THE WAKSMAN FOUNDATION OF JAPAN INC. 　

Report of Researchesin2013

Dr. Selman A.Waksman

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THE WAKSMAN FOUNDATION

OF JAPAN INC.

Published by THE WAKSMAN FOUNDATION OF JAPAN INC.

30-8 Daikyo-cho, Shinjuku-ku, Tokyo, Japan

THE WAKSMAN FOUNDATIONOF JAPAN INC.

Report of Researches in 2013

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THE WAKSMAN FOUNDATIONOF JAPAN INC.

Report of Researches in 2013

THE WAKSMAN FOUNDATION OF JAPAN INC. 　

Report of Researchesin2013

Dr. Selman A.Waksman

●スミ版●シアン版を紺色（前回と同色）に 2色刷り

THE WAKSMAN FOUNDATION

OF JAPAN INC.

Published by THE WAKSMAN FOUNDATION OF JAPAN INC.

30-8 Daikyo-cho, Shinjuku-ku, Tokyo, Japan

THE WAKSMAN FOUNDATIONOF JAPAN INC.

Report of Researches in 2013

2 0 1 4

THE WAKSMAN FOUNDATIONOF JAPAN INC.

Report of Researches in 2013

THE WAKSMAN FOUNDATIONOF JAPAN INC.

Honorary Patron

H.I.H. Prince Akishino

Board of Directors

Chairman : Ichiro Kitasato, Adviser, The Kitasato Institute.Former Chairman of The Board, Meiji Seika Kaisha, Ltd.

Shogo Sasaki, Prof. Emeritus, Keio Univ.

Teruhiko Beppu, Prof. Emeritus, Tokyo Univ.

Takeshi Ishikawa, Vice PresidentRetirement Allowance Foundation of Private Colleges and Universities.

Tadakatsu Shimamura, Prof. Emeritus, Showa Univ.

Tadayoshi Shiba, Chairman Emeritus, The Kitasato Institute.

ManagingDirector : Takeji Nishikawa, Prof. Emeritus, Keio Univ.

Comp-troller : Yoshiharu Wakiyama, Senior Adviser, Kaken Pharmaceutical, Co., Ltd.

Shirow Enoki, Former President, Seikagaku Corporation.

Councilors

　Ryoichi Mori, Prof. Emeritus, Kyushu Univ.

　Keizo Takemi, Member,The House of Councilors.

　Koichi Yamanishi, Director General,The Research Foundation for Microbial Diseases of Osaka Unoversity.

　Sachiko Goto, Prof. Emeritus, Toho Univ.

　Isao Uchida, Senior Adviser, Yokogawa Electric Corporation.

　Shigeo Koyasu, D. Sc. Director RIKEN Center for Integrative Medical Sciences.

　Takashi Shoda, Senior Corporate Adviser, Daiichi Sankyo Co., Ltd.

　Yuko Kitagawa, Prof., Keio Univ. Sch. Med.

Special Adviser

　Akira Uehara, Chairman & CEO, Taisho Pharmaceutical Holdings Co., Ltd.

　Osamu Nagayama, Chairman & CEO, Chugai Pharmaceutical Co., Ltd.

　Haruo Naito, President & CEO, Eisai Pharmaceutical Co., Ltd.

　Seiichi Sato, President & CEO, Sato Pharmaceutical Co., Ltd.

　Yoshihiro Miwa, President & CEO, Kowa Co., Ltd.

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ii

Preface to the First Report (1962)

It is indeed a privilege to take this opportunity to write a few words of introduction to the first report of the Waksman Foundation of Japan Inc., covering five years of its activities and comprising the results of the work of the first two years of research carried out by various scholars in Japan in the fields of microbiology and medical science, supported by this Foundation.

In 1952, I accepted the invitation from Keio University and the Kitasato Institute, to deliver the centennial lecture in honor of the great Japanese bacteriologist, Shibasaburo Kitasato. Before departing for Japan, I proposed to the trustees of the Rutgers Research and Educational Foundation which owned the patents on streptomycin, to share the royalties under the patent in Japan, for the support of research in microbiology and allied fields in that country. The trustees heartily approved my recommendation that I make such announcement to that effect.

Soon upon my arrival in Japan (December 17, 1952), I invited a group of eminent microbiologists, biochemists, and clinical investigators to meet with me in order to discuss the plan. Everyone present was very enthusiastic about the proposal. It was decided that a proper committee be selected to work out the plan of a Foundation under which the royalties were to be received and distributed for the support of Japanese investigators working in different universities in Japan and elsewhere, in the fields of microbiology and medical research. The committee recommended that a Board of Directors be selected and the proposed Foundation be named THE WAKSMAN FOUNDATION OF JAPAN INCORPORATION.

The Rutgers Research and Educational Foundation approved at once the above recommendations and issued a statement, signed by Dr. Lewis Webster Jones, President of the Foundation, to the effect that

‘‘The Rutgers Research and Educational Foundation desires to emphasize that its principal concern is the advancement of scientific knowledge in the public interest and that it confidently expects that the Waksman Foundation for Microbiology and Medical Research in Japan will be similarly motivated, thereby serving the peoples of both countries.’’

This announcement was received with enthusiasm both by the scientific world and the popular press in Japan and in the United States. It took several years before the Waksman Foundation of Japan Inc. was properly organized, and before applications were received and approved. In 1958, I had the privilege of participating in the first official meetings of the Board of Directors of the Japanese Foundation and to greet personally the first group of scholars to whom grants had been made.

In summarizing these brief remarks in connection with the first cinqueannual report of the Waksman Foundation of Japan Inc., I would like to enphasize that this example of collaboration between universities and scientists of the United States and Japan may serve to encourage collaboration between scientific workers throughout the world towards a better understanding between men and women and towards a happier and healthier human race, so that all the nations on this earth can live in peace and that man may finally ‘‘break

iii

his swords and build out of them plowshares’’ for the betterment of mankind as a whole.

Selman A. Waksman

Professor EmeritusRutgers-State University N. J., U. S. A.

The ‘‘Waksman Foundation of Japan Inc.’’ was established in 1957 with the spirit of humanity by Dr. S.A. Waksman, Professor of Microbiology, Rutgers University, U.S.A. The Foundation’s operations are possible only because Dr. S.A. Waksman and the Rutgers Research and Educational Foundation donated patent royalties he received from the production in Japan of the discovery, Streptomycin.

Because of these royalties, each year many Japanese scholars and research workers in the fields of Microbiology and medical science are encouraged and find it possible to continue their work. Especially, in accordance with Dr. Waksman’s suggestion, the funds are distributed to scholars in local and economically hampered schools and laboratories and to those developing research workers who are endeavoring to expand in their fields. This thought of Dr. Waksman’s is most appreciated, as it matches our Oriental phylosophy, and results in the search for a jewel among ordinary stones, which is the highest work of the science-leader.

Some five years have now passed since the start of this Foundation, and many persons have received aid through this period.

The reports which are presented herein cover the first and second group of research workers who received financial assistance from the Foundation.

Toshio Katow, M. D.

Executive Director

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v

Contents

Hirotaka Kanuka:

Genetic dissection of intermediate host and tapeworm interaction ………………………… 1

Jun-ichi Wachino:

Horizontal gene transfer of an amikacin resistant 16S rRNA methyltransferase gene from multidrug resistant Acinetobacter baumannii …………………………………… 7

Hayato Takahashi, M.D., Ph.D.:

Analysis on the mechanism of T cell peripheral tolerance against pemphigus autoantigen, desmoglein 3 ……………………………………………………………… 15

Hitomi Mimuro:

Helicobacter pylori regulate host non-coding RNA expression to increase the proliferation of gastric epithelium during chronic infection ………………………… 23

── Report of Researches in 2013──

Genetic dissection of intermediate host and tapeworminteraction

Hirotaka Kanuka

Department of Tropical Medicine, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan

ABSTRACT Dwarf tapeworm, Hymenolepis nana, which belongs to phylum Cyclophyllidea, is the most com-mon cestode of human. Its intermediate host is arthropods, in particular, beetles. Once the intermediate host ingeststapeworm eggs, oncospheres immediately hatch and pass through insect gut wall. Cysticercoids develop within thehemocoel where they survive without loss of infectivity until the intermediate host is ingested by a definitive host.To dissect the interaction between tapeworm and intermediate host, we employed a reverse genetic approach withred flour beetle, Tribolium castaneum, in which a robust systemic RNA interference (RNAi) response is observed,as a model system to explore host responses to tapeworm infection. Adult knock-down phenotypes in T. castaneumwere induced by injection of double-stranded RNA (dsRNA) into late instar larvae. We performed RNAi screeningtargeting several gene transcripts of Toll and immune deficiency (IMD) pathways, which are two major signal-ing pathways of humoral immune response in insects. Reduction of Toll pathway function, which was inducedby RNAi-mediated silencing of MyD88, Dif1, and Dif2, in addition to JAK/STAT and JNK components, causedincrease of burden of cysticercoids. On the other hand, RNAi-mediated knockdown of IMD pathway components,dredd and imd, had no significant difference on cysticercoid load. Our findings suggest a pivotal role of specificpathway such as Toll signaling in regulating resistance to tapeworm infection.

Introduction

Understanding the molecular machinery of theresponses of disease-transmitting arthropods (vectors)against pathogens is of great importance for currentefforts to develop novel strategies for control ofvector-borne diseases. Arbovirus, protozoan parasites,and parasitic nematodes undergo substantial stage-specific losses during those developments in the vectormosquito using a certain defense system, which insome cases lead to complete refractoriness of themosquito against those pathogens. On the other hand,susceptible mosquitoes have a greater tendency to holdsufficient number of parasites for transmission thanrefractory strains, acting as a “cargo of pathogen”.The ability of vector arthropods to possess humanpathogens (i.e., virus, protozoan parasites, and

worms) without showing any pathological symptomsis commonly referred to as the “vector paradox”,which remains the important and unanswered questionconcerning dual strategies of host (vector) defense:resistance and tolerance

Defense against pathogenic micro-organisms andother parasites can be divided into two conceptuallydifferent components: resistance, a character thatreduces the pathogen’s opportunity of successfulinfection through an impact on pathogen fitnesspotentially via the host’s rate of pathogen clearance,and tolerance, the host’s ability to cope with thehealth impacts of a pathogenic encounter without aconsequent reduction in fitness to the host. In vectorarthropods, a distinguishing feature between these twostrategies for dealing with pathogen spreading is thatresistance has a negative effect on pathogens, whereastolerance does not, as a result, their relative importance

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in vector species may have substantial consequences forthe evolution of vector-pathogen interactions. Solvingand ultimately manipulating the vector paradox tocontrol the vector-borne diseases is in many respectsthe “Holy Grail” for all of scientists in the researcharea. By continuing to unravel the mysteries thatunderlie the mutual prosperity between pathogen andvector, we believe that it may one day be possible toselectively attenuate the tolerance activities of vectorsin addition to enhance the resistance and, consequently,to “minimize” vector fitness/health in such a waythat vector itself is converted from a life-threateningarthropods to one that is a fragile blood-sucking bugwith a weak constitution unable to endure the pathogentransmission.

Human tapeworms, such as Hymenolepis nana,require insect as intermediate host to infect definitivehost. The tapeworm, which belongs to phylumCyclophyllidea, is the most common cestode of human.Its intermediate hosts are grain pest beetles andcockroaches. Humans get infected with tapewormby accidental ingestion of infected insects. Whenthe intermediate host insect ingests tapeworm eggs,oncospheres immediately hatch and pass through insectgut wall. Cysticercoids develop in the hemocoel wherethey survive without loss of infectivity until theintermediate host is ingested by a definitive host. Forthis reason, it has been supposed that the responseof intermediate host insects to tapeworm is absent orweak.

Here we focus to determine the signal transductionpathways for tolerance, by using insect genetic system,and more importantly, to identify and characterizethe manner through which artificial aberrations inthe tolerance process contribute to the disruptionof pathogen spreading by vectors. To dissect thetolerance in addition to resistance system, we employeda reverse genetic approach with red flour beetle,Tribolium castaneum, in which a robust systemicRNA interference (RNAi) response is observed, as amodel system to explore host responses to pathogensinfection. Our approach with specific aims designedwill contribute to uncover common features in thetolerance system that may have in tern contributed tothe certain specific characteristic of vector species.

Materials and Methods

Beetle Cultures

The beetle cultures were reared on whole wheatflour [+5% (wt/wt) yeast] at 30◦C in a temperature andhumidity controlled incubator. An enhancer trap linepu11, which has enhanced yellow fluorescent protein(EGFP) expression in the hindwing and elytron discs,was used for all RNAi experiments.

Tapeworm Parasite

Hymenolepis nana used in this study is parasitictapeworm of human and rodent. The tapeworm isrelatively small (∼ 4 cm) and has short life cycle (∼ 1month). The tapeworm does not need intermediatehost to complete its experimental life cycle becauseof direct ingestion of tapeworm eggs. The passageof this tapeworm is accomplished by using mice asexperimental host. H. nana (a gift from Dr. KazuhitoAsano, Showa University) has been maintained inlaboratory for more than 20 years. Preparation of H.nana eggs and oral infection of mice were performedas previously described (Asano and Muramatsu, IntJ Parasitol 27(11): 1437-43 (1997)). Briefly, micewere challenged orally with about 1000 H. nana eggsand killed 4 days later. All parasites derived from theprimary infection were recovered as lumen-dwellingadult worms.

Gene Cloning and dsRNA Synthesis

The Tribolium ortholog of Drosophila gene wasidentified via BLAST analysis. Tribolium pupal cDNAwas used to clone the cDNA fragment into pT7 vector(Invitrogen). The dsRNA templates were synthesizedby PCR using the vector primer, or gene specificprimers with the T7 polymerase promoter sequenceat the 5’ end. dsRNAs were synthesized by in vitrotranscription (Megascript T7; Ambion) using 1.5 μgof templates. The resulting dsRNA samples were thenpurified by Megaclear kit (Ambion). Specificity of theproducts was confirmed via agarose gel electrophoresis.

