Environmental triggers in IBD: a review of progress and evidence

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1 Envionmntal tiggs in IBD: a viw of pogss and vidnc Ashwin N. Ananthakishnan 1, Chals N. Bnstin 2, Dimitios Iliopoulos 3, Andw Macphson 4, Makus F. Nuath 5, Raja A. Raja Ali 6, Stphan R. Vavicka 7 and Claudio Fiocchi 8 Abstact A numb of nvionmntal factos hav bn associatd with th dvlopmnt of IBD. Altation of th gut micobiota, o dysbiosis, is closly linkd to initiation o pogssion of IBD, but whth dysbiosis is a pimay o sconday vnt is uncla. Nvthlss, aly-lif vnts such as bith, bastfding and xposu to antibiotics, as wll as lat childhood vnts, a considd potntial isk factos fo IBD. Ai pollution, a consqunc of th pogssiv contamination of th nvionmnt by countlss compounds, is anoth facto associatd with IBD, as paticulat matt o oth componnts can alt th host s mucosal dfncs and tigg immun sponss. Hypoxia associatd with high altitud is also a facto und invstigation as a potntial nw tigg of IBD flas. A ky issu is how to tanslat nvionmntal factos into mchanisms of IBD, and systms biology is incasingly cognizd as a statgic tool to unavl th molcula altations lading to IBD. Envionmntal factos add a substantial lvl of complxity to th undstanding of IBD pathognsis but also pomot th fundamntal notion that complx disass such as IBD qui complx thapis that go wll byond th cunt singl-agnt tatmnt appoach. This Rviw dscibs th cunt concptualization, vidnc, pogss and diction suounding th association of nvionmntal factos with IBD. REVIEWS IBD Two disass (Cohn s disas and ulcativ colitis) affcting pimaily th digstiv tact, chaactizd by chonic inflammation. Micobiota Community of micooganisms compising bactia, viuss, fungi, Achaa and ukayotic micooganisms. Cospondnc to A.N.A. Massachustts Gnal Hospital, Havad Mdical School, 165 Cambidg Stt, Boston, Massachustts 02114, USA. aananthakishnan@ mgh.havad.du doi: /ngasto Publishd onlin 11 Oct 2017 Th changing pidmiology of IBD (Cohn s disas and ulcativ colitis) acoss tim and gogaphy suggsts that nvionmntal factos hav a majo ol in inducing o modifying disas xpssion 1 3. Considing that IBD mgd in Wstn countis thoughout th middl of th 20th cntuy, th mgnc of IBD in dvloping nations ov th past 25 yas suggsts that this pidmiological volution is latd to wstnization of lifstyl and industialization 2,3. Ubanization of socitis is associatd with changs in dit, antibiotic us, hygin status, micobial xposus and pollution, which hav bn implicatd as potntial nvionmntal isk factos fo IBD (FIG. 1). Envionmntal isk factos fo individual, familial, community-basd, county-basd and gionally basd oigin could all contibut to th pathognsis of IBD 1,4 6. Lnding futh suppot to th citical impotanc of nvionmntal influncs is cognition of th cntal ol of th gut micobiota in th dvlopmnt and popagation of inflammation in IBD 7. Although host gntics might patly dtmin gut micobial stuctu, xtnal nvion mntal xposus fom th tim of bith to adulthood continu to alt th composition, stuctu and function of th gut micobiom, thby dynamically alting th isk and natual histoy of disas thoughout lif 8,9. Discoving how nvion mntal factos influnc th onst of IBD and contibut to its pathognsis could, ultimatly, hlp to dtmin how individuals can duc thi isk of disas o hav a mild clinical cous. Th sach fo pathognic nvion mntal factos is also impotant, as many unmt thaputic nds and suboptimal outcoms in IBD main. Mchanistic insights obtaind fom obustly dfining nvionmntal influncs could also lad to idntification of nw thaputic tagts and tatmnt statgis. In this Rviw, w summaiz th latst litatu on vaious nvionmntal influncs in IBD and discuss how such factos could povid insights into IBD pathognsis and potntial thaputic pathways. Th gut micobiota in IBD Dysbiosis is dfind as a chang in th nomal micobial cology, considd mainly fo th intstin in th contxt of IBD. Infction can b dfind as th invasion and multiplication of micooganisms in body tissus by a causativ agnt o agnts (idntifid ith though classic cultu o indictly though thi gnomic signatus) and cdibl pathognicity. Nomally, th gut NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY ADVANCE ONLINE PUBLICATION MacmilanPublishsLimitd,patofSpingNatu.Alightssvd.

