P eriodontal diseases are mainly chronic infectious diseases result from response to a complex dental plaque

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1 OPEN SUBJECT AREAS: PERIODONTITIS METABOLIC DISORDERS Received 15 Jnury 214 Accepted 9 April 214 Pulished 6 My 214 Correspondence nd requests for mterils should e ddressed to K.Y. (kz@dent. niigt-u.c.jp) Orl pthoiont induces systemic inflmmtion nd metolic chnges ssocited with ltertion of gut microiot Kei Arimtsu 1,2, Hitomi Ymd 1,2, Hrun Miyzw 1,2, Tkyoshi Mingw 1,2, Myuk Nkjim 1,2, Mrk I. Ryder 3, Kzuyoshi Gotoh 4, Disuke Motook 4, Shot Nkmur 4, Tetsuy Iid 4 & Kzuhis Ymzki 1 1 Lortory of Periodontology nd Immunology, Division of Orl Science for Helth Promotion, Niigt University Grdute School of Medicl nd Dentl Sciences, Niigt, Jpn, 2 Division of Periodontology, Deprtment of Orl Biologicl Science, Niigt University Grdute School of Medicl nd Dentl Sciences, Niigt, Jpn, 3 Division of Periodontology, Deprtment of Orofcil Sciences, School of Dentistry, University of Cliforni, Sn Frncisco, USA, 4 Deprtment of Infection Metgenomics, Reserch Institute for Microil Diseses, Osk University, Osk, Jpn. Periodontitis hs een implicted s risk fctor for metolic disorders such s type 2 dietes, therosclerotic vsculr diseses, nd non-lcoholic ftty liver disese. Although cteremis from dentl plque nd/or elevted circulting inflmmtory cytokines emnting from the inflmed gingiv re suspected mechnisms linking periodontitis nd these diseses, direct evidence is lcking. We hypothesize tht disturnces of the gut microiot y swllowed cteri induce metolic endotoxemi leding metolic disorders. To investigte this hypothesis, chnges in the gut microiot, insulin nd glucose intolernce, nd levels of tissue inflmmtion were nlysed in mice fter orl dministrtion of Porphyromons gingivlis, representtive periodontopthogens. Pyrosequencing reveled tht the popultion elonging to Bcteroidles ws significntly elevted in -dministered mice which coincided with increses in insulin resistnce nd systemic inflmmtion. In -dministered mice lood endotoxin levels tended to e higher, wheres gene expression of tight junction proteins in the ileum ws significntly decresed. These results provide new prdigm for the interreltionship etween periodontitis nd systemic diseses. P eriodontl diseses re minly chronic infectious diseses result from response to complex dentl plque microiome contining vrious periodontopthic cteri species. Periodontl diseses destroys the toothsupporting tissues nd leds to tooth loss if not dequtely treted. Advnced form of the diseses ffects,1% to 15% of dults worldwide 1. Epidemiologicl evidence suggests tht periodontl infection is ssocited with n incresed risk of vriety of diseses such s therosclerotic vsculr diseses 2,3, type 2 dietes 4,5, nd non-lcoholic ftty liver disese 6. Among the vrious periodontopthic cteri, considerle reserch hs focused on the role of Porphyromons gingivlis in possile mechnisms linking periodontl diseses nd other humn diseses, due to its unique pthogenicity 7 nd its ssocition with these vrious diseses. Once n inflmmtory lesion in the periodontium is estlished, cteri from the dentl plque cn invde into the gingivl tissue through the ulcerted sulculr epithelil lining of periodontl pockets nd then disseminte into the systemic circultion 8. In ddition, vrious proinflmmtory cytokines re produced in the inflmed periodontl tissue 9 which cn lso enter the systemic circultion. Although the precise mechnisms hve not een clrified, the resulting cteremi nd increse in circulting inflmmtory meditors my potentite n inflmmtory rection in other tissues or orgns. In fct, numer of studies hve demonstrted elevted levels of serum hs-crp 1 nd IL-6 11,12 in periodontitis ptients when compred with periodontlly helthy sujects. By contrst, there is little evidence for n incresed detection nd/or numers of orl cteri in the lood of periodontitis ptients when compred with periodontlly helthy sujects 13. In ddition, we hve shown tht repeted orl inocultion of induced elevtion of serum inflmmtory mrkers (serum myloid A nd IL-6) in SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 1

2 mice. However, no ws detected in the lood t ny time point of these experiments 14. Moreover, in this mouse model, limited inflmmtory cell infiltrte ws seen in the gingivl tissue. It is well-known tht oesity, chronic disese chrcterized y n excessive growth of dipose tissue, increses the risk of insulin resistnce, dietes, therosclerosis, hypertension, chronic kidney disese, nd crdiovsculr moridity nd mortlity Interestingly, mny of these diseses re lso ssocited with inflmmtory periodontl diseses. Insulin resistnce, key feture of oesity, is stte in which the sensitivity of trget cells to respond to ordinry levels of insulin is reduced. This insulin sensitivity is modulted in prt y dipokines. In oesity, dipokine production is either up-regulted or down-regulted. Specificlly, insulin resistnce-ssocited dipokines re usully up-regulted wheres insulin-sensitivity-ssocited dipokines re down-regulted 18. Oesity is reported to e ssocited with periodontl diseses. Sito et l. first reported tht oese Jpnese sujects were more likely to hve periodontl disese thn thin sujects 19. Although lter studies