RESEARCH ACTIVITIES

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Research in this division is directed toward understanding the complex interactionsthat occur between pathogenic bacteria and their human hosts at very early stageof bacterial infectious processes. Our special interest is focused upon the molecu-lar pathogenicity of enteropathogenic bacteria, such as Shigella, Helicobacter py-lori, enteropathogenic E. coli and enterohemorrhagic E. coli. We are also search-ing for effective methods to protect or regulate bacterial infection by using knowl-edge accumulated.

1. Helicobacter pylori CagA Phosphorylation-Independent Function in Epithelial Prolif-eration and Inflammation

Masato Suzuki, Hitomi Mimuro, Kotaro Kiga,Makoto Fukumatsu, Nozomi Ishijima, HanakoMorikawa, Shigenori Nagai1, Shigeo Koyasu1,Robert H. Gilman2, Dangeruta Kersulyte3,Douglas E. Berg3 and Chihiro Sasakawa: 1De-partment of Microbiology and Immunology,Keio University School of Medicine, 35 Shi-nanomachi, Shinjuku-ku, Tokyo 160-8582, Ja-pan, 2Department of Microbiology, Facultad deMedecina, Universidad Peruana Cayetano He-redia, Lima 31, Peru, 3Department of Molecu-lar Microbiology , Washington UniversitySchool of Medicine, St. Louis, MO 63110, USA

CagA, a major virulence factor of Helicobacterpylori (Hp), is delivered into gastric epithelialcells and exists in phosphorylated and nonphos-phorylated forms. The biological activity of thephosphorylated form is well established; how-ever, function(s) of the nonphosphorylated formremain elusive. Here, we report that a conservedmotif in the C-terminal region of CagA, which is

distinct from the EPIYA motifs used for phos-phorylation and which we designate CRPIA(conserved repeat responsible for phosphory-lation-independent activity), plays pivotal rolesin Hp pathogenesis. The CRPIA motif in non-phosphorylated CagA was involved in interact-ing with activated Met, the hepatocyte growthfactor receptor, leading to the sustained activa-tion of phosphatidylinositol 3-kinase/Akt signal-ing in response to Hp infection. This in turn ledto the activation of β-catenin and NF-κB signal-ing, which promote proliferation and inflamma-tion, respectively. Thus, nonphosphorylatedCagA activity contributes to the epithelial prolif-erative and proinflammatory responses associ-ated with development of chronic gastritis andgastric cancer.

2. Transient Shielding of Intimin and theType III Secretion System of Enterohemor-rhagic and Enteropathogenic Escherichiacoli by a Group 4 Capsule’

Yulia Shifrin1, Adi Peleg1, Ophir Ilan1, ChenNadler1, Simi Kobi1, Kobi Baruch1, GalYerushalmi1, Tatiana Berdichevsky1, Shoshy

Department of Microbiology and Immunology

Division of Bacterial Infection細菌感染分野

Professor Chihiro Sasakawa, Ph.D.Assistant Professor Hitomi Mimuro, Ph.D.Assistant Professor Michinaga Ogawa, Ph.D.Assistant Professor Masato Suzuki, Ph.D.

教 授 医学博士 笹 川 千 尋講 師 医学博士 三 室 仁 美助 教 医学博士 小 川 道 永助 教 医学博士 鈴 木 仁 人

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Altuvia1, Maya Elgrably-Weiss1, Cecilia Abe2,Stuart Knutton2, Chihiro Sasakawa, JenniferM. Ritchie3, Matthew K. Waldor3 and Ilan Ro-senshine1: 1Department of Molecular Geneticsand Biotechnology, The Hebrew UniversityFaculty of Medicine, POB 12272, Jerusalem91120, Israel, 2Institute of Child Health, Uni-versity of Birmingham, Whittall Street, Bir-mingham B4 6NH, United Kingdom, 3TheChanning Laboratory , Harvard MedicalSchool, 181 Longwood Ave., Boston Massachu-setts 02115

Enterohemorrhagic and enteropathogenic Es-cherichia coli (EHEC and EPEC, respectively)strains represent a major global health problem.Their virulence is mediated by the concerted ac-tivity of an array of virulence factors includingtoxins, a type III protein secretion system(TTSS), pili, and others. We previously showedthat EPEC O127 forms a group 4 capsule (G4C),and in this report we show that EHEC O157also produces a G4C, whose assembly is de-pendent on the etp, etk, and wzy genes. We fur-ther show that at early time points postinfection,these G4Cs appear to mask surface structuresincluding intimin and the TTSS. This maskinginhibited the attachment of EPEC and EHEC totissue-cultured epithelial cells, diminished theircapacity to induce the formation of actin pedes-tals, and attenuated TTSS-mediated proteintranslocation into host cells. Importantly, wefound that Ler, a positive regulator of intiminand TTSS genes, represses the expression of thecapsule-related genes, including etp and etk.Thus, the expression of TTSS and G4C is con-versely regulated and capsule production is di-minished upon TTSS expression. Indeed, at later

time points postinfection, the diminishing cap-sule no longer interferes with the activities of in-timin and the TTSS. Notably, by using the rabbitinfant model, we found that the EHEC G4C isrequired for efficient colonization of the rabbitlarge intestine. Taken together, our results sug-gest that temporal expression of the capsule,which is coordinated with that of the TTSS, isrequired for optimal EHEC colonization of thehost intestine.

3. Control of epithelial cell structure and de-velopmental fate: Lessons from Helicobac-ter pylori

Hitomi Mimuro, Douglas E. Berg1, ChihiroSasakawa: 1Departments of Molecular Microbi-ology, Genetics and Medicine, Washington Uni-versity School of Medicine, MO, USA

Valuable insights into eukaryotic regulatorycircuits can emerge from studying interactionsof bacterial pathogens such as Helicobacter pyloriwith host tissues. H. pylori uses a type IV secre-tion system (T4SS) to deliver its CagA virulenceprotein to epithelial cells, where much of it be-comes phosphorylated. CagA’s phosphorylatedand non-phosphorylated forms each interactwith host regulatory proteins to alter cell struc-ture and cell fate. Kwok and colleagues showedthat CagA destined for phosphorylation is deliv-ered using host integrin as receptor and H. py-lori’s CagL protein as an integrin-specific adhe-sin, and that CagL-integrin-binding activates thekinase cascade responsible for CagA phospho-rylation. This research contributes to under-standing infectious disease and the control ofcell fates.

Publications

Takeshima, E., Tomimori, K., Kawakami, H.,Ishikawa, C., Sawada, S., Tomita, M., Senba,M., Kinjo, F., Mimuro, H., Sasakawa, C., Fu-jita, J. & Mori, N. NF-kappaB activation byHelicobacter pylori requires Akt-mediated phos-phorylation of p65. BMC Microbiol 9, 36(2009).

Ogawa, M., Nakagawa, I., Yoshikawa, Y., Hain,T., Chakraborty, T. & Sasakawa, C. Strepto-coccus- , Shigella-, and Listeria-induced auto-phagy. Methods Enzymol 452, 363-81 (2009).

Takeshima, E., Tomimori, K., Teruya, H.,Ishikawa, C., Senba, M., D’Ambrosio, D.,Kinjo, F., Mimuro, H., Sasakawa, C.,Hirayama, T., Fujita, J. & Mori, N. Helicobacterpylori-Induced Interleukin-12 p40 Expression.Infect Immun (2009).

Suzuki, M., Mimuro, H., Kiga, K., Fukumatsu,M., Ishijima, N., Morikawa, H., Nagai, S.,Koyasu, S., Gilman, R.H., Kersulyte, D., Berg,D.E. & Sasakawa, C. Helicobacter pylori CagAphosphorylation-independent function in epi-thelial proliferation and inflammation. CellHost Microbe 5, 23-34 (2009).

Bronte-Tinkew, D.M., Terebiznik, M., Franco, A.,Ang, M., Ahn, D., Mimuro, H., Sasakawa, C.,Ropeleski, M.J., Peek, R.M., Jr. & Jones, N.L.Helicobacter pylori cytotoxin-associated gene Aactivates the signal transducer and activatorof transcription 3 pathway in vitro and invivo. Cancer Res 69, 632-9 (2009).

Ishigame, H., Kakuta, S., Nagai, T., Kadoki, M.,Nambu, A., Komiyama, Y., Fujikado, N.,Tanahashi, Y., Akitsu, A., Kotaki, H., Sudo,

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K., Nakae, S., Sasakawa, C. & Iwakura, Y. Dif-ferential roles of interleukin-17A and -17F inhost defense against mucoepithelial bacterialinfection and allergic responses. Immunity 30,108-19 (2009).

Shifrin, Y., Peleg, A., Ilan, O., Nadler, C., Kobi,S., Baruch, K., Yerushalmi, G., Berdichevsky,T., Altuvia, S., Elgrably-Weiss, M., Abe, C.,Knutton, S., Sasakawa, C., Ritchie, J.M., Wal-dor, M.K. & Rosenshine, I. Transient shieldingof intimin and the type III secretion system ofenterohemorrhagic and enteropathogenic Es-cherichia coli by a group 4 capsule. J Bacteriol190, 5063-74 (2008).

Mimuro, H., Berg, D.E. & Sasakawa, C. Controlof epithelial cell structure and developmentalfate: lessons from Helicobacter pylori . Bioessays30, 515-20 (2008).

Klionsky, D.J., Abeliovich, H., Agostinis, P.,Agrawal, D.K., Aliev, G., Askew, D.S., Baba,M., Baehrecke, E.H., Bahr, B.A., Ballabio, A.,Bamber, B.A., Bassham, D.C., Bergamini, E.,Bi, X., Biard-Piechaczyk, M., Blum, J.S., Brede-sen, D.E., Brodsky, J.L., Brumell, J.H., Brunk,U.T., Bursch, W., Camougrand, N., Cebollero,E., Cecconi, F., Chen, Y., Chin, L.S., Choi, A.,Chu, C.T., Chung, J., Clarke, P.G., Clark, R.S.,Clarke, S.G., Clave, C., Cleveland, J.L., Co-dogno, P., Colombo, M.I., Coto-Montes, A.,Cregg, J.M., Cuervo, A.M., Debnath, J., De-marchi, F., Dennis, P.B., Dennis, P.A., Deretic,V., Devenish, R.J., Di Sano, F., Dice, J.F., Di-figlia, M., Dinesh-Kumar, S., Distelhorst, C.W.,Djavaheri-Mergny, M., Dorsey, F.C., Droge,W., Dron, M., Dunn, W.A., Jr., Duszenko, M.,Eissa, N.T., Elazar, Z., Esclatine, A., Eskelinen,E.L., Fesus, L., Finley, K.D., Fuentes, J.M.,Fueyo, J., Fujisaki, K., Galliot, B., Gao, F.B.,Gewirtz, D.A., Gibson, S.B., Gohla, A., Gold-berg, A.L., Gonzalez, R., Gonzalez-Estevez, C.,Gorski, S., Gottlieb, R.A., Haussinger, D., He,Y.W., Heidenreich, K., Hill, J.A., Hoyer-Hansen, M., Hu, X., Huang, W.P., Iwasaki, A.,Jaattela, M., Jackson, W.T., Jiang, X., Jin, S., Jo-hansen, T., Jung, J.U., Kadowaki, M., Kang,C., Kelekar, A., Kessel, D.H., Kiel, J.A., Kim,