2 Ky points Gut micobiota composition is known to b impotant in maintaining halth and mdiating disas Dysbiosis, a chang in th nomal micobial cology, occus in th intstin in th contxt of IBD Gut inflammation in IBD is chaactizd by a ducd divsity of micobiota, which could nd th host mo suscptibl to colonization with pathogns o pathobionts Envionmntal factos pobably hav a majo ol in IBD; antibiotic us, childbith mod, bastfding, ai pollution, NSAID us, hypoxia o high altitud, dit and uban nvionmnts hav bn studid Futu studis should adopt a multi-omic big data appoach, intgating sval lays of data on clinical paamts, nvionmntal xposus, gntics, pigntics, immunological function and micobial stuctu Xnobiotics Substancs that a foign to th body. Pathobionts Micooganisms associatd with chonic inflammatoy disass. Achaa A kingdom of singl-cll micooganisms without a nuclus o mmbanbound oganlls. micobiota vais longitudinally and tansvsly in th gastointstinal tact in a singl individual, and th micobial consotia vay btwn diffnt individuals, with vidnc of clusts of distinct micobial communitis in diffnt individuals 10. Ths wll-balancd host micobial symbiotic stats pobably spond diffntly to pisods of infction, ditay changs, dug intak and xposu to xnobiotics. Th intstinal micobiota is known to b impotant in both maintaining halth and mdiating disas 8,9, and its composition is influncd by both nvionmntal and host factos 8,9,11. Appoximatly on-thid of facal bactial taxa a hitabl 11. In 1,098 individ uals in th discovy cohot individuals, 58 singl nuclotid poly mophisms (SNPs) w idnti fid as associatd with th lativ abundanc of 33 taxa. Among ths SNPs, fou loci w plicatd in a scond cohot of 463 individ uals: s (nast gn UBR3) associatd with Riknlla ca, s (CNTN6) associatd with Facalibactium, s (DMRTB1) associatd with Lachnospia, and s (SALL3) associatd with Eubactium 11. That th gut micobiota is altd duing intstinal inflammation is wll known. In animal modls of intstinal infction (fo xampl, Salmonlla infction), solution of th infction only occus onc th nomal micobiota is -stablishd 12. Multipl studis hav now invstigatd th diffnc in gut micobial composition btwn patints with IBD and halthy individ uals. Gut inflammation in IBD is chaact izd by a ducd divsity of micobiota, which could nd th host mo suscptibl to colonization with patho gns o pathobionts 7,13. Th undlying uninflamd mucosa in IBD has a diffnt micobiota than halthy mucosa 7,13. Hnc, gut dysbiosis can tigg futh changs in th gut micobiota of individuals with IBD, o altnativly, th gut pithlium of individ uals with IBD could b conduciv to dvlopmnt of an abant micobiota 14. An lgant study xamind sampls fom multipl gastointstinal locations collctd bfo tatmnt in nw-onst padiatic Cohn s disas 13. An axis dfind by an incasd abundanc in ctain bactia, which includd Entobactiaca, Pastu llaca, Villo nllaca and Fuso bactiaca, and dcasd abundanc in Eysiplotichals, Bactoidals, and Clostidials (FIG. 2) colatd stongly with psnc of disas. Micobiom compaison btwn patints with Cohn s disas with and without antibiotic xposu indicatd that anti biotic us amplifis micobial dys biosis associatd with th condition 13. In addition to altations in th com position and function of th bac tial taxa, studis hav also suggstd a potntial ol fo ntic viuss, fungi and Achaa 7,15. In a mous modl, muin noo vius infction was ncssay in addition to mutations in th autophagy gn ATG16L1 to caus altd Panth cll phnotyps that p disposd to colitis 16. Nith th gntic vaiant no ntic infctions alon w sufficint to sult in a pathognic chang. Altations in th human gut micobiom that dvlop as aly as infancy could contibut to th pathognsis of IBD 17. Som of th alist xposus ptubing th infant gut micobiota a mod of bith, bastfding and xposu to antibiotics, all of which hav bn xamind as factos pdisposing to th dvlopmnt of IBD and a xplod hin. Ealy-lif factos Antibiotics. Halthy