supported the positive ssocition etween oesity nd periodontitis 2 22, there re lso studies showing no ssocition etween these two conditions 23. While there re severl epidemiologicl studies demonstrting this ssocition, there re few reports on the mechnism linking oesity nd periodontl diseses. One proposed mechnism involves the dipocyte hypertrophy found in oesity. This dipocyte hypertrophy leds to infiltrtion of mcrophges nd other inflmmtory cells. This phenomenon results in low-grde chronic inflmmtion nd enhnced proinflmmtory cytokine production 24,25. Among the mny dipokines, TNF- is considered key plyer in inflmmtory cell ctivtion nd recruitment 26.TNF- produced y the dipocytes together with TNF- produced in the periodontl tissue my induce further periodontl tissue destruction. However, severl reports hve shown tht serum TNF- levels of periodontitis ptients re either not elevted or lower in periodontitis ptients compred with non-periodontitis sujects 27. Thus, the pthogenic mechnisms for the effects of periodontl disese ffecting systemic disese nd the specific effects of the infection with periodontopthic cteri on the pthology of vrious tissues remin to e elucidted. In this study, we propose tht endotoxemi is responsile for inflmmtion of vrious orgns nd tissues in this mouse model through orl dministrtion. However, this endotoxemi my not e induced directly y cteri from the orl cvity, ut rther through chnges in gut microiot induced y inocultion of cteri from the orl cvity into the gut through swllowing. Susequent ctivtion of proinflmmtory genes within lood vessels, dipose tissue nd liver could increse the risk of therosclerosis, insulin resistnce nd non-lcoholic ftty liver disese (NAFLD), respectively. Results Locl nd systemic inflmmtion in -dministered mice. Our previous study nd others demonstrted tht repeted orl gvge dministrtion of induced lveolr one resorption. However, whether this could e due to locl inflmmtion of gingivl tissue is still unresolved. Although serum IL-6 levels significntly incresed in -dministered mice compred with shm-dministered mice, no difference of gingivl inflmmtion, which ws miniml, etween -dministered nd shmdministered mice ws oserved (Fig. 1). dministrtion induces insulin resistnce, liver stetosis, nd mcrophge infiltrtion in dipose tissue. To explore the effects of orl gvge dministrtion of on insulin resistnce, we performed glucose nd insulin tolernce tests. As shown in Fig. 2, oth glucose tolernce nd insulin sensitivity ws diminished in P. gingivlis-dministered mice compred with shm-dministered mice, despite similr ody weight chnges during the experimentl period (Supplementry Fig. S1). Although the effect of dministrtion ws reltively wek, significnt difference ws oserved t 15 min for the insulin tolernce test, nd n overll effect ws evident s shown in Figs. 2 nd 2d. However, there were no differences in lood insulin levels etween -dministered mice nd shm-dministered mice (Supplementry Fig. S2). Orl gvge dministrtion of promoted mcrophge infiltrtion into the dipose tissue nd typicl crown-like structure ws oserved histologiclly (Fig. 3). Histologicl nlysis showed tht dministered mice hd ccumulted much higher mounts of heptic ft (Fig. 3) nd triglyceride (Fig. 3c), s compred to the shmdministered mice. dministrtion induces n inflmmtory response in dipose tissue. Next we compred gene expression profiles of epididyml dipose tissue etween -dministered nd shm-dministered mice (Fig. 4). In -dministered mice, expression of proinflmmtory genes Tnf, Ccl2, Il6, nd Il1 were significntly upregulted, wheres the genes tht improve insulin sensitivity Pprc, Ppr, C1qtnf9, Irs1, Sirt1, nd Slc24 were downregulted. In ddition to proinflmmtory genes, expression of Angptl4 which is involved in insulin resistnce ws lso upregulted. These results suggest tht orl gvge dministrtion of induces n inflmmtory response nd insulin resistnce in dipose tissue. There ws no difference in the expression of three regultors of insulin signlling, Grn, Retn, nd Lep, etween P. gingivlis-dministered mice nd shm-dministered mice. dministrtion induces n inflmmtory response in the liver nd n inflmmtion-relted gene response. Orl dministrtion of led to incresed mrna expression of the proinflmmtory cytokines TNF- nd IL-6, s well s of Fitm2 nd Plin2, oth of which re strongly ssocited with lipid droplet formtion in the liver. Furthermore, Acc nd G6pc, which positively regulte ftty cid synthesis nd gluconeogenesis respectively, were lso upregulted. On the other hnd, mrna expression of the molecules hving potentilly nti-inflmmtory properties Pprc nd Sirt1, were downregulted. In ddition, expression of the insulin signlling gene Irs1 ws lso decresed compred with shm-dministered mice. We oserved tht not only gene expression (Fig. 5) ut lso protein levels of TNF- were incresed in the liver of -dministered mice (Fig. 5). Orl dministrtion of lters the gut microil ecology. After 1 cycles of orl dministrtion of live W83, the contents of the distl 3cm of the smll intestine (ileum) were recovered immeditely fter euthniztion y mnul extrusion. We defined the extent to which orl dministrtion of ltered the composition of the microiot y pyrosequencing the 16S riosoml RNA genes in the ileum contents. In totl, 41,557 (men 6,926 reds/smple) nd 32,18 (men 5,351 reds/smple) sequencing reds for the -dministered smples nd the shmdministered smples, respectively, were otined y two sequencing runs. The mjority of ligned reds were determined to e from the phyl Firmicutes nd Bcteroidetes, representing 55.4% nd 38.7% of the flor respectively in -dministered mice, nd 72.8% nd 17.