H.P., Kimchi, A., Kinsella, T.J., Kiselyov, K.,Kitamoto, K., Knecht, E., Komatsu, M., Komi-nami, E., Kondo, S., Kovacs, A.L., Kroemer,G., Kuan, C.Y., Kumar, R., Kundu, M., Lan-dry, J., Laporte, M., Le, W., Lei, H.Y., Le-nardo, M.J., Levine, B., Lieberman, A., Lim, K.L., Lin, F.C., Liou, W., Liu, L.F., Lopez-Berestein, G., Lopez-Otin, C., Lu, B., Macleod,K.F., Malorni, W., Martinet, W., Matsuoka, K.,Mautner, J., Meijer, A.J., Melendez, A., Mich-els, P., Miotto, G., Mistiaen, W.P., Mizushima,N., Mograbi, B., Monastyrska, I., Moore, M.N.,Moreira, P.I., Moriyasu, Y., Motyl, T., Munz,C., Murphy, L.O., Naqvi, N.I., Neufeld, T.P.,Nishino, I., Nixon, R.A., Noda, T., Nurnberg,B., Ogawa, M., Oleinick, N.L., Olsen, L.J., Oz-polat, B., Paglin, S., Palmer, G.E., Papassideri,I., Parkes, M., Perlmutter, D.H., Perry, G., Pi-acentini, M., Pinkas-Kramarski, R., Prescott,M., Proikas-Cezanne, T., Raben, N., Rami, A.,Reggiori, F., Rohrer, B., Rubinsztein, D.C.,Ryan, K.M., Sadoshima, J., Sakagami, H.,Sakai, Y., Sandri, M., Sasakawa, C., Sass, M.,Schneider, C., Seglen, P.O., Seleverstov, O.,Settleman, J., Shacka, J.J., Shapiro, I.M., Si-birny, A., Silva-Zacarin, E.C., Simon, H.U., Si-mone, C., Simonsen, A., Smith, M.A., Spanel-Borowski, K., Srinivas, V., Steeves, M., Sten-mark, H., Stromhaug, P.E., Subauste, C.S.,Sugimoto, S., Sulzer, D., Suzuki, T., Swanson,M.S., Tabas, I., Takeshita, F., Talbot, N.J., Tal-loczy, Z., Tanaka, K., Tanaka, K., Tanida, I.,Taylor, G.S., Taylor, J.P., Terman, A., Tet-tamanti, G., Thompson, C.B., Thumm, M.,Tolkovsky, A.M., Tooze, S.A., Truant, R., Tu-manovska, L.V., Uchiyama, Y., Ueno, T.,Uzcategui, N.L., van der Klei, I., Vaquero, E.C., Vellai, T., Vogel, M.W., Wang, H.G., Web-ster, P., Wiley, J.W., Xi, Z., Xiao, G., Yahalom,J., Yang, J.M., Yap, G., Yin, X.M., Yoshimori,T., Yu, L., Yue, Z., Yuzaki, M., Zabirnyk, O.,Zheng, X., Zhu, X. & Deter, R.L. Guidelinesfor the use and interpretation of assays formonitoring autophagy in higher eukaryotes.Autophagy 4, 151-75 (2008).

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The goal of our Department is to elucidate the cellular defense system and thecounteracting viral strategy at the level of gene regulation and to establish newapproaches for modulating human immunological response and for suppressinghuman pathogenic viruses. We have been studying epigenetical regulation of hu-man and HIV genomes by a chromatine remodeling factor, SWI/SNF complex andalso analyzing genome-wide networks formed between microRNAs and such im-portant transcriptional factors as AP-1 and NF-kappaB to explore the cause of hu-man diseases. Using these results, we also develop new retrovirus/lentivirus vec-tors that express proteins, short hairpin RNAs, miRNAs and efficient inhibitoryRNAs targeting specific miRNAs (Tough Decoy RNAs) for human gene therapyand basic researches.

1. Epigenetical regulation of host and retrovi-ral genes.

The SWI/SNF complex contributes to theregulation of gene expression by altering thechromatin structure, and plays many importantroles in epigenetic regulation in many organ-isms. In mammals, this complex is composed ofabout 10 subunits, and each of the complex con-tains a single molecule of either Brm or BRG1,but not both. These two proteins are the cata-lytic subunits and have DNA-dependent ATPaseactivity that drives remodeling of nucleosomes.We previously reported that Brm and BRG1have clear differences in their biological activi-ties; Brm is essential for maintenance of murineleukemia virus (MuLV)-based retroviral gene ex-pression, whereas BRG1 is not. Therefore, celllines that do not express detectable levels ofBrm protein undergo very rapid retroviral genesilencing that occurs stochastically and discon-tinuously.

Whereas SWI/SNF complex has been recog-nized as regulator at the transcriptional level,this year, we have demonstrated that this com-plex in conjugation with p54nrb can subsequentlyfunction as a splicing regulator for the transcriptproducts. Considering that p54nrb/PSF was pre-viously reported to regulate HIV mRNA stabil-ity, this finding would be quite important toelucidate post-transcriptional regulation of HIV(a). In 2007, we showed a clear correlation be-tween Brm-deficiency and undifferentiatedstatus of gastric cancer, and further demon-strated that Brm-type, but not BRG1-type SWI/SNF complex is required for villin expression.By intensive analysis on the human villin pro-moter, we have shown that Cdx2 regulates in-testinal villin expression through recruiting Brm-type SWI/SNF complex to the villin promoter.We are now convinced that Cdx2 is a key linkerbetween the loss of Brm and undifferentiatedstatus of gastic cancer (b). Like chromatin re-modeling, DNA methylation plays crucial roles

Department of Microbiology and Immunology

Division of Host-Parasite Interaction宿主寄生体学分野

Professor Hideo Iba, Ph.D.Assistant Professor Shigeru Minoguchi, Ph.D.Assistant Professor Taketoshi Mizutani, Ph.D.Assistant Professor Nobutake Yamamichi, Ph.D.

教 授 理学博士 伊 庭 英 夫助 教 医学博士 箕 口 滋助 教 医学博士 水 谷 壮 利助 教 医学博士 山 道 信 毅

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in human epigenetical regulation. We here alsopresent evidence that mouse ES (embryonicstem) cell clones that were transduced withMSCV (murine stem cell virus) do show vari-egated proviral expression. We found that the fi-delity of DNA methylation among the genomicsequences that flank the proviral integrationsites would be the determinant of this reversibil-ity. These results would reflect a unique and in-teresting feature of ES cell epigenome regulation(c).

a. Brm transactivates the telomerase reversetranscriptase (TERT) gene and modulatesthe splicing patterns of its transcripts inconcert with p54nrb

Taiji Ito, Hirotaka Watanabe, NobutakeYamamichi, Shunsuke Kondo, Toshio Tando,Takeshi Haraguchi , Taketoshi Mizutani ,Kouhei Sakurai, Shuji Fujita, Tomonori Izumi1

Toshiaki Isobe1 and Hideo Iba: 1Division ofProteomics Research, Institute of Medical Sci-ence, University of Tokyo

We report that a DBHS (for Drosophila behav-ior, human splicing) family protein, p54nrb, bindsboth BRG1 and Brm, catalytic subunits of SWI/SNF chromatin remodeling complex, and alsoanother core subunit of this complex, BAF60a.The N-terminal region of p54nrb is sufficient topull-down other core subunits of the SWI/SNFcomplex, suggesting that p54nrb binds SWI/SNF-like complexes. Polypyrimidine tract-bindingprotein (PTB)-associated splicing factor (PSF),another DBHS family protein known to directlybind p54nrb, was also found to associate with theSWI/SNF-like complex. When short hairpin (sh)RNAs targeting Brm were retrovirally expressedin a BRG1-deficient human cell line (NCI-H1299), the resulting clones showed downregula-tion of the telomerase reverse transcriptase (TERT)gene and an enhancement of ratios of exon-7-and-8-excluded TERT mRNA that encodes aninactive protein. All of these clones displaygrowth arrest within two months of the Brm-knockdown. In NCI-H1299 cells, Brm, p54nrb,PSF, and RNA polymerase II phosphorylated onCTD serine 2, specifically co-localize at a regionincorporating an alternative splicing acceptorsite of TERT exon7. These findings suggest thatat the TERT gene locus in human tumor cellscontaining functional SWI/SNF complex, Brm,and possibly BRG1, in concert with p54nrb wouldinitiate efficient transcription and could be in-volved in the subsequent splicing of TERT tran-scripts by accelerating exon-inclusion, whichpartly contributes to the maintenance of activetelomerase.

b. Cdx2 and the Brm-type SWI/SNF complexcooperatively regulate villin expression ingastrointestinal cells.

Nobutake Yamamichi, Ken-ichi Inada2, ChihiroFurukawa, Kouhei Sakurai, Toshio Tando,Aya Ishizaka, Takeshi Haraguchi, TaketoshiMizutani, Mitsuhiro Fujishiro3, Ryoichi Shi-momura2, Masashi Oka4, Masao Ichinose4, Yu-taka Tsutsumi2, Masao Omata3, and Hideo Iba:21st Department of Pathology, Fujita HealthUniversity School of Medicine, 3Department ofGastroenterology, Faculty of Medicine, Univer-sity of Tokyo, 4Second Department of InternalMedicine, Wakayama Medical College.

In our recent study showing a correlation be-tween Brm-deficiency and undifferentiatedstatus of gastric cancer, we found that the Brm-type SWI/SNF complex is required for villin ex-pression. To elucidate intestinal villin regulationmore precisely, this year, we have analyzedstructure and function of the promoter of hu-man villin . About 1.1kb upstream of the deter-mined major TSS, we identified a highly con-served region (HCR-Cdx) among mammals,which contains two binding sites for Cdx. Ex-pression analyses of 30 human gastrointestinalcell lines suggested that villin is regulated byCdx2. Introduction of Cdx family genes into col-orectal SW480 cells revealed that villin isstrongly induced strongly by Cdx2, and theknockdown of Cdx2 in SW480 cells caused aclear downregulation of villin . Reporter assaysshowed that HCR-Cdx is crucial for Cdx2-dependent and Brm-dependent villin expression.Immunohistochemical analyses of gastric intesti-nal metaplasia and gastric cancer revealed thatvillin and Cdx2 expression are tightly coupled.GST pull-down assays demonstrated a direct in-teraction between Cdx2 and several SWI/SNFsubunits . Chromatin immunoprecipitationanalyses further showed the recruitment of Cdx2 and Brm around the HCR-Cdx. From these re-sults, we concluded that Cdx2 regulates intesti-nal villin expression through recruiting Brm-type SWI/SNF complex to the villin promoter.

c. Instability of retroviral DNA methylation inembryonic stem cells

Shigeru Minoguchi and Hideo Iba

The epigenetic status of pluripotent stem cellshas been demonstrated to be extremely unstable.In our current study, we have attempted to fur-ther investigate the epigenetic dynamics of thestem cell genome by monitoring the expressionof the murine stem cell virus (MSCV) retroviral

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vector in embryonic stem (ES) cells. WhilstMSCV is progressively silenced by proviralDNA methylation in ES cells, a substantial num-ber of MSCV-transduced ES cell clones do showvariegated proviral expression. This expressionprofile is due in part to the transient and revers-ible properties of MSCV silencing. However, thespontaneous reactivation rates of the silencedproviruses differ significantly between thesevariegated clones, indicating that the reversibil-ity of silencing is dependent on the proviral in-tegration site. Our current data suggest that thefidelity of DNA methylation among the genomicsequences that flank the proviral integrationsites may be the determinant of this reversibilityof MSCV silencing. Given that the adjoining epi-genome environment affects the epigenetic regu-lation of proviral DNA, the reversible MSCV si-lencing effect is thus likely to reflect a uniqueand interesting feature of ES cell epigenomeregulation that has not previously been re-vealed.

2. Regulatory networks formed between tran-scriptional factors and miRNAs and devel-opment of new powerful tools for networkanalysis and for human therapy.

Recently new layers of molecular mechanismsare shown to be involved in the gene regulation;many RNA transcripts of exogenous as well asendogenous genes are regulated at post-transcriptional level by a mechanism designatedas RNA interference (RNA silencing). In humancells, major players of RNA interference are en-dogenous micro (mi)RNAs as well as short in-terfering (si)RNAs that are produced from eitherhost or viral aberrant RNAs rich in dsRNA re-gions.