humans bfo, duing and aft a 5 day cous of oal cipofloxacin dmonstatd dcasd divsity, ichnss and vnnss of th facal micobiota 18. Th abundanc of oughly on-thid of th bactial taxa changd duing cipofloxacin administation, and, although th micobiota lagly tund to ptatmnt community composition at 4 wks aft tatmnt, sval bactial taxa faild to cov within 6 months, suggsting a psistnt ffct of vn a shot cous of oal anti biotics 19. In anoth study 18,19, th distal gut micobiota of th individuals xamind ov 10 months and spanning two couss of cipo floxacin dmonstatd that th ffct of th anti biotic on gut Autho addsss 1 Massachustts Gnal Hospital, Havad Mdical School, 165 Cambidg Stt, Boston, Massachustts 02114, USA. 2 Univsity of Manitoba IBD Clinical and Rsach Cnt, 804 F-175 McDmot Avnu, Winnipg Manitoba R3E 3P4, Canada. 3 Cnt fo Systms Biomdicin, Vatch & Tama Manoukian Division of Digstiv Disass, Dpatmnt of Mdicin, UCLA, 650 Chals E. Young Div South CHS , Los Angls, Califonia , USA. 4 Gastontology/UVCM, Inslspital, Fibugstass 8, 3010 Bn, Switzland. 5 I. Dpatmnt of Mdicin, Univsity of Elangn- Nünbg, Univsity Hospital, Ulmnwg 18, Elangn, Gmany. 6 Th National Univsity of Malaysia, UKM Mdical Cnt, Jalan Yaacob Latif, Kuala Lumpu, Malaysia. 7 Timli Hospital, Dpatmnt of Gastontology & Hpatology, Bimnsdofstass 497, 8063 Zuich, Switzland. 8 Dpatmnt of Pathobiology, Ln Rsach Institut, and Dpatmnt of Gastontology & Hpatology, Digstiv Disass and Sugy Institut, Th Clvland Clinic Foundation, 9500 Euclid Avnu, Clvland, Ohio 44195, USA. 2 ADVANCE ONLINE PUBLICATION

3 Smoking Infctions Micobiota Education lvl Pollution Psychological stat Mod of bith IBD Dit Socioconomic factos Bastfding Gogaphy Excis Modn lifstyl Figu 1 Envionmntal factos contibuting to IBD pathognsis. Envionmntal factos including concption, matnal influncs on th ftus, mod of bith, aly childhood and dit and lifstyl xposus duing adulthood modify th isk of dvlopmnt of Cohn s disas and ulcativ colitis. micobiota was pofound and apid, with a loss of divsity and a shift in community composition occuing within 3 4 days of stating tatmnt. On wk aft th nd of ach cous, communitis bgan to tun to thi initial stat, but th tun was oftn incomplt. In all individuals, th composition of th gut micobiota stabilizd by th nd of th xpimnt, but was altd fom its initial stat 19. As antibiotics hav bn widly usd in both dvloping and dvlopd nations, and a incasingly bing usd in dvloping nations, it is asonabl to consid that antibiotic us could b a ky pdisposing facto in IBD patho gnsis. Misus and abus of antibiotics as wll as us in livstock might futh compound this issu. Sval studis hav xamind whth antibiotic us aly in lif pdisposs to IBD and hav consistntly dmonstatd this association in Wstn populations 20. A Canadian IBD pidmiological databas study compad antibiotic us in th fist ya of lif btwn 36 individuals with IBD and 360 halthy contols. Of childn with IBD, 58% had on o mo antibiotic dispnsations in thi fist ya of lif compad with 39% of contols; moov, thos civing on o mo dispnsations of antibiotics w naly th tims as likly to b diagnosd with IBD 21. In a spaat analysis using a much lag data st of 294 childn with IBD matchd to 2,377 halthy contols to xamin whth a diagnosis of otitis mdia (sving as a poxy fo antibiotic us) was associatd with IBD, th invstigatos found that individuals with an otitis mdia diagnosis by ag 5 yas w naly th tims as likly to hav IBD (95% CI ; P = 0.001), poviding futh suppot fo th association of aly antibiotic us with ultimat dvlopmnt of IBD 22. Anoth study fom Manitoba, Canada, found that adults diagnosd with IBD w also mo likly to hav bn pscibd antibiotics 2 5 yas bfo thi diagnosis 23. Antibiotic dispnsations w associatd with both Cohn s disas and ulcativ colitis, with th association nomin ally stong in Cohn s disas cass fo 1 and 2 dispnsations, whas 3 dispnsations was associatd with 1.5 tims th liklihood