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EGFP-expressing transgenic line (Pu-11)

full genome sequence (2008)

Tribolium castaneum

amount of dsRNA

5 days after 30 days after

buffer 500ng100ng buffer 500ng 100ng1ng 100pg

EGFP dsRNA

RNA interference (RNAi) effect in T. castaneum

amount of dsRNA

Figure 1. Tribolium castaneum exhibits a robust systemic RNA interference response.pu11 larvae, pupae, and adults after injection with buffer early in the last larval stage. Green fluorescent protein (GFP)expression in these individuals is identical to non-injected pu11 beetles. pu11 larvae injected with various concentrationsof GFP dsRNA. Injection of 100 pg dsRNA in each larva induces almost perfect silencing in most larvae

Concentration of dsRNA ranged from 5 to 7 μg/uL.

dsRNA Injection into Beetle

Injections were performed in the last larval stageor penultimate larval stage. EGFP expression in pu11developing wing and elytron discs was used to selectappropriate larval stages. Stereomicroscopes (Leica)were used for the injections. At least 50 larvae wereused for each set of injections. Approximately 0.7–0.9 μL of 1 μg/μL dsRNA solution was injected intoeach larva. After injection, the larvae were kept inflour at 30◦C until they became adults for phenotypicanalysis.

Parasite Infection into Beetle

Adult loss-of-function phenotypes were examinedby injection of 100 ng dsRNA into late instar larvaeor penultimate larvae of pu11 beetles expressingEGFP as described above. After 3 days starvation,artificial infection was performed by direct feeding ofadult tapeworms to the beetle, which was collectedfrom small intestine of infected mice. Twelve intacttapeworms were fed to thirty beetles. After 10 daysfrom infection, beetles were dissected to quantify thenumber of cysticercoid.

Results

To investigate signaling regulation interactionbetween parasitic worm and intermediate host, weperformed a loss-of-function (RNAi-based) screeningto find genes that affect tolerance and/or resistancein beetles infected with tapeworm. No observabledecrease in tapeworm load in prolonged survival strainsagainst infection will be taken as an indication of ashift in the balance between resistance and tolerance ofhost defense to a state of coexistence with the invadingparasite.

To dissect interaction between tapeworm andintermediate host, we employed RNAi screening withT. castaneum, as a model system to explore hostresponses to tapeworm infection. The RNAi effect canbe elicited in any tissue and any stage by the injection ofdsRNA into the hemocoel, and injection of dsRNA intoadult females can even be used to identify phenotypesin offspring. In order to confirm RNAi effect in T.castaneum, dsRNA for EGFP was injected to larvae ofpu11 beetles (EGFP expressing enhancer trap line). Thesilencing effect was observed even at 100 pg/larva andsustained for 30 days (Figure 1).

Transcriptomes of beetle midgut, which iscrucial interface between beetles and tapeworms, wereanalyzed using next-generation sequencing (Figure2). This transcriptome analysis resulted in theidentification of 203 differentially expressed genes

— 3 —

Figure 2. Tribolium castaneum infection increases expression of immune-related genes.Transcriptomes of T. castaneum midgut, which is crucial interface between beetles and tapeworms, were analyzed usingnext-generation sequencing. Pie chart represents the classification of 203 differentially expressed genes with statisticalsignificance based on GO biological process.

0

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Dif2EGFP

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MyD88EGFP0

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012

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spätzle1EGFP

<0.01p p p

Figure 3. Toll pathway is involved in resistance to tapeworm infection in Tribolium castaneum.Reduction of Toll pathway function, which was induced by RNAi-mediated silencing of MyD88, Dif1, Dif2 and Spatzle1,caused increase of burden of cysticercoids. For scatter plot, the each dot represents cysticercoid counts from individualbeetles and the horizontal black bar indicates the median infection level. dsRNA for EGFP-injected groups were used asnegative control.

upon tapeworm infection. Furthermore, tapeworminfection caused elevated expression of 5 genesencoding antibacterial peptides. This result suggeststhat tapeworm infection induces innate immunitygenerating antibacterial peptides. We then selected 101genes for primary RNAi screening, not only innateimmunity-related genes but also Tribolium ortholog ofhost defense-related genes (e.g., JNK and JAK/STATgenes), which were previously reported in otherarthropods.

Reduction of Toll pathway function, which wasinduced by RNAi-mediated silencing of MyD88, Dif1,Dif2 and Spatzle1, caused increase of burden of

cysticercoids (Figure 3). These results suggest thatinnate immunity induced by Toll pathway signalingsuppresses tapeworm infection. The contribution ofToll pathway to parasite infection was already reportedin mosquito-plasmodium studies, thus these resultsconfirm the validity of this RNAi screening method.On the other hand, RNAi-mediated knockdown of IMDpathway components, dredd and imd, had no significantdifference on cysticercoid load (data not shown).

RNAi-mediated depletion of JAK/STAT pathwaycomponents, Domeless (encoding a receptor for aligand stimulating JAK/STAT) and Hopscotch (JAKkinase), increased cysticercoid burden. On the other

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p<0.01

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p<0.01

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Figure 4. JAK/STAT pathway is involved in resistance to tapeworm infection in Tribolium castaneum.RNAi-mediated depletion of JAK/STAT pathway components, Domeless and Hopscotch, increased cysticercoid burden.The silencing of negative regulator of this pathway, SOCS36E, reduced cysticercoid load. For scatter plot, the each dotrepresents cysticercoid counts from individual beetles and the horizontal black bar indicates the median infection level.dsRNA for EGFP-injected groups were used as negative control.

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basket1

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p<0.01

Figure 5. JNK pathway regulates susceptibility to tapeworm infection in Tribolium castaneum.Knock-down of the JNK pathway components, basket1, basket2 and slipper, enhanced tapeworm infection. For scatterplot, the each dot represents cysticercoid counts from individual beetles and the horizontal black bar indicates the medianinfection level. dsRNA for EGFP-injected groups were used as negative control.

hand, silencing of negative regulator of this pathway,SOCS36E, reduced cysticercoid load (Figure 4). Theseresults suggest that JAK/STAT pathway facilitatestapeworm infection. We also found that deletion of theJNK pathway components, basket1, basket2 (encodingJNK, respectively), and slipper (encoding upstreamkinase of JNK pathway), enhanced tapeworm infection(Figure 5), suggesting that the host response inducedby JNK pathway signaling reduces tapeworm burden.Eiger, the TNF-superfamily ligand in T. castaneum,induces cell death through the activation of JNKsignaling. Eiger plays a central role in cell competition,which is a mechanism to keep tissue homeostasis.RNAi-mediated silencing of Tribolium Eiger genecaused increase of the number of cysticercoid (Figure

6). These results suggest that JNK pathway-inducedstress response, which is independent of innateimmunity, limits tapeworm infection.

To get deeper mechanistic insights about tolerantmechanism in insects and to apply these findingsinto vector species, it is necessary to describe theconserved tolerance system via analyzing the knock-down phenotypes of candidate tolerance-related genesin particular p38 MAP kinase family in Tribolium inaddition to the fly (Shinzawa et al., Cell Host Microbe6(3): 244-52 (2009)). Elucidating the mechanisms bywhich vector-specific traits arose may shed light onone of the general principles of host tolerance diversity.However, we determined the p38 loss-of-functionphenotypes in Tribolium and observed significant effect

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010203040506070

p<0.01

the

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ber o

f cys

ticer

coid

EigerEGFP

Figure 6. TNF ligand is involved in defense response totapeworm infection in Tribolium castaneum.Eiger, the TNF-superfamily ligand in T. castaneum,induces cell death through the RNAi-mediatedsilencing of T. castaneum Eiger gene encodinga TNF-superfamily ligand caused increase of thenumber of cysticercoid. The each dot representscysticercoid counts from individual beetles and thehorizontal black bar indicates the median infectionlevel. dsRNA for EGFP-injected groups were usedas negative control.

on neither cysticercoid burden nor survival rate of hostbeetle (data not shown), suggesting that there could bealternative pathways controlling host tolerance againsttapeworm infection.

Our findings in this report indicate that JAK/STATand JNK pathways play pivotal role to regulateresistance to tapeworm infection in addition to Toll-mediate innate immunity. It is supposed that theintegration of several host responses may be importantto define the proper competency of intermediate hostfor tapeworm transmission.

Discussion

Effective control strategies of parasitic diseasestargeting intermediate host currently include insecticidetreatment delivered through spraying of housesor insecticide-impregnated insect nets. While thesemethods are effective at decreasing arthropod numbers,they also contribute to the rise of insecticide-resistanceintermediate host species. An alternative strategyinvolves inhibition of parasite development withinthe intermediate host. One such strategy involvesrelease of transgenic intermediate host resistant toparasite that could compete with wild populations.

However, transgenes have proven difficult to fix inpopulations. The finding that Toll and JAK/STATpathways in immune response tissues was capableof conferring resistance to tapeworm H. nana wassurprising considering traditional roles for the Toll andJAK/STAT pathways; the JAK/STAT pathway has beenthought to be activated in response to virus while theToll pathway was responsible for immunity to Gram-positive bacteria and fungi. Recently this view of insectinnate immunity has begun to change with numerousfindings contradicting traditional views. Certainly, thisstudy provides further evidence for a modified viewof innate immunity against large parasite such astapeworm.

While this study clearly demonstrates a novelrole for JNK and JAK/STAT pathways in conferringresistance to beetles infected with tapeworm, a rolefor these factors in resistance cannot be ruled out.Indeed, survival of an infected organism likely dependson a combination of both resistance to that organismand tolerance to the infection. Such a combination ofmechanisms can lead to a total of 9 different states of anorganism ranging from high tolerance coupled with lowresistance to low tolerance coupled with high resistanceand all states in between. The JNK and JAK/STAT-mediated defense seen in this study potentially fallsinto the class represented by high resistance and lowtolerance. In order to shed light on such complicateddefense mechanisms comprehensive work investigatingthe synergistic effects of both tolerance and resistanceto host fitness will be required.

Conclusion

Invertebrate model systems such as Tribolium arerecognized as a powerful means of studying conservedmechanisms and genes relevant to human health. In thisresearch proposal, through bridging from Triboliumto vector species, our work will greatly extend theknowledge of the molecular basis of tolerance inpathogen transmitting vectors. Our findings will offerimportant insights and inroads in understanding thisimportant and essential question that clearly underliesthe biological and pathological actions of vectortolerance.

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Horizontal gene transfer of an amikacin resistant 16SrRNA methyltransferase gene from multidrug resistantAcinetobacter baumannii

Jun-ichi Wachino

Department of Bacteriology, Nagoya University Graduate School of Medicine, 65 Tsurumaicho, Showa-ku,Nagoya, Aichi 466-8550, Japan

Introduction

Acinetobacter baumannii is an opportunisticGram-negative pathogen that causes severe infectionssuch as ventilator-associated pneumonia in hospitalizedpatients and is frequently associated with nosocomialoutbreaks (1). A. baumannii is becoming a seriousthreat to public health due to the acquisition ofmultidrug-resistant genes. This has resulted in theemergence of A. baumannii strains resistant tocarbapenems, aminoglycosides, and fluoroquinolones,which limits the choice of antimicrobial agents forthe treatment of clinical infections. The antimicrobialstigecycline and colistin are used as a last resort in thetreatment of multidrug-resistant (MDR) A. baumannii.However, even tigecycline and colistin resistant strainshave recently emerged (2, 3). The rapid spread of MDRA. baumannii has been attributed to the transmissionof several international strains, which are assigned toInternational clones (IC) I and II (4). Of these, the IC-II is emerging as the world's dominant population ofMDR A. baumannii.

One of the aminoglycoside resistance mechanismsidentified in MDR A. baumannii is the aminoglycosideresistance 16S rRNA methyltransferase gene (armA),which prevents aminoglycoside from binding to theribosome through modification within the aminoacyl-tRNA binding site of the 16S rRNA (5). Aminoglyco-side resistance has been widely disseminated throughthe acquisition of armA among A. baumannii isolates.

Galimand et al. has shown that the translocation ofarmA associated with Tn1548 could occur from a non-conjugative plasmid of a Klebsiella pneumoniae clini-cal isolate using a genetically tractable Escherichia colisystem (6). It is therefore possible that the translocationof the armA gene could also occur in A. baumannii asseen in K. pneumoniae.

Recently, the complete genome sequences for anumber of endemic MDR A. baumannii belongingto the IC-II have become available. Genome analysisrevealed that the armA gene is located withinthe chromosome for the majority of the IC-IIstrains. We hypothesized that the IC-II is a majorsource for the distribution of the armA gene toother bacterial species, including antibiotic sensitivenon-baumannii Acinetobacter species. Acinetobacterspecies are naturally transformable (7), an attributewhich is thought to accelerate the dissemination ofantibiotic resistance genes. However, the contributionof this feature to the emergence of MDR A. baumanniihas yet to be determined. The aim of this study wasto elucidate the molecular mechanisms behind thetransfer of antibiotic resistance genes, focusing on theaminoglycoside resistance gene armA, in A. baumannii.

Materials and methods

Bacterial strains and growth conditions

The A. baumannii clinical strain ARS60, whichwas isolated from a patient sputum sample in 2004,

— 7 —

was used in this study. The antibiotic susceptible A.baumannii laboratory strain ATCC17978 was used asthe recipient strain. The bacterial strains were grown inLuria-Bertani (LB) agar and broth, with the addition ofamikacin (25 μg/ml) when required.

Genome sequencing of A. baumannii strains

Genomic DNA was extracted from the A.baumannii strains using a QIAamp DNA Minikit. The genomic DNA was sheared to a size ofapproximately 500 bp, performed by Covaris. Thesheared genomic DNA was purified using a MinElutePCR purification kit. Pyrosequencing was performedon a 454 GS junior. The A. baumannii ARS60strain reads were assembled into contigs by the denovo assembler (Newbler Assembler version 2.3). TheA. baumannii ATCC17978ΩarmA reads were firstmapped to the reference genome of the A. baumanniiATCC17978 strain using a reference mapper program.The remaining unmapped reads were further assembledinto contigs by the de novo assembler. This allowed theidentification of foreign DNA inserted into the genomeof A. baumannii ATCC17978.