%, respectively in shm-dministered mice. The difference of the proportion of Bcteroidetes nd Firmicutes etween -dministered mice nd shm-dministered mice ws sttisticlly significnt (Fig. 6). Notly, opertionl txonomy unit (OTU)-sed cteril diversity nlysis reveled tht the popultion elonging to Bcteroidles ws significntly elevted in -dministered mice compred with shm-dministered mice (Fig. 6 nd 6c). Becuse elongs to the order Bcteroidles, incresed proportions of the order Bcteroidles could e due to the dministered. To clrify this possile mechnism, DNA smples SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 2

3 2 *** IL-6 (pg/ml) Figure 1 Systemic nd locl inflmmtion in -dministered nd shm-dministered mice (N 5 8 in ech group). () Serum level of IL-6. All dt re mens 6 SD. Significnt differences were oserved etween the -dministered groups nd the shm-dministered group (***p,.1, Mnn-Whitney U-test). () Histologicl findings of gingivl tissues of -dministered nd shm-dministered mice. Sections of the periodontium round the disto-uccl root of the first molr were H-E stined. Right pnels re mgnified views of the oxed res. No difference of gingivl inflmmtion ws oserved etween -dministered nd shm-dministered mice. of the ileum contents were mplified using -specific primers. However, no -specific DNA ws detected (Supplementry Fig. S3). dministrtion increses serum endotoxin level. As it hs een reported tht chnges in the gut microiot influence metolic endotoxemi, we compred the serum endotoxin levels etween -dministered mice nd shm-dministered mice. As shown in Fig. 7, endotoxin levels were very low fter overnight fsting, with no difference etween -dministered mice nd shm-dministered mice. However, the endotoxin levels drmticlly incresed if the mice were fed d liitum irrespective of dministrtion. The endotoxin level tended to e higher in -dministered mice compred to shm-dministered mice. However, this difference ws not sttisticlly significnt (Fig. 7). In order to further exmine the effect of orl dministrtion of on endotoxemi, the serum endotoxin levels were monitored t 1, 3, 12 hrs. fter single dministrtion of P. Blood glucose (mg/dl) c Blood glucose (%) Time (min) ** -1, 6 9 Time(min) d AUC (min mg/dl) Inverse AUC of ITT (min %) 4, 3, 2, 1, -2, -3, -4, -5, * ** Figure 2 induced insulin resistnce. () Intrperitonel glucose tolernce test. Blood glucose levels were determined t the indicted times fter intrperitonel lod of glucose (1 g/kg). () Are under lood concentrtion curve (AUC) of the Fig. 2. (c) Intrperitonel insulin tolernce test (ITT). Blood glucose levels were determined t the indicted times fter intrperitonel lod of insulin (.5 unit/kg). (N 5 6 in ech group). (d) Insulin sensitivity ws ssessed y inverse AUC of ITT. All dt re mens 6 SD. (*p,.5; **p,.1, Mnn-Whitney U-test). SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 3

4 c Triglyceride (nmol/μg) ** Figure 3 Reltionship etween dministrtion nd inflmmtory chnges of epididyml dipose tissue nd liver. () Epididyml dipose tissues were fixed 1% formlin, sectioned, nd stined with rt nti-mouse F4/8 primry ntiody (Ad Serotec). Arrow heds indicte F4/8 positive mcrophges. () Oil red O stining of the liver tissue re shown. Incresed numer of lipid contining heptocytes (rrow heds) re seen in -dministered mice. (c) dministrtion incresed heptic triglyceride content. (N 5 8 in ech group). All dt re mens 6 SD. (**p,.1, Mnn-Whitney U-test). gingivlis. Serum endotoxin levels incresed s erly s 1 hr. fter dministrtion, peked t 3 hrs., nd mintined these higher levels until 12 hrs. (Fig. 7). One hour fter orl dministrtion, ws detected in the jejunum nd ileum, nd its proportion in the microil flor decresed t 3 hrs. Although DNA ws detected in the colon t 16 hrs, the proportion of DNA ws extremely low ecuse of fr lrger quntities of other cteri in the colon (Supplementry Fig. S4). At these time points, no ws detected in the lood smples of -dminstered mice wheres other cteril DNA ws detected (Supplementry Fig. S5). Chnges in gene expression profiles in the intestine y orl dministrtion of. Next we exmined the effect of P. gingivlis dministrtion on the expression of genes tht ply importnt role for prevention of metolic syndrome nd metolic endotoxemi in the smll intestine (Fig. 8) nd inflmmtory responses in the lrge intestine (Fig. 8). The expression of genes tht code intestinl lkline phosphtse (Akp3) nd tight junction protein (Tjp1) in the smll intestine were oth downregulted in P. gingivlis-dministered mice compred to shm-dministered mice. In the smples of lrge intestine, mrna expression of the proinflmmtory cytokines IL-6, IL-12, IFN-c nd IL-17c were significntly upregulted in -dministered mice compred to shm-dministered mice. However, there ws no difference in expression of IL-1 nd TNF-. Discussion Periodontl disese is chronic inflmmtory disese where groups of periodontopthic cteri such s plys mjor role in Reltive gene expression CTRP9 (ng/mg) 1,5 1, 5 **. Figure 4 Effect of orl dministrtion of on the gene nd protein expressions in epididyml dipose tissue. () Comprison of reltive gene expression levels in the dipose tissue etween the -dministered nd the shm-dministered mice (N 5 8 in ech group). The reltive quntity of experimentl mrna ws normlized to the reltive quntity of glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna. () CTRP9 contents in the epididyml dipose tissue were determined y ELISA of tissue lystes. All dt re mens 6 SD. (*p,.5; **p,.1; ***p,.1, Mnn-Whitney U-test). SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 4