of bing diagnosd with ulcativ colitis. A dos dpndnt lationship btwn th numb of antibiotic dispnsations and th isk of IBD was obsvd, futh implicating antibiotic us as a pdisposing facto in IBD pathognsis 23. A mta-analysis invstigating antibiotic xposu as a isk facto fo dvloping IBD analysd 11 obsvational studis (ight cas contol and th cohot) including 7,208 patints diagnosd with IBD. All antibiotics w associatd with IBD, with th xcption of pnicillin. Exposu to mtonidazol (OR 5.01, 95% CI ) o fluooquinolons (OR 1.79, 95% CI ) was most stongly associatd with nw-onst IBD, paticulaly Cohn s disas, and this association was mo makd in childn 20. Oth aly-lif influncs. Studis xamining 16S ibosomal RNA (RNA) gn squncing dmonstatd that, by th nd of th fist ya of lif, th idiosyncatic micobial cosystms in ach baby, although still distinct, had convgd towads a pofil chaactistic of th adult gastointstinal tact 24, mphasizing th potntial impotanc of influncs in this piod on subsqunt isk of IBD. Intstingly, babis bon by casaian sction a dpivd of contact with th matnal gut o vaginal micobiota, and thi micobiotas a chaactizd by a lack of stict anaobs and th psnc of facultativ anaobs such as Clostidium spcis 25,26. Infants bon via casaian sction hav a mo slowly divsifying micobiota than thos bon vaginally. In a study using in situ hybidiza tion of facal sampls fom 7 ya-old childn, signifi cantly high numbs of clostidia w found in thos dlivd vaginally compad with casaian-bon childn (P = ), indicating that an abnomal intstinal micobiota potd aft casaian sction dlivy could continu vn byond infancy 27. Howv, dspit data dmonstating that mod of bith xts a stong influnc on th intstinal micobiom, an association with IBD has not bn dmonstatd in pidmiological studis. A population-basd analysis to dtmin whth mod of dlivy (casaian vsus vaginal dlivy) affcts isk of IBD was caid out on data fom 1,671 individuals with IBD and 10,488 halthy contols linkd to moths obsttical cods. Ovall, th was no diffnc in th pcntag of individuals with IBD bon by casaian sction (11.6%) vsus contols (11.7%, P = 0.93). Futhmo, individuals with IBD w no mo likly to hav bn bon by casaian sction than w thi siblings without IBD (1,740 siblings fom 1,615 familis; 11.6% vsus 11.3%; P = 0.79) 28. NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY ADVANCE ONLINE PUBLICATION 3

4 REVIEWS Bastfding Pgnancy vs bottl fding Gntics Dugs Envionmnt Dit Micobiota Changs in taxonomic composition Rducd alpha divsity Dcasd lvls of Fimicuts and Bactoids Incasd abundanc of adhnt-invasiv Eschichia coli, paticulaly in ilal Cohn s disas Dcasd lvls of Facalibactium pausnitzii Incasd lvls of Gammapotobactia Changs in micobial function Rducd shot-chain fatty acid poduction and amino acid biosynthsis Incasd oxidativ stss Incasd sulfat tanspot Incasd auxotophy Dvlopmnt of IBD Natu Rviws fom Gastontology Hpatology Figu 2 Liflong influncs on th gut micobiom concption to& adult lif lad to dysbiosis and dvlopmnt of IBD. Th gut micobiom is suscptibl to th influnc of host gntics and nvionmntal influncs thoughout childhood and adult lif. Th sultant altations in taxonomic composition and function contibut to th dvlopmnt of intstinal inflammation in IBD. Studis hav dmonstatd a stong and duabl ffct of bastfding on composition of th infant gut micobiota. Th Canadian Halthy Infant Longitudinal Dvlopmnt (CHILD) bith cohot study collctd facal sampls at 4 months of ag and xamind th associ ation btwn gut micobiota composition (though high-thoughput DNA squncing) and pi natal factos including infant fding 25. Actinobactia and Fimicuts w th most common phyla obsvd in both goups, with low bactial divsity obsvd in bastfd infants than fomula-fd infants. Fomula-fd infants had an incasd abundanc of Pptostptococcus and notably Clostidium difficil, which has bn associ atd with atopic spons and allgic snsitization25. Exposu to human milk duing th dvlopmnt