L-2
Identifying of the causative genes in spontaneous mutant mouse strains by forward-reverse genetics ○Seiya Mizuno Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan
The forward genetic analysis with spontaneous mutant gives us new insights of gene function in vivo. I also had found three mutant strains (TAS, Moja, and WS) in our laboratory.The TAS is a transgenic mouse carried rtTA and tTS. Although these transgenes does not affect brain development, TAS mouse showed agenesis of the corpus callosum (ACC). The FISH analyses revealed that both rtTA and tTS were inserted between 11 and 13 Mb on Chr. 18. I then found abnormal Cables1 gene expression in TAS. The exon4 of Cables1 was deleted by transgene insertion. Interestingly, spliced exon 1-3 of Cables1 was fused to transgene in mRNA level. I concluded ACC in TAS is caused by effect of mutated Cables1 fused to transgenes.The Moja mouse is spontaneous mutant which showed long hair. Because the phenotypes of Maja were very similar to Fgf5 null mutant mouse, we analyzed Fgf5 gene locus in Moja. As expected, Fgf5 was not expressed in Moja skin caused by lack of exon3 of Fgf5 by natural LTR insertion mutation.The WS mutant embryos failed to implantation. FISH and SNPs analyses revealed that approximately 1.2 Mb genomic region containing 9 genes (from Kit to Cep135) was deleted in WS. Since the Exoc1, one of these 9 genes, was highly expressed in early embryos, I selected the Exoc1 as first candidate of causative gene for early embryonic death in WS. As expected, Exoc1 null mutant showed embryonic death same as WS. These results indicate lethal phenotype in WS is caused by Exoc1 deletion.
S1-1
Germfree and gnotobiotic animals ○Kazuhiro Hirayama Department of Veterinary Public Health, The University of Tokyo, Tokyo, Japan
Intestinal microbiota plays important roles in health and diseases. However, it is often very difficult to study the role of intestinal microbiota, partly due to its extremely diverse composition and very special environment in the intestine including highly anaerobic condition. By comparing conventional and germfree animals, with and without intestinal microbiota, role of microbiota and their effects on hosts, both beneficial and harmful, can be studied in vivo. At the very beginning of the germfree animal research, major interest on the great efforts to establish germfree animal colonies was whether life without bacteria would be possible or not. Since germfree technology has been established, germfree animals have been used to study physiology and pathology of the host without interference of indigenous microbiota. Infectious diseases have been one of the most important subjects in germfree research. Germfree animals have also been playing significant contributions to the understanding of nutrition and immunological phenomena. Germfree animals inoculated with known bacterial strain(s), gnotobiotic animals, have revealed the role of particular bacterial strain(s) or species. Recently, germfree genetically modified rodents which are used as disease models demonstrated important role of intestinal microbiota for expression of pathophysiology.
—S 4—
S1-2
Gut micrrobiome-derived short chain fatty acids in host defense and immunity ○Hiroshi Ohno RIKEN Cener for Integrative Medical Sciences
Animal gut, is colonized with a huge number of commensal bacteria; for example, the human colon is colonized with more than 100 trillions of commensal microbes classified into 500~1,000 species, collectively called gut microbiota. These microbes closely communicate each other to constitute complex metabolic pathways and further interact with the host to establish the unique gut ecosystem, which deeply impacts host physiology and pathology including host defense and immunity. However, the underlying mechanisms of how gut ecosystem influences host defense and immune system have poorly understood.We have proposed an integrated multiomics approach, where different levels of exhaustive analyses such as (meta)genomics, (meta)transcriptomics and metabolomics are combined.By applying the integrative multi-omics approach, we have shown that Bifidobacterium-derived acetate can modify gene expression of the colonic epithelium to confer resistance against enterohemorrhagic Escherichia coli O157, which ultimately protects mice from O157-infectious death. We have also found that butyrate produced by gut micirobiota can enhance differentiation of colonic regulatory T (Treg) cells from naive T cells, via epigenetic modification through its histone deacetylase inhibitory ability.Therefore, the integrative multi-omics approach is a suitable way to understand the complex gut ecosystem.