Transposon detection using PCR

The presence of a circular intermediate (CI) wasdetected by inverse PCR, using the primer sets, P1(5'-ccatcacatgtatgaccaga-3') and P2 (5'-gtttttccagtac-tacgcca-3'). Evidence of genetic rejoining following ex-cision of a composite transposon was confirmed byPCR, using the primer sets P3 (5'-aattgggctgatgagtacgg-3') and P4 (5'-gctgcacagtggttttctga-3').

Susceptibility testing

The minimum inhibitory concentrations (MICs)were determined. Bacteria samples were adjusted to108cfu/ml, and spread on a Mueller-Hinton agar plate.The Etest strip was placed onto the plate and incubatedat 35◦C for 18 h.

Purification of the component responsible for thetransfer of armA

The A. baumannii ARS60 strain was cultured inLB broth and the bacterial cells were removed bycentrifugation. The supernatant was passed through0.45 μm filter and ultracentrifugation was carriedout at 150,000 × g for 90 min at 4◦C. Afterultracentrifugation, the pellets were resuspended withphosphate buffered saline (PBS), passed through0.22 μm filter and stored at −80◦C until required.

Transformation

The purified component from the bacterial super-natant was combined with A. baumannii ATCC17978and incubated on LB agar plates. After incubation, thebacteria growth was scraped off with a sterile loop, re-suspended in LB broth and spread onto LB LB agarplates supplemented with 25 μg/ml amikacin.

Results

Antibiotic resistance genes

The draft genome sequence of the A. baumanniiARS60 strain consisted of 144 contigs, correspondingto 3,963,199 bp, with an N50 contig size of 60,507bp. The web-based tool, ResFinder version 1.3 wasused to interrogate the draft genome sequence, inorder to identify acquired antibiotic resistance genes.The analysis showed that several antibiotic resistancegenes were embedded within mobile genetic elements,located on the chromosome of the ARS60 strain (Table1). Due to the acquisition of these antibiotic resistancegenes, A. baumannii ARS60 has become a multidrugresistant strain. The A. baumannii ARS60 strain hasbeen assigned to ST208 by the Bartual scheme, ST2by Pastuer Institute Scheme and belongs to the IC-II.

Genetic environment of armA

The genetic platform containing the armA gene,located on the chromosome of the A. baumannii ARS60

— 8 —

Table 1. Antibiotic resistance genes found in A. baumannii ARS60 strain

Resistance gene Function Resistance Phenotypeaac(3)-Ia acetyltransferase aminoglycosideaadA1 adenylyltransferase aminoglycosideaac(6')-Ib acetyltransferase aminoglycosideaph(3')-Ic phosphotransferase aminoglycosidestrA phosphotransferase aminoglycosidestrB phosphotransferase aminoglycosidearmA 16S rRNA methyltransferase aminoglycosideblaOXA−66 β -lactamase β -lactamblaADC−25 β -lactamase β -lactamblaTEM−1D β -lactamase β -lactammph phosphotransferase macrolidemel efflux transporter macrolidecatB3 acetyltransferase chloramphenicolsul1 dihydropteroate synthase sulfonamidetetB efflux transporter tetracycline

strain (Fig. 1), was revealed following analysis of thedraft genome sequence and the use of the PCR to closethe gaps between contigs. Upstream of the armA genelies a putative transposase gene, ISCR1 (previouslycalled orf513). The classical class 1 integron includesΔ intI1 at its 5'-conserved segment (CS), the antibioticresistance gene cassettes aac(6')-Ib, catB3, aadA andqacEΔ1-sul1 at its 3'-CS. Downstream of the armAgene, are macrolide resistance genes mel and mph, ahypothetical protein, IS66, and a putative rep gene.This 18.3 kb region containing the armA gene wasflanked by two IS6 elements, with 14-bp invertedrepeats (IRL and IRR) at the terminal ends, indicatinga composite transposable element. The same 8 bpnucleotide sequences, CTCATCCT was located at bothterminal sides of the composite transposable element.The presence of these 8 bp target sites would be anindication that insertion of the transposon bearing thearmA gene had occurred at this position.

Detection of circular transposon DNA and postexcisionsite

It is likely that the chromosomally located 18.3 kbcomposite transposable element containing the armAgene could form a non-replicative CI, before lateral

transposon transfer. To confirm the CI formation,inverse PCR was performed using the genomic DNAextracted from the A. baumannii ARS60 strain. If thecomposite transposon excises and forms a CI, a PCRproduct would be generated. The 1.2 kb PCR productgenerated by the P1 and P2 primer sets, confirmedthe presence of the CI (Fig. 2). In addition, wehypothesized that there would be evidence for the generejoining following the excision of transposon. To testthis we used PCR and confirmed the presence of a1.5 kb fragment produced by P3 and P4 primer sets,indicating the left IS6 element (Fig. 2). These resultssuggest that CI formation occurred among a smallproportion of A. baumannii ARS60 population

Transfer of the armA gene in vitro

Components purified from the A. baumanniiARS60 strain supernatant by ultracentrifugation, werecombined with the A. baumannii ATCC17978 strain.After incubation, the ATCC17978 transformants thathad acquired amikacin resistance were selected on LBagar plates containing amikacin, as shown in Fig. 3. Theacquisition of amikacin resistance was demonstratedwhen the components purified from the A. baumanniiARS60 supernatant following 6 h of growth, were

— 9 —

A1S_2192

A1S_2193

A1S_2194

A1S_2196

A1S_2197

A1S_2198

A1S_2199

A1S_2200

A1S_2201

A1S_2202

A1S_2203

A1S_2204

A1S_2205

A1S_2206

A1S_2207

A1S_2208

A1S_2209

A1S_2210

A1S_2211

A1S_2212

A1S_2213

A1S_2214

A1S_2215

60_2192

60_2193

60_2194

60_2196

60_2197

60_2199

60_2200

60_2201

60_2202

60_2203

60_2204

60_2205

60_2206

60_2207

60_2208

60_a

60_b

60_2198

IS6

rep

IS66

mph

mel

hyp

armA

tmpAcp1

ISCR1

sul1qacE 1aadA1

catB3

intI1

IS6

19-kb (composite transposon)

4.3-kb

60_2212

60_2213

60_2214

60_2215

60_2200

60_2201

60_2202

60_2203

60_2204

60_2205

60_2206

60_2207

60_2208

A1S_2192

A1S_2193

A1S_2194

A1S_2196

A1S_2197

A1S_2198

1-kb

AT

CC

17978

AR

S60

17978arm

A

A1S_2209

A1S_2210

A1S_2211

A1S_2212

A1S_2213

A1S_2214

A1S_2215

60_2199

Fig. 1. Comparison of the region surrounding the armA gene in A. baumannii ARS60 and A. baumannii ATCC17978ΩarmA. Thegenes for A. baumannii ATCC17978 are shown with white arrows and those for A. baumannii ARS60 are shown with lightgreen arrows.

mixed with the A. baumannii ATCC17978 strain,and increase to 24 h, and then decrease to 48 h.When the A. baumannii ATCC17978 strain was notcombined with the purified components from the A.baumannii ARS60 strain supernatant, resistant colonydid not appear. Several colonies which had grownon agar plates supplemented with amikacin weresubjected to PCR, and the presence of the armA

gene was confirmed. PFGE and Southern hybridizationrevealed that the armA gene was integrated into A.baumannii ATCC17978 chromosome (data not shown).The transformants (named 17978ΩarmA) showed thesame high levels of amikacin resistance as the ARS60parent strain (Fig. 4).

— 10 —

circular intermediate

18.3-kb

IS6

armA

A. baumannii ARS60

1.5 1.0

(kb)

X

23.1 9.4

2.01.51.0

0.5

(kb) (kb)

P3

armA

IS6P4

P3

P4

Fig. 2. Detection of circular intermediate carrying the armA gene and the site following excision.

Fig. 3. Results of amikacin susceptibility testing by the Etest for A. baumannii ARS60 strain, A. baumannii ATCC17978ΩarmA,and A. baumannii ATCC17978.

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10-9

10-8

10-7Tr

ansf

orm

atio

n ef

ficie

ncy

3 6 12 24 48

hours

Fig. 4. Transformation efficiency for A. baumanniiATCC17978 following transformation with pu-rified components from A. baumannii ARS60supernatant. Error bars indicate the standard deviation.

Acquisition of the ARS60 strain DNA by thetransformant 17978ΩarmA

The genome sequence of the transformant17978ΩarmA was determined by NGS analyses andthe results are shown in Fig. 1. The transformant17978ΩarmA acquired a DNA fragment approximately34 kb in size, which contained a composite transposoncarrying the armA gene derived from the A. baumanniiARS60 strain.

Discussion

A. baumannii isolated from clinical settingshave rapidly acquired resistance to a number ofantimicrobials, through the uptake of antibioticresistance genes. It is assumed that horizontal genetransfer (HGT) plays a crucial role in acquiringantibiotic resistance genes by this organism, asAcinetobacter species have an inherent ability toefficiently acquire exogenous DNA. The A. baumanniiclinical isolate was also expected to take up antibioticresistance genes through HGT; however, the detailshave not yet been fully elucidated. We hypothesized

that A. baumannii has a specific system to efficientlydeliver resistance genes in order to bestow resistance toantibiotic sensitive Acinetobacter species.

In this study, we have succeeded in purifyingcomponents, which include DNA fragments encodingantibiotic resistance genes, from the culture medium.We have used these purified components to transforman antibiotic sensitive laboratory strain, and gener-ated aminoglycoside resistant bacteria. Rumbo et alreported that MDR A. baumannii clinical isolates re-leased outer membrane vesicle (OMV), which con-tained small plasmids bearing carbapenem resistancegenes. These OMVs played a crucial role in transfer-ring antibiotic resistance genes to other A. baumanniistrains (8). The armA gene of the A. baumannii ARS60strain was carried by a composite transposon, whichcould be circularized (Fig. 2). It was speculated that cir-cularized transposons carrying the armA gene might bepresented in the OMV delivered to other A. baumanniistrains as suggested by Rumbo et al (8). We confirmedthat the CI carried the armA gene in the ARS60 strain.However, the aminoglycoside resistant transformant,17978ΩarmA acquired a DNA fragment approximately34 kb larger than the composite transposon carrying thearmA gene, probably through a homologous recombi-nation event. Therefore, the CI carrying the armA genedoes not appear to be directly involved in the dissemi-nation of the armA gene, at least under the experimentalconditions used in this study.

Our results indicate the presence of another mech-anism by which HGT is achieved in A. baumannii. Wewere able to further purify the supernatant components,which included DNA fragments, using sucrose densitygradient centrifugation and gel filtration chromatog-raphy, and succeeded in transferring the armA genein vitro. Purified components were resistant to DNasetreatment, suggesting that the DNA fragments may beprotected by OMV. Our future research aims includeuncovering the mechanism responsible for the HGT ofantibiotic resistance genes in A. baumannii.

References

(1) Howard, A., O'Donoghue, M., Feeney, A., etal. 2012. Acinetobacter baumannii: an emerging

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opportunistic pathogen. Virulence 3: 243-250.(2) Vila, J., and Pachon, J. 2012. Therapeutic

options for Acinetobacter baumannii infections:an update. Expert Opin Pharmacother 13: 2319-2336.

(3) Sun, Y., Cai, Y., Liu, X., et al. 2013. Theemergence of clinical resistance to tigecycline. IntJ Antimicrob Agents 41: 110-116.

(4) Zarrilli, R., Pournaras, S., Giannouli, M., etal. 2013. Global evolution of multidrug-resistantAcinetobacter baumannii clonal lineages. Int JAntimicrob Agents 41: 11-19.

(5) Wachino, J., and Arakawa, Y. 2012. Exogenouslyacquired 16S rRNA methyltransferases foundin aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat

15: 133-148.(6) Galimand, M., Sabtcheva, S., Courvalin, P., et

al. 2005. Worldwide disseminated armA amino-glycoside resistance methylase gene is borne bycomposite transposon Tn1548. Antimicrob AgentsChemother 49: 2949-2953.

(7) Overballe-Petersen, S., Harms, K., Orlando, L.A., et al. 2013. Bacterial natural transformation byhighly fragmented and damaged DNA. Proc NatlAcad Sci USA 110: 19860-19865.

(8) Rumbo, C., Fernandez-Moreira, E., Merino, M.,et al. 2011. Horizontal transfer of the OXA-24carbapenemase gene via outer membrane vesicles:a new mechanism of dissemination of carbapenemresistance genes in Acinetobacter baumannii.Antimicrob Agents Chemother 55: 3084-3090.

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Analysis on the mechanism of T cell peripheral toler-ance against pemphigus autoantigen, desmoglein 3

Hayato Takahashi, M.D., Ph.D.

Department of Dermatology, Keio University School of Medicine, Shinjuku, Tokyo 160-8582, Japan

Introduction

Pemphigus vulgaris (PV) is a life-threateningbullous disease. In patients with PV, anti-desmoglein3 (Dsg3) IgG autoantibodies are detected in theserum (1). Dsg3 is a cadherin-type transmembraneglycoprotein that is expressed in keratinocyte andcritical in cell-cell adhesion of the cells. Theautoantibodies bind keratinocyte cell surfaces in vivoand disturb cell adhesion function of Dsg3, resultingin acantholysis that is loss of cohesion betweenkeratinocytes characteristically seen in PV histology.Since Dsg3 is expressed not only in the skin butalso mucosal membrane including oral mucosa andesophagus and so on, anti-Dsg3 antibodies can induceerosion and blister not only in the oral mucosa andesophagus but also in the skin in combination with anti-Dsg1 autoantibodies as clinical manifestations (2).