MicroRNAs (miRNAs) are endogenous 20-24nt RNAs known to mediate the repression oftarget mRNAs by suppressing translation orpromoting mRNA decay in animal. More than700 species of miRNAs have now been identi-fied in human, and also predicted to targetroughly 30％ of the total coding genes in hu-man. A significant body of evidence has accu-mulated that they are involved in cellular devel-opment, differentiation, innate and acquired im-munity induction, and anti-viral responses bypost-transcriptional regulation of important tar-get mRNAs. In spite of the strong impact ofmiRNAs on regulatory network comparable totranscription factors, it still remains largely un-known how human miRNA expression itself isregulated at the transcriptional level, althoughthe vertebrate miRNA genes are thought to begenerally transcribed by RNA polymerase II(pol II) to produce a pri-miRNA containing a 5’-

cap structure and polyA tail. To elucidategenome-wide interplay between miRNAs andtranscriptional factors, this year, we developedan algorithm that predicts promoter region ofhuman miRNA genes that are involved in im-portant regulatory systems evolutionarily con-served among vertebrates (a). We have furtherscrutinized an important regulatory system in-volving, miR-21, which has been reported to beexpressed at high levels in almost all the humancancers and further to be involved in differentia-tion of several kinds of stem cells. By detailedanalysis on the promoter of the miR-21 gene, wefound that an evolutionarily conserved double-negative feedback regulation, which involvesmiR-21, NFIB protein and miR-21 promoter,would be operating as a mechanism to substainmiR-21 expression (b). It is also quite importantto develop efficient methods for the stable sup-pression of specific microRNA activity. We fi-nally show that unique RNA decoys developedhere (designated Tough Decoy: TuD) achievethe efficient and long-term-suppression of spe-cific miRNA (c).

a. Putative promoter regions of miRNA genesinvolved in evolutionarily conserved regu-latory systems among vertebrates.

Shuji Fujita and Hideo Iba

Just as transcription factors, miRNA genesmodulate global patterns of gene expressionduring differentiation, metabolic activation,stimulus response and also carcinogenesis.However, little is currently known how themiRNA gene expression itself is regulated ow-ing to lack of basic information of their genestructure. Global prediction of promoter regionsof miRNA genes would allow us to explore themechanisms underlying gene-regulatory mecha-nisms involving these miRNAs. We speculatethat if specific miRNA molecules are involved inevolutionarily conserved regulatory systems invertebrates, this would entail a high level ofconservation of the promoter of miRNAgene aswell as the miRNA molecule. By our currentscreening of putative promoter regions ofmiRNA genes (miPPRs) on this base, we identi-fied 59 miPPRs that would direct production of79 miRNAs. We present both biochemical andbioinformatical verifications of these putativepromoters.

b. miR-21 gene expression triggered by AP-1is sustained through a double negativefeedback mechanism.

Shuji Fujita, Taiji Ito, Taketoshi Mizutani,

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Shigeru Minoguchi, Nobutake Yamamichi,Kouhei Sakurai and Hideo Iba

miR-21 has been reported to be highly ex-pressed in various cancers and also to be induc-ible in a human promyelocytic cell line, HL-60,after PMA treatment. To examine molecularmechanisms involved in miR-21 expression, weanalyzed structure of the miR-21 gene by deter-mining its promoter and primary transcripts.We show that AP-1 activates the miR-21 tran-scription in conjugation with SWI/SNF complex,after PMA stimulation, through the conservedAP-1 and PU.1 binding sites in the promoteridentified here. The previous findings of en-hanced miR-21 expression in several cancersmay therefore reflect the elevated AP-1 activityin these carcinomas. A single precursor RNAcontaining miR-21 was transcribed just down-stream from the TATA box in this promoter,which is located in an intron of a coding geneTMEM49 . Importantly, expression of this over-lapping gene is completely PMA-independentand all its transcripts are polyadenylated beforereaching the miR-21 hairpin embedding region,indicating that miRNAs could have their ownpromoter even if overlapped with other genes.By available algorithms that predict miRNA tar-get using a conservation of sequence comple-mentary to the miRNA seed sequence, we nextpredicted and confirmed that the NFIB mRNAis a target of miR-21. NFIB protein usually bindsthe miR-21 promoter in HL-60 cells as a nega-tive regulator and is swept off from the miR-21promoter during PMA-induced macrophage dif-ferentiation of HL-60. The translational repres-sion of NFIB mRNA by miR-21 acceleratesclearance of NFIB in parallel with the simultane-

ous miR-21-independent transcriptional repres-sion of NFIB after PMA stimulation. Since ex-ogenous miR-21 expression moderately inducedendogenous miR-21, an evolutionarily conserveddouble negative feedback regulation would beoperating as a mechanism to sustain miR-21 ex-pression.

c. Vectors expressing efficient RNA decoysachieve the long-term suppression of spe-cific microRNA activity in mammaliancells.

Takeshi Haraguchi, Yuka Ozaki and Hideo Iba

Whereas the strong and stable suppression ofspecific microRNA activity would be essentialfor the functional analysis of these molecules,and also for the development of therapeutic ap-plications, effective inhibitory methods toachieve this have not yet been fully established.In our current study, we tested various RNA de-coys which were designed to efficiently exposeindigestible complementary RNAs to a specificmiRNA molecule. These inhibitory RNAs wereat the same time designed to be expressed inlentiviral vectors and to be transported into thecytoplasm after transcription by RNA polym-erase III. We report the optimal conditions thatwe have established for the design of such RNAdecoys (we term these molecules TuD RNAs;tough decoy RNAs). We demonstrate that TuDRNAs induce specific and strong biological ef-fects and also show that TuD RNAs achieve theefficient and long-term-suppression of specificmiRNAs for over one month in mammaliancells.

Publications

Fujita, S. and Iba, H. Putative promoter regionsof miRNA genes involved in evolutionarilyconserved regulatory systems among verte-brates. Bioinformatics, 24 (3): 303-308 (2008)

Ito, T., Watanabe, H., Yamamichi, N., Kondo, S.,Tando, T., Haraguchi, T., Mizutani, T.,Sakurai, K., Fujita, S., Izumi, T., Isobe, T., andIba, H. Brm transactivates the telomerase re-verse transcriptase (TERT) gene and modulatesthe splicing patterns of its transcripts in con-cert with p54nrb. Biochemical J ., 411: 201-209(2008)

Kayama, H., Ramirez-Carrozzi, V.R., Yamamoto,M., Mizutani, T., Kuwata, H., Iba, H., Matsu-moto, M., Honda, K., Smale, S.T., and Takeda,K. Class-specific regulation of pro-inflamma-tory genes by MYD88 pathways and IκBζ. J.

Biol. Chem ., 283: 12468-12477 (2008)Minoguchi, S. and Iba, H. Instability of retrovi-

ral DNA methylation in embryonic stem cells.Stem Cells , 26: 1166-1173 (2008)

Fujita, S., Ito, T., Mizutani, T., Minoguchi, S.,Yamamichi, N., Sakurai, K. and Iba, H. Mir-21gene expression triggered by AP-1 is sus-tained through a double negative feedbackmechanism. J. Mol. Biol , 378: 492-504 (2008)

Haraguchi, T., Ozaki, Y., and Iba, H. Vectors ex-pressing efficient RNA decoys achieve thelong-term suppression of specific microRNAactivity in mammalian cells. Nucleic Acids Res.in press

Yamamichi, N., Inada, K., Furukawa, C., Saku-rai, K., Tando, T., Ishizaka, A., Haraguchi, T.,Mizutani, T., Fujishiro, M., Shimomura, R.,

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Oka, M., Ichinose, M., Tsutsumi, Y., Omata,M., and Iba, H. Cdx2 and the Brm-type SWI/SNF complex cooperatively regulate villin ex-pression in gastrointestinal cells. Exp. Cell Res.in press

Akagi, T., Fukagawa, T., Kage, Y., To, H., Mat-sunaga, N., Koyanagi, S., Uchida, A., Fujii, A.,Iba, H., Ikemura, T., Aramaki, H., Higuchi, S.,and Ohdo, S. Role of glucocorticoid receptorin the regulation of cellular sensitivity to iri-notecan hydrochloride. J. Pharmacol. Sci . inpress

Yamamichi, N., Shimomura, R., Inada, K.,Sakurai, K., Haraguchi, T., Ozaki, Y., Fujita,S., Mizutani, T., Furukawa, C., Fujishiro, M.,Ichinose, M., Shiogama, K., Tsutsumi, Y.,

Omata, M., and Iba, H. LNA in situ hybridi-zation analysis of miR-21 expression duringcolorectal cancer development. Clinical CancerResearch in press山道信毅，伊庭英夫：クロマチン構造変換因子と癌―Brm型SWI／SNF複合体と胃癌 臨床検査５２�：６９３―６９７，２００８．藤田修二，伊庭英夫：miRNAの遺伝子制御ネットワーク 蛋白質 核酸 酵素 ５３�：１７８１―１７８６，２００８．伊庭英夫：miRNAと転写制御因子が形成するネットワーク ゲノム医学 in press原口健，伊庭英夫；miR-２１の発現亢進と発癌―亢進は癌の原因か，結果か？ またいかに亢進を抑制するのか？ 実験医学 印刷中

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Viruses can cause devastating diseases. The long-term goal of our research is tounderstand the molecular pathogenesis of viral diseases, using influenza andEbola virus infections as models. Interactions between viral and host gene prod-ucts during viral replication cycles determine the consequences of infection (i.e.,the characteristics of disease manifestation, whether limited or widespread);hence, our research has centered on such interactions in theses viral infections.

1. A naturally occurring deletion in its NSgene contributes to the attenuation of anH5N1 swine influenza virus in chickens

Zhu Q, Yang H, Chen W, Cao W, Zhong G,Jiao P, Deng G, Yu K, Yang C, Bu Z,Kawaoka Y, Chen H.

In 2001 and 2003, we isolated two H5N1 vi-ruses, A/swine/Fujian/1/01 (SW/FJ/01) andA/swine/Fujian/1/03 (SW/FJ/03), from pigs inFujian Province, southern China. Genetically,these two viruses are similar, although the NSgene of the SW/FJ/03 virus has a 15-nucleotidedeletion at coding positions 612 to 626. TheSW/FJ/01 virus is highly lethal for chickens,whereas the SW/FJ/03 virus is nonpathogenicfor chickens when administrated intravenouslyor intranasally. To understand the molecular ba-sis for the difference in virulence, we used re-verse genetics to create a series of single-generecombinants of both viruses. We found that arecombinant virus containing the mutated NSgene from the SW/FJ/03 virus in the SW/FJ/01

virus background was completely attenuated inchickens. We also found that viruses expressingthe mutant NS1 protein of SW/FJ/03 did notantagonize the induction of interferon (IFN) pro-tein. Conversely, only the recombinant viruscontaining the wild-type SW/FJ/01 NS gene inthe SW/FJ/03 background was lethal in chick-ens and antagonized IFN protein levels. Further,we proved that the NS1 genes of the two vi-ruses differ in their stabilities in the host cellsand in their abilities to interact with the chickencleavage and polyadenylation specificity factor.These results indicate that the deletion of aminoacids 191 to 195 of the NS1 protein is critical forthe attenuation of the SW/FJ/03 virus in chick-ens and that this deletion affects the ability ofthe virus to antagonize IFN induction in hostcells.

2. A novel approach to the development ofeffective H5N1 influenza A virus vaccines:the use of M2 cytoplasmic tail mutants

Watanabe T, Watanabe S, Kim JH, Hatta M,

Department of Microbiology and Immunology

Division of Virologyウイルス感染分野

Professor Yoshihiro Kawaoka, D.V.M., Ph.D.Associate Professor Taisuke Horimoto, D.V.M., Ph.D.Assistant Professor Hideo Goto, D.V.M., Ph.D.Assistant Professor Masayuki Shimojima, D.V.M., Ph.D.Research Associate Yuko Sakai-Tagawa, Ph.D.Project Assistant Professor Kiyoko Iwatsuki-Horimoto, D.V.M., Ph.D.

教 授 獣医学博士 河 岡 義 裕准教授 農学博士 堀 本 泰 介助 教 獣医学博士 五 藤 秀 男助 教 獣医学博士 下 島 昌 幸助 手 医学博士 坂井（田川）優子特任助教 獣医学博士 岩附（堀本）研子

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Kawaoka Y.