S1-3
Gut microbial metabolites, short-chain fatty acids and obesity ○Ikuo Kimura Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
Food intake regulates energy balance and its dysregulation leads to metabolic disorder, such as obesity and diabetes. During feeding, gut microbiota affects host nutrient acquisition and energy regulation and can influence the development of obesity and diabetes. Short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate, which are produced by gut microbial fermentation of dietary fiber, are recognized as essential host energy sources and act as signal transduction molecules via G-protein coupled receptors (GPR41, GPR43, OLFR78, GPR109A) and as epigenetic regulators of gene expression by the inhibition of histone deacetylase. Recent evidence suggests that dietary fiber and the gut microbial-derived SCFAs exert multiple beneficial effects on the host energy metabolism not only by improving the intestinal environment, but also by directly affecting various host peripheral tissues. We report that GPR41 regulates sympathetic activity, and GPR43 regulates adipose-insulin signaling by sensing SCFAs produced by gut microbiota. We believe that these results will provide valuable insights into therapeutic targets for treating metabolic disorder and the use of probiotics to control gut microbiota.
—S 5—
S1-4
The mechanism of obesity-associated liver cancer development through gut microbial metabolite ○Naoko Ohtani The Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science
Over the past few decades, obesity has become more prevalent in most developed countries, and is increasingly recognized as a risk factor of cancer. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Recently, we found that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumor-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to certain aspects of obesity-associated HCC development in humans as well.
S1-5
Intestinal microbiota and allergy and inflammation ○Akira Shibuya Department of Immunology,Faculty of Medicine, Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan
Imbalances in gut microbiota composition, described as dysbiosis, are caused by many factors, including host genetics, lifestyle, and exposure to microorganisms or various medical procedures. Dysbiosis has been associated not only with intestinal inflammation but also with many diseases outside the gut, such as atopic dermatitis, allergy, obesity, and diabetes. However, how dysbiosis in gut microbiota composition influences the diseases outside the gut. In this symposium, we show that overgrowth of gut fungi induced by antibiotics (Abx )-treatment promoted alternatively activated M2 macrophage polarization in the lung. Abx-treated mice exhibited increased plasma concentration of prostaglandin E2. Treatment with an antifungal drug or a cyclooxygenase inhibitor aspirin, as well as serum amyloid P, suppressed M2 macrophage polarization and attenuated allergic airway inflammation of Abx-treated mice. These results suggest that control of M2 macrophage activation is a promising therapeutic approach for a certain patients with dysbiosis-related allergic inflammatory disorders.
—S 6—
S1-6
New strategy of IBD treatment using microbiota ○Takanori Kanai Department and Hepatology, Keio University School of Medicine,Tokyo,Japan
Both the incidence of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s diseases (CD), are rapidly increasing, not only in western countries, but also in developed Asian countries. In Japan, approximately 160,000 patients with UC and 40,000 with CD are currently registered by Japanese Health, Labour and Welfare Ministry. Although biologic agents targeting the immune system have been effective in patients with IBD, cessation of treatment leads to relapse in almost all patients, suggesting that immune dysregulation is an effect, not a cause, of IBD. Advances in next-generation gene sequencing technology have resulted in the identification of over 160 IBD-associated susceptibility genes within the past 10 years. These susceptibility genes, however, are unlikely to be the main cause of IBD, because in the past 50 years the numbers of IBD patients have increased 100-fold in Japan. It is more likely that dramatic changes in the social environment, especially dietary habits leading to the dysregulated composition of intestinal microbiota or dysbiosis, are fundamental causes of IBD. In Japan, upgrading of the water supply and sewerage systems, abuse of antibiotics, and especially dietary habits are now very similar to those in developed western countries. This review talk will provide current topics regarding new treatment using microbiota including probiotics.