Some previous reports support that autoreactiveT cells are involved in and regulate the anti-Dsg3antibody production from autoreactive B cells. Forexample, nucleotide sequencing of anti-Dsg3 antibodygene isolated from PV patients detected somatichyper-mutations in CDR3 region of the antibody,revealing T cell-dependent affinity maturation ofautoantibodies (3). PV is associated with HLA-DR14/DQB1*0503 in Japanese and HLA-DRB1*0402in Jewish, suggesting that CD4+ T cells recognize Dsg3peptide presented in the MHC class II molecules (4-6).These results indicate that Dsg3-reactive T cells play acritical role in PV pathogenesis and can be a therapeutictarget to control the disease.

Previously PV mouse model is generated by trans-fer of lymphocytes from Dsg3-/- mouse into Rag2-/-

mouse (7). In Dsg3-/- mouse, tolerance mechanismagainst Dsg3 is not established because of absence ofDsg3. Therefore, transferred lymphocyte can efficientlystart to respond to Dsg3 and induce anti-Dsg3 antibodyproduction and PV phenotype in Rag2-/- mouse thatphysiologically expresses Dsg3. Recently we estab-lished Dsg3-reactive T cell clones from Dsg3-/- mouseand demonstrated the pathogenicity of the clones byconfirming PV phenotype induction after transferringthe clones with Dsg3-/- B cells into Rag2-/- mouse (8).Furthermore, blocking IL-4, which is detected as a sig-nificantly up-regulated cytokine in pathogenic clonesbut not in non-pathogenic ones, ameliorates PV pheno-type. These results clearly support that Dsg3-reactive Tcells is critical in PV mouse model.

While biologics, in addition to conventional drugsuch as steroid and immunosuppressant, are newlyintroduced for treatment of autoimmune diseases,antigen-specific therapy that harbors much less sideeffect is not clinically available yet. The purpose ofthis study is to understand molecular mechanism ofimmunological tolerance against Dsg3 and providethe basis to invent new therapeutic approach targetingautoreactive T cells. Especially it would be moreimportant to understand peripheral tolerance butnot central one since peripheral tolerance is moreapproachable than thymus when considering practicaltreatment in patients. In this study, we generatedand analyzed Dsg3-specific T cell receptor transgenicmouse to understand behavior of Dsg3-specific T cellsin immunological tolerance.

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Materials and Methods

Mice

C57BL/6 mice (H-2b, Ly-9.2) and 129/SV (H-2b, Ly-9.1) were purchased from CLEA Japan (Tokyo,Japan) and Sankyo Labo Service Corporation (Tokyo,Japan). C57BL/6 Rag-2-/- mice were purchased fromCentral Institute for Experimental Animals (Tokyo,Japan). Dsg3-/- mice with a mixed genetic backgroundof 129/SV and C57BL/6J, and 129/SV Dsg3-/- micewere obtained by mating male and female Dsg3-/-

mice (Jackson Laboratory, Bar Harbor, ME) (7).Dsg1tg/tg-Dsg3-/- mice with C57BL/6 background weregenerated as described in the other article (9). Animalswere housed under specific pathogen-free conditions.The Keio University Ethics Committee for AnimalExperiments approved all experiments in this study.

Transgenic vector

Genes for TCR α and β chains of pathogenicDsg3-reactive T cell clone, 140#27, were subclonedinto transgenic expression cassettes for TCR α andβ chains, which were kindly provided by Dr. DianeMathis (Harvard University) (10) following appropriatetreatment with restriction enzymes.

Generation of Dsg3-reactive TCR transgenic mice

Linearized transgenes of TCR α and β chains fora T cell clone, 140#27, were injected into fertilizedoocyte of C57BL/6J (H-2b) mice to generate TCRtransgenic mice, C57BL/6J-Tg(Dsg3TCR140), whichwere maintained by mating C57BL/6J mice.

Peptide

111 overlapping 15-mer peptides covering EC1 toEC3 of Dsg3 extracellular domain (1 - 394 aa) weresynthesized and purchased from Sigma-Aldrich Japan(Tokyo, Japan).

In vitro reconstitution of Dsg3-reactive TCR

T cell hybridoma, TG40-CD4, which was kindlyprovided by Dr. Takashi Saito (Riken Research Cen-ter for Allergy and Immunology), looses intrinsicTCR expression and expresses mouse CD4, whichwas retrovirally introduced. 10 μg linearized vec-tors for TCR α and β chain expression in addi-tion to pSV2-hph (ATCC, Rockville, MD) contain-ing hygromycin-resistance gene were electrically intro-duced into TG40-CD4. Stable transfectants were se-lected by using hygromycin after electroporation. Ex-pression of transduced vectors was confirmed by flowcytometry detecting co-expression of TCRβ and CD3,which is expressed as a complex with both TCR α andβ chains. Stable transfectants were sorted by MACScell isolation system using anti-TCRβ6 Ab-PE (BectonDickinson, San Diego, CA) in combination with anti-PE Ab-microbeads (Miltenyi Biotech, Bergisch Glad-bach, Germany).

Reactivity of T cells and T cell hybridomas againstDsg3 peptides

Single cell suspension of 4 × 104 cells wereprepared from the spleen of TCR transgenic mice andcultured with 1×105 40 Gy-irradiated splenocytes and10 μg/ml peptide in 96-well round bottom plate andDsg3-specific T cell proliferation was measured by 3H-thymidine uptake as previously described (11). 2×104

T cell hybridoma cells were cultured with 1 × 106

40 Gy-irradiated splenocytes and peptide of indicatedconcentration in 96-well flat bottom plate for 24 hours.Culture supernatants were subsequently subjected toIL-2 ELISA (BD). Some of the experiments wereperformed in combination with anti-MHC class II mAb(M5/114) or isotype-matched rat mAb (BD).

Flow cytometry

Single cell suspension of thymus, spleen, orlymph nodes from mice were appropriately stainedby CD4-FITC, CD44-FITC, TCRβ -PE, TCRVβ6-PE, CD4-PerCP-Cy5.5, CD8-PerCP-Cy5.5, 7-AAD,

— 16 —

CD45-APC/Cy7, CD4-PE/Cy7, CD62L-biotin, andCD229.1-biotin in combination with streptavidin-APC.

Generation of bone marrow chimera mice

CD3-depleted BM cells from Dsg3H1 mice wereprepared by using CD3-microbeads (Miltenyi Biotech)and intravenously transferred into 7-Gy irradiated129/SV mice or 129/SV Dsg3-/- mice. Two monthslater, the recipient mice were used for furtherexperiments.

Adoptive transfer

Dsg3-/- B cells were prepared from Dsg3-/- miceas previously described (8). CD4+Vβ6+ T cells wereprepared from the spleen and LN of Dsg3H1 or Dsg3L3mice by depleting B220+ and CD8+ cells, followed bythe positive selection of Vβ6+ cells using the MACScell separation system (Miltenyi Biotech). 5 × 106

Dsg3-/- B cells and 2.5× 106 CD4+Vβ6+T cells wereintravenously transferred into Rag2-/- mice. In someexperiments, retrovirally transduced CD4+ T cells weretransferred into Rag2-/- mice in combination with orwithout Dsg3-/- B cells. Naıve Ly9.1−CD4+ T cellsderived from wild type or Dsg3-/- mice with bonemarrow transfer from Dsg3H1 mouse were preparedby depleting Ly9.1+, B220+, CD8+, Gr-1+, CD11b+,DX5+, and CD44+ cells from splenocytes and LNcells by magnetic beads and 3 ∼ 15×105 T cells weretransferred with Dsg3-/- B cells into Rag2-/- mice.

Anti-Dsg3 antibody detection

Anti-Dsg3 IgG antiobody was quantified byELISA and detected by living cell staining as describedpreviously (7).

Histological analysis

Formalin-fixed tissue was stained with hema-toxylin and eosin, and observed with an inverted mi-croscope TE2000-U (Nikon, Tokyo, Japan). For im-munofluorescent staining, 10-μm cryosections of thepalate were fixed with acetone, and subsequently

stained with anti-mouse IgG Ab-Alexa488 (MolecularProbes, Eugene, OR), anti-TCRβ Ab-PE, and TOTO3(Molecular Probes). Sections were observed undera confocal laser fluorescence microscope FV1000(Olympus, Tokyo, Japan).

Results

Dsg3H T cell hybridoma cells respond to Dsg3 peptide

Nucleotide sequence of TCR α and β chain genesderived from pathogenic Dsg3-reactive T cells wereanalyzed and the results showed that α chain consistsof AV8S13 and J21 and β chain consists of BV6S1and Jβ1.3. T cell hybridoma cells (Dsg3H1 hybridomacell), in which these genes are reconstituted, were co-cultured with Dsg3 overlapping peptide and Dsg3H1hybridoma cells respond to Dsg3 (301-315) peptide byIL-2 production (data not shown). Antigenic epitope ofDsg3H1 TCR was confirmed at peptide levels.

CD4+ T cells from Dsg3-specific TCR transgenicmouse respond to Dsg3 peptide

Using genes encoding Dsg3-specific TCR, Dsg3-spcific TCR transgenic mouse (Dsg3H1 mouse) wasgenerated. It was confirmed that Vβ6+ thymocyteswere able to develop to CD4+CD8− single positivecells in the thymus (Figure 1a). In the secondarylymphoid tissues, about 30% of CD4+ cells were Vβ6+

cells, that is a higher proportion than that in wildtype mouse (usually seen in less than 10%, Figure1b). To evaluate whether these transgenic T cells areanergic or reactive to Dsg3, the splenocytes werecultured with antigenic Dsg3 peptide and Dsg3-specificresponse could be detected (Figure 2). Therefore, itwas confirmed that Dsg3H1 mouse was expectedlygenerated harboring Dsg3-reactive T cells.

Transgenic T cells from Dsg3H1 mouse induceinterface dermatitis in vivo

Since Dsg3H TCR was originally cloned frompathogenic Dsg3-reactive T cells that promoted anti-Dsg3 IgG production from B cells and pemphigus

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CD

4

Vβ6+ thymocytes

CD8

25 35

8

Spleen LN

TCRVβ6

# ce

ll

(Gated in CD4+ cells)

37 27

a b

Figure 1. Flow cytometric analysis of thymus, spleen and lymph nodes (LN) in Dsg3H1 mouse. Single cell suspension was preparedfrom thymus (a), spleen and LN (b) of Dsg3H1 mouse and analyzed by flow cytometry. For the thymocytes analysis, dotplots analyzed by CD and CD8 expression were shown after gating on Vβ 6+ cells. For the analysis of spleen and LN,histograms for Vβ 6 expression were shown after gating on CD4+ cells. Dsg3H1 T cells developed into CD4+ singlepositive cells in thymus and detected as CD4+Vβ 6+ cells in the secondary lymphoid tissues. Data from reference (12).

0 1000 2000 3000 4000

1

2Dsg3(301-315)

Dsg3(223-237)

Splenocytes from Dsg3H1 mice

3H-thymidine uptake (cpm)

Figure 2. Dsg3-reactivity of splenocytes from Dsg3H1 mouse. Splenocytes were prepared from Dsg3H1 mouse and cultured withantigenic Dsg3 (301-315) peptide and control Dsg3 (223-237) peptide. cells were harvested 16 hours after 3H-thymidinewere added into culture medium and 3H-thymidine uptake was evaluated. Dsg3H1 splenocytes responded to antigenicDsg3 peptide. Data from reference (12).

phenotype in vivo after adoptive transfer with Dsg3-/-

B cells into Rag2-/- mouse, transgenic T cells (Dsg3H1T cells) were expected to have the same pathogenicity.To confirm the expectation, Dsg3H1 T cells wereisolated from Dsg3H1 mouse and transferred withB cells into Rag2-/- mouse. The transferred mouseclinically developed erythema, hair loss and erosionin the skin as well as swelling in the limbs (Figure3a) that started to appear around 2 - 3 weeks after thetransfer. Histologically, acantholysis, a specific findingfor pemphigus, was not observed in the palate andskin, instead many lymphocytes infiltrating at dermal-epidermal junction with liquefaction were observed(Figure 3b). In addition, lymphocytes further showed

intraepidermal infiltration and degeneration changeof keratinocytes were observed (Figure 3c). Thesehistological findings together demonstrated that thepathological change induced by Dsg3H1 T cells isinterface dermatitis, a distinctively identified type ofskin inflammation with unknown etiology. No anti-Dsg3 antibody production was detected by ELISA inthe mouse (data not shown).

Development of Dsg3H1 T cells is regulated bytolerance mechanism against Dsg3

To analyze how Dsg3H1 T cells are influencedfrom tolerance mechanism against Dsg3, we evaluate

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Figure 3. Pathogenic activity of Dsg3H1 T cells to induce interface dermatitis. CD4+ T cells were prepared from Dsg3H1 mouseand transferred with Dsg3-/- B cells into Rag2-/- mouse. Skin phenotype were observed (a). Hair loss and erosion andswelling in the skin were apparently seen. Histopathology of the palate revealed that lymphocytes infiltrated at dermal-epidermal junction with liquefaction degeneration (b) and degenerated keratinocytes were observed (c). Scale bar; 50 μm.Data from reference (12).

17% 97%

TCRVβ6

Dsg3 (+) Dsg3 (-)

Figure 4. Dsg3H1 T cells undergo tolerance mechanism against Dsg3 and partial deletion. Bone marrow transfer was performedfrom Dsg3H1 mouse to wild type (left) or Dsg3-/- mouse (right). Thymocytes from recipient mice were analyzed by flowcytometry and histogram was shown after gating on CD4+ cells. Almost all CD4+ cells were Vβ 6+ cells in the absenceof Dsg3. Only 20% of CD4+ cells were detected as Vβ 6+ cells in the presence of Dsg3. The results clearly indicated thattolerance mechanism against Dsg3 exists in mouse. Data from reference (12).

the development of Dsg3H1 T cells in the presenceor absence of Dsg3 after bone marrow cells weretransferred from Dsg3H1 mouse into wild type orDsg3-/- mice. As a result, Dsg3H1 T cells fullydeveloped in the absence of Dsg3 since almost allof CD4+ T cells were Vβ6+ cells in the lymphnodes (Figure 4). On the other hand, Dsg3H1 Tcells underwent partial deletion in the presence ofDsg3 since only about 20% of CD4+ T cells wereVβ6+ cells. Dsg3H1 mouse were further crossbred

with Rag2-/- mouse to avoid from endogenous TCRexpression and its development was similarly analyzedby BMT into wild type or Dsg3-/- mice. Dsg3H1 T cellscould again fully develop in the absence of Dsg3, whilethe cells failed to develop and completely disappearedin the presence of Dsg3 (data not shown). These resultsdemonstrated that tolerance mechanism against Dsg3exists in mouse and Dsg3H1 mouse is a useful tool toclearly visualize the mechanism in future studies.

