known to be related to the development and function of various immunological compartments in the liver, such as Th17 cells, natural killer T (NKT) cells, and innate lymphoid cells (ILCs). We evaluated the roles of RORγt-expressing cells in mouse acute hepatitis model using RORγt deficient (RORγt^-/-) mice and RAG-2 and RORγt double deficient (RAG-2^-/- × RORγt^-/-) mice. Acute hepatitis was induced in mice by injection with carbon tetrachloride (CCl₄), to investigate the regulation of liver inflammation by RORγt-expressing cells. We detected RORC expression in three compartments, CD4⁺ T cells, NKT cells, and lineage marker-negative SCA-1⁺Thy1^high ILCs, of the liver of wild type (WT) mice. CCl₄-treated RORγt^-/- mice developed liver damage in spite of lack of RORγt-dependent cells, but with reduced infiltration of macrophages compared with WT mice. In this regard, ILCs were significantly decreased in RAG-2^-/- × RORγt^-/- mice that lacked T and NKT cells. Surprisingly, RAG-2^-/- × RORγt^-/- mice developed significantly severer CCl₄-induced hepatitis compared with RAG-2^-/- mice, in accordance with the fact that hepatic ILCs failed to produce IL-22. Lastly, anti-Thy1 monoclonal antibody (mAb), but not anti-NK1.1 mAb or anti-asialo GM1 Ab administration exacerbated liver damage in RAG-2^-/- mice with the depletion of liver ILCs. Collectively, hepatic RORγt-dependent ILCs play a part of protective roles in hepatic immune response in mice.</p>

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In vitro expansion of single Lgr5 + liver stem cells induced by Wnt-driven regeneration

Huch Meritxell, Dorrell Craig, Boj Sylvia F., Van Es Johan H., Li Vivian S W, Van De Wetering Marc, Sato Toshiro, Hamer Karien, Sasaki Nobuo, Finegold Milton J., Haft Annelise, Vries Robert G., Grompe Markus, Clevers Hans

Nature 494 ( 7436 ) 247 - 250 2013.02

Research paper (scientific journal), Joint Work, ISSN 0028-0836

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5⁺ stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ + cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5⁺ cells generate hepatocytes and bile ducts in vivo. Single Lgr5⁺ cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah-/-mice. These findings indicate that previous observations concerning Lgr5⁺ stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.

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Dendritic cells administered intrarectally penetrate the intestinal barrier to break intestinal tolerance via Th2-medeiated colitis in mice

Handa Tango, Kanai Takanori, Sato Toshiro, Mikami Yohei, Sujino Tomohisa, Hayashi Atsushi, Mizuno Shinta, Matsumoto Atsuhiro, Hisamatsu Tadakazu, Hibi Toshifumi

Immunology Letters 150 ( 1-2 ) 123 - 129 2013.02

Research paper (scientific journal), Joint Work, ISSN 0165-2478

Intestinal lamina propria dendritic cells (LPDCs) in mice are known to extend dendrites between the intestinal epithelia and the luminal side when processing luminal antigens. We conducted intrarectal cell transfer experiments of bone marrow-derived dendritic cells (BMDCs) in mice to assess dendritic cell penetration of the intestine. Intrarectally administered GFP⁺ BMDCs localized in the colonic LP within 3h and the spleen within 12h after administration. 72h after administration, recipient C57BL/6 mice showed acute diarrhea, and administration of BMDCs (once weekly for 3 weeks) induced intestinal inflammation with increased numbers of recipient macrophages and CD4⁺ T cells exhibiting a Th2-mediated immune response. These results demonstrate that DCs actively communicate across the intestinal barrier, and highlight a potential technique for controlling colonic immune tolerance.