Outbreaks of highly pathogenic H5N1 influ-enza viruses in avian species began in Asia andhave since spread to other continents. Concernregarding the pandemic potential of these vi-ruses in humans is clearly warranted, and thereis an urgent need to develop effective vaccinesagainst them. Previously, we and others demon-strated that deletions of the M2 cytoplasmic tailcaused a growth defect in A/WSN/33 (H1N1)influenza A virus in vitro. We therefore testedthe feasibility of using M2 tail mutants as liveattenuated vaccines against H5N1 virus. Firstwe generated a series of highly pathogenic H5N1 (A/Vietnam/1203/04 [VN1203]) M2 cytoplas-mic tail deletion mutants and examined theirgrowth properties in vitro and in vivo. Wefound that one mutant, which contains an 11-amino-acid deletion from the C terminus (M2del11 virus), grew as well as the wild-type virusbut replicated in mice less efficiently. We thengenerated a recombinant VN1203M2del11 viruswhose hemagglutinin (HA) gene was modifiedby replacing sequences at the cleavage site withthose of an avirulent type of HA (M2del11-HAavir virus). This M2del11-HAavir virus pro-tected mice against challenge with lethal dosesof homologous (VN1203; clade 1) and antigeni-cally distinct heterologous (A/Indonesia/7/2005; clade 2) H5N1 viruses. Our results suggestthat M2 cytoplasmic tail mutants have potentialas live attenuated vaccines against H5N1 influ-enza viruses.

3. Limited compatibility between the RNA po-lymerase components of influenza virustype A and B

Iwatsuki-Horimoto K, Hatta Y, Hatta M, Mu-ramoto Y, Chen H, Kawaoka Y, Horimoto T.

Reassortants between type A and B influenzaviruses have not been detected in nature, al-though both viruses co-circulate in humanpopulations. One explanation for this may befunctional incompatibility of RNA transcriptionand replication between type A and B viruses.To test this possibility, we constructed type A/Bmosaic polymerase machinery, containing PB2,PB1, PA and nucleoprotein from each of the twovirus types, and assessed their polymerase ac-tivities with a type A promoter in a reporter as-say. Type B polymerase machinery containinghomologous components was functional withthe type A promoter albeit to various extentsdepending on the segments from which the re-gions downstream of the promoter sequencewere derived, indicating functional compatibility

between the type A promoter and B polymerasemachinery. However, all of the A/B mosaic po-lymerase machinery, except that containing PAfrom a type A and the others from a type B vi-rus strain, did not function with the type A pro-moter, indicating limited compatibility amongpolymerase components of both types. Taken to-gether, these data suggest that incompatibilityamong components of the polymerase machin-ery for RNA transcription and replication aloneis not responsible for the lack of heterotypic re-assortants.

4. A vaccine prepared from a non-pathogenicH7N7 virus isolated from natural reservoirconferred protective immunity against thechallenge with lethal dose of highly patho-genic avian influenza virus in chickens

Sakabe S, Sakoda Y, Haraguchi Y, Isoda N,Soda K, Takakuwa H, Saijo K, Sawata A,Kume K, Hagiwara J, Tuchiya K, Lin Z,Sakamoto R, Imamura T, Sasaki T, KokumaiN, Kawaoka Y, Kida H.

During 2001-2004, 41 H7 influenza viruses (2H7N1 and 39 H7N7 strains) were isolated fromfecal samples of migratory ducks that flew fromSiberia in the autumn of each year to Japan andMongolia. A phylogenetic analysis of the he-magglutinin (HA) genes of the nine representa-tive isolates revealed that they belonged to theEurasian lineage and the deduced amino acidsequence at the cleavage site of the HAs repre-sented apathogenic profiles. One of the H7 iso-lates A/duck/Mongolia/736/02 (H7N7) waschosen from these H7 isolates for the prepara-tion of the test vaccine. To improve the growthpotential of A/duck/Mongolia/736/02 (H7N7)in chicken embryos, A/duck/Hokkaido/Vac-2/04 (H7N7) was generated by genetic reassort-ment between A/duck/Mongolia/736/02 (H7N7) as the donor of the PB2, PB1, PA, HA, NA,and NS genes and A/duck/Hokkaido/49/98 (H9N2) as that of NP and M genes. The test vac-cine was prepared as follows; A/duck/Hok-kaido/Vac-2/04 (H7N7) was propagated inchicken embryos and the virus in the allantoicfluid was inactivated and adjuvanted to form anoil-in-water emulsion. The test vaccine conferredimmunity to chickens, completely protecting themanifestation of clinical signs against the chal-lenge with lethal dose of H7 highly pathogenicavian influenza virus. These results indicate thatinfluenza viruses isolated from natural reser-voirs are useful for vaccine strains.

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5. Comparison of the clinical effectiveness ofoseltamivir and zanamivir against influ-enza virus infection in children

Sugaya N, Tamura D, Yamazaki M, IchikawaM, Kawakami C, Kawaoka Y, Mitamura K.

We compared the clinical effectiveness ofoseltamivir and zanamivir in children with in-fluenza A (H1N1) virus, influenza A (H3N2) vi-rus, and influenza B virus infections. Total feb-rile period and the duration of fever after thestart of treatment were compared between anoseltamivir-treated group (mean age, 8.9 years;range, 4.0-15.9 years) and a zanamivir-treatedgroup (mean age, 10.0 years; range, 4.0-15.7years) in the pediatric outpatient clinics of ourhospitals. Oseltamivir was used to treat 91 chil-dren with influenza A (H3N2) infection and 24children with influenza A (H1N1) infection.Zanamivir was used to treat 35 children with in-fluenza A (H3N2) infection and 12 children withinfluenza A (H1N1) infection. Oseltamivir wasalso used to treat 128 children with influenza Bvirus infection, and zanamivir was used to treat59 with influenza B virus infection. There wasno statistically significant difference in total feb-rile period or duration of fever after the start oftreatment between the oseltamivir-treated groupand the zanamivir-treated group of childrenwith influenza A (H3N2) infection (mean dura-tion of febrile period, 2.40 days vs. 2.39 days;mean duration of fever after the start of treat-ment, 1.35 days vs. 1.40 days), influenza A (H1N1) (mean duration of febrile period, 2.60 daysvs. 2.46 days; mean duration of fever after thestart of treatment, 1.79 days vs, 1.54 days), or in-fluenza B (mean duration of febrile period, 2.95days vs. 2.84 days; mean duration of fever afterthe start of treatment, 1.86 days vs. 1.67 days).Oseltamivir was more effective against influenzaA (H3N2) than against influenza A (H1N1) orinfluenza B. Oseltamivir and zanamivir wereequally effective in reducing the febrile periodof children with influenza A (H1N1), influenzaA (H3N2), and influenza B virus infection.

6. Drosophila RNAi screen identifies hostgenes important for influenza virus repli-cation

Hao L, Sakurai A, Watanabe T, Sorensen E,Nidom CA, Newton MA, Ahlquist P, KawaokaY.

All viruses rely on host cell proteins and theirassociated mechanisms to complete the viral lifecycle. Identifying the host molecules that partici-pate in each step of virus replication could pro-

vide valuable new targets for antiviral therapy,but this goal may take several decades toachieve with conventional forward geneticscreening methods and mammalian cell cultures.Here we describe a novel genome-wide RNA in-terference (RNAi) screen in Drosophila1 that canbe used to identify host genes important for in-fluenza virus replication. After modifying influ-enza virus to allow infection of Drosophila cellsand detection of influenza virus gene expres-sion, we tested an RNAi library against 13,071genes (90％ of the Drosophila genome), identify-ing over 100 for which suppression in Droso-phila cells significantly inhibited or stimulatedreporter gene (Renilla luciferase) expressionfrom an influenza-virus-derived vector. The rele-vance of these findings to influenza virus infec-tion of mammalian cells is illustrated for a sub-set of the Drosophila genes identified; that is,for three implicated Drosophila genes, the corre-sponding human homologues ATP6V0D1, COX6A1 and NXF1 are shown to have key functionsin the replication of H5N1 and H1N1 influenzaA viruses, but not vesicular stomatitis virus orvaccinia virus, in human HEK 293 cells. Thus,we have demonstrated the feasibility of usinggenome-wide RNAi screens in Drosophila toidentify previously unrecognized host proteinsthat are required for influenza virus replication.This could accelerate the development of newclasses of antiviral drugs for chemoprophylaxisand treatment, which are urgently needed giventhe obstacles to rapid development of an effec-tive vaccine against pandemic influenza and theprobable emergence of strains resistant to avail-able drugs.

7. Cross-clade protective immunity of H5N1influenza vaccines in a mouse model

Murakami S, Iwasa A, Iwatsuki-Horimoto K,Ito M, Kiso M, Kida H, Takada A, Nidom CA,Le QM, Yamada S, Imai H, Sakai-Tagawa Y,Kawaoka Y, Horimoto T.

H5N1 highly pathogenic avian influenza vi-ruses evolved into several clades, leading to ap-preciably distinct antigenicities of their hemag-glutinins. As such, candidate H5N1 pre-pandemic vaccines for human use should besought. Here, to evaluate fundamental immuno-genic variations between H5N1 vaccines, weprepared four inactivated H5N1 test vaccinesfrom different phylogenetic clades (clade 1, 2.1,2.2, and 2.3.4) in accordance with the WHO rec-ommendation, and tested their cross-clade im-munity in a mouse model by vaccination fol-lowed by challenge with heterologous virulentviruses. All H5N1 vaccines tested provided full

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or partial cross-clade protective immunity, ex-cept one clade 2.2-based vaccine, which did notprotect mice from clade 2.3.4 virus challenge.Among the test vaccines, a clade 2.1-based vac-cine possessed the broadest-spectrum cross-immunity. These results suggest that currentlystockpiled pre-pandemic vaccines, especiallyclade 2.1-based vaccines, will likely be useful asbackup vaccines in a pandemic situation, evenone involving antigenic-drifted viruses.

8. Local not systemic modulation of dendriticcell S1P receptors in lung blunts virus-specific immune responses to influenza

Marsolais D, Hahm B, Edelmann KH, WalshKB, Guerrero M, Hatta Y, Kawaoka Y,Roberts E, Oldstone MB, Rosen H.

The mechanism by which locally deliveredsphingosine analogs regulate host response tolocalized viral infection has never been ad-dressed. In this report, we show that intratra-cheal delivery of the chiral sphingosine analog( R ) -2-amino-4- ( 4-heptyloxyphenyl ) -2-methyl-butanol (AAL-R) or its phosphate ester inhibitsthe T-cell response to influenza virus infection.In contrast, neither intraperitoneal delivery ofAAL-R nor intratracheal instillation of the non-phosphorylatable stereoisomer AAL-S sup-pressed virus-specific T-cell response, indicatingthat in vivo phosphorylation of AAL-R andsphingosine 1-phosphate (S1P) receptor modula-tion in lungs is essential for immunomodulation.Intratracheal delivery of water-soluble S1P1 re-ceptor agonist at doses sufficient to induce sys-temic lymphopenia did not inhibit virus-specificT-cell response, indicating that S1P1 is not in-volved in the immunosuppressive activities ofAAL-R and that immunosuppression acts inde-pendently of naive lymphocyte recirculation.Accumulation of dendritic cells (DCs) in drain-ing lymph nodes was inhibited by intratrachealbut not intraperitoneal delivery of AAL-R. Di-rect modulation of DCs is demonstrated by theimpaired ability of virus-infected bone marrow-derived DCs treated in vitro with AAL-R to trig-ger in vivo T-cell response after adoptive trans-fer to the airways. Thus, our results suggest thatlocally delivered sphingosine analogs induce im-munosuppression by modulating S1P receptorsother than S1P1 or S1P2 on dendritic cells in thelungs after influenza virus infection.

9. Compatibility among polymerase subunitproteins is a restricting factor in reassort-ment between equine H7N7 and human H3N2 influenza viruses

Li C, Hatta M, Watanabe S, Neumann G,Kawaoka Y.

Reassortment is an important driving force forinfluenza virus evolution, and a better under-standing of the factors that affect this processcould improve our ability to respond to futureinfluenza pandemics and epidemics. To identifyfactors that restrict the generation of reassortantviruses, we cotransfected human embryonic kid-ney cells with plasmids for the synthesis of viralRNAs of both A/equine/Prague/1/56 (Prague;H7N7) and A/Yokohama/2017/03 (Yokohama;H3N2) viruses together with the supporting pro-tein expression plasmids. Of the possible 256genotypes, we identified 29 genotypes in 120randomly plaque-picked reassortants examined.Analyses of these reassortants suggested that theformation of functional ribonucleoprotein (RNP)complexes was a restricting factor, a finding thatcorrelated with the activities of RNP complexescomposed of different combinations of the pro-teins from the two viruses, as measured in aminigenome assay. For at least one nonfunc-tional RNP complex (i.e., Prague PB2, PraguePB1, Yokohama PA, and Prague NP), the lack ofactivity was due to the inability of the three po-lymerase subunit proteins to form a het-erotrimer. Adaptation of viruses possessing agene encoding a chimera of the PA proteins ofthe two viruses and the remaining genes fromPrague virus resulted in compensatory muta-tions in the PB2 and/or PA protein. These re-sults indicate substantial incompatibility amongthe gene products of the two test viruses, a criti-cal role for the RNP complex in the generationof reassortant viruses, and a functional interac-tion of PB2 and PA.