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Discussion

The goal of this study is to clarify peripheraltolerance mechanism against Dsg3, an autoantigen ofpemphigus, and provide the immunological basis forfuture development of antigen-specific immunotherapy.We generated Dsg3-specific TCR transgenic mouse,Dsg3H1 mouse, those transgenic T cells were able toundergo tolerance mechanism (12). Especially in thesetting of Rag2-/- background, Dsg3H1 T cells canno longer develop in the presence of Dsg3 but fullydevelop in the absence of Dsg3, clearly showing “allor none” response. Using this mouse as sensor, itwill be possible to clearly visualize where tolerancemechanism is functional, how much the mechanism canbe manipulated and what kind of cells or moleculesmediate the strict mechanism, by evaluating Dsg3H1T cell development.

Through this study, we apparently demonstratedthat Dsg3H1 mouse is a very useful mouse to monitortolerance mechanism against Dsg3 and will utilizethe mouse in further analysis for peripheral tolerance.Hopefully the new molecular or cellular pathwaymediating peripheral tolerance would be discoveredthrough this study and applied to creation of new ideafor treatment of autoimmune diseases.

Conclusion

Dsg3-specific TCR transgenic mouse is useful tomonitor tolerance mechanism against Dsg3.

Acknowledgements

This study was supported by the WaksmanFoundation of Japan Inc.

References

(1) Amagai M, Klaus-Kovtun V and Stanley JR(1991) Autoantibodies against a novel epithelialcadherin in pemphigus vulgaris, a disease of celladhesion. Cell 67: 869-877.

(2) Amagai M, Tsunoda K, Zillikens D, Nagai T

and Nishikawa T (1999) The clinical phenotypeof pemphigus is defined by the anti-desmogleinautoantibody profile. J Am Acad Dermatol 40:167-170.

(3) Payne AS, Ishii K, Kacir S, Lin C, Li H, et al.(2005) Genetic and functional characterization ofhuman pemphigus vulgaris monoclonal autoanti-bodies isolated by phage display. J Clin Invest 115:888-899.

(4) Niizeki H, Inoko H, Mizuki N, Inamoto N,Watababe K, et al. (1994) HLA-DQA1, -DQB1and -DRB1 genotyping in Japanese pemphigusvulgaris patients by the PCR-RFLP method.Tissue Antigens 44: 248-251.

(5) Ahmed AR, Mohimen A, Yunis EJ, Mirza NM,Kumar V, et al. (1993) Linkage of pemphigusvulgaris antibody to the major histocompatibilitycomplex in healthy relatives of patients. J ExpMed 177: 419-424.

(6) Ahmed AR, Wagner R, Khatri K, Notani G,Awdeh Z, et al. (1991) Major histocompatibilitycomplex haplotypes and class II genes innon-Jewish patients with pemphigus vulgaris.Proceedings of the National Academy of Sciencesof the United States of America 88: 5056-5060.

(7) Amagai M, Tsunoda K, Suzuki H, Nishifuji K,Koyasu S, et al. (2000) Use of autoantigen-knockout mice in developing an active autoim-mune disease model for pemphigus. J Clin Invest105: 625-631.

(8) Takahashi H, Amagai M, Nishikawa T, FujiiY, Kawakami Y, et al. (2008) Novel systemevaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigusvulgaris. J Immunol 181: 1526-1535.

(9) Hata T, Nishifuji K, Shimoda K, Sasaki T, YamadaT, et al. (2011) Transgenic rescue of desmoglein3 null mice with desmoglein 1 to develop asyngeneic mouse model for pemphigus vulgaris.Journal of dermatological science 63: 33-39.

(10) Kouskoff V, Signorelli K, Benoist C and Mathis D(1995) Cassette vectors directing expression of Tcell receptor genes in transgenic mice. J ImmunolMethods 180: 273-280.

(11) Kuwana M, Medsger TA, Jr. and Wright TM

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(1995) T cell proliferative response induced byDNA topoisomerase I in patients with systemicsclerosis and healthy donors. J Clin Invest 96:586-596.

(12) Takahashi H, Kouno M, Nagao K, Wada N, HataT, et al. (2011) Desmoglein 3-specific CD4+ Tcells induce pemphigus vulgaris and interfacedermatitis in mice. J Clin Invest 121: 3677-3688.

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Helicobacter pylori regulate host non-coding RNAexpression to increase the proliferation of gastricepithelium during chronic infection

Hitomi Mimuro

Division of Bacteriology, Department of Infectious Diseases Control, International Research Center for InfectiousDiseases, Institute of Medical Science, The University of Tokyo 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639,Japan

Introduction

Chronic inflammation is a critical risk factor forand has been estimated to cause 20% of all humancancers. Specific pathogens have been identified ascausal risks for particular types of cancer; Helicobacterpylori in gastric cancer, Hepatitis B and C viruses inliver cancer, Papilloma virus for ovarian cancer, EBvirus for malignant lymphoma, adult T-cell leukemiavirus for ATL, and human herpes virus 8 for Ka-posi’s sarcoma. However, the molecular mechanismsby which these microbial infections trigger malig-nancies remain largely unknown. Understanding thesemechanisms would therefore contribute to the molecu-lar basis for development of novel preventive, therapeu-tic, and diagnostic approaches against cancer.

Chronic Helicobacter pylori (Hp) infection ofthe gastric epithelium is strongly associated withthe development of gastritis, peptic ulcers, mucosa-associated lymphoid tissue lymphoma, and gastriccancer. In gastric cancer, nearly 60% of all casesin developed countries and 75% in developingcountries are attributed to chronic infection withHp. During persistent Hp colonization of the gastricmucosa, sustained inflammation and aberrant epithelialproliferation are considered to be major factors thatlead to Hp-associated gastric diseases, although themechanisms underlying disease progression remainelusive.

An accumulation of genetic and epigeneticalterations in normal tissues triggers carcinogenesis.For example, DNA methylation of specific genescan be found in up to several percent of the cellsin noncancerous gastric mucosae, and the rate ofgene methylation is considered to be correlated withan increased risk of gastric cancer. Importantly, Hpinfection in the stomach has been shown to potentiatethe induction of aberrant DNA methylation in thegastric epithelium, along with chronic inflammation.Notably, methylation of promoter sites leads to geneexpression silencing.

On the other hand, cumulative evidence suggeststhat microRNAs (miRNAs) might play importantroles in the initiation and progression of varioushuman diseases. In this context, we sought toidentify miRNAs that are causally involved ingastric malignancies associated with Hp infection byusing several systematic and bioinformatic approachesand studying gastric epithelial cell lines, stomachtissue from Mongolian gerbils, and human stomachbiopsy specimens. In this comprehensive study, wedemonstrate that miR-210 is a critical miRNA,which regulates gastric epithelial cell proliferationby targeting potential oncogenes. Furthermore, ourwork provides substantial evidence for the causalinvolvement of epigenetic silencing of miR-210 indevelopment of gastric malignancies induced bychronic Hp infection.

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Materials and Methods

Strains and cell culture

The Hp strain ATCC 43504, which was describedpreviously, was passaged on 5% sheep blood agarplates (Nippon Becton Dickinson Co., Ltd.) andincubated in an atmosphere containing 5% O2 for2-3 days at 37◦C. Bacterial cultures were grown inBrucella broth (Difco Labs., Inc.) supplemented with5% fetal bovine serum (FBS) (Gibco-BRL) under thesame conditions for 12–24 hrs at 37◦C with gentleagitation. HeLa, 293T, MKN1, SH-10-Tc, and AZ521cells were maintained in Dulbecco’s modified Eagle’smedium (DMEM, Sigma-Aldrich) containing 10%FBS. OCUM-1, OCUM-2D, OCUM-2M, OCUM-8,OCUM-9, and OCUM-12 cells were maintained inDMEM containing 2 mM glutamine, 0.5 mM sodiumpyruvate, and 10% FBS. AGS, MKN45, MKN74, andNCI-N87 cells were maintained in RPMI-1640 (Sigma-Aldrich) containing 10% FBS. Cultured cells wereinfected with Hp at a multiplicity of infection (MOI) of0.2-125. To incubate cells or bacteria under anaerobicconditions or microaerophilic conditions, AnaeroPackSystems (Mitsubishi Gas Chemical Co., Inc.) wereused.

Human biopsy samples and Hp infection of Mongoliangerbils

Primary gastric mucosa samples from 35 healthyvolunteers were obtained to investigate miR-210, IL-8, STMN1, DIMT1, U6 and ACTB expression andto analyze DNA methylation levels in Hp-infectedgastric tissue. The study included 19 Hp-positivecases and 16 Hp-negative cases. An additional 85primary gastric mucosa samples were obtained tomeasure the levels of miR-210 and U6. These sampleswere scored depending on the severity of neutrophilinfiltration, mononuclear cell infiltration, intestinalmetaplasia, atrophy, and Hp content, according to theupdated Sydney system. These samples were acquiredfrom the archives of the Department of Pathologyat the University of Tokyo Hospital. Hp infection

was diagnosed in the biopsies by H&E staining andconfirmed by real-time PCR analysis of Hp rRNA fromtotal RNA. The 20 gastric tumor samples and pairedgastric normal tissues were obtained to analyse miR-210 and U6 expression levels. Tissues were fixed inneutral buffered formalin and embedded in paraffinblocks for RNA extraction. Formalin-fixed, paraffin-embedded tissues (FFPE) were sectioned to a 10 μmthickness for RNA or DNA extraction. Total RNA,including miRNA, or total DNA was extracted fromthe paraffin-embedded tissues of surgically resectedsamples with a RecoverAll Total Nucleic Acid IsolationKit for FFPE Tissues (Ambion). All aspects of thisstudy were approved by the University of Tokyo EthicsCommittee.

For the in vivo infection model, six-week-old maleMGS/Sea Mongolian gerbils (Meriones unguiculatus;CLEA Japan Inc.) were intragastrically inoculated witha bacterial culture containing 109 CFU of Hp (ATCC43504) in Brucella broth containing 5% (v/v) FCS.Control animals were orally administered Brucellabroth containing only 5% (v/v) FCS. To isolateRNA, the gastric mucosal tissues were immediatelyharvested and frozen in liquid nitrogen. Total RNA wasextracted using ISOGEN after the stomach tissue washomogenized. All animal experiments were conductedin accordance with the guidelines of the Universityof Tokyo for the care and use of laboratory animalsand were approved by the ethics committee for animalexperiments of the University of Tokyo.

cDNA microarray analysis

AGS or MKN45 cells were transfected with 50 nMof pre-miRNA Negative control #1 (miR-Nega) (Lifetechnologies, Ambion) or pre-miRNA miR-210 for36 hrs using Lipofectamine RNAiMAX (Invitrogen).Total RNA was purified using an RNeasy Kit(Qiagen) according to the manufacturer’s instructions.Gene expression profiling was performed using theAffymetrix Human Genome U133A Plus 2.0 Array(Affymetrix), to analyze over 47,000 transcripts,including ∼21,000 well-characterized human genes,according to the manufacturer’s instructions. The datawere normalized and analyzed by GeneSpring GX

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7.3 (Agilent Technologies). Target genes containingmiR-210 seed sequences were identified by HumanTargetScan 5.1 (http://www.targetscan.org/). Geneswith expression levels lower than 40% of themiR-Nega transfected control cells, after miR-210overexpression, and containing miR-210 target sites intheir 3’UTRs, were considered to be candidates formiR-210 targeting.

microRNA array

Mongolian gerbils were inoculated with a bacte-rial culture containing 109 CFU of Hp (ATCC 43504),and total RNA containing the small RNA fractionwas purified from gastric tissues at 63 days post-infection using the miRNeasy Mini Kit (Qiagen), ac-cording to the manufacturer’s instructions. OCUM-9cells were treated with 1 μM 5-aza-dC for 4 days.Total RNA was purified as described above. The to-tal RNA was reverse transcribed for 1st strand syn-thesis using PCRMegaplexTM Primer Pool RT primers(Applied Biosystems). The miRNA expression pro-file of Hp-infected AGS cells or control cells was de-termined using Human TaqMan R© Array MicroRNACards A (Applied Biosystems). PCR reactions wereperformed using TaqMan Universal PCR Master MixNo AmpErase UNG (2x) with a 7900HT thermocy-cler (Applied Biosystems) and the manufacturer’s rec-ommended program. The expression profile for eachmiRNA was analyzed using the ΔΔCt method andthe expression levels were calculated using U6 expres-sion as an internal control. For the miRNA array withOCUM-9 cells, SNU48 was used as an internal control.Heat maps were produced with Java TreeView software（http://jtreeview.sourceforge.net/）

Luciferase assay

For transfection, 3 × 104 293T cells were platedin 96-well plates in 100 μ l of culture medium.After overnight culture, the cells were co-transfectedwith 0.02 μg of the indicated plasmids and a 50nMconcentration of siRNA, or 50 nM of pre-miRNAusing 0.5 μ l of Lipofectamine 2000 (Invitrogen),according to the manufacturer’s recommendations. For

the luciferase assays, cell lysates were prepared 36 hrspost-transfection using the passive lysis buffer providedby the manufacturer and then assayed for luciferaseactivity using the Dual-Luciferase R© Assay System(Promega) with a Veritas Microplate LuminometerTM

(Promega). Luciferase activity of the fusion proteinswas measured and normalized to the activity of Renillaluciferase. The results are expressed as the mean valuesof each sample group.