of th infant immun systm could also conf tolanc to dit ay and micobial antigns. Intapatum anti biotics in casaian and vaginal dlivy a associatd with infant gut micobiota dysbiosis, and bastfding modifis som of ths ffcts26. In animal studis, Il10 / mic dmonstatd ducd lvls of systmic TNF and IFNγ and ducd svity of histological inflamma tion in th intstin whn fd mous bastmilk29. Th possibl association btwn bastfding and th inci dnc of ulcativ colitis in humans was stablishd as long ago as 1961 (REF. 30). A mta-analysis of 17 lvant aticls xamind th associa tion btwn bastfding and IBD and dmonstatd a stong invs association with both Cohn s disas (OR 0.45, 95% CI ) and ulcativ colitis (OR 0.56, 95% CI )31. A systmatic viw focusing on padiatic IBD simi laly dmonstatd a stong invs association with aly-onst disas32. Oth studis hav dmonstatd that long duation of bastfding is associatd with a stong potctiv influnc, with dcasing isk of IBD whn infants w bastfd fo duations of 3, 6, o 12 months6. Whth aly-lif influncs such as bastfding continu to dmonstat an ffct vn in individuals with stablishd disas is lss wll known. A singl pot fom a fal cnt of 333 patints with Cohn s disas and 270 patints with ulcativ colitis found that a histoy of bing bast fd in infancy was associatd with a dcasd isk of Cohn s disas latd sugy 33. Togth, th dscibd data suppot th hypothsis that altations in th gut micobiota both in th fist ya of lif following dlivy and subsquntly could b citical isk factos fo IBD, xting thi influncs by alting th dvloping gut micobiota. Ubanization, pollution and dit Ubanization. Epidmiological studis hav consistntly shown that IBD is mo common in uban cnts34. In a Swiss cohot study, living in an uban zon was associ atd with both Cohn s disas and ulcativ colitis (lativ isk (RR) 1.49, P <0.001; RR 1.63, P <0.001, spctivly)35. Fom 1960 to 2005, a piod with incas ing ubanization, th hav bn incass of 2.4% (95% CI %; P <0.001) and 3.6% (95% CI %; P <0.001) p annum in th pvalnc of ulcativ colitis and Cohn s disas, spctivly, in industializd countis34. Th ffct of uban nvionmnts on dis as isk is paticulaly appant in countis that hav witnssd industializa tion and wstnization ov th past fw dcads. Although th ovall incidnc and pvalnc mains makdly low than in th Wstn Hmisph, whn availabl, data on scula tnds in disas budn suggst faily apid incass in inci dnc fo both Cohn s disas and ulcativ colitis in th dvlopd and dvloping countis in Asia. Elgant, comphnsiv data fom th Japan Ministy of Halth dmonstatd a substantial (mo than tnfold) incas in th incidnc of both Cohn s disas and ulcativ colitis sinc th 1970s, confimd by simila data fom South Koa, China and India36. Th ffct of ubaniza tion on dvlopmnt of IBD might b mdiatd by a vaity of changs that accompany such dvlopmnt, including changs in lifstyl and bhaviou, xposu to nvionmntal pollution, and altation in dit, all of which a xplod hin. Envionmntal pollution. Vaious componnts of ambi nt ai pollution hav bn associatd with dtimntal halth conditions in uban nvionmnts. Shot-tm xposu of th gut to high lvls of aibon paticu lat matt sults in incasd gut pmability and hight nd innat immun spons in th small intstin, whil chonic xposu sults in incasd xpssion of po-inflammatoy cytokins and altations in mico biota composition and function in th colon in mic37. In addition, long-tm xposu xacbatd colitis in an Il10 / mous modl37. Howv, pidmio logical data xamining th association btwn ai pollution and IBD hav yildd mixd sults fo paticu lat matt 4 ADVANCE ONLINE PUBLICATION d v s s t h g i l l A. u t a N g n i p S f o t a p, d t i m i L s h s i l b u P n a l l i m c a M