5 Reltive gene expression TNF-α (pg/mg) 5 *** Figure 5 Effect of orl dministrtion of on the gene nd protein expressions in liver. () Comprison of reltive gene expression levels in the liver tissue etween the -dministered nd the shm-dministered mice (N 5 8 in ech group). The reltive quntity of experimentl mrna ws normlized to the reltive quntity of glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna. () TNF- levels in the liver tissue were determined y ELISA of tissue lystes. All dt re mens 6 SD. (*p,.5; **p,.1; ***p,.1, Mnn-Whitney U-test). its initition nd progression. A numer of epidemiologicl studies hve suggested tht periodontl disese is risk fctor for vrious systemic diseses nd conditions, including crdiovsculr disese, type 2 dietes, NAFLD, nd rheumtoid rthritis 28. Interestingly, oesity increses the risk of these diseses In ddition, oesity is lso ssocited with n incresed risk of periodontl disese. Therefore, it is possile tht periodontl disese nd oesity hve similr effects on these systemic conditions. The common systemic effect of oesity nd periodontl disese is considered to e low-grde inflmmtion 13. It is well-known tht diet-induced oesity is implicted in systemic low grde inflmmtion. Nutritionl ftty cids ctivte Toll-like receptor 4 (TLR4) signlling in dipocytes nd mcrophges. In ddition, the cpcity of ftty cids to induce inflmmtory signlling in dipocytes or mcrophges is lunted in the sence of TLR4 32,33. Furthermore, dipose tissue lipolysis from hypertrophied dipocytes, could serve s nturlly occurring lignd for TLR4 to induce inflmmtion. It is pprent tht gut microil ecology could e n importnt fctor in the development of oesity y ffecting energy homeostsis. For exmple, Cni et l. elegntly demonstrted tht the composition of the gut microiot is influenced y high-ft diet- nd geneticlly oese o/o-induced metolic endotoxemi 34. Components originting from the gut microiot, such s lipopolyscchride, lipoteichoic cid, peptidoglycn, flgellin, nd cteril DNA cn cuse immune system ctivtion nd susequent inflmmtion. Proposed mechnisms for periodontl diseses inducing systemic inflmmtion hve included: (i) the direct effect of infectious gents or their products, nd (ii) incresed expression of cytokines, chemokines, nd cell dhesion molecules produced in periodontitis lesions 35. Additionl mechnisms my include, (iii) trnsloction of swllowed from the gut to the circultiong system, nd (iv) ltertion of gut microil composition-induced increses in gut epithelil permeility y swllowed. The first hypothesis is sed on the lesion size of periodontl disese (periodontitis). It is reported tht the men dentogingivl epithelil surfce re of periodontitis ptients where sugingivl iofilm is contcting thinning nd/or ulcerted gingivl sulculr epithelium is pproximtely 2 cm This re is considered to ct s n entrnce of periodontopthic cteri into the systemic circultion. This mechnism is supported y numer of studies tht hve demonstrted n ssocition etween periodontl disese nd endotoxemi. However, periodontl tretment-induced endotoxemi is detectle s erly s 5 min fter instrumenttion nd disppers t 3 min 37,38. In our study, increses in the serum endotoxin level ws oserved 1 hr fter single dministrtion of nd peked t 3 hrs. For the second hypothesis, there is no direct evidence tht incresed inflmmtory mrkers in periodontitis ptients re in fct derived from inflmed periodontl tissues. For the third possiility, lthough ws detected in the jejunl nd ilel contents, nd colonic contents up to 3 hrs nd 16 hrs fter single dministrtion, respectively, it ws not detected in the lood of P. ginigivlis-dministered mice wheres other cteril DNA ws detected in the lood. Therefore, it is highly likely tht detected endotoxins re not derived from. Given tht recent findings hve implicted n ltered gut microiot s contriutor of not only metolic diseses such s therosclerosis 39,4, type 2 dietes 41, nd NAFLD 42 ut lso immune diseses such s rheumtoid rthritis 43 tht re lso ssocited with periodontitis, it is resonle to ssume tht the systemic inflmmtory chnges seen in -dministered mice could e ttriutle to this ltered gut microiot. Furthermore, periodontl diseses themselves could e modulted y ltertion of gut microiot-induced systemic inflmmtion. In support of this hypothesis, it hs een reported tht high-ft diet-induced oesity incresed lveolr one resorption, chrcteristic feture of periodontitis 44,45. The swllowed sliv of periodontitis ptients is reported to contin up to 1 9 cteri/ml, in L/dy 46 48, mking totl of greter thn 1 12 cteri/dy. Since the cteril flor of the orl cvity is distinct from tht of the