MS1-1 AVMA Guidelines for the Euthanasia of Animals: 2013 Edition ○Makoto Suzuki Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
Article 40 of the Law for Humane Treatment and Management of Animals, mandates, ‟In case an animal must be destroyed, a method that minimizes pain and distress to the animal as much as possible shall be used.” The Guidelines for euthanasia of Animals state, ‟Administrator and operator should understand physiology and ecology, and should have appropriate euthanasia methods to minimize distress.” They further direct, ‟When euthanizing an animal, chemical or physical methods for euthanasia must render animals unconscious with minimal pain and distress, and they should stop cardiac or respiratory function.” However, these regulations include no description of euthanasia techniques, even though the Guidelines for Proper Conducts of Animal Experiments require detailed methods regarding final disposition of experimental animals. IACAC reviews euthanasia protocols from the point of scientific justification and humanness, while allowing the gathering of valid data. IACUC investigates whether animals are unconscious prior to death, whether euthanasia processes employed prevent unnecessary pain and distress, and whether they minimize risk to people and animals. ‟AVMA Guidelines for the Euthanasia of Animals: 2013” cover euthanasia methods for mammals, birds, reptiles, amphibians and finned fishes. Here I will introduce appropriate euthanasia methods for laboratory animals of different species or age.
—S 7—
Identifying of the causative genes in spontaneous mutant mouse strains by forward-reverse genetics ○Seiya Mizuno Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan
The forward genetic analysis with spontaneous mutant gives us new insights of gene function in vivo. I also had found three mutant strains (TAS, Moja, and WS) in our laboratory.The TAS is a transgenic mouse carried rtTA and tTS. Although these transgenes does not affect brain development, TAS mouse showed agenesis of the corpus callosum (ACC). The FISH analyses revealed that both rtTA and tTS were inserted between 11 and 13 Mb on Chr. 18. I then found abnormal Cables1 gene expression in TAS. The exon4 of Cables1 was deleted by transgene insertion. Interestingly, spliced exon 1-3 of Cables1 was fused to transgene in mRNA level. I concluded ACC in TAS is caused by effect of mutated Cables1 fused to transgenes.The Moja mouse is spontaneous mutant which showed long hair. Because the phenotypes of Maja were very similar to Fgf5 null mutant mouse, we analyzed Fgf5 gene locus in Moja. As expected, Fgf5 was not expressed in Moja skin caused by lack of exon3 of Fgf5 by natural LTR insertion mutation.The WS mutant embryos failed to implantation. FISH and SNPs analyses revealed that approximately 1.2 Mb genomic region containing 9 genes (from Kit to Cep135) was deleted in WS. Since the Exoc1, one of these 9 genes, was highly expressed in early embryos, I selected the Exoc1 as first candidate of causative gene for early embryonic death in WS. As expected, Exoc1 null mutant showed embryonic death same as WS. These results indicate lethal phenotype in WS is caused by Exoc1 deletion.
S1-1
Germfree and gnotobiotic animals ○Kazuhiro Hirayama Department of Veterinary Public Health, The University of Tokyo, Tokyo, Japan
Intestinal microbiota plays important roles in health and diseases. However, it is often very difficult to study the role of intestinal microbiota, partly due to its extremely diverse composition and very special environment in the intestine including highly anaerobic condition. By comparing conventional and germfree animals, with and without intestinal microbiota, role of microbiota and their effects on hosts, both beneficial and harmful, can be studied in vivo. At the very beginning of the germfree animal research, major interest on the great efforts to establish germfree animal colonies was whether life without bacteria would be possible or not. Since germfree technology has been established, germfree animals have been used to study physiology and pathology of the host without interference of indigenous microbiota. Infectious diseases have been one of the most important subjects in germfree research. Germfree animals have also been playing significant contributions to the understanding of nutrition and immunological phenomena. Germfree animals inoculated with known bacterial strain(s), gnotobiotic animals, have revealed the role of particular bacterial strain(s) or species. Recently, germfree genetically modified rodents which are used as disease models demonstrated important role of intestinal microbiota for expression of pathophysiology.