5 xposu, indicating that whn th is an associ ation, oth componnts of ai pollution could hav a ol in disas pathognsis. Kaplan t al. 38 studid whth ambint ai pollution lvls w associatd with th incidnc of IBD. Individuals who sidd in gions with NO 2 concntations in th highst quintil w mo likly to b diagnosd with Cohn s disas bfo 23 yas of ag than individuals in gions in th lowst quintil. Individuals siding in aas of high SO 2 concntations w mo likly to dvlop ulc ativ colitis than thos in aas with low SO 2 (REF. 38). In a Euopan nstd cas contol study, xposu to fin paticulat matt was invsly associatd with isk of IBD but not individually fo Cohn s disas o ulcativ colitis 39. By contast, poximity to havy taffic load was associatd with incasd isk of disas, and oth ai pollutants such as nitous oxids showd a tnd towads positiv associations with IBD 39. An cological analysis colating missions and numb of hospitalizations fo IBD by aa cod in Wisconsin, USA, idntifid a dict colation btwn total dnsity of ai pollutant missions and adult IBD hospitalizations 40. Each 1 log incas in th dnsity of total citia pollutant mission was associatd with a 40% incas in th at of IBD (Cohn s disas o ulcativ colitis). Analysis of individual pollutants suggstd statistically significant associations with CO, NO 2, SO 2 and fin paticulat matt (PM 2.5 ) 40. Dit. Evidnc indicats that th composition of th gut micobiota can influnc suscptibility to ulcativ colitis and Cohn s disas. Ditay changs a common in modn human socity and caus altations in th composition of th gut micobiota that, in tun, can lad to an abant intstinal immun spons and, vntually, IBD Dysbiosis can induc colitis in mic 44, paallling what is obsvd in patints with IBD who display ducd micobial divsity, with nichmnt of bactia of th family Entobactiaca and dpltion in bactia fom th phylum Bactoidts and ctain bactia fom th phylum Fimicuts 7,13. Th mchanisms by which dysbiosis can tigg IBD a not fully undstood, but th incas in invasiv bactial spcis coupld with a dcas in potctiv bactia could disupt local immun homo stasis, incas mucosal pmability and caus loss of immun tolanc 45. Th ol of dit in th pathognsis of Cohn s disas and ulcativ colitis was initially infd though tospctiv cas contol studis, lying on call fo dtmination of p-illnss dit. A padiatic cas contol study by Am t al. 46 potd an invs association btwn intak of fuits and vgtabls and isk of Cohn s disas. Moov, pospctiv cohot studis fom Euop and Noth Amica hav povidd mo obust stimats of th ffcts of ditay maconutints and miconutints on disas isk. In a lag pospctiv cohot of 170,776 womn followd fo 26 yas, womn in th highst quintil of intak of ditay fib w signifi cantly lss likly to dvlop incidnt Cohn s disas (OR 0.59, 95% CI ) compad with thos in th lowst quintil 47. Futhmo, in addition to th total intak of fib, th ffct diffd basd on th souc of fib. Th highst quintil of intak of ditay fib fom fuits, and to a lss xtnt vgtabls, was associatd with a ducd isk of Cohn s disas; by contast, fib fom whol gains, cal o ban was not associatd with a modifid disas isk 47. In two pospctiv cohot studis, ditay n-3 polyunsatuatd fatty acid (PUFA) intak has bn invsly associatd with isk of ulcativ colitis, whas ditay n-6 PUFA intak is associatd with incasd isk of incidnt ulcativ colitis In a study of spontanous and NSAID-inducd colitis in mic, ditay n-3 PUFAs ducd th clinical svity of colitis 51. In addition, th poduction of TNF by splnic CD4 + T clls was suppssd. As TNF plays a pivotal pat in IBD pathognsis, ths findings a consistnt with pvious pots documnting th pophylactic ffct of n-3 PUFAs on xpimntal colitis 51. Th association of incidnt IBD with intak of cabohydats and potin has bn mo mixd, with som pidmiological cohots, but not oth studis, poting a high isk of incidnt IBD with a dit ich in animal potin 52,53. Th association btwn dit and isk of IBD sms to xist not just fo cnt dit bfo diagnosis of illnss but also adolscnt dit duing high school, indpndnt of dit duing adulthood 54. In addition to ditay maconutints, mico nutints might also plausibly modify isk of IBD. Vaious miconutints a cognizd to hav impotant ols as co nzyms involvd in maintaining