gut 49, it is possile tht swllowed cteri could ffect the composition of the gut microflor. In fct, it hs een reported tht orl proiotic intervention lters gut cteril composition 5. Thus, it is unlikely tht orl cteri lone could e custive gents of endotoxemi in periodontitis ptients. In the present study, orl dministrtion of induced chnge of cteril composition in the ilel microflor. Although severl studies hve shown the eneficil effect of Bcteroidetes phylum on the gut 51,52, we oserved n incresed proportion of the order Bcteroidles elonging Bcteroidetes phylum, ccompnied y insulin resistnce nd the ltertion of gene expression in dipose tissue nd liver. The expression of severl proinflmmtory genes nd nti-inflmmtory or insulin sensitivity-improving genes were upregulted or downregulted, respectively, in the dipose tissue nd the liver of -dministered mice. In ddition, expression of Sirt1 which is reported to induce n increse in glucose uptke nd insulin signlling ws lso downregulted. It hs lso een reported tht SIRT1 expression is inversely relted to inflmmtory gene expression, prticulrly TNF-. These chnges in proinflmmtory nd nti-inflmmtory gene expression my contriute to increses in serum glucose levels nd in insulin intolernce. Although the effect of is not roust, continuous deteriortion of glucose metolism could hve significnt effect. SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 5

6 c (/) Figure 6 Comprison of the gut microiot etween -dministerd nd shm-dministered mice y 16S rrna sequencing nlysis. Reltive undnces of ech cteril group in Phylum () nd OTU () level re indicted y oxplot. Ech defined OTU otined from 16S rrna deep sequencing ws compred to the genome dtse (GenomeDB) from NCBI using lstin (c). Close reltive species nd percent similrities re shown. Fold increses indicte the men rtio of reltive undnce of the OTU in the gut microiot from the -dministered group to shmdministered group. (*p,.5, **p,.1, Mnn-Whitney U-test). One of the possile resons for the discrepncy of inflmmtionssocited chnge of microiot etween previous studies nd our study could e the difference of the site from where the smples were otined. Previous studies nlyzed fecl or cecl smples, wheres we nlysed ilel contents. The ileum is considered to e n importnt orgn ecuse chylomicron is formed into smll vesicles in the epithelil cells of the ileum, nd lipid is minly sored from the ileum. In ddition, Peyer s Ptches re locted in the ilel wll. Therefore, s previously demonstrted with cecl cteril 53, the cteril composition in the ileum my hve significnt impct on systemic inflmmtion. It is lso noteworthy tht in humns, Bcteroidetes my promote type 2 dietes through n endotoxininduced inflmmtory response 54. Heno-Meji et l., demonstrted tht significnt expnsion of Porphyrmondcee ws found following HFD or with methioninecholine-deficient diet dministrtion in the fecl microiot in the inflmmsome-deficient setting nd ws ssocited with progression of NAFLD nd oesity 55. We propose tht dministered is not directly responsile for the increse of the fmily Porphyromondcee in the gut, s we did not detect in the gut y using specific primers. However, cteri elonging to this fmily of cteri my ply role in the induction of endotoxemi nd susequent inflmmtory responses. Alterntively, my suppress inflmmsome ctivtion y other cteri. For exmple, it hs een reported tht suppresses inflmmsome ctivity through inhiition of endocytosis 56. Another interesting finding from this study ws tht mrna expression of lkline phosphtse ws downregulted in the ileum of -dministered mice. Klnnen et l. previously demonstrted tht defect in intestinl lkline phosphtse (IAP), ws ssocited with high-ft diet-induced metolic syndrome, nd endogenous nd orlly supplemented IAP inhiited endotoxin sorption, s well s reversed metolic syndrome in mice 57. Therefore, IAP is considered to ply n importnt role in the SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 6

7 Serum endotoxin (EU/ml) Serum endotoxin (EU/ml) * * Overnight fsting Non-fsting Figure 7 Effect of orl dministrtion of on the serum endotoxin levels. () Serum endotoxin (LPS) concentrtion (EU/ml) were determined fter 1 cycles of dministrtion or shm dministrtion (N 5 8 in ech group). All dt re mens 6 SD. () Serum endotoxin (LPS) concentrtion (EU/ml) were determined t the indicted times fter single dministrtion of (N 5 6 in ech group). All dt re mens 6 SD. (*p,.5, one-wy ANOVA). suppression of endotoxemi. Although the underlying mechnisms y which orl dministrtion of nd/or ltertion of gut microiot downregulte the mrna of lkline phosphtse hs not een clrified, downregultion of the Akp3 gene my e fctor for elevted systemic inflmmtion. Moreover, gene expression nlysis of the smll intestine demonstrted downregulted mrna expression of the tight junction protein ZO-1 in -dministered mice. In previous studies on mouse models, the endotoxemi following high-ft diet dministrtion ws ssocited with reduced expression of genes encoding for ZO-1 nd occludin 34. These results suggest tht dministered lters the gut microiot, nd lters the gut epithelil cell rrier function, resulting in incresed gut permeility. However, the mechnism for how this chnge in the gut microiot impirs gut rrier function hs not een elucidted. P. gingivlis