10. Establishment of Canine RNA Polym-erase I-Driven Reverse Genetics for Influ-enza A Virus: Its Application for H5N1 Vac-cine Production

Murakami S, Horimoto T, Yamada S,Kakugawa S, Goto H, Kawaoka Y.

In the event of a new influenza pandemic,vaccines whose antigenicities match those of cir-culating strains must be rapidly produced. Here,we established an alternative reverse geneticssystem for influenza virus using the canine po-lymerase I (PolI) promoter sequence that worksefficiently in the Madin-Darby canine kidneycell line, a cell line approved for human vaccineproduction. Using this system, we were able togenerate H5N1 vaccine seed viruses more effi-ciently than can be achieved with the currentsystem that uses the human PolI promoter in

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African green monkey Vero cells, thus improv- ing pandemic vaccine production.

Publications

Murakami, S., Horimoto, T., Yamada, S.,Kakugawa, S., Goto, H., Kawaoka, Y. Estab-lishment of canine RNA polymerase I-drivenreverse genetics for influenza A virus: its ap-plication for H5N1 vaccine production. J. Vi-rol. 82: 1605-1609, 2008.

Zhu, Q., Yang, H., Chen, W., Cao, W., Zhong,G., Jiao, P., Deng, G., Yu, K., Yang, C., Bu, Z.,Kawaoka, Y., Chen, H. A naturally occurringdeletion in its NS gene contributes to the at-tenuation of an H5N1 swine influenza virusin chickens. J. Virol. 82: 220-228, 2008.

Itoh, Y., Ozaki, H., Tsuchiya, H., Okamoto, K.,Torii, R., Sakoda, Y., Kawaoka, Y., Ogasawara,K., Kida, H. A vaccine prepared from a non-pathogenic H5N1 avian influenza virus strainconfers protective immunity against highlypathogenic avian influenza virus infection incynomolgus macaques. Vaccine 26: 562-572,2008.

Watanabe, T., Watanabe, S., Kim, J.H., Hatta,M., Kawaoka, Y. A novel approach to the de-velopment of effective H5N1 influenza A vi-rus vaccines: the use of M2 cytoplasmic tailmutants. J. Viro.l 82: 2486-2492, 2008.

Halfmann, P., Kim, J.H., Ebihara, H., Noda, T.,Neumann, G., Feldmann, H., Kawaoka, Y.Generation of biologically contained Ebolavi-ruses . Proc. Natl. Acad. Sci. USA 105: 1129-1133, 2008.

Yamayoshi, S., Noda, T., Ebihara, H., Goto, H.,Morikawa, Y., Lukashevich, I.S., Neumann,G., Feldmann, H., Kawaoka, Y. Ebola virusmatrix VP40 protein uses the COPII transportsystem for its intracellular transport. Cell Host& Microbe 3: 168-177, 2008.

Iwatsuki-Horimoto, K., Hatta, Y., Hatta, M., Mu-ramoto, Y., Chen, H., Kawaoka, Y., Horimoto,T. Limited compatibility between the RNApolymerase components of influenza virustype A and B. Virus Res. 135: 161-165, 2008.

Sakabe, S., Sakoda, Y., Haraguchi, Y., Isoda, N.,Soda, K., Takakuwa, H., Saijo, K., Sawata, A.,Kume, K., Hagiwara, J., Tuchiya, K., Lin, Z.,Sakamoto, R., Imamura, T., Sasaki, T., Koku-mai, N., Kawaoka, Y., Kida, H. A vaccine pre-pared from a non-pathogenic H7N7 virus iso-lated from natural reservoir conferred protec-tive immunity against the challenge with le-thal dose of highly pathogenic avian influenzavirus in chickens. Vaccine 26: 2127-2134, 2008.

Sawai, T., Itoh, Y., Ozaki, H., Isoda, N.,Okamoto, K., Kashima, Y., Kawaoka, Y.,Takeuchi, Y., Kida, H., Ogasawara, K. Induc-

tion of cytotoxic T-lymphocyte and antibodyresponses against highly pathogenic avian in-fluenza virus infection in mice by inoculationof apathogenic H5N1 influenza virus particlesinactivated with formalin. Immunology 124:155-165, 2008.

Sugaya, N., Tamura, D., Yamazaki, M.,Ichikawa, M., Kawakami, C., Kawaoka, Y.,Mitamura, K. Comparison of the clinical effec-tiveness of oseltamivir and zanamivir againstinfluenza virus infection in children. Clin. In-fect. Dis. 47: 339-345, 2008.

Hao, L., Sakurai, A., Watanabe, T., Sorensen, E.,Nidom, C.A., Newton, M.A., Ahlquist, P.,Kawaoka, Y. Drosophila RNAi screen identi-fies host genes important for influenza virusreplication. Nature 454: 890-893, 2008.

Murakami, S., Iwasa, A., Iwatsuki-Horimoto, K.,Ito, M., Kiso, M., Kida, H., Takada, A., Ni-dom, C.A., Le, Q.M., Yamada, S., Imai, H.,Sakai-Tagawa, Y., Kawaoka, Y., Horimoto, T.Cross-clade protective immunity of H5N1 in-fluenza vaccines in a mouse model. Vaccine26: 6398-6404, 2008.

Marsolais, D., Hahm, B., Edelmann, K.H.,Walsh, K.B., Guerrero, M., Hatta, Y.,Kawaoka, Y., Roberts, E., Oldstone, M.B., Ro-sen, H. Local not systemic modulation of den-dritic cell S1P receptors in lung blunts virus-specific immune responses to influenza. Mol.Pharmacol. 74: 896-903, 2008.

WHO/OIE/FAO H5N1 Evolution WorkingGroup. Toward a unified nomenclature sys-tem for highly pathogenic avian influenza vi-rus (H5N1). Emerg. Infect. Dis. 14: e1, 2008.

Jia, B., Shi, J., Li, Y., Shinya, K., Muramoto, Y.,Zeng, X., Tian, G., Kawaoka, Y., Chen, H.Pathogenicity of Chinese H5N1 highly patho-genic avian influenza viruses in pigeons.Arch. Virol. 153: 1821-1826, 2008.

Li, C., Hatta, M., Watanabe, S., Neumann, G.,Kawaoka, Y. Compatibility among polymerasesubunit proteins is a restricting factor in reas-sortment between equine H7N7 and human H3N2 influenza viruses. J. Virol. 82: 11880-11888, 2008.

Sakuma, T., Noda, T., Urata, S., Kawaoka, Y.,Yasuda, J. Inhibition of Lassa and Marburg vi-rus production by tetherin. J. Virol. 83: 2382-2385, 2009.

Murakami, S., Horimoto, T., Le, Q.M., Nidom,C.A., Chen, H., Muramoto, Y., Yamada, S.,Iwasa, A., Iwatsuki-Horimoto, K., Shimojima,M., Iwata, A., Kawaoka, Y. Growth determi-

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nants for H5N1 influenza vaccine seed virusesin MDCK cells. J. Virol. 82: 10502-10509, 2009.

Watanabe, T., Watanabe, S., Shinya, K., Kim, J.H., Hatta, M., Kawaoka, Y. Viral RNA polym-erase complex promotes optimal growth of1918 virus in the lower respiratory tract of fer-rets. Proc. Natl. Acad. Sci. USA 106: 588-592,2009.

Marsolais, D., Hahm, B., Walsh, K.B., Edelmann,K.H., McGavern, D., Hatta, Y., Kawaoka, Y.,Rosen, H., Oldstone, M.B. A critical role forthe sphingosine analog AAL-R in dampeningthe cytokine response during influenza virusinfection. Proc. Natl. Acad. Sci. USA 106:1560-1565, 2009.

Ozawa, M., Maeda, J., Iwatsuki-Horimoto, K.,Watanabe, S., Goto, H., Horimoto, T.,Kawaoka, Y. Nucleotide sequence require-ments at the 5’ end of the influenza A virusM RNA segment for efficient virus replication.J. Virol. in press .

Kakugawa, S., Shimojima, M., Goto, H., Hori-moto, T., Oshimori, N., Neumann, G.,Yamamoto, T., Kawaoka, Y. The MAPK-activated kinase RSK2 plays a role in innateimmune responses to influenza virus infec-tion. J. Virol. in press .

Fan, S., Deng, G., Song, J., Tian, G., Suo, Y., Ji-ang, Y., Guan, Y., Bu, Z., Kawaoka, Y., Chen,H. Two amino acid residues in the matrixprotein M1 contribute to the virulence differ-ence of H5N1 avian influenza viruses in mice.

Virology in press .Halfmann, P., Ebihara, H., Marzi, A., Hatta, Y.,

Watanabe, S., Suresh, M., Neumann, G., Feld-mann, H., Kawaoka, Y. Replication-DeficientEbolavirus as a Vaccine Candidate. J. Virol. inpress .

Li, Z., Watanabe, T., Hatta, M., Watanabe, S.,Nanbo, A., Ozawa, M., Kakugawa, S., Shimo-jima, M., Yamada, S., Neumann, G., Kawaoka,Y. Mutational analysis of conserved amino ac-ids in the influenza A virus nucleoprotein. J.Virol. in press .

Neumann, G., Watanabe, S., Kawaoka, Y. Char-acterization of Ebolavirus Regulatory GenomicRegions. Virus Res. in press .

Le, Q.M., Sakai-Tagawa, Y., Ozawa, M., Ito, M.,Kawaoka, Y. Selection of H5N1 influenza vi-rus PB2 during replication in humans. J. Virol.in press .

Tamura, D., Mitamura, K., Yamazaki, M., Fu-jino, M., Nirasawa, M., Kimura, K., Kiso, M.,Shimizu, H., Kawakami, C., Hiroi, S., Taka-hashi, K., Hatta, M., Minagawa, H., Kimura,Y., Kaneda, S., Sugita, S., Horimoto, T.,Sugaya, N., Kawaoka, Y. Oseltamivir-ResistantInfluenza A Viruses Circulating in Japan. J.Clin. Micro. in press .

Takano, R., Nidom, C.A., Kiso, M., Muramoto,Y., Yamada, S., Shinya, K., Sakai-Tagawa, Y.,Kawaoka, Y. A comparison of the pathogenic-ity of avian and swine H5N1 influenza vi-ruses in Indonesia. Arch. Virol. in press .

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Immune cells express multiple Toll-like receptors (TLRs) that are concomitantly ac-tivated by a variety of pathogen products during microbial and viral infection.There is presumably a need to coordinate the expression function of TLRs in indi-vidual cells. Recent reports also have indicated that the balance of TLRs re-sponses has an important role in inducing autoimmune diseases. Our researchmain focuses on molecular regulatory mechanism to coordinate pathogen recogni-tion by TLRs.