th gut pithlial bai, o dictly influncing intstinal immun sponss o gut micobial composition. Fo xampl, zinc is an impotant cofacto fo vaious intstinal mtallo potinass and, in cll cultu modls, zinc dficincy is associatd with ducd bai intgity and incasd pmability 55. In a pospctiv cohot study, high intak of zinc was invsly associatd with isk of Cohn s disas in womn 56. Th duction in isk was notd up to a daily zinc intak of 16 mg p day, twic th commndd daily intak. In stablishd disas, low sum lvls of zinc w associatd with incasd isk of hospitalizations, sugy and disas-latd complications in both Cohn s disas and ulcativ colitis 57. Futhmo, nomalization of zinc lvls was associatd with impovmnt in outcoms. In a small intvntional study, zinc supplmntation was associatd with duction in intstinal pmability as masud using th lactulos:mannitol atio 55. Vitamin D was initially cognizd fo its ol in bon halth, but mging pidmiological, xpimntal and intvntional data suppot a plausibl ol in pathognsis of Cohn s disas. In a pospctiv cohot study, womn with high lvls of pdictd plasma vitamin D had a significantly ducd likli hood of dvloping incidnt Cohn s disas vn aft adjusting fo lvant confounds (HR 0.38, 95% CI ) 58. In a study of 3,217 patints with stablishd IBD, individuals with sum 25 hydoxy vitamin D lvls <20 ng/ml had high isk of IBD-latd sugy and hospitalizations than thos with lvls >30 ng/ml (REF. 59). Impotantly, thos who w initially dficint but subsquntly attaind nomal lvls had a substantially low liklihood of sugy than thos who maind dficint in vitamin D. NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY ADVANCE ONLINE PUBLICATION 5

6 Hypoxia A stat of ducd oxygnation in th tissus. Finally, th association btwn dit and isk of incidnt disas might not b though nutitiv factos alon but also though oth componnts addd duing food pocssing, such as mulsifis. In lgant xpimntal studis, two mulsifis, caboxymthyl cllulos (CMC) and polysobat 80 (P80), w shown to alt th gut micobiota towads a po-inflammatoy stat by incasing lvls of bioactiv flagllin within 1 day 60,61. Th po-inflammatoy micobiota was associatd with altations in gn xpssion and dvlopmnt of colitis. Tansf of stool fom such mic to gm-f mic poducd th changs in both micobial composition and intstinal xpssion of po-inflammatoy gns 60,61. Oth factos NSAIDs. NSAIDs a among th most fquntly consumd mdications, and thi association with gastic o duodnal ulcations is wll cognizd. Howv, vidnc has also linkd thm to th dvlopmnt of IBD 62. Sval xplanations hav bn offd as th potntial mchanism fo th association btwn NSAIDs and IBD. Fist, non-slctiv inhibition of cyclooxygnas (COX) ducs synthsis of potctiv postaglandins. Il10 / mic administd NSAIDs had a 75% duction in potctiv postaglandin E 2 poduction not sn with slctiv COX2 inhibition 63. Scond, cytoskltal disuption and incasd mucosal pmability both involv activation by gut bactia and uncoupling of mitochondial oxidativ phosphoylation 64,65. A pospctiv cohot study assssd th associ ation btwn aspiin and NSAID us and incidnc of Cohn s disas and ulcativ colitis 62. A high isk of both conditions was obsvd with th highst fquncy of NSAID us. Compad with non-uss, womn who usd NSAIDs at last 15 days p month had an incasd isk of both Cohn s disas (HR, 1.59, CI, ) and ulcativ colitis (multivaiat HR, 1.87; 95% CI ). Similaly, incasd isk of IBD was sn with polongd duation of NSAID us (>6 yas) 62. Oth studis hav xamind if NSAID us similaly has a dltious ffct in stablishd IBD. Long t al. 66 xamind outcoms in 791 individuals with IBD in mission with baslin and follow up data availabl fo analysis. Of ths, 247 patints with Cohn s disas (43.2%) and 89 ulcativ colitis patints (40.6%) potd NSAID us. Patints with Cohn s disas with NSAID us 5 tims p month had gat isk of activ disas at follow up (23% vsus 15%, P = 0.04) 66. No ffct was obsvd in patints with ulcativ colitis (22% vsus 21%, P = 0.98). In a pospctiv tial, patints with quiscnt Cohn s disas and ulcativ colitis w andomly assignd to civ th non-nsaid analgsic paactamol (n = 26) and th convntional NSAIDs napoxn (n = 32), diclofnac (n = 29), and indomthacin (n = 22) fo 4 wks. Non-slctiv NSAIDs, but not paactamol, w associatd with a 17 28% laps at within 9 days of ingstion 67. Bonn t al. 68 showd high doss of NSAIDs to b associ atd with an incasd numical disas activity indx sco among patints with Cohn s disas with colonic involvmnt. Th safty of slctiv COX2 inhibitos in 222 patints with ulcativ colitis in mission was xamind in a study publishd in 2006 (REF. 69). Patints w andomly assignd to civ oal clcoxib (200 mg) o placbo twic daily fo 14 days. In th clcoxib goup, 3% xpincd disas xacbation though day 14, compad with 4% in th placbo goup (P = 0.719), suggsting that slctiv inhibition of COX2 is not associatd with disas laps in IBD 69. High altitud and hypoxia. Hypoxia is known to induc inflammatoy sponss in immun clls and ndothlial clls: it is associatd with an accumulation of inflammatoy clls in multipl ogans and lvatd cytokins in xpimntal mous modls aft shot-tm xposu to low oxygn concntations In human studis, lvls of ciculating IL 6, IL 1a and C activ potin a upgulatd in spons to hypobaic hypoxic conditions such as high altitud, and th systmic incass in ths inflammatoy maks could flct local inflammation in th intstin 76. Mammals hav oxygn snsing mch anisms that hlp thm adapt quickly to hypoxia by incasing spiation, blood flow and suvival sponss 72. If an inadquat supply of oxygn psists, additional mchanisms attmpt to sto oxygnation o hlp th body adapt to hypoxia. Cllula adaptations to hypoxia ly on th tansciption facto hypoxiainducibl facto (HIF), which is inactiv whn oxygn is abundant but is activatd in hypoxic conditions 72. Binding of HIF to th hypoxia spons lmnt of sval HIF-gulatd gns sults in th incasd tansciption of sval potins involvd in angio gnsis (such as vascula ndothlial gowth facto, VEGF), glycolysis (glucos tanspot potins), and ythopoisis 72. Sological studis hav suggstd that sum concntations of VEGF, a potnt angiognic facto, a incasd in patints with IBD 77. Vmuln t al. 78 potd that patints with ulcativ colitis o Cohn s disas hav incasd xpssion of HIF 1. Patints with IBD also hav incasd colonic mrna xpssion of glyco lytic nzyms, which is tiggd by hypoxia though th tansciption facto HIF 1 (REF. 78). Effct of high altitud on halthy volunts. In pliminay findings of a study of halthy volunts (mountains aft apid ascnt to high altitud), atial oxygn satuation (so 2 ) valus w masud at th diffnt altituds (bas at 490 m, 3,650 m and at 4,559 m). As xpctd, so 2 valus w significantly low at both high-altitud locations than at th bas location (P <0.001). Pptic mucosal lsions on ndo scopy w psnt in 28% of individuals on day 2 and in 61% on day 4, dmonstating that, vn in halthy individuals, high altitud can induc ulcations and inflammation in th gastointstinal tact 79. Hypoxia in IBD. Vavicka t al. 80 invstigatd whth flights and/o jounys to gions lying at an altitud of >2,000 m abov sa lvl a associatd with fla-ups in patints with IBD, within 4 wks of th tip. Patints with IBD with at last on fla up duing a 12 month obsvation piod w compad with a goup of patints in mission. Patints with IBD xpincing fla-ups 6 ADVANCE ONLINE PUBLICATION

7 Disas volution Pimay factos Exposom Gnom Micobiom Immun factos Bfo IBD Exposom Th ntity of all nvionmntal xposus of an individual in a liftim. Epignom DNA mthylation and histon modifications that gulat xpssion of gns within a cll. Ealy IBD Lat Aft IBD Sconday factos Epignom DAMPs Nuondocin systm Cll diffntiation Altd tissu homostasis Adipos tissu Oths Figu 3 IBD a continuously volving biological pocss. IBD is a continually volving biological pocss, suscptibl to vaious -om influncs that sult in sconday biological altations in th individual, alting