—S 4—
S1-2
Gut micrrobiome-derived short chain fatty acids in host defense and immunity ○Hiroshi Ohno RIKEN Cener for Integrative Medical Sciences
Animal gut, is colonized with a huge number of commensal bacteria; for example, the human colon is colonized with more than 100 trillions of commensal microbes classified into 500~1,000 species, collectively called gut microbiota. These microbes closely communicate each other to constitute complex metabolic pathways and further interact with the host to establish the unique gut ecosystem, which deeply impacts host physiology and pathology including host defense and immunity. However, the underlying mechanisms of how gut ecosystem influences host defense and immune system have poorly understood.We have proposed an integrated multiomics approach, where different levels of exhaustive analyses such as (meta)genomics, (meta)transcriptomics and metabolomics are combined.By applying the integrative multi-omics approach, we have shown that Bifidobacterium-derived acetate can modify gene expression of the colonic epithelium to confer resistance against enterohemorrhagic Escherichia coli O157, which ultimately protects mice from O157-infectious death. We have also found that butyrate produced by gut micirobiota can enhance differentiation of colonic regulatory T (Treg) cells from naive T cells, via epigenetic modification through its histone deacetylase inhibitory ability.Therefore, the integrative multi-omics approach is a suitable way to understand the complex gut ecosystem.
S1-3
Gut microbial metabolites, short-chain fatty acids and obesity ○Ikuo Kimura Department of Applied Biological Science, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
Food intake regulates energy balance and its dysregulation leads to metabolic disorder, such as obesity and diabetes. During feeding, gut microbiota affects host nutrient acquisition and energy regulation and can influence the development of obesity and diabetes. Short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate, which are produced by gut microbial fermentation of dietary fiber, are recognized as essential host energy sources and act as signal transduction molecules via G-protein coupled receptors (GPR41, GPR43, OLFR78, GPR109A) and as epigenetic regulators of gene expression by the inhibition of histone deacetylase. Recent evidence suggests that dietary fiber and the gut microbial-derived SCFAs exert multiple beneficial effects on the host energy metabolism not only by improving the intestinal environment, but also by directly affecting various host peripheral tissues. We report that GPR41 regulates sympathetic activity, and GPR43 regulates adipose-insulin signaling by sensing SCFAs produced by gut microbiota. We believe that these results will provide valuable insights into therapeutic targets for treating metabolic disorder and the use of probiotics to control gut microbiota.
—S 5—
S1-4
The mechanism of obesity-associated liver cancer development through gut microbial metabolite ○Naoko Ohtani The Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science
Over the past few decades, obesity has become more prevalent in most developed countries, and is increasingly recognized as a risk factor of cancer. Although several events were proposed to be involved in obesity-associated cancer, the exact molecular mechanisms that integrate these events have remained largely unclear. Recently, we found that senescence-associated secretory phenotype (SASP) has crucial roles in promoting obesity-associated hepatocellular carcinoma (HCC) development in mice. Dietary or genetic obesity induces alterations of gut microbiota, thereby increasing the levels of deoxycholic acid (DCA), a gut bacterial metabolite known to cause DNA damage. The enterohepatic circulation of DCA provokes SASP phenotype in hepatic stellate cells (HSCs), which in turn secretes various inflammatory and tumor-promoting factors in the liver, thus facilitating HCC development in mice after exposure to chemical carcinogen. Notably, blocking DCA production or reducing gut bacteria efficiently prevents HCC development in obese mice. Similar results were also observed in mice lacking an SASP inducer or depleted of senescent HSCs, indicating that the DCA-SASP axis in HSCs has key roles in obesity-associated HCC development. Moreover, signs of SASP were also observed in the HSCs in the area of HCC arising in patients with non-alcoholic steatohepatitis, indicating that a similar pathway may contribute to certain aspects of obesity-associated HCC development in humans as well.