1. A single base mutation in the PRAT4Agene reveals differential interaction ofPRAT4A with Toll-like receptors

Takashi Kiyokawa1,2 , Sachiko Akashi-Takamura1, Takuma Shibata1, Fumi Matsu-moto1, Chiaki Nishitani3, Yoshio Kuroki3,Yasuyuki Seto2 and Kensuke Miyake1: 1Divisionof Infectious Genetics, The Institute of MedicalScience and 2Department of GastrointestinalSurgery, Graduate School of Medicine, TheUniversity of Tokyo, Tokyo, Japan, 3Depart-ment of Biochemistry, School of Medicine, Sap-poro Medical University, Sapporo, Japan

Toll-like receptors (TLRs) play an essentialrole in defense responses. Immune cells expressmultiple TLRs which are simultaneously acti-vated by microbial pathogens. PRAT4A (PRoteinAssociated with Tlr4 A) is a chaperone-like en-doplasmic reticulum (ER)-resident protein re-quired for the proper subcellular distribution ofmultiple TLRs. PRAT4A－／－ mice show impairedexpression of TLR2/4 on the cell surface and thelack of ligand-induced TLR9 relocation from theER to endolysosome. Consequently, TLR re-

sponses to whole bacteria as well as to TLR2, 4,and 9 ligands are impaired. We here comparethe interaction of these TLRs with PRAT4A. As-sociation of endogenous PRAT4A was easily de-tected only with TLR4. The TLR4 region respon-sible for strong interaction with PRAT4A is veryclose to the site necessary for interaction withMD-2. By using transient expression, we wereable to detect PRAT4A interaction with TLR2and TLR9. The PRAT4A single-nucleotide mu-tant replacing methionine 145 with lysine (M145K) associates with TLR9 but does not rescueligand-dependent TLR9 trafficking. By contrast,the M145K mutant weakly, if at all, associateswith TLR2 and TLR4. The M145K mutant ap-preciably rescues cell surface TLR2 expressionand its responses in PRAT4A－／－ bone marrow-derived dendritic cells, whereas little if any res-cue of cell surface TLR4/MD-2 expression andits responses occurs. These results demonstratethat PRAT4A differentially interacts with eachTLR, and suggest that a single-nucleotidechange in the PRAT4A gene influences not onlythe strength of TLR responses but can also alterthe relative activity of each TLR.

Department of Microbiology and Immunology

Division of Infectious Genetics感染遺伝学分野

Professor Kensuke Miyake, M.D., Ph.D.Assistant Professor Sachiko Akashi-Takamura, M.D., Ph.D.Assistant Professor Takahisa Furuta, D.V.M., Ph.D.Assistant Professor Shin-Ichiroh Saitoh, Ph.D.

教 授 医学博士 三 宅 健 介助 教 医学博士 高村（赤司）祥子助 教 農学博士 古 田 隆 久助 教 医学博士 齋 藤 伸一郎

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2. Detectin of soluble PRAT4A (PRotein As-sociated with Toll-like receptor 4) by novelanti-PRAT4A monoclonal antibodies

Takuma Shibata, Sachiko Akashi-Takamura,Richard Jennings, and Kensuke Miyake

Immunocompetent cells express multiple Toll-like receptors (TLRs) which play sentinel rolesin detecting microbial products. OrchestratedTLR activation induces appropriate immune re-sponses. It is known that TLR associating mole-cules, such as MD-2, CD36, UNC93B1, HMGB-1,gp96, play essential role in ligand recognition byTLRs. It is important to clarify how the TLR ac-cessory molecules function in immune cells tocorrectly understand TLR-dependent immuneresponses.

We have previously reported that a novelprotein ‘PRotein Associated with TLR4 (PRAT4A)’ is required for multiple TLR responses bymodulating TLR trafficking. And mRNA level ofPRAT4A significantly decreased after variousTLR stimulation. These results suggest thatPRAT4A is essential for controlling multipleTLR responses. Furthermore, we have recog-nized by immunoprecipitation assay that Ba/F3cells over-expressing PRAT4A secreted PRAT4Ain the culture supernatant without any stimula-tion.

To delineate the undetected function of endo-genous PRAT4A in immune cells, we have es-tablished several monoclonal anti-PRAT4A anti-bodies and studied about the dynamics of endo-genous PRAT4A. We could detect secreted en-dogenous PRAT4A by immunoprecipitation as-say in the normal mice serum and the culturesupernatant of Bone Marrow derived Macro-phages (BMMfs), but not of BM derived Den-dritic cells (BMDCs).

In addition, we found by FACS analysis thatPRAT4A was detectable on the cell surface ofBMMfs, but not of BMDCs, after various TLRligand stimulation, especially TLR4 and TLR9ligand stimulation.

There is possibility that PRAT4A is not only aTLR associating molecule but also a modulatorof immune responses induced by TLR activa-tion. Further study is under way to reveal a roleof soluble PRAT4A in immune responses.

3. Soluble MD-1 detection using novel mono-clonal antibodies

Richard Jennings, Sachiko Akashi-Takamura,Shibata Takuma, Toshihiko Kobayashi, andKensuke Miyake

In the absence of MD-1, cells are unable to ex-

press RP105 on their surface, and thusly RP105loses function. Current data indicates thatRP105/MD-1 has an inhibitory role in myeloidderived cells but not in B cells, where resultshave shown RP105/MD-1 to positively regulateTLR stimulation. This apparent functional di-chotomy between cell types is, as of yet, unex-plained. There is the possibility that this dichot-omy is explained not through the complex itself,but either through associated molecules, or dis-tinctive functions of RP105 and MD-1, outside ofthe RP105/MD-1 complex. Previous experimen-tation has been unable to distinguish the func-tions of MD-1 and RP105 as individual mole-cules. In this study, through the production of anovel series of antibody clones (JR clones), weare able to show, for the very first time, that un-der physiological conditions soluble MD-1 existsextra-cellularly.

MD-1KO BALB/c mice were immunized withBa/F3 cells stably transfected to express myc-tagged murine MD-1 on their surface. Initialscreening utilized the immunogen cell line. La-teer screening utilized splenic cells harvestedfrom B6 background wild type, MD-1KO, andRP105KO mice. 7 antibody clones were ob-tained, these antibodies all show the ability torecognize MD-1 on the surface of the immuno-gen cell line, and vary in their ability to recog-nize cell surface MD-1 on splenic cells. Selectiveclones show mitogenic properties, although co-stimulation with RP14 results in the blunting ofthis mitogenic property. Using these antibodies,MD-1 was successfully immuno-precipitatedfrom cell lysates, RAW cell supernatant, BALB/cWT serum, and BALB/c RP105KO serum. Thisclearly shows that MD-1 exists extra-cellularly,and that this property of MD-1 is not dependenton RP105 expression. Further experiment onextra-cellular MD-1 may lead to a more exhaus-tive understanding of it’s immunobiology.

4. Involvement of mast cells and mast cell-derived TNF in the pathogenic mecha-nisms of superantigen-mediated diseases

Takahisa Furuta1, Ken’ichi Imanishi3, HidehiroUeshiba4, Taichi Ezaki5, Yoichiro Iwakura2,Yasukazu Ohmoto7, Naohiro Watanabe6, andTakehiko Uchiyama3,8: 1Division of InfectiousGenetics, Department of Microbiology and Im-munology, Institute of Medical Science, Univer-sity of Tokyo, Tokyo, Japan 2Division of CellBiology, Center for Experimental Medicine, In-stitute of Medical Science, University of Tokyo,Tokyo, Japan 3Department of Microbiologyand Immunology, 4Institute of Laboratory Ani-mals, 5Department of Anatomy and Develop-mental Biology, Tokyo Women’s Medical Uni-

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versity School of Medicine, Tokyo, Japan. 6De-partment of Tropical Medicine, Jikei Univer-sity School of Medicine, Tokyo, Japan. 7FreeRadical Research Institute, Otsuka Pharmaceu-tical Co., Ltd., Tokushima, Japan. 8Departmentof Human Science, Tokiwa University, Ibaraki,Japan.

Roles of mast cells and mast cell-derived cy-tokines in induction of a lethal response in miceinjected with a combined injection of staphylo-coccal enterotoxin A (SEA) and D-Galactosamine(GalN) were examined to further understand thepathogenic mechanisms of the superantigen-mediated diseases. All of mast cell-deficient W/Wv mice were survived upon injection of SEAand GalN, whereas the control littermate ＋/＋mice were died with high serum TNF level anddegranulation of mast cells upon the lethal chal-lenge. The results suggested that mast cell andmast cell-derived TNF were critically importantin the induction of the SEA-induced lethal re-sponse. This was confirmed by reconstitutionexperiments with bone marrow-derived mastcells (BMMCs). W/Wv mice reconstituted withBMMCs of ＋/＋ mice were recovered the sus-ceptibility to the lethal challenge and died.However, W/Wv mice reconstituted withBMMCs of TNF－／－ mice were not recovered thesusceptibility to the lethal challenge and sur-vived. In vitro studies showed that increasedTNF level was observed when BMMCs from＋/＋ or IFN－／－ but not TNF－／－ were culturedwith Antigen Presenting Cell and CD4＋ T cells.These findings propose a novel viewpoint thatmast cells and mast cells-derived TNF play im-portant roles in the pathogenic mechanism ofthe superantigen-mediated diseases.

5. Roles for LPS-dependent interaction andrelocation of TLR4 and TRAM in TRIF-signaling

Natsuko Tanimura, Shinichiroh Saitoh, FumiMatsumoto, Sachiko Akashi-Takamura, andKensuke Miyake

Toll-like receptor 4 (TLR4) activates two dis-tinct signaling pathways including productionof proinflammatory cytokines or type I interfer-ons (IFNs), respectively. Two adaptor molecules,MyD88 and TIRAP/Mal, are essential for theformer but not for the latter signaling pathway,which instead requires two adaptors, TRIF/TICAM-1 and TRAM/TICAM-2. TIRAP is asorting adaptor molecule recruiting MyD88 toactivated TLR4 in the plasma membrane. TRAMis thought to serve as a signaling molecule be-tween TLR4 and TRIF by associating with these

molecules. Little is known, however, howTRAM interacts with TLR4 or with TRIF duringLPS responses. Here we show that LPS inducesupregulation of TLR4-association with TRAM,TRIF recruitment to the plasma membrane, andtheir subsequent internalization into endosome/lysosome. The internalized signaling complexconsisting of TLR4 and TRAM co-localizes withTRAF3, a signaling molecule downstream ofTRIF, in endosome/lysosome. These results sug-gest that TLR4 activates TRIF-signaling pathwayin endosome/lysosome.

6. Unc93B1 biases Toll-like receptor-mediated responses in dendritic cells to-wards DNA- and against RNA-sensing

Ryutaroh Fukui1, Shin-ichiroh Saitoh1, FumiMatsumoto1, Hiroko Kozuka-Hata2, MasaakiOyama2, Koichi Tabeta3, Bruce Beutler4, &Kensuke Miyake1: 1Division of Infectious Ge-netics, 2Medical Proteomics Laboratory, the In-stitute of Medical Science, the University of To-kyo, Shirokanedai, Tokyo 108-8639, Japan. 3

Center for Transdisciplinary Research, NiigataUniversity, Gakkocho-Dori, Niigata 951-8514,Japan. 4Department of Genetics, The ScrippsResearch Institute, La Jolla, CA92037, UnitedStates.

Toll-like receptors (TLR) 3, 7, and 9 recognizemicrobial nucleic acids in endolysosomes, andinitiate innate and adaptive immune responses.TLR7/9 in dendritic cells (DC) also responds toself-derived RNA/DNA, respectively, and driveautoantibody production. Remarkably, TLR7and TLR9 appear to have mutually opposing,pathogenic or protective, impacts on lupus ne-phritis in MRL/lpr mice. Little is known, how-ever, about a contrasting relationship betweenTLR7 and TLR9. We here show that TLR7 andTLR9 are inversely linked by Unc93B1, a multi-ple membrane-spanning ER protein. Comple-mentation cloning with a TLR7-unresponsive,but TLR9-responsive cell line revealed that D34in Unc93B1 repressed TLR7-mediated responses.D34A mutation rendered Unc93B1-deficient DCshyperresponsive to TLR7 ligand but hypore-sponsive to TLR9 ligand with TLR3 responsesunaltered. Unc93B1 associates with and deliversTLR7/9 from the ER to endolysosomes. D34Amutation biased Unc93B1 association againstTLR9 and towards TLR7. Taken together, Toll-like receptor response to DNA and RNA in DCsis biased towards DNA-sensing by Unc93B1.

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Publications

Youn, HS., Lee, JK., Choi, YJ., Saitoh, SI., Mi-yake, K., Hwang, DH. and Lee, JY. Cinnamal-dehyde suppresses toll-like receptor 4 activa-tion mediated through the inhibition of recep-tor oligomerization. Biochem. Pharmacol. 75:494-502, 2008.

Matsumoto, F., Saitoh, S., Fukui, R., Kobayashi,T., Tanimura, N., Konno, K., Kusumoto, Y.,Akashi-Takamura, S. and Miyake, K. Cathep-sins are required for Toll-like receptor 9 re-sponses. Biochem. Biophys. Res. Commun.367: 693-699, 2008.