dministrtion-induced ltertions of the gut microiot lso induced upregulted mrna expression of vrious proinflmmtory cytokines. It is not known whether these inflmmtory chnges of the lrge intestine ffect systemic inflmmtory responses. In conclusion, in the present study, we demonstrted tht orl dministrtion of induced ltertion of gut microiot s well s inflmmtory chnges in vrious tissues nd orgns. These chnges re considered to e ttriutle to increses in levels of endotoxin in the lood. However, s with previous oservtions on the influence of high-ft diet-induced metolic endotoxemi induced chnges of the gut microiot, it remins to e elucidted whether cuse-nd-effect reltionship exists etween orl dministrtion of -induced systemic inflmmtion nd chnges in the gut microiot. Also, further investigtions re needed to exmine whether other orl cteri hve similr effects on the systemic metolism. Becuse the composition of the orl microflor nd gut microflor re quite distinct, the considerle flow of lrge quntities of orl cteri into gut during the frequent ct of swllowing could distur the lnce of the gut microflor. Furthermore, cteril components responsile for the ltertion of gut cteril composition hve lso not een elucidted. Therefore, further studies re needed to clrify the exct mechnisms of how induces shifts in the gut microiot towrds the production of metolic products nd shifts in the composition of cteril species responsile for the induction of metolic syndrome. Methods Mice. All experiments were performed in ccordnce with the Regultions nd Guidelines on Scientific nd Ethicl Cre nd Use of Lortory Animls of the Science Council of Jpn, enforced on June 1, 26, nd pproved y the Institutionl Reltive gene expression ** Akp3 * Tjp1 Reltive gene expression Figure 8 Comprison of reltive gene expression levels in the smll intestine () nd colon tissue () etween the shm-dministered group nd the P. gingivlis-dministered group (N 5 8 in ech group). The reltive quntity of experimentl mrna ws normlized to the reltive quntity of glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna. All dt re mens 6 SD. (*p,.5; **p,.1, ***p,.1, Mnn-Whitney U-test). SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 7

8 Animl Cre nd Use Committee t Niigt University (permit numer 39). Sixweek-old mle C57BL/6N mice were otined from Jpn SLC, Inc. (Shizuok, Jpn). The mice were cclimtized under specific pthogen-free conditions nd fed regulr chow nd sterile wter until the commencement of infection t 8 weeks of ge. Bcteril cultures. strin W83 ws cultured in modified Gifu neroic medium (GAM) roth (Nissui, Tokyo, Jpn) in n neroic jr (Becton Dickinson Microiology System, Cockeysville, MD) in the presence of n AneroPckTM (Mitsuishi Gs Chemicl Co. Inc., Tokyo, Jpn) for 48 hours t 37uC. Bcteril suspensions were prepred in phosphte-uffered sline (PBS) without Mg 21 /C 21 using estlished growth curves nd spectrophotometric nlysis. The numer of CFUs ws stndrdized y mesuring opticl density (6 nm). Orl dministrtion. The murine experimentl periodontitis model ws developed ccording to Bker et l. 58 with slight modifictions. A totl of 1 9 CFU s of live P. gingivlis suspended in 1 ml of PBS with 2% croxymethyl cellulose (Sigm- Aldrich, St. Louis, MO) ws given to ech mouse vi feeding needle. This suspension ws given 2 times week for 5 weeks. The control group ws shm-dministered without the. During the experimentl period, ll mice were llowed to et nd drink d liitum. One dy fter the finl tretment, orl sws were otined nd tested for the presence of s previously descried 59. Mice were then euthnized with CO 2, nd their tissues were removed. Detection of in lood nd intestinl contents. Whole lood ws tken t t 15 min, 3, 12, nd 24 hrs fter single dministrtion of. Intestinl contents were otined t 1, 3, nd 16 hrs fter dministrtion of.dna ws extrcted from whole lood nd intestinl contents using QIAmpDNA Blood Mini Kit (Qigen, Hilden, Germny) nd QIAmp DNA Stool Mini Kit (Qigen), respectively. Quntittive rel-time PCR ws performed on LightCyclerH 96 System (Roche) using Fst Strt Essentil DNA Green Mster (Roche). Universl 16S rrna ws mplified using forwrd primer 59-ACTCCTACGGGAGGCAGCAGT-39 nd reverse primer 59-ATTACCGCGGCTGCTGGC S rrna ws mplified using forwrd primer 59- AGGCAGCTTGCCATACTGCG-39nd reverse primer 59- ACTGTTAGCAACTACCGATGT-39. Gut microiot nlysis y pyrosequencing. The ileum ws removed nd the ilel contents were collected using sterile wter (Bio-Rd Lortories, Hercules, CA). The otined ilel contents were homogenised nd DNA ws extrcted using PowerSoilH DNA isoltion kit (MO BIO, Crlsd, CA). PCR ws performed using primer set (784F:59-AGGATTAGATACCCTGGTA-39 nd 161R: 59- CRRCACGAGCTGACGAC-39) trgeting the V5-V6 region of the 16S rrna genes 6. To mplify the trgeted region, 1 ml of extrcted DNA served s the templte in 5-ml rections using KAPA HiFi HS Redy Mix (KAPA Biosystems, Wourn, MA). The PCR protocols were 95uC for 3 min, 25 cycles of 98uC for 2 s, 6uC for 15 s, nd 72uC for 15 s nd 72uC for 1 min. Two 1 ml of 3-cycle reconditioning PCR rections were performed per smple to eliminte heteroduplexes, with 1-ml liquots of the initil PCR product mixture s the templte nd other PCR conditions unchnged. Products of the two reconditioning PCR rections per