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Primary mouse small intestinal epithelial cell cultures

Sato Toshiro, Clevers Hans

Methods in Molecular Biology (Springer) 945 319 - 328 2013

Joint Work, Accepted

<p>The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently shown that Lgr5 (Leucine-rich repeat-containing G protein-coupled receptor) is expressed in intestinal stem cells by an in vivo genetic lineage tracing strategy. In the past, extensive efforts have been made to establish primary small intestinal culture systems. However, no defined, reproducible and robust culture system had been developed. To establish such a system, we screened for optimal growth factor combinations based on genetic evidence of self-renewal regulation, differentiation, and carcinogenesis of intestinal stem cells. Here, we describe methods that we have established for the isolation and culture of primary small intestinal epithelial stem cells. In this culture system, isolated crypts form organoid structures with a histological hierarchy recapitulating in vivo small intestinal epithelium. Single isolated Lgr5+ intestinal stem cells also form these organoid structures, in which stem cells are maintained by self-renewal and give rise to all lineages of the intestinal epithelium. This culture system is particularly useful for studying the regulation of intestinal stem cell self-renewal and differentiation.</p>

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Growing self-organizing mini-guts from a single intestinal stem cell

Sato Toshiro, Clevers Hans

Science 340 ( 6137 ) 1190 - 1194 2013

Research paper (scientific journal), Joint Work, ISSN 0036-8075

Recent examples have highlighted how stem cells have the capability to initiate morphogenesis in vitro; that is, to generate complex structures in culture that closely parallel their in vivo counterparts. Lgr5, the receptor for the Wnt-agonistic R-spondins, marks stem cells in multiple adult organs of mice and humans. In R-spondin-based three-dimensional cultures, these Lgr5 stem cells can grow into ever-expanding epithelial organoids that retain their original organ identity. Single Lgr5 stem cells derived from the intestine can be cultured to build epithelial structures that retain hallmarks of the in vivo epithelium. Here, we review the mechanisms that support this notable example of self-organization and discuss applications of this technology for stem cell research, disease modeling (e.g., for colorectal cancer and cystic fibrosis), and regenerative medicine.

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Dysregulated balance of retinoid-related orphan receptor γt-dependent innate lymphoid cells is involved in the pathogenesis of chronic DSS-induced colitis

Kimura Kayoko, Kanai Takanori, Hayashi Atsushi, Mikami Yohei, Sujino Tomohisa, Mizuno Shinta, Handa Tango, Matsuoka Katsuyoshi, Hisamatsu Tadakazu, Sato Toshiro, Hibi Toshifumi

Biochemical and Biophysical Research Communications 427 ( 4 ) 694 - 700 2012.11

Research paper (scientific journal), Joint Work, ISSN 0006-291X

Retinoid-related orphan receptor (ROR) γt-expressing and IL-22-producing NKp46⁺ innate lymphoid (ILC22) cells reside in the lamina propria of the intestine in mice, suggesting that ILC22 cells contribute to host defense during intestinal damage in models of colitis in mice. Nevertheless, another set of pathological interferon (IFN)-γ and/or IL-17A-producing innate lymphoid cells (ILC1 and ICL17) may participate in the pathogenesis in different models of colitis. We here showed that RORγt⁺IL-22⁺ ILC22 cells were localized in Thy-1^highSCA-1^high and/or Thy-1^highSCA-1^low subpopulations of the intestine in normal and dextran sodium sulfate (DSS)-induced colitic RORγt-sufficient Rag-2^-/- mice. RORγt-deficient Rag-2^-/- mice developed more severe DSS-induced colitis accompanied with lower expression of REG3β and REG3γ in the colon, but with a lower ratio and absolute number of IFN-γ-producing ILC1 cells as compared to control RORγt-sufficient Rag-2^-/- mice. Collectively, not only the presence of ILC22 cells but also the balance of protective and pathogenic ILCs may be involved in the prevention of colitis.