Tanimura, N., Saitoh, S., Matsumoto, F., Akashi-Takamura, S. and Miyake, K. Roles for LPS-dependent interaction and relocation of TLR4and TRAM in TRIF-signaling. Biochem. Bio-phys. Res. Commun. 368: 94-99, 2008.

Shawkat, S., Karima, R., Tojo, T., Tadakuma, H.,Saitoh, S., Akashi-Takamura, S., Miyake, K.,Funatsu, T. and Matsushima, K. Visualizationof the molecular dynamics of lipopolysaccha-ride on the plasma membrane of murinemacrophages by total internal reflection fluo-rescence microscopy. J. Biol. Chem. 283:22962-22971, 2008.

Furuta, T., Imajo-Ohmi, S., Fukuda, H., Kano, S.,Miyake, K. and Watanabe. N. Mast cell-mediated immune responses through IgE anti-body and Toll-like receptor 4 by malarial per-oxiredoxin. Eur. J. Immunol. 38: 1341-1350,2008.

Kuraoka, M., Furuta, T, Matsuwaki, T., Omatsu,T., Ishii, Y., Kyuwa, S. and Yoshikawa, Y. Di-rect experimental occlusion of the distal mid-dle cerebral artery induces high reproducibil-ity of brain ischemia in mice. Exp Anim. 2009.58: 19-29.

Hiratsuka, S., Watanabe, A., Sakurai, Y., Akashi-Takamura, S., Ishibashi, S., Miyake, K.,Shibuya, M., Akira, S., Aburatani, H., Maru,Y. The S100A8-serum amyloid A3-TLR4paracrine cascade establishes a pre-metastaticphase. Nat. Cell Biol. 10: 1349-1355, 2008.

Kiyokawa, T., Akashi-Takamura, S., Shibata, T.,Matsumoto, F., Nishitani, C., Kuroki, Y., Seto,Y., Miyake, K. A single base mutation in thePRAT4A gene reveals differential interactionof PRAT4A with Toll-like receptors. Int. Im-munol. 20: 1407-1415, 2008.

Akashi-Takamura, S., Miyake, K. TLR accessorymolecules. Curr Opin Immunol. 20: 420-425,2008.

Kobayashi, T., Takahashi, K., Nagai, Y., Shibata,T., Otani, M., Izui, S., Akira, S., Gotoh, Y., Ki-yono, H., Miyake, K. Tonic B cell activationby Radioprotective 105/MD-1 promotes dis-ease progression in MRL/lpr mice. Int Immu-nol 20: 881-891, 2008.

Miyake, K. Nucleic acid-sensing Toll-like recep-tors: beyond ligand search. Adv Drug DelivRev 60(7): 782-5, 2008.

Kikuchi, Y., Koarada, S., Nakamura, S., Yone-mitsu, N., Tada, Y., Haruta, Y., Morito, F.,Ohta, A., Miyake, K., Horiuchi, T., Nagasawa,K. Increase of RP105-lacking activated B cellsin the peripheral blood and salivary glands inpatients with Sjogren’s syndrome. Clin. Exp.Rheumatol. 26: 5-12, 2008.

Akahoshi, M., Nakashima, H., Sadanaga, A., Mi-yake, K., Obara, K., Tamari, M., Hirota, T.,Matsuda, A., Shirakawa, T. Promoter poly-morphisms in the IRF3 gene confer protectionagainst systemic lupus erythematosus. Lupus17: 568-74, 2008.

Yamashita, Y., Kouro, T., Miyake, K., Takatsu,K., Kido, MA., Tanaka, T., Goto, M., Kincade,PW. Participation of intercellular adhesionmolecule-2 (CD102) in B lymphopoiesis. Im-munol Lett 120: 79-86, 2008.

Tada, Y., Koarada, S., Morito, F., Mitamura, M.,Inoue, H., Suematsu, R., Ohta, A., Miyake, K.,Nagasawa, K. Toll-like receptor homolog RP105 modulates the antigen-presenting cellfunction and regulates the development ofcollagen-induced arthritis. Arthritis Res. Ther.10: R121, 2008.

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The mucosal immune system not only plays an important role as the first line ofimmunological defense for preventing the host from invasion of microorganisms,but also contributes to the establishment and maintenance of mucosal homeosta-sis. Our major focus is the elucidation and understanding of molecular and cellularnature of the mucosal immune system for the development of mucosal vaccineagainst infectious diseases and mucosal immune therapy for mucosa-associateddiseases, such as food allergy and inflammatory diseases.

1. M Cell Biology

Tomonori Nochi1, Masato Yoshida1, GemilsonS. Pontes1, Kazutaka Terahara1, Osamu Iga-rashi1, Shiho Kurokawa1, Mio Mejima1, YukoTakahashi1, Koji Hase2, Hiroshi Ohno2, AnsonW. Lowe3, Yoshikazu Yuki1 and Hiroshi Ki-yono1: 1Division of Mucosal Immunology, De-partment of Microbiology and Immunology,The Institute of Medical Science, The Univer-sity of Tokyo, 2Laboratory for Epithelial Im-munobiology, Research Center for Allergy andImmunology, RIKEN, 3Department of Medi-cine, Stanford University

Membranous or microfold cells (M cells) lo-cated in the follicle-associated epithelium (FAE)of Peyer’s patches (PPs) play a pivotal role inup-taking the luminal antigens for initiation ofantigen-specific immune responses in both sys-temic and mucosal compartments. We recentlyestablished the purification strategy of isolatedM cells from PPs with our previously developedM cell-specific monoclonal antibody (NKM 16-2-4) and subsequently performed DNA microarrayusing NKM 16-2-4＋ M cells to identify the re-

sponsible genes which involve in antigen-sampling and cell differentiation. As results,1329 genes were raised up as M cell-specificgene candidates and our subsequent in situ hy-bridization analysis clearly showed that gly-coprotein 2 (GP2) and MARCS-like protein(MLP) mRNAs were specifically expressed byPP M cells. In addition, immunohistochemicalanalysis with newly established GP2-specificmonoclonal antibody (10F5-9-2) and MLP-specific polyclonal antibody also confirmed thespecific expression in M cells at protein level.Since our current effort is aimed to elucidate thebiological role of two M cell-specific molecules,the genome-wide assessment of gene expressionwould facilitate to understand the M cell im-munobiology.

2. Mucosal vaccine development

Tomonori Nochi1, Yoshikazu Yuki1, YukoKatakai2, Tomoko Kohda3, Daisuke Tokuhara1,Mio Mejima1 , Yuko Takahashi1 , ShihoKurokawa1, Hiroaki Shibata2, Sunji Kozaki3,Kenji Terao2 and Hiroshi Kiyono1: 1Division ofMucosal Immunology, Department of Microbi-

Department of Microbiology and Immunology

Division of Mucosal Immunology炎症免疫学分野

Professor Hiroshi Kiyono, D.D.S., Ph.D.Lecturer Jun Kunisawa, Ph.D.Assistant Professor Yoshikazu Yuki, M.B.A., Ph.D.Assistant Professor Shintaro Sato, Ph.D.

教 授 医学博士 清 野 宏講 師 薬学博士 國 澤 純助 教 医学博士 幸 義 和助 教 医学博士 佐 藤 慎太郎

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ology and Immunology, The Institute of Medi-cal Science, The University of Tokyo, 2TsukubaPrimate Research Center, National Institute ofBiomedical Innovation, 3Graduate School ofLife and Environmental Sciences, Osaka Pre-fecture University

Because needle-free vaccine delivery has a po-tential to lead to significant advances, develop-ment of the vaccine is one of the defined as“grand challenges in global health”. In order todevelop effective and preventive needle-freevaccine, we chose a recombinant form of non-toxic fragment of C-terminal half of heavy chain(Hc) of botulinum neurotoxin type A (BoNT/A)and tested the potential as vaccine against botu-lism on two mucosal administration routes incynomologus macaques. Nasal immunization of0.5mg Hc without adjuvant induced brisk toxin-specific antibody immune responses with neu-tralizing activity in both systemic and mucosalcompartment. In contrast, sublingual immuniza-tion of 1.0mg Hc without adjuvant induced cer-tain toxin-specific systemic antibody with weakneutralizing activity and no mucosal immuneresponses. Furthermore, the nasal immunizationwith Hc protected these macaques from chal-lenging with BoNT/A of 35,000 fold of lethalamount of monkey, without any clinical signs ofdisease for 30 days. The results show that nasalimmunization with Hc is an attractive strategyfor the protection of botulism, which is naturallyoccurred or stemmed from acts of bioterrorism.

3. Rice-based vaccine: MucoRiceTM

Daisuke Tokuhara1, Yoshikazu Yuki1, To-monori Nochi1, Hiroshi Yasuda2, Mio Mejima1,Shiho Kurokawa1, Yuko Takahashi1, NobuhiroKataoka1, Ushio Nakanishi1, Fumio Takaiwa3

and Hiroshi Kiyono1: 1Division of Mucosal Im-munology, Department of Microbiology andImmunology, The Institute of Medical Science,The University of Tokyo, 2National Agricul-tural Research Center for Hokkaido Region,3National Institute of Agrobiological Sciences

Rice-expressed cholera toxin B subunit(MucoRiceTM-CTB) was previously constructedand proved to be an effective cold-chain freeoral vaccine for the induction of enterotoxinneutralizing immunity. Our further investiga-tions revealed that oral MucoRiceTM-CTB immu-nization protected against live Vibrio cholera-and Enterotoxigenic Escherichia coli-induced di-arrhea in mice. This finding indicated thatMucoRiceTM-CTB could use as a cold-chain freevaccine against cholera as well as travellers’ di-arrhea in humans. We also created a second

generation of rice-based vaccine expressing non-toxic double mutant cholera toxin (dmCT). Oralimmunization of MucoRiceTM-dmCT inducedCTB-specific serum IgG and mucosal IgA anti-bodies with neutralizing activity but not anyCTA-specific serum and mucosal antibodies. Al-though the potency of MucoRice-dmCT as a CTvaccine was almost equal to that of MucoRice-CTB, these results suggest that the MucoRicesystem has potential for the development ofmulticomponent vaccines.

4. Marine Vaccine Project

Ei Lin Ooi1, Noel Verjan1, Christopher M.A.Caipang2, Ikumi Haraguchi1, Takeo Oshima1,Tomonori, Nochi1, Hidehiro Kondo2, Ikuo Hi-rono2, Takashi Aoki3, Hiroshi Kiyono1 andYoshikazu Yuki1: 1Division of Mucosal Immu-nology, Department of Microbiology and Im-munology, The Institute of Medical Science,The University of Tokyo, 2Laboratory ofGenome Science, Graduate School of MarineScience and Technology, Tokyo University ofMarine Science and Technology

Because many of fisheries stocks have de-clined in the worldwide, fish farming, especiallyshrimp and salmon farming has boomed. Thus,marine industry needs vaccines for these fishesto protect against infectious diseases instead ofantibiotics due to food safety and ecological con-cerns from consumers. Based on a collaborationwith Tokyo University of Marine Science andTechnology, we started a marine project for thedevelopment of fish vaccines. In this study, wefound that in vivo immunostimulatory effects ofa recombinant Atlantic salmon (Salmo salar)interferon-α (rIFN-α). A minimal injection doseof 0.1 µg of rIFN-α per g fish significantly pro-tected trout against a lethal dose of infectioushaematopoietc necrosis virus (IHNV) andshowed that a salmonid rIFN can modulate theinnate immune response of salmon and mediateearly antiviral protection from many of viruses’threat. We also showed that a potential oral vac-cine against white spot syndrome virus (WSSV),a major threat to shrimp culture worldwide, toassess the efficacy of the oral administration ofrecombinant VP28, an envelope protein ofWSSV, in providing protection in shrimp,Penaeus japonicus upon challenge with WSSV.