RNA interference and transient transfection

AGS or MKN45 cells were transfected with theappropriate siRNAs at a final concentration of 50 nMusing Lipofectamine RNAiMAX (Invitrogen) and thereverse transfection method according to the manufac-turer’s recommendations. All siRNAs for target geneswere synthesized, purified, and duplexed by RNAi Co.,Ltd., Tokyo, Japan. Pre-miRNA210 (miR-210) or Anti-miRNA (miR-Nega) (Life technologies, Ambion) wastransiently transfected into AGS, MKN45, OCUM-9 orOCUM-2D cells using the same conditions as describedfor the siRNAs.

Cell cycle analysis and cell proliferation assay

For cell cycle analysis, MKN45 cells were fixedin 70% ethanol overnight at −20◦C. After washingwith PBS, the cells were incubated with 10 mg/mlRNase A (Sigma) and 50 μg/ml propidium iodide inPBS for 2 hrs on ice in the dark. Cell cycle datawas collected with a FACSCalibur flow cytometer(BD Biosciences immunocytochemistry system) andanalyzed with CellQuest and FlowJo software. Forthe cell proliferation assay, transiently transfectedMKN45, MKN74, AGS or NCI-N87 cells werecultured under normal conditions. After the indicatedtime period, the culture medium was replaced withmedium containing 10 μ l/well of the substrate for theCell Counting Kit-8 (Dojindo Laboratories). Briefly,the plates were shaken for 10 sec and incubated at37◦C for 1 hr. Then, the absorbance at 450 nm for eachwell was measured using a Varioskan Flash (ThermoScientific). The morphological changes of each cellwere observed using an Axiovert 135-SENSYS (Carl

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Zeiss MicroImaging, Inc.).

Quantitative real-time PCR

To measure gene expression levels, including pri-miR-210, total RNA was reverse transcribed into cDNAwith oligo (dT) primers and random hexamer primersusing ReverTra Ace R© (TOYOBO Life Science). Real-time PCR was performed using a LightCycler R© DX400(Roche) and SYBR R© Premix Ex TaqTM II (TaKaRa)according to the manufacturer’s protocols. RelativemRNA expression was calculated using the human β -actin gene as an endogenous reference standard. Thespecificity of the PCR products was confirmed bymelting curve analysis. To determine the mature miR-210 expression levels, total RNA was isolated fromcell lines using an miRNeasy Mini Kit (Qiagen). TotalRNA was used in reverse transcriptase and subsequentpre-amplification reactions using the miScript ReverseTranscription Kit and the manufacturer’s protocol(Qiagen). miRNA real-time PCR was performed todetect miRNAs and U6 using a miScript SYBR GreenPCR Kit (Qiagen). The specificity of the PCR productswas confirmed by melting curve analysis. The miRNAlevels were normalized relative to the U6 expressionlevels. The results are expressed as the means.

Bisulfite conversion, bisulfite sequencing andmethylation-specific PCR (MSP)

Genomic DNA was extracted from gastric biopsytissue sections and subjected to sodium bisulfitemodification using EpiTect Bisulfite Kits (Qiagen)following the manufacturer’s protocols. For bisulfitesequencing, sodium bisulfite-treated DNA was PCRamplified using primers common to methylated andunmethylated DNA sequences. The purified PCRproducts were separated into the Hp-infected anduninfected groups. The mixed PCR products in eachgroup were cloned into the pGEM-T Easy vector(Promega), and over 40 clones were sequenced.For MSP, the methylated and unmethylated humanDNA obtained from the EpiTect PCR Control DNASet (Qiagen) was used to generate methylation-specific primers and PCR annealing temperatures that

specifically amplify only methylated or unmethylatedDNA. Quantitative SYBR Green MSP was performedto determine the levels of miR-210 CpG DNAmethylation using a LightCycler R© DX400 (Roche) andSYBR R© Premix Ex TaqTM II (TaKaRa). The DNAmethylation levels of the miR-210 CpG island werecalculated as the amount of methylated DNA relative tothe total levels of methylated and unmethylated DNA.

DNA constructs

The DNA sequence surrounding miR-210 onhuman chromosome 11p15.5 was subcloned into thepGL4.20 vector (Promega) from the human BAC cloneRP11-392J11 (Invitrogen) using restriction enzymesSalI and NotI to create the pmiR-210-upstream-3399-Luc vector. For in vitro DNA methylationanalyses, the CpG island sequence in miR-210 wasgenerated from human BAC clone RP11-392J11 usingrestriction enzymes StuI and KpnI, and then subclonedinto a promoter-eliminated pcDNA3. This miR-210CpG island vector was treated with SssI, HhaI orHpaII methyltransferase (New England Biolabs) for3 hrs at 37◦C, and purified using a QIAquick PCRpurification kit (Qiagen). Complete DNA methylationwas confirmed by digesting with the HpaII restrictionenzyme (New England Biolabs). A CMV promoter-driven miR-132 expression vector was created bycloning the genomic region, including the pre-miR-132 sequence and its 5’- and 3’-flanking regions, intothe MCS of pcDNA3. To generate a reporter vectorbearing 12 human 3’UTR sequences (ATP11A, DIMT1,H2AFY, INPP5A, NDUFA4, PPP1R2, RAB27B,SH3BGRL, STMN1, TNPO3, VAMP7, and WLS),the full-length 3’UTR of each gene was amplifiedfrom AGS cDNA using specific primer pairs withrestriction endonuclease sites for XhoI or NheI (senseprimer) and Sbf I, SalI or XbaI (antisense primer).The products were directly digested with the indicatedrestriction enzymes and cloned into the pmirGLOvector (Promega) according to the manufacturer’sinstructions. pmirGLO 3’UTR mutant vectors forSTMN1 and DIMT1 were generated by amplifyingthe plasmids with phosphorylated primers containingthe mutant sequences. The linearized amplified target

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plasmid with the desired mutations was ligated usingLigation High (TOYOBO) to generate a circularplasmid. All mutants were sequenced to confirm thepresence of the desired mutations.

In Situ Hybridization (ISH)

The tissues of Mongolian gerbils were dissected,fixed with Tissue Fixative (Genostaff), and thenembedded in paraffin, and sectioned at 5 μm. ForISH, tissue sections were de-waxed with xylene, andre-hydrated through an ethanol series and PBS. Thesections were fixed with 4% paraformaldehyde in PBSfor 15 min and then washed with PBS. The sectionswere treated with 8 μg/ml Proteinase K in PBS for30 min at 37◦C, washed with PBS, re-fixed with 4%paraformaldehyde in PBS, again washed with PBS,and placed in 0.2 N HCl for 10 min. After washingwith PBS, the sections were acetylated by incubationin 0.1 M tri-ethanolamine-HCl, pH 8.0, 0.25% aceticanhydride for 10 min. After washing with PBS, thesections were treated with or without PBS at 80◦Cfor 5 min. Hybridization was performed with probes(miRCURY LNA microRNA Detection Control probe,miRCURY LNA miR-210 Detection probe (EXIQON),or Mongolian gerbils STMN1 detection probe) at50◦C for 16hrs. After hybridization, the sections werewashed in 5xHybriWash (Genostaff), equal to 5xSSC,at 50◦C for 20 min and then in 50% formamide, 2 xHybriWash at 60◦C, followed by RNase treatment in50 μg/ml RNaseA in 10 mM Tris-HCl, pH 8.0, 1MNaCl and 1 mM EDTA for 30 min at 37◦C. Thenthe sections were washed twice with 2xHybriWashfor 20min, twice 0.2xHybriWash for 20min, and oncewith TBST (0.1% Tween20 in TBS). After treatmentwith 0.5% blocking reagent (Roche) in TBST for 30min, the sections were incubated with anti-DIG APconjugate (Roche) diluted 1:1000 with TBST for 2 hrsat RT. The sections were washed twice with TBST andthen incubated in 100 mM NaCl, 50 mM MgCl2, 0.1%Tween 20, 100 mM Tris-HCl, pH 9.5. Color reactionswere performed with NBT/BCIP solution (Sigma)overnight and then washed with PBS. The sectionswere counterstained with Kernechtrot stain solution(Mutoh), and mounted with CC/Mount (Diagnostic

Biosystem). Images were acquired from the stainedspecimens using a Micro Imager.M1 microscope (CarlZeiss), Zeiss AxioVision and MosaiX Softwares.

Immunofluorescence Microscopy

Anguilla anguilla agglutinin (AAA; Sigma-Aldrich) was Cy3 labeled using Cy3 monofunctionalreactive dye (Amersham Bioscience). Immunofluo-rescence staining was performed as described pre-viously (Mimuro et al., 2007). For PCNA staining,acetone-fixed sections were stained with anti-PCNAantibody after epitope retrieval by heating in citratebuffer (10 mM sodium citrate buffer [pH 6.0]). FITC-conjugated secondary antibody was used to visualizerabbit antibody. Cy3-labeled AAA was used to visu-alize pit cells. The stained specimens were examinedwith a confocal laser-scanning microscope (LSM510;Carl Zeiss) equipped with LSM510 version 3.2 soft-ware (Carl Zeiss).

Results

Several miRNA genes are epigenetically regulated byDNA methylation during chronic infection with Hp.

Hp infection of the stomach has been shownto potentiate silencing of genes by aberrant DNAmethylation in the gastric epithelium, due to chronicinflammation. We hypothesized that chronic Hpinfection could also lead to miRNA gene silencingby DNA methylation in gastric epithelia. To thisend, we first examined miRNA expression levels inuninfected and Hp-infected gastric epithelial cells.Mongolian gerbils were infected intragastrically with109 CFU Hp ATCC43504 and gastric epithelial cellswere examined for changes in miRNA expression levels63 days after infection using a Taqman microRNAarray. Several miRNAs, such as miR-210, miR-375,and miR-99a, were found to have reduced expressionin the gastric epithelium of chronically Hp-infectedgerbils, as compared to expression levels in uninfectedgerbils (Fig. 1a).

Although DNA methylation-mediated epigeneticsilencing of miRNAs has previously been demon-

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Grade of intestinal metaplasia

Score 0 1 2 3(n = 77)(n = 24)(n = 17)(n = 2)

0

0.2

0.4

0.6

0.8

1

1.2

TumorNormal(n = 20) (n = 20)

Grade of neutrophil infiltration

Score 0 1 2 3

miR

-210

/ U

6ex

pres

sion

(n = 52)(n = 14)(n = 46)(n = 8)Grade of mononuclear cell infiltration

Score 0 1 2 3(n = 8) (n = 49)(n = 56) (n = 7)

00.20.40.60.8

11.21.41.61.8

Grade of atrophy

Score 0 1 2 3(n = 26)(n = 49)(n = 42) (n = 3)

00.20.40.60.8

11.21.41.61.8

Grade of Hp content

Score 0 1 2 3(n = 56)(n = 34)(n = 25) (n = 5)

c

d

miR

-210

/ U

6ex

pres

sion

miR

-210

/ U

6ex

pres

sion

***

**

*** ****

24

25

26

27

28

29

30

31

32

33

34

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

miR

-210

/ U

6 ex

pres

sion

miR

-210

Ct v

alue

Hp (−) (n = 16) (n = 19)

Hp (+) Hp (−) (n = 16) (n = 19)

Hp (+)

00.20.40.60.8

11.21.41.61.8

00.20.40.60.8

11.21.41.61.8

00.20.40.60.8

11.21.41.61.8

P = 0.0066

Hp-infectedMongolian gerbilFold-change < 0.5

a b5-aza-dC treatedOCUM-9 cellsFold-change > 2.0

44 246

Hp (-) Hp (+) 5aza

-

5aza

+

MicroRNAs

miR-210

miR-375

miR-99a

miR-328

miR-411

let-7c

5x10-3 1x10-5

Mongoliangerbil

stomach

OCUM-9

0.21

0.25

0.28

0.29

0.29

0.46

25.4

3.2

3.2

3.1

47.2

2.3

Fold-change

Fold-change

Fig. 1. miR-210 is down-regulated during chronic Hp infection.(a) Mongolian gerbils were inoculated with Hp for 63 days. The gastric mucosae from uninfected (n=4) or infected (n=4)gerbils were subjected to a TaqMan-based miRNA microarray. OCUM-9 cells were treated with 1 μM 5-aza-dC for 4days and subjected to a TaqMan-based miRNA microarray. The diagram represents the number of genes whose expressionexhibited more than a 2-fold decrease in Hp-infected gerbils, as well as a more than 2-fold increase in 5-aza-dC-treatedOCUM-9 cells. Gene numbers were obtained by examining gene lists generated by a 2-fold, P < 0.001 criterions. Six genesoverlapped with both groups were listed with a heat map. (b) The expression levels of miR-210 in human gastric mucosawere measured by real-time PCR. The Mann-Whitney U test was used to calculate the P-values. (c) The levels of miR-210 expression was assessed against degrees of neutrophil infiltration, mononuclear cell infiltration, intestinal metaplasia,atrophy, and Hp content, according to the updated Sydney system21 in 120 human gastric biopsy specimens. The miR-210expression levels were measured by real-time PCR. The horizontal bars indicate the mean value of each sample group.*P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney U test. (d) The levels of miR-210 of 20 paired gastric normal andtumor tissues. The miR-210 expression levels were measured by real-time PCR. The red horizontal bars indicate the meanvalue of each sample group.