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Dll1 + secretory progenitor cells revert to stem cells upon crypt damage

Van Es Johan H., Sato Toshiro, Van De Wetering Marc, Lyubimova Anna, Yee Nee Annie Ng, Gregorieff Alex, Sasaki Nobuo, Zeinstra Laura, Van Den Born Maaike, Korving Jeroen, Martens Anton C M, Barker Nick, Van Oudenaarden Alexander, Clevers Hans

Nature Cell Biology 14 ( 10 ) 1099 - 1104 2012.10

Research paper (scientific journal), Joint Work, ISSN 1465-7392

Lgr5 + intestinal stem cells generate enterocytes and secretory cells. Secretory lineage commitment requires Notch silencing. The Notch ligand Dll1 is expressed by a subset of immediate stem cell daughters. Lineage tracing in Dll1 GFP-ires-CreERT2 knock-in mice reveals that single Dll1 high cells generate small, short-lived clones containing all four secretory cell types. Lineage specification thus occurs in immediate stem cell daughters through Notch lateral inhibition. Cultured Dll1 high cells form long-lived organoids (mini-guts) on brief Wnt3A exposure. When Dll1 high cells are genetically marked before tissue damage, stem cell tracing events occur. Thus, secretory progenitors exhibit plasticity by regaining stemness on damage.

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On the biomechanics of stem cell niche formation in the gut - Modelling growing organoids

Buske Peter, Przybilla Jens, Loeffler Markus, Sachs Norman, Sato Toshiro, Clevers Hans, Galle Joerg

FEBS Journal 279 ( 18 ) 3475 - 3487 2012.09

Research paper (scientific journal), Joint Work, ISSN 1742-464X

In vitro culture of intestinal tissue has been attempted for decades. Only recently did Sato et al. [Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J., et al. (2009) Nature459, 262-265] succeed in establishing long-term intestinal culture, demonstrating that cells expressing the Lgr5 gene can give rise to organoids with crypt-like domains similar to those found in vivo. In these cultures, Paneth cells provide essential signals supporting stem cell function. We have recently developed an individual cell-based computational model of the intestinal tissue [Buske, P., Galle, J., Barker, N., Aust, G., Clevers, H. & Loeffler, M. (2011) PLoS Comput Biol7, e1001045]. The model is capable of quantitatively reproducing a comprehensive set of experimental data on intestinal cell organization. Here, we present a significant extension of this model that allows simulation of intestinal organoid formation in silico. For this purpose, we introduce a flexible basal membrane that assigns a bending modulus to the organoid surface. This membrane may be re-organized by cells attached to it depending on their differentiation status. Accordingly, the morphology of the epithelium is self-organized. We hypothesize that local tissue curvature is a key regulatory factor in stem cell organization in the intestinal tissue by controlling Paneth cell specification. In simulation studies, our model closely resembles the spatio-temporal organization of intestinal organoids. According to our results, proliferation-induced shape fluctuations are sufficient to induce crypt-like domains, and spontaneous tissue curvature induced by Paneth cells can control cell number ratios. Thus, stem cell expansion in an organoid depends sensitively on its biomechanics. We suggest a number of experiments that will enable new insights into mechano-transduction in the intestine, and suggest model extensions in the field of gland formation.

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CCR9⁺ macrophages are required for eradication of peritoneal bacterial infections and prevention of polymicrobial sepsis

Mizukami Takahiro, Kanai Takanori, Mikami Yohei, Hayashi Atsushi, Doi Tomomitsu, Handa Tango, Matsumoto Atsuhiro, Jun Li, Matsuoka Katsuyoshi, Sato Toshiro, Hisamatsu Tadakazu, Hibi Toshifumi