smple were comined nd purified using DNA clen nd Concentrtor-5 (ZYMO RESEARCH, Irvine, CA). Pyrosequencing of the 16S rrna mplicons ws crried out on the 454 GS Junior pltform (Roche, Bsel, Switzerlnd). Denoising, txonomic ssignments, nd estimting reltive undnce of sequencing dt were performed y the nlysis pipeline of the QIIME softwre pckge 61. An opertionl txonomic unit (OTU) ws defined t 97% similrity. OTU indicting reltive undnce of under.1% ws filtered to remove noise. Glucose nd insulin tolernce tests. GTTs were performed in mice orlly dministered nd shm-dministered mice. Mice were injected i.p. with single dose of 1 g of glucose per kg of ody weight. For the insulin tolernce test, insulin (.5 unit/kg) ws dministered y n i.p. injection. Blood smples were collected through the til vein efore glucose or insulin injection nd t 15, 3, 6, 9, nd 12 min. Blood glucose concentrtions were immeditely determined y the Glucose Pilot ssy (Aventir Biotech, LLC CA). The serum insulin concentrtion ws determined for y ELISA (Moring, Tokyo, Jpn). Periodontl tissue, liver nd dipose tissue histology. The fixed mndiles were dissected, declcified, emedded, nd sectioned s descried previously 62. Seril sections 5 mm thick were otined in the sgittl direction long the long xis of the teeth nd stined with hemtoxylin nd eosin. Mouse livers were emedded in Tissue- Tek OCT (Finetechnicl Skur, Tokyo, Jpn) nd frozen in liquid nitrogen. Frozen 7 mm sections were otined in cryostt (LEICA, Wetzlr, Germny), dried t room temperture for 6 min, fixed in 1% formldehyde for 1 min, stined with oil red O for 2 min, nd counter-stined with hemtoxylin. Adipose tissue smples from shm-dministered nd -dministered mice were fixed in 1% formlin for immunohistochemistry. Briefly, smples were emedded in prffin, sectioned, nd stined with rt nti-mouse F4/8 ntiody (ABd Serotec, Rleigh, NC; 155 dilution). Cytokine ssy. Proteins were extrcted using T-PER Mmmlin Protein Extrction Regent (Pierce Biotechnology, Rockford, IL). C1q/TNF-relted protein 9 (CTRP9) nd TNF- were mesured using the Mouse CTRP9 ELISA kit (Aviscer Bioscience, Snt Clr, CA) nd the Mouse TNF ELISA kit (Thermo Scientific, Settle, WA), respectively. The totl protein concentrtions were mesured y the Pierce BCA Protein Assy kit (Thermo Scientific). Individul protein concentrtions were clculted s the undnce of specific protein constituents divided y the totl protein concentrtions. Serum IL-6 level ws determined y using commercil ELISA kit (Thermo Scientific). Endotoxin ssy. Endotoxin levels were determined in ser collected t 16 hrs fter finl dministrtion of from the til veins of mice using limulus moeocyte lyste test (QCL-1TM, BioWhittker, Wlkersville, MD) ccording to the mnufcturer s instruction. Serum smples were diluted t 1 to 4 for the ssy. Opticl densities were mesured using n ELISA plte reder (Model 68, Bio-Rd Lortories) t 45 nm. Liver triglyceride content. Liver triglycerides were determined with Triglyceride Quntifiction Colorimetric kit (Bio Vision, Milpits, CA). Triglyceride vlues were expressed s the concentrtion of triglycerides divided y the totl protein concentrtion. The totl protein concentrtions were mesured y the Pierce BCA Protein Assy kit (Thermo Scientific). Anlysis of gene expression in dipose tissue, liver, nd intestine. Totl RNA from dipose tissue, liver, smll intestine nd lrge intestine smples ws extrcted using n RNesy Mini kit nd treted with DNse I (Qigen, Germntown, MD) ccording to the mnufcturer s instructions. Aliquots of RNA were then reverse-trnscried to cdna using rndom primers (Tkr Bio Inc., Shig, Jpn) nd M-MLV reverse trnscriptse (Life Technologies Corportion, Crlsd, CA). Primers nd proes specific for rel-time PCR were purchsed from Life Technologies Corportion. Rections were crried out in 25-ml mixture in LightCyclerH 96 System(Roche) using TqMn Gene Expression Assys (Life Technologies Corportion) contining 9 nm primer nd 25 nm proe. The rections consisted of 1-minute incution t 95uC, followed y 4 cycles of two-step mplifiction procedure consisting of nneling/extension t 6uC for 1 minute nd denturtion for 15 seconds t 95uC. LightCyclerH 96 softwre (Roche) ws used to nlyze the stndrds nd crry out the quntifictions. The reltive quntity of ech mrna ws normlized to the reltive quntity of glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna. Sttisticl nlysis. Nonprmetric dt were evluted using the Mnn-Whitney U- test for two-group comprisons, wheres comprisons of three or more groups were nlysed y one-wy ANOVA with d hoc Bonferroni post tests using Grphpd PrismH (GrphPd Softwre, Inc., L Joll, CA). A proility vlue of p,.5 ws considered sttisticlly significnt. For ssessing significnt differences etween the P. gingivlis-dministered nd shm-dministered smples, we used the Mnn Whitney U-test from the R pckge ( 1. Petersen, P. E. & Ogw, H. Strengthening the prevention of periodontl disese: the WHO pproch. J Periodontol. 76, (25). 2. Bhekr, A. A., Singh, S., Sh, S., Molnr, J. & Aror, R. The prevlence nd incidence of coronry hert disese is significntly incresed in periodontitis: met-nlysis. Am Hert J. 154, (27). 3. Humphrey, L. L., Fu, R., Buckley, D. I., Freemn, M. & Helfnd, M. Periodontl disese nd coronry hert disese incidence: systemtic review nd metnlysis. J Gen Intern Med. 23, (28). 