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strated, the specific conditions of and mechanisms bywhich individual miRNA genes are silenced by methy-lation are not well described. To explore miRNA genesregulated by DNA methylation, we performed miRNAexpression profiling in several samples of the OCUM-9gastric cell line. A Taqman-based array analysis iden-tified several genes, such as miR-411 and miR-210,with significantly increased expression in OCUM-9cells treated with 5-aza-dC, a DNA methyltransferaseinhibitor, suggesting that these miRNAs are probablysilenced by DNA methylation in OCUM-9 cells(Fig. 1a).We then reasoned that miRNAs which are both down-regulated in Hp-infected gerbils and up-regulatedin 5-aza-dC-treated OCUM-9 cells are likely tobe silenced by DNA methylation during chronicHp infection. The genes that occur at the con-vergence of these two pools include miR-210,miR-375, miR-99a, miR-328, miR-411 and let-7c(Fig. 1a). Among them, we found miR-210 to bean intriguing candidate that might be regulated byepigenetic control during chronic Hp infection, asit appeared to be the most sensitive to DNA methy-lation (significantly down-regulated during chronicinfection up-regulated when methylation was inhibited),Together, the data presented here demonstrates thatexpression of several miRNA genes is epigeneticallyregulated by DNA methylation during chronic Hpinfection.

miR-210 is down-regulated in gastric epithelium frompatients with Hp-chronic infection

To examine the in vivo expression of miR-210 during the development of Hp-associated gastricillness, we further investigated epithelial cells informalin-fixed, paraffin-embedded (FFPE) humangastric specimens from patients with or without Hpinfection. RNA samples from the gastric epitheliaof FFPE specimens were examined for miR-210expression using quantitative RT-PCR. The levelsof miR-210 in Hp-positive gastric specimens weredecreased compared to those in Hp-negative gastricspecimens (Fig. 1b), consistent with a previous studyof human gastric biopsy specimens. To evaluate theclinical link between miR-210 down-regulation and the

progression of Hp-mediated gastritis, we collected 120Hp-positive human gastric specimens and characterizedthe severity of gastritis according to the Sydneysystem (Fig. 1c). We found that the levels of miR-210 expression decreased in parallel with increasedgrades of neutrophil infiltration, mononuclear cellinfiltration, atrophy, and Hp content (Fig. 1c). Inaddition, we assessed miR-210 expression in distinctnormal and tumour areas in biopsies from 20 gastriccancer patients, and found significantly higher miR-210 levels in normal regions as compared to intumor regions (Fig. 1d). As reported previously, somecancerous regions displayed higher miR-210 levelsthan did normal regions. As miR-210 is known to beup-regulated in hypoxic conditions, this is perhaps dueto the occurrence of local hypoxia inside solid tumors.We could therefore surmise that miR-210 expressionis suppressed concurrently with the progression of Hp-mediated gastric lesions.

A CpG island on the miR-210 gene locus undergoesmethylation during the course of Hp-associated chronicgastritis

Given the down-regulation of miR-210 in Hp-mediated gastric lesions, we sought to examine thepossible regulation of miR-210 by DNA methylationduring chronic Hp infection. The miR-210 genomiclocus contains a CpG island that overlaps withthe miR-210 coding sequence and extends upstreaminto the miR-210 promoter region (Fig. 2a). Totest if the methylation status of this CpG islandchanges during chronic Hp infection, we examinedthe stomach FFPE specimens obtained from Hp-positive or Hp-negative adults, to assess the extentof CpG methylation within miR-210 by Bisulfitesequencing. We measured two distinct regions, namedBisulfite sequencing 1 and Bisulfite sequencing 2,within the CpG island (Fig. 2a and 2b). Region1 was found to have a higher degree (50.3%) ofmethylation in Hp-positive stomach tissue than inHp-negative stomachs (31.1%) (Fig. 2b). Analysisof the second region displayed a similar trend inmethylation status (40.9% in Hp-positive and 13.6% inHp-negative tissues, Fig. 2b). In addition, we analyzed

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4 distinct regions by methylation-specific PCR (MSP),and all four, MSP1, MSP2, MSP3 and MSP4, werefound to be more methylated in Hp-positive stomachspecimens than in Hp-negative stomachs (Fig. 2aand 2c). Together our analyses strongly pointed to asignificantly higher rate of CpG methylation withinthe miR-210 locus in Hp-positive vs. Hp-negativeindividuals. Increased miR-210 methylation was alsoobserved in human inflammatory bowel disease (IBD)specimens, indicating that a persistent inflammatoryprocess is probably important for the induction of DNAmethylation.

To elucidate the causal link between miR-210down-regulation and higher DNA methylation at theCpG island of the miR-210 locus, we cloned thesequence encompassing miR-210 and part of the CpGisland onto a plasmid vector, to further investigate theeffect of DNA methylation on miR-210 expression invitro (Fig. 2d). The miR-210-containing vectors weretreated with DNA methyltransferases SssI, HhaI, orHpaII, prior to transfection into epithelial cells. Whenthe CpG island containing miR-210 was completelymethylated using SssI (CG → mCG), miR-210expression levels were markedly reduced in epithelialcells (Fig. 2d). On the other hand, a partially methylatedCpG-miR-210, resulting from HhaI or HpaII treatment(GCGC → CmCGG), had a moderate to none effecton miR-210 expression (Fig. 2d), strongly suggestingthat miR-210 expression can be influenced by themethylation status of several CpG sites in a site-specificand dose-dependent manner. Additionally, there wasa modest inverse correlation between the miR-210expression levels and the methylation levels of the CpGisland around the miR-210 coding region in variousgastric epithelial cell lines, including MKN7, AGS,MKN1, OCUM-8, SH-10-Tc, NCI-N87, OCUM-1,OCUM-2D, MKN74, MKKN45, OCUM-2M, AZ521,OCUM-12, and OCUM-9. Altogether, we concludethat the level of DNA methylation occurring withinthe CpG island affects miR-210 expression, and thatreduced miR-210 expression in Hp-infected stomachtissue results, at least in part, from DNA methylationof the miR-210 CpG island.

miR-210 activity suppresses gastric cell proliferation

To pursue the biological impact of deregulatedmiR-210 expression on the gastric epithelium dur-ing Hp infection, we introduced double-stranded miR-210 into various gastric epithelial cell lines, includingMKN45, AGS, OCUM-9 and OCUM-2D, and investi-gated the effect of high levels of miR-210 expressionon epithelial cell proliferation. Cell viability was sig-nificantly decreased by miR-210 overexpression after96 hrs of incubation (Fig. 3a). In contrast, there was amodest increase in proliferation of AGS and OCUM-2D cells transfected with the miR-210 inhibitor anti-miR-210 for 96 hrs, as compared to control cells trans-fected with anti-miR-Nega (Fig. 3b). Levels of miR-210 were reduced by approximately 40% in anti-miR-210-overexpressing AGS cells, measured by real-timePCR analysis. To confirm the specific action of miR-210, we studied the effects of miR-210 overexpressionin MKN45 cells by transfecting miR-210 or miR-210mut (mutations introduced into the seed sequence, re-quired for target gene recognition). Relative miR-210expression levels and viable cells were measured af-ter 96 hours of transfection (Fig. 3c). Cells overex-pressing miR-210 but not miR-210 mut displayed de-creased viability in a dose-dependent manner (Fig. 3c).To verify the effects of miR-210 on the cell cycle, wetransfected miR-210, miR-210 mut or miR-Nega con-trol into MKN45 cells. Two days post transfection, cellswere stained with propidium iodide and analyzed byflow cytometry to visualize nuclei and thereby assesscell-cycle progression. Cells overexpressing miR-210but not the negative control or the miR-210 mut con-struct were found largely to be in the G1 phase, andless so in G2/M and S phases (Fig. 3d). Together, theseresults strongly suggest that reduced miR-210 expres-sion might lead to cell cycle induction, dependent on afunctional seed sequence.

miR-210 targets STMN1 and DIMT1 genes, leading togastric cell proliferation

miRNAs bind to the 3’ untranslated region (UTR)of target gene mRNAs and enhance their degradation.

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13.6% 40.9%

31.1% 50.3%Hp (−)

Unmethylated CpGMethylated CpGHuman stomach

Hp (+)

Bis

ulfit

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quen

cing

1B

isul

fite

sequ

enci

ng 2

0

20

40

60

80

100

100 bp

AK123483miR-210

CpG island

MSP4

CpG

Homo sapiens Chromosome 11p15.5

MSP1

Met

hyla

tion

leve

l (%

)

Hp

02468

1012

NS

Rel

ativ

e m

iR-2

10

expr

essi

on

MSP3MSP2

MSP4MSP1 MSP3MSP2

Hp positive (n = 19)Hp negative (n = 16)

P = 0.11

Promoter-lesspcDNA3

miR-210poly ACpG island

CpG-miR-210

miR-132expression

vector(internalcontrol)

Cell lines

Quantitative real-time PCR(miR-210/miR-132)

24hrs incubation

MCS

100bp

StuI KpnI

miR-210poly ACpG island

CG site (161)GCGC site (22)CCGG site (25)

CpG-miR-210

SssIHhaIHpaII

0

1

2

3

4

HeLa AGS

a

b c

d

Hp (−) Hp (+)

Treated with in vitroDNA methylstransferase(SssI, HhaI and HpaIII)

(−) (+) (−) (+)(−) (+)(−) (+)

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Bisulfite sequencing 1 Bisufite sequencing 2

****

***

***

***

***

Fig. 2. miR-210 expression is epigenetically silenced during chronic H. pylori infection.(a) A schematic diagram of the CpG island and CpG sites in the miR-210 locus on human chromosome 11p15.5. miR-210 and the transcription start site of pri-miR-210 are in the CpG island. The green bar, CpG island; open rectangles,methylation-specific PCR (MSP) regions; closed arrowheads and bars (purple, blue, green or orange), bisulfite sequencedand methylation specific PCR regions. (b) Bisulfite sequencing of the miR-210 CpG island in human gastric biopsy samples.Each box indicates the methylation status of the CpG site. Over 40 clones from each mixed sample were sequenced. Eachrow represents an individual sequenced DNA strand. The percent methylation in each sequenced region was indicated. (c)MSP analysis on the DNA methylation levels of miR-210 in human gastric mucosa biopsies. The ratios were calculatedby the number of methylated CGs versus the number of unmethylated CGs. *P < 0.05, **P < 0.01, ***P < 0.001 byMann-Whitney U test. (d) A schematic representation of the assay system for DNA methylation-dependent regulationof miR-210 expression. The CpG-miR-210 plasmid contains the miR-210 CpG island and an artificially added poly Asite within the multi cloning site (MCS) of promoter-less pcDNA3 plasmid. The CpG island sequence was derived fromhuman chromosome 11: 557,848 - 559,554 (NCBI36/hg18). The black vertical bars indicate the CpG methylation sitesrecognized in vitro by each DNA methyltransferase. The CpG-miR-210 expression vector was treated in vitro with eachDNA methyltransferase of SssI (CG to mCG), HhaI (GCGC to GmCGC) and HpaII (CCGG to CmCGG). Each DNA wastransfected into HeLa or AGS cells together with a CMV-derived miR-132 expression vector (internal control). The relativeexpression of miR-210/miR-132 was quantified by real-time PCR. The error bars indicate SD (n=3). *P < 0.05, **P < 0.01,***P < 0.001, NS (not significant) by Student’s t test.

— 31 —

miR

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96h (miR-210 mut)

Fig. 3. miR-210 regulates gastric cell proliferation.(a) The indicated gastric cells were transfected with 50 nM of miR-210 and assessed for cell viability 4 days later. The dataindicates average values calculated from triplicates. Error bars show SD (n=3). (b) The gastric cell lines were transfectedwith 50 nM of anti-miR-Nega or anti-miR-210 and assessed for cell viability 4 days later. The data shown is average valuescalculated from triplicates. Error bars show SD (n=3). (c) MKN45 cells were transfected with the indicated concentration ofmiR-210 or mutant miR-210 and assessed for cell viability 4 days later. The error bars indicate SD (n=3). (d) MKN45 cellswere transfected with an miRNA negative control, miR-210 or a mutant miR-210. After 2 days, the cells were stained withpropidium iodide and analyzed by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant by Student’st test.

We screened for miR-210 target genes in gastricepithelial cells by performing a cDNA microarray andbioinformatics analysis (Fig. 4a). We overexpressedmiR-210 by introducing 50 nM pre-miR-210 intoMKN or AGS cells and collected total RNA 36hrs after transfection. We screened a total of 20,886genes using a cDNA microarray, and found 146 and237 genes in MKN45 and AGS cells, respectively,which had lower expression levels in pre-miR-210transfected cells compared with 60% in miR-Negatransfected control cells, (Fig. 4a). Using TargetScanver. 6.2 (http://www.targetscan.org/), we were able to

identify genes containing a putative miR-210 seedsequence in the 3’UTR. There are three characteristicsof miR-210 seed sequences, called 8-mer, 7mer-m8and 7mer-A1. These categories have hierarchy of siteefficacy: 8-mer > 7mer-m8 > 7mer-A1. Of the 28genes suppressed by miR-210 in both MKN45 andAGS cells, 12 genes possessed a miR-210 targetsequence (Fig. 4a).

We examined these 12 genes using real-time PCRand found that all 12 genes, ATP11A, DIMT1, H2AFY,INPP5A, NDUFA4, PPP1R2, RAB27B, SH3BGRL,STMN1, TNPO3, VAMP7, and WLS, were strongly

— 32 —

siRNA

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(-)pre-miR-Negapre-miR-210

00.20.40.60.8

11.21.4 si-GFP

miR-210miR-210 mut

Rel

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1)

0

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209118 28

Downregulated by miR-210 (lower than 40%)

12 genes

Target genes containing seed sequence in the 3’UTR were selected

Targeting analysis(Fig. 4c) P < 0.05

10 genes

Cell proliferation analysis(Fig. 4d) P < 0.05

2 genes

STMN1 stathmin 1/oncoprotein 18DIMT1 DIM1 dimethyladenosine

transferase 1 homolog (S. cerevisiae

)

Via

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**

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*

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7

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*

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_2

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LS_2

MKN45 AGS

Fig. 4. miR-210 regulates gastric cell proliferation by targeting STMN1, DIMT1L and METTL13.(a) A schematic diagram of the strategy used to identify miR-210 target genes involved in gastric cell proliferation. (b)The expression levels of 12 genes in MKN45 or AGS cells transfected with pre-miR-210 were measured by real-time PCR.The fold induction of each gene was calculated by normalizing to the gene expression level of cells transfected with thepre-miRNA-negative control. All genes were significantly down-regulated by pre-miR-210 (n=3, P < 0.05 by Student’s ttest). (c) The 3’UTR sequence of each gene was cloned downstream of the luciferase reporter vector. Each reporter plasmidand the small RNAs indicated in the figure were transfected into 293T cells, and luciferase assays were performed 36 hrslater. The levels of luciferase activity in 293T cells overexpressing pre-miR-210 or miR-210 were compared to each controlof pre-miR-Nega or miR-210 mut, respectively, by Student’s t test. Error bars show SD (n=4). *P < 0.05 by Student’s ttest. (d) MKN45 or AGS cells were transfected with the indicated siRNAs, and cell viability was measured 4 days later.Error bars show SD (n=3). (e) The relative mRNA levels of STMN1 and DIMT1 in miR-210, miR-210 mut, or miR-Nega-transfected AGS cells are shown. The data indicate the means ± SD (n=3). *P < 0.001 by Student’s t test. (f) Left panel;Luciferase vectors containing the 3’UTR (3’UTR WT) of each gene and mutant vectors possessing mutated target sites (3’UTR mut) were constructed. The reporter vectors and miR-210 were co-transfected into 293T cells for 36 hrs and subjectedto luciferase assays. The means ± SD (n=4) are shown. Right panel; Part of the luciferase vector sequences for the STMN1and DIMT1L 3’UTRs. The sequences in red characters are the mutated miR-210 target sites. *P < 0.05, **P < 0.001 byStudent’s t test. (g) STMN1 and DIMT1L regulate the cell cycle of MKN45 cells. MKN45 cells were transfected with theindicated siRNAs at a final concentration of 50 nM. The cell cycle phase was analyzed using propidium iodide staining andflow cytometry.