S1-5
Intestinal microbiota and allergy and inflammation ○Akira Shibuya Department of Immunology,Faculty of Medicine, Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan
Imbalances in gut microbiota composition, described as dysbiosis, are caused by many factors, including host genetics, lifestyle, and exposure to microorganisms or various medical procedures. Dysbiosis has been associated not only with intestinal inflammation but also with many diseases outside the gut, such as atopic dermatitis, allergy, obesity, and diabetes. However, how dysbiosis in gut microbiota composition influences the diseases outside the gut. In this symposium, we show that overgrowth of gut fungi induced by antibiotics (Abx )-treatment promoted alternatively activated M2 macrophage polarization in the lung. Abx-treated mice exhibited increased plasma concentration of prostaglandin E2. Treatment with an antifungal drug or a cyclooxygenase inhibitor aspirin, as well as serum amyloid P, suppressed M2 macrophage polarization and attenuated allergic airway inflammation of Abx-treated mice. These results suggest that control of M2 macrophage activation is a promising therapeutic approach for a certain patients with dysbiosis-related allergic inflammatory disorders.
—S 6—
S1-6
New strategy of IBD treatment using microbiota ○Takanori Kanai Department and Hepatology, Keio University School of Medicine,Tokyo,Japan
Both the incidence of inflammatory bowel diseases (IBD), including ulcerative colitis (UC) and Crohn’s diseases (CD), are rapidly increasing, not only in western countries, but also in developed Asian countries. In Japan, approximately 160,000 patients with UC and 40,000 with CD are currently registered by Japanese Health, Labour and Welfare Ministry. Although biologic agents targeting the immune system have been effective in patients with IBD, cessation of treatment leads to relapse in almost all patients, suggesting that immune dysregulation is an effect, not a cause, of IBD. Advances in next-generation gene sequencing technology have resulted in the identification of over 160 IBD-associated susceptibility genes within the past 10 years. These susceptibility genes, however, are unlikely to be the main cause of IBD, because in the past 50 years the numbers of IBD patients have increased 100-fold in Japan. It is more likely that dramatic changes in the social environment, especially dietary habits leading to the dysregulated composition of intestinal microbiota or dysbiosis, are fundamental causes of IBD. In Japan, upgrading of the water supply and sewerage systems, abuse of antibiotics, and especially dietary habits are now very similar to those in developed western countries. This review talk will provide current topics regarding new treatment using microbiota including probiotics.
MS1-1 AVMA Guidelines for the Euthanasia of Animals: 2013 Edition ○Makoto Suzuki Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan
Article 40 of the Law for Humane Treatment and Management of Animals, mandates, ‟In case an animal must be destroyed, a method that minimizes pain and distress to the animal as much as possible shall be used.” The Guidelines for euthanasia of Animals state, ‟Administrator and operator should understand physiology and ecology, and should have appropriate euthanasia methods to minimize distress.” They further direct, ‟When euthanizing an animal, chemical or physical methods for euthanasia must render animals unconscious with minimal pain and distress, and they should stop cardiac or respiratory function.” However, these regulations include no description of euthanasia techniques, even though the Guidelines for Proper Conducts of Animal Experiments require detailed methods regarding final disposition of experimental animals. IACAC reviews euthanasia protocols from the point of scientific justification and humanness, while allowing the gathering of valid data. IACUC investigates whether animals are unconscious prior to death, whether euthanasia processes employed prevent unnecessary pain and distress, and whether they minimize risk to people and animals. ‟AVMA Guidelines for the Euthanasia of Animals: 2013” cover euthanasia methods for mammals, birds, reptiles, amphibians and finned fishes. Here I will introduce appropriate euthanasia methods for laboratory animals of different species or age.
—S 7—