4. Chávrry, N. G., Vettore, M. V., Snsone, C. & Sheihm, A. 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PLoS One 5, e985 (21). 55. Heno-Meji, J. et l. Inflmmsome-medited dysiosis regultes progression of NAFLD nd oesity. Nture 482, (212). 56. Txmn, D. J. et l. Porphyromons gingivlis medites inflmmsome repression in polymicroil cultures through novel mechnism involving reduced endocytosis. J Biol Chem. 287, (212). 57. Klinnn, K. et l. Intestinl lkline phosphtse prevents metolic syndrome in mice. Proc Ntl Acd Sci U S A. 11, (213). 58. Bker, P. J., Evns, R. T. & Roopenin, D. C. Orl infection with Porphyromons gingivlis nd induced lveolr one loss in immunocompetent nd severe comined immunodeficient mice. Arch Orl Biol. 39, (1994). 59. Ashimoto, A., Chen, C., Bkker, I. & Slots, J. Polymerse chin rection detection of 8 puttive periodontl pthogens in sugingivl plque of gingivitis nd dvnced periodontitis lesions. Orl Microiol Immunol. 11, (1996). 6. Andersson, A. F. et l. Comprtive nlysis of humn gut microiot y rcoded pyrosequencing. PLoS One 3, e2836 (28). 61. Cporso, J. G. et l. QIIME llows nlysis of high-throughput community sequencing dt. Nt Methods 7, (21). 62. Sulniute, R., Lindh, T., Wilczynsk, M., Li, J. & Ny, T. Plsmin is essentil in preventing periodontitis in mice. Am J Pthol. 179, (211). Acknowledgments This work ws supported y JSPS KAKENHI Grnt Numers nd , nd Sunstr Inc. (Osk, Jpn). The uthors thnk Dr. Bruce Beutler (Center for Genetics of Host Defense, University of Texs Southwestern Medicl Center, Dlls, TX) nd Dr. Tomoyuki Hond (Division of Periodontology, Deprtment of Orl Biologicl Science, Niigt University Grdute School of Medicl nd Dentl Sciences, Niigt, Jpn) for the criticl reding nd preprtion of the mnuscript, respectively. Author contriutions K.A. reserched dt nd wrote the mnuscript. H.Y., H.M., T.M., M.N., K.G. nd D.M. reserched dt. S.N. reserched dt nd contriuted to discussion. M.R. nd T.I. contriuted to discussion nd reviewed/edited mnuscript. K.Y. wrote the mnuscript. All uthors reviewed the mnuscript. Additionl informtion Supplementry informtion ccompnies this pper t scientificreports Competing finncil interests: The uthors declre no competing finncil interests. How to cite this rticle: Arimtsu, K. et l. Orl pthoiont induces systemic inflmmtion nd metolic chnges ssocited with ltertion of gut microiot. Sci. Rep. 4, 4828; DOI:1.138/srep4828 (214). This work is licensed under Cretive Commons Attriution 3. Unported License. Theimgesinthisrticlereincludedintherticle scretivecommonslicense, unless indicted otherwise in the imge credit; if the imge is not included under the Cretive Commons license, users will need to otin permissionfrom the license holder in order to reproduce the imge. To view copy of this license, visit SCIENTIFIC REPORTS 4 : 4828 DOI: 1.138/srep4828 9

10 1 Supplementry informtion Orl pthoiont induces systemic inflmmtion nd metolic chnges ssocited with ltertion of gut microiot Kei Arimtsu 1,2, Hitomi Ymd 1,2, Hrun Miyzw 1,2, Tkyoshi Mingw 1,2, Myuk Nkjim 1,2, Mrk I. Ryder 3, Kzuyoshi Gotoh 4, Disuke Motook 4, Shot Nkmur 4, Tetsuy Iid 4 nd Kzuhis Ymzki 1* 1 Lortory of Periodontology nd Immunology, Division of Orl Science for Helth Promotion, Niigt University Grdute School of Medicl nd Dentl Sciences, Niigt, Jpn 2 Division of Periodontology, Deprtment of Orl Biologicl Science, Niigt University Grdute School of Medicl nd Dentl Sciences, Niigt, Jpn 3 Division of Periodontology, Deprtment of Orofcil Sciences, School of Dentistry, University of Cliforni, Sn Frncisco, USA 4 Deprtment of Infection Metgenomics, Reserch Institute for Microil Diseses, Osk University, Osk, Jpn * Correspondence: Prof. Kzuhis Ymzki, Lortory of Periodontology nd Immunology, Division of Orl Science for Helth Promotion, Niigt University Grdute School of Medicl nd Dentl Sciences, 5274 Gkkocho 2-n-cho, Chuo-ku, Niigt , Jpn Tel: , Fx: , E-mil: kz@dent.niigt-u.c.jp

11 Body weight (g) Supplementry Figure S Dy Dy24 Dy39 Chnges in ody weight during experimentl period (N = 8 in ech group).

12 Serum insulin (ng/ml) Supplementry Figure S Insulin levels of -dministered nd shm-dministered mice. Fsting serum insulin level were determined y ELISA (N = 1 in ech group).

13 Supplementry Figure S3 Detection of in the ilel smples of -dministered nd shmdministered mice. Representtive 1.2% grose gels showing the results of PCR mplifiction of DNA extrcted from ilel smples for detection of 16S rrna.

14 16S rrna / Universl 16S rrna Supplementry Figure S Jejunum Ileum Colon Trnsloction of dministered in the intestinl trct. After single dministrtion of, intestinl contents were recovered from the jejunum, ileum, nd colon t 1, 3, nd 16 hrs. Reltive undnce of -specific 16S rrna gene to universl 16S rrna genes re shown.

15 Supplementry Figure S Detection of -specific 16S rrna gene () or universl 16S rrna genes () in the lood smples of -dministered nd shm-dministered mice. After single dministrtion of orlly or intrvenously (infroritl vein), lood drws were tken from left ventricle of the hert t, 3, 12, nd 24 hrs or 15 min, respectively. Representtive results of one of the three independent experiments re shown. The numer of PCR cycles is shown ove lnes.