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suppressed by miR-210 in both MKN45 and AGS cells(Fig. 4a and b). Importantly, 11 out of the 12 genespossessed either 7mer-m8 or the 8mer seed sequence intheir 3’UTR sequence. We subsequently asked whetherthe suppression of these 12 genes by miR-210 wasmediated by 3’UTR targeting (Fig. 4c). To this end,the 3’UTR sequences of the 12 miR-210 targeted geneswere fused with the firefly luciferase gene in a reporterplasmid and luciferase activity was measured in 293Tcells overexpressing exogenously co-expressing miR-210 for 36 h. The results suggested that all genesexcept for ATP11A and PPP1R2 can be suppressed bymiR-210 expression (Fig. 4a and c). We subsequentlyscreened these 12 genes for effects on gastric epithelialcell proliferation by siRNA against each miR-210target. Four days post transfection with 50nM of eachsiRNA, we measured the extent of cell proliferationin MKN45 and AGS cells. Inhibition of some genes,especially STMN1 (also termed stathmin1, oncoprotein18, or metablastin) and DIMT1 (also termed DIM1,demethyladenosine transferease 1), showed a decreasein cell proliferation in both cell lines (Fig. 4a andd). STMN1 is a well-known protein up-regulated insolid tumors, and is involved in the initiation of tumordevelopment. However, the biological functions ofDIMT1, which belongs to the S-adenosylmethionine-dependent methyltransferase superfamily, are unknownin cancer. Thus, having experimentally identified 10genes as novel miR-210 targets in this study, wedetermined that at least two, STMN1, and DIMT1, mayplay specific roles in tumorigenesis.

STMN1 and DIMT1 are key players in modulating cellproliferation

We sought to further characterize the biologicalrole of STMN1 and DIMT1 as miR-210 target genes,and first studied the effect of miR-210 overexpressionon target gene expression. AGS cells transfected withmiR-210 or miR-210 mut (a mutant of the seedsequence) were assessed for STMN1 and DIMT1 geneexpression. Expression of miR-210 but not miR-210mut led to suppressed levels of STMN1 and DIMT1(Fig. 4e) We also created a series of luciferase reporterplasmids encoding wildtype (WT) and mutated forms

of the 3’UTR of STMN1 or DIMT1 (Fig. 4f). Each ofthe reporter plasmids were co-transfected with miR-210 vectors into 293T cells, and luciferase activitywas measured 36 hrs post transfection. Expressionlevels of STMN1 and DIMT1 with WT 3’UTR regionswere dampened in the presence of miR-210, but the3’UTR mutants were not (Fig. 4f), implying that miR-210 regulation of STMN1 and DIMT1 gene expressionoccurs via their 3’UTR seed sequences. Indeed, thisnotion was further supported by a bioinformaticsapproach using microRNA target prediction programsprovided by TargetScan, miRanda, Diana Micro-T,and PITA. Furthermore, overexpression of STMN1or DIMT1 in MKN45 cells led to increased cellproliferation as compared to control cells. Lastly, weinvestigated whether siRNA expression against each ofthe miR-210 target genes would lead to cell cycle arrest.We detected an accumulation of cells in the G1 phasewhen MKN45 cells were treated with siRNA againsttarget genes for 2 days (Fig. 4g). Based on these results,we conclude that miR-210 modulation of STMN1 andDIMT1 gene expression may be the mechanism bywhich chronic Hp infection promotes proliferation ofgastric epithelial cells.

Increased expression of STMN1 and DIMT1 in Hp-positive gastric epithelium

To test the hypothesis that miR-210 regulatescell proliferation through STMN1 and DIMT1 inthe stomachs of patients suffering from Hp chronicinfection, we examined the mRNA expression levelsof these three genes in FFPE Hp-positive and Hp-negative gastric human sections using RT-PCR. Asshown in Fig. 5, mRNA levels of both STMN1 andDIMT1 were up-regulated in Hp-positive stomachs(n=19) as compared to Hp-negative control stomachs(n=16). To confirm this, we employed our Mongoliangerbil stomach model to investigate miRNA expressionlevels in gastric epithelial cells. Gerbils were infectedintragastrically with 109 CFU of Hp ATCC43504,miR-210 expression levels were measured 63 dayspost infection. miR-210 expression was found to belower in Hp-infected gerbil stomachs as compared touninfected stomachs (Fig. 6a and b). In contrast to

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0

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Rel

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essi

on

expr

essi

on

Hp (−) Hp (+) (n = 16) (n = 19)

* **

Fig. 5. Expression levels of STMN1 and DIMT1L areincreased in gastric biopsy samples from Hp-infectedindividuals.mRNA expression levels were quantified by real-timePCR. *P < 0.05, **P < 0.01 by Mann-Whitney Utest.

WT infection, we found that gerbils infected with acagA deletion mutant strain (ΔcagA) of ATCC43504showed no decrease in miR-210 expression in thestomach, as compared to uninfected stomachs (Fig.6a), CagA is a major pathogenic factor for Hp-induced inflammation, and our results suggest thatCagA also plays an important role in suppression ofmiR-210. Moreover, expression of Stmn1 and Dimt1was much higher in the gastric tissue of Hp-infectedgerbil stomachs, whereas miR-210 was barely detected(Figs. 6a, b and c). Importantly, PCNA (ProliferatingCell Nuclear Antigen)-positive epithelial cells weresignificantly more abundant in Hp-positive gastrictissue than in Hp-negative gastric tissue (Fig. 6d).Furthermore, STMN1 and PCNA protein levels wereincreased in the gastric mucosa of Hp-infected gerbilsas compared to uninfected gerbils. Taken together,we can conclude that miR-210 plays a key role inregulating proliferation of gastric epithelial cells, bytargeting a set of genes including STMN1 and DIMT1.

Discussion

Our current study identified down-regulationof miR-210 as a risk factor for developmentof Hp-associated gastric diseases including cancer.Furthermore, we have provided substantial evidencesupporting the causal link between suppression ofmiR-210 expression and epigenetic modification ofthe DNA sequence encompassing the miR-210 gene,highlighting DNA methylation as a mechanism forepigenetic silencing of miRNA genes during chronic

inflammation. Since aberrant DNA methylation hasalso been reported in other chronic inflammatorydiseases, including ulcerative colitis and HVB-associated chronic hepatitis, that are causative forcancers, we predict that similar inflammation-inducedDNA methylation leading to miRNA gene silencingcould be an underlying tumorigenic mechanismassociated with those diseases. Therefore, it will be ofparticular interest to identify miRNA genes sensitive topathogenic DNA methylation, as well as the affectedmiRNA-target genes in specific disease states. Inaddition, the molecular mechanisms of how chronicinflammation causes an increase in DNA methylationis an important question to pursue in future studies.

There is some discrepancy concerning the statusof miR-210 as a carcinogenic miRNA. Some studiesfind augmented miR-210 expression in solid tumors,while others see reduced expression in uterine cancerIn our study, miR-210 levels were lower in Hp-positivehuman stomachs than in Hp-negative human stomachs(Fig. 1c). The varying levels of miR-210 found indifferent solid tumors could be explained in part by thedifferent states of hypoxia present during distinct stagesof cancer. After enhanced formation of a solid tumor, alow oxygen state might be dominant, leading to higherlevels of miR-210. As robust methylation is inducedby prolonged inflammation, miR210 expression levelsmight be affected by an altered balance of hypoxia andinflammation in distinct cancer states. Our data showsthat miR-210 levels are reduced in Hp-positive gastricspecimens, while the miR-210-associated CpG islandis hypermethylated (Figs. 2b and c). This suggeststhat sustained down-regulation of miR-210 by CpGisland methylation could cause prolonged aberrantproliferation of gastric epithelial cells during chronicHp infection, thus supporting a micro-tumorigenicenvironment.

Although previous studies have reported an as-sociation between inflammatory diseases and aberrantexpression of miRNAs, some of which have beenalso shown to be DNA methylated, few groups havedemonstrated in vivo evidence for the causal link be-tween affected miRNAs and their targets during diseaseprogression. Our comprehensive analysis identified 12genes that are down regulated in response to miR-210

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AAAAA

Target mRNA(STMN1, DIMT1 etc.)

pri-miR-210

miR-210

CH3 CH3 CH3 CH3

Cell prolifera

AAAAAAAAAA

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Hp (−)/Stmn1 Anti-sense

Hp (+)/Stmn1 Anti-sense

Hp (+)/Stmn1 Sense

Hp (−) Hp (+)

PCNA / AAA

Fig. 6. Decreased miR-210 expression is associated with increased levels of Stmn1 and proliferation-related marker in Hp-infectedMongolian gerbils.(a) Mongolian gerbils were inoculated with Hp, and 63 days later, gastric mucosa tissues were isolated from uninfected(Hp (-), n=5) and infected (Hp (+), n=3 for miR-210 and n=6 for mRNA) gerbils. The expression levels of miR-210, Stmn1and Dimt1l were quantified by real-time PCR analysis. The Mann-Whitney U test was used to calculate P-values. (b) Insitu hybridization detection of miR-210 in the gastric sections from Hp-infected Mongolian gerbils. Gastric sections fromuninfected (Hp (-)) or infected (Hp (+)) were stained with the indicated probes, and counterstained with Kernechtrot stain.The scramble probe was used for background staining. Scale bar, 100 μm. (c) In situ hybridization of Stmn1 in the stomachsections from Hp-infected animals. The sense probe was used for background staining. Scale bar, 100 μm. (d) Nine weeksafter infection, the gerbils were sacrificed, and stomach sections were immunostained with PCNA (proliferating cells, green)and AAA (pit cells, red). Scale bar, 100 μm. (e) Gene silencing of miR-210 upregulates target gene expression and promotesaberrant gastric epithelial cell proliferation during chronic Hp infection.

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overexpression (Figs. 4a and b), most of which had notbeen previously characterized as miR-210 targets. ThesiRNA inhibition experiments described in this studysuggest that the activities of STMN1 and DIMT1 reg-ulate gastric epithelial proliferation more profoundlythan other genes (Fig. 4d). STMN1 has been character-ized as an oncoprotein (also known as oncoprotein 18),associated with gastric and other various types of can-cer. Therefore, our discovery of STMN1 up-regulationin Hp-positive stomachs, where miR-210 levels arevery low, suggests that it is an important factor in thedevelopment of gastric cancer upon chronic Hp infec-tion. Given that the increase in DNA methylation of themiR-210 gene, is correlated with disease progression(Fig. 1c), we propose a novel pathophysiological path-way promoting aberrant gastric epithelial cell prolifer-ation upon chronic infection by Hp (Fig. 6e).

In conclusion, our results demonstrate thatepigenetic silencing of miR-210 following Hp infectionpromotes the proliferation of gastric epithelial cells.Our findings should therefore help to further understandhow chronic Hp infection of the gastric mucosabecomes a risk for gastric cancer, and to identify targetsfor future research which will be aimed at developingnew drugs and markers for tumor differentiation.

Conclusion

Persistent colonization of the gastric mucosa byHelicobacter pylori (Hp) elicits chronic inflammationand aberrant epithelial cell proliferation, whichincreases the risk of gastric cancer. We examined

the ability of microRNAs to modulate gastric cellproliferation in response to persistent Hp infection andfound that epigenetic silencing of miR-210 plays akey role in gastric disease progression. Importantly,DNA methylation of the miR-210 gene was increasedin Hp-positive human gastric biopsies as comparedto Hp-negative controls. Moreover silencing of miR-210 in gastric epithelial cells promoted proliferation.We identified STMN1 and DIMT1 as miR-210 targetgenes and demonstrated that inhibition of miR-210expression augmented cell proliferation by activatingSTMN1 and DIMT1. Together, our results highlightinflammation-induced epigenetic silencing of miR-210as a mechanism of induction of chronic gastric diseases,including cancer, during Hp infection.

Acknowledgments

Hitomi Mimuro is grateful to Drs. KotaroKiga, Masato Suzuki, Aya Shinozaki-Ushiku,Taira Kobayashi, Takahito Sanada, Minsoo Kim,Michinaga Ogawa, Yuka W. Iwasaki, Hiroyuki Kayo,Yoko Fukuda-Yuzawa, Masakazu Yashiro, MasashiFukayama, Taro Fukao, and Chihiro Sasakawa fortheir contribution to this study and helpful discussions.This study was supported in part by grants from theWaksman Foundation of Japan Inc. and the Grants-in-Aid for Scientific Research from the Ministry ofEducation, Culture, Sports, Science and Technology(MEXT) and Japan Society for the Promotion ofScience (JSPS)

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