5. Mucosal Trafficking

Jun Kunisawa, Masashi Gohda, YosukeKurashima, Morio Higuchi, Yuki Kagiyama,Atsuhiro Matsumoto, Akihiro Uozumi, To-mokazu Ishizuka, Izumi Ishikawa, Ikuko Oga-

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hara, Eri Yoshikawa, and Hiroshi Kiyono: Di-vision of Mucosal Immunology, Department ofMicrobiology and Immunology, The Instituteof Medical Science, The University of Tokyo

A lipid mediator, sphingosine 1-phosphate (S1P), is known to regulate lymphocyte traffickingat systemic immune compartments (e.g. , thy-mus) and we have recently reported that uniquegut-associated immunocompetent cells such asintraepithelial T lymphocytes and pathogenic Tand mast cells causing food allergy also utilizeS1P for their trafficking. In this study, we haveidentified the role of S1P in the regulation of in-nate and acquired gut IgA production. InPeyer’s patches, major sites for acquired-typeIgA production pathway, B cells change the S1Preceptor expression during their differentiationto IgA＋ plasmablasts, which enable them to de-termine whether they stay in or emigrate outfrom Peyer’s patches. Thus, inhibition of S1P-mediated pathway by FTY720, an S1P1 modula-tor, resulted in the selective accumulation of S1Preceptor＋ IgA＋ plasmablasts in the Peyer’spatches and consequent impairment of intestinalIgA responses against orally immunized proteinantigen. We also found that S1P played a keyrole in the innate intestinal IgA responses byshowing that peritoneal B cells used S1P in theirtrafficking into the intestine. In this pathway,NF-κB-inducing kinase (NIK) expressed in peri-toneal stromal cells is essential. Thus, althoughperitoneal B cells from NIK-mutated alym-phoplasia (aly) mice expressed substantial levelsof S1P receptor and normally migrated toward S1P in vitro and in vivo, aly mice showed de-creased sensitivity to FTY720. We found thatNIK-mutated stromal cells showed aberrant ex-pression of VCAM-1, ICAM-1, and CXCL13,leading to the impaired ability to support S1P-mediated peritoneal B cell emigration in alymice. Thus, transfer of wild-type stromal cellsinto the peritoneum restored S1P-mediated traf-ficking of aly peritoneal B cells. We are now ex-tending these studies by investigating the in-volvement of gut environmental factors (e.g.,dietary materials and commensal microbiota) inthe regulation of gut immune system becausethese factors affect the generation of lipid me-diators.

6. Food Allergy

Yosuke Kurashima1, Haruyo Adachi1, NaokoTakayama1, Mi-Na Kweon2, Jun Kunisawa1,and Hiroshi Kiyono1: 1Division of Mucosal Im-munology, Department of Microbiology andImmunology, The Institute of Medical Science,The University of Tokyo, 2Mucosal Immunol-

ogy Section, International Vaccine InstituteTo establish novel and effective strategies to

regulate intestinal allergic diseases, we have elu-cidated molecular and cellular mechanisms ofthe development of food allergy using ovalbu-min-specific food allergy murine model. In thisyear, we have revealed the involvement ofregulatory-type T cells and a lipid mediator inthe development of food allergy. In the formercase, we found that Peyer’s patches (PPs) pos-sessed regulatory function in allergic diarrheaby showing that mice lacking PPs were moresusceptible to the allergy development. Addi-tional analyses revealed that PPs contained IL-10 producing regulatory CD4＋CD25＋ Foxp3＋ Tcells (Treg) and inhibited the development of al-lergic diarrhea. Indeed, the treatment with anti-CD25 or anti-IL-10 antibody resulted in therapid and severe development of allergic diar-rhea. These studies demonstrate that PP is thesite to induce IL-10-producing Treg cells for thecontrol of intestinal allergic responses. In the lat-ter study, we showed that sphingosine 1-phosphate (S1P), a lipid mediator, played im-portant roles in the development of food allergyby regulating the trafficking of pathogenic Tand mast cells. When mice were treated withFTY720, a S1P receptor modulator, the incidenceof allergic diarrhea was markedly inhibited. Inthose mice, the number of mast cells and acti-vated CD4＋ Th2 cells was decreased in the in-testinal compartment. These findings suggestthat S1P-mediated trafficking of mast cells andactivated T cells could be an effective target forthe prevention and treatment of food allergy.Based of these findings, we are now trying toelucidate the detailed molecular and cellularmechanisms of induction, activation, and traf-ficking of allergen-specific CD4＋ Th2 cells, mu-cosal mast cell and Treg cells to develop the im-munotherapy against intestinal allergic diseases.

7. Non-canonical second lymphoid organo-genesis

Takahiro Nagatake1 , Satoshi Fukuyama1 ,Dong-Young Kim1,2 , Kaoru Takamura1,3 ,Osamu Igarashi1, Kazunari Okada1, ShintaroSato1, Jun Kunisawa1, Anton M. Jetten4, Yoshi-fumi Yokota5, and Hiroshi Kiyono1: 1Division ofMucosal Immunology, Department of Microbi-ology and Immunology, Institute of MedicalScience, The University of Tokyo, 2Departmentof Otorhinolaryngology, Seoul National Univer-sity College of Medicine, 3Department of Otor-hinolaryngology, The Shimane UniversitySchool of Medicine, 4Cell Biology Section,Laboratory of Respiratory Biology, NationalInstitute of Environmental Health Sciences,

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National Institutes of Health, 5Department ofMolecular Genetics, School of Medicine, Uni-versity of Fukui

Mucosa-associated lymphoid tissue plays akey role in the regulation of mucosal immunesystem. Nasopharynx-associated lymphoid tis-sue (NALT) and Peyer’s patch (PP) develop atrespiratory tract and intestine, respectively. Wehave recently identified that tear duct-associatedlymphoid tissue (TALT) developed at murinelacrimal sac. TALT was shown to take up ocularantigens and generated antigen-specific T cellresponse as well as germinal center reaction andimmunoglobulin-class switch recombination .CD3－CD4＋CD45＋ lymphoid tissue inducer cells(LTi) initiate developmental program of lym-phoid organs. Transcriptional regulators, inhibi-tor of DNA binding/differentiation (Id)2 andretinoic acid receptor-related orphan receptor(ROR)γt regulate the differentiation of LTi. Wecould reveal the presence of three distinct LTisubsets for mucosa-associated lymphoid tissueorganogenesis. Based upon the expression levelof CD4 and the requirement of Id2 and RORγt,PP inducer was determined as Id2- and RORγt-dependent CD3－CD4highCD45＋ cells, whereasNALT inducer was Id2-dependent, RORγt-independent CD3－CD4lowCD45＋ cells. Newlyidentified TALT was induced by Id2- andRORγt-independent CD3－CD4lowCD45＋ cells .Collectively, organogenesis of mucosa-associatedlymphoid tissues (e.g., TALT, NALT, PP) is initi-ated by distinct inducer population.

8. Molecular and Cellular Analysis of Host-Microflora Interaction

Takashi Obata1, Yoshiyuki Goto1, Jun Kuni-sawa1, Shintaro Sato1, Naoko Shibata1, Yoshi-nori Umesaki2, Yoshimi Benno3 and Hiroshi Ki-yono1: 1Division of Mucosal Immunology, De-partment of Microbiology and Immunology,The Institute of Medical Science, The Univer-sity of Tokyo, 2Yakult Central Institute for Mi-crobiological Research, 3Microbe Division/Ja-

pan Collection of Microorganisms, RIKENBioResource Center,

Our mucosa acts directly as an immunologicalinterface between the external environment andthe host. The surface area of the mucosa is over200 times larger than that of the skin. Of thesemucosal areas, the intestine is most frequentlyexposed to a huge number and a wide varietyof environmental antigens including bacteriaand food products. It has been demonstratedthat gut microbiota are ingested into Peyer’spatches (PPs) and induce host immune re-sponses in the experiment with culurable Entero-bacter cloacae. However, the behavior of intesti-nal bacteria is still well understood becausemore than 90％ of the intestinal microbes havenot been cultured. In this study, we revealed thebacterial composition in the various intestinalcompartments by using 16S rRNA gene clone li-brary analysis. For example, Lactobacillius andsegmented filamentous bacteria (SFB) weredominated at the surface of PPs. In contrast,some kinds of opportunistic bacteria were pre-dominantly existed inside PPs.

We also revealed that gut microflora in the il-eal villous parts consists of unique bacterialpopulation and is involved in the induction ofintestinal epithelial fucosylation, which hasshown to be utilized by some kinds of commen-sal bacteria as a nutrient. Germ-free (GF) micehad low numbers of fucosylated epithelial cells(F-ECs) compared with conventional mice andthe F-ECs were restored when GF mice wereconventionalized. These epithelial fucosylation isregulated by fucosyltransferase 2 (Fut2) inducedby commensal bacteria. We also found Fut2-deficient mice, which are defective in F-ECs,have aberrant intestinal bacterial population inthe ileum, in particular, the frequency of Lactoba-cillus decreased. These results suggest a novelsymbiotic relationship between host and gut mi-crobiota. We are now trying to reveal the contri-bution of site-specific indigenous gut microbiotato the host mucosal immune system.

Publications

Mestecky, J., Nguyen, H., Czerkinsky, C., andKiyono, H. Curr. Opin. Gastroenterol. 24: 713-719, 2008.

Fehervari, Z., and Kiyono, H. Trends Immunol.29: 503-504, 2008.

Kunisawa, J., Nochi, T., and Kiyono, H. TrendsImmunol. 29: 505-513, 2008.

Caipang, C.M., Verjan, N., Ooi, E.L., Kondo, H.,Hirono, I., Aoki, T., Kiyono, H., and Yuki, Y.

Fish Shellfish Immunol. 25: 315-320, 2008.Terahara, K., Yoshida, M., Igarashi, O., Nochi,

T., Pontes, G.S., Hase, K., Ohno, H.,Kurokawa, S., Mejima, M., Takayama, N.,Yuki, Y., Lowe, A.W., and Kiyono, H.J. Immu-nol. 180: 7840-7846, 2008.

Uematsu, S., Fujimoto, K., Jang, M.H., Yang, B.G., Jung, Y.J., Nishiyama, M., Sato, S., Tsu-jimura, T., Yamamoto, M., Yokota, Y., Kiyono,

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H., Miyasaka, M., Ishii, K.J., and Akira, S.Nat. Immunol. 9: 769-776, 2008.

Verjan, N., Ooi, E.L., Nochi, T., Kondo, H., Hi-rono, I., Aoki, T., Kiyono, H., and Yuki, Y.Fish Shellfish Immunol. 25: 170-180, 2008.

Kobayashi, T., Takahashi, K., Nagai, Y., Shibata,T., Otani, M., Izui, S., Akira, S., Gotoh, Y., Ki-yono, H., and Miyake, K. Int. Immunol. 20:881-891, 2008.

Ooi, E.L., Verjan, N., Haraguchi, I., Oshima, T.,Kondo, H., Hirono, I., Aoki, T., Kiyono, H.,and Yuki, Y. Dev. Comp. Immunol. 32: 1211-1220, 2008.

Momoi, F., Hashizume, T., Kurita-Ochiai, T.,Yuki, Y., Kiyono, H., and Yamamoto, M. In-fect. Immun. 76: 2777-2784, 2008.

Gohda, M., Kunisawa, J., Miura, F., Kagiyama,Y., Kurashima, Y., Higuchi, M., Ishikawa, I.,Ogahara, I., and Kiyono, H.J. Immunol. 180:5335-5343, 2008.

Chang, S.Y., Cha, H.R., Igarashi, O., Rennert, P.

D., Kissenpfennig, A., Malissen, B., Nanno,M., Kiyono, H., and Kweon, M.N.J. Immunol.180: 4361-4365, 2008.

Ooi, E.L., Verjan, N., Hirono, I., Nochi, T.,Kondo, H., Aoki, T., Kiyono, H., and Yuki, Y.Fish Shellfish Immunol. 24: 506-513, 2008.

Kunisawa, J., Gohda, M., Kurashima, Y.,Ishikawa, I., Higuchi, M., and Kiyono, H.Blood. 111: 4646-4652, 2008.

Chang, S.Y., Cha, H.R., Uematsu, S., Akira, S.,Igarashi, O., Kiyono, H., and Kweon, M.N.J.Immunol. 180: 1609-1618, 2008.

Hashizume, T., Togawa, A., Nochi, T., Igarashi,O., Kweon, M.N., Kiyono, H., and Yamamoto,M. Infect. Immun. 76: 927-934, 2008.

Kiyono, H., Kunisawa, J., McGhee, J.R., andMestecky, J. The mucosal immune system. InFundamental Immunology (Edited by WilliamE. Paul). Lippincott-Raven, Philadelphia, pp.983-1030, 2008.

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