Immunology Letters 147 ( 1-2 ) 75 - 79 2012.09

Research paper (scientific journal), Joint Work, ISSN 0165-2478

Sepsis is a systemic inflammatory response to infection associated with multiple organ dysfunction syndrome and a high mortality rate. In septic shock induced by severe peritonitis, early response of peritoneal macrophages against infected microbes is vital in preventing the spread of infection. We found that the mucosal homing receptor CCR9, is induced in peritoneal macrophages in response to inflammatory stimulation. We used a cecal ligation and puncture (CLP) model of sepsis to determine the role of CCR9 with respect to peritoneal macrophages, and controlling peritoneal infection and systemic inflammation. CCR9^-/- mice showed aggravated septic shock with higher mortality rates compared with wild-type (WT) mice. Six hours after CLP, CCR9^-/- mice demonstrated a greater inflammatory response. This was associated with higher production of inflammatory cytokines, such as IL-6, TNF and IP-10 in peritoneal lavage compared with WT mice. Although the numbers of peritoneal bacteria were elevated in CCR9^-/- mice subjected to CLP compared with WT mice, this was normalized in CCR9^-/- mice subjected to CLP through the adoptive transfer of WT peritoneal macrophages. We conclude that CCR9 is required for recruitment of peritoneal macrophages in the steady state to control systemic sepsis during early phases of peritoneal infection.

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The use of infliximab in the prevention of postsurgical recurrence in polysurgery Crohn's disease patients

Sakuraba Atsushi, Sato Toshiro, Matsukawa Hidehiko, Okamoto Susumu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

International Journal of Colorectal Disease 27 ( 7 ) 947 - 952 2012.07

Research paper (scientific journal), Joint Work, ISSN 0179-1958

Purpose Crohn's disease (CD) commonly recurs after surgery, and a number of patients need repeated surgery, especially smokers and those with repeated surgeries or penetrating disease. Whether infliximab prevents postsurgical recurrence in high-risk CD remains unknown. In the present pilot openlabeled study, we investigated the safety and efficacy of scheduled infliximab, which was started early after surgery, in maintaining remission of CD patients who have undergonemultiple surgeries due to penetrating disease. Methods Eleven patients (nine male, two female; age range, 26-48 years) who had undergone repeated surgeries (median, 4; range, 2-5) for penetrating disease were enrolled. Two to 4 weeks after surgery, the patients were started on intravenous infliximab (5 mg/kg) at an 8-week interval. The primary end points were the proportion of patients in clinical remission at the end of the study, the rate of endoscopic/ radiologic remission at 24 months, and the rate of adverse effects. Results One patient dropped out due to non-compliance, and ten patients were eligible for analysis. Clinical remission was maintained in six of ten patients (60.0%) at the end of the study. At 24 months, four out of ten patients were in endoscopic or radiological remission (40.0%). Two patients experienced adverse effects (18.2%), one of whom elected to withdraw from the study. Conclusion The findings of no major safety concern and possible clinical benefit in our study suggest that further investigation of infliximab as a treatment for prevention of postsurgical recurrence in high-risk CD is warranted.

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Intermittent Granulocyte and Monocyte Apheresis Versus Mercaptopurine for Maintaining Remission of Ulcerative Colitis

Sakuraba Atsushi, Sato Toshiro, Morohoshi Yuichi, Matsuoka Katsuyoshi, Okamoto Susumu, Inoue Nagamu, Takaishi Hiromasa, Ogata Haruhiko, Iwao Yasushi, Hibi Toshifumi

Therapeutic Apheresis and Dialysis 16 ( 3 ) 213 - 218 2012.06

Research paper (scientific journal), Joint Work, ISSN 1744-9979

The effect of granulocyte and monocyte adsorption apheresis (GMA) on prevention of relapse of ulcerative colitis (UC) is not clear. This was a pilot open-labeled, prospective, randomized, unblinded study to compare the tolerability and efficacy of intermittent GMA (once every 2weeks) with mercaptopurine to maintain remission of UC. Twenty-one patients with UC, who had achieved remission by induction therapies were randomly assigned to receive either intermittent GMA (N=10) or oral mercaptopurine (0.5mg/kg per day; N=11). The study period was 24months. The rate of the patients maintaining remission and the incidences of adverse effects were compared between the two groups. At 24months, seven of 10 patients (70.0%) on intermittent GMA and seven of 11 patients (63.6%, P=1.00) on oral mercaptopurine were still in remission. Three patients relapsed in each group. One patient taking mercaptopurine, but none receiving intermittent GMA, dropped out because of adverse effects. Intermittent therapy with GMA was well tolerated and a substantial proportion of patients maintained remission. Intermittent GMA therapy in maintaining remission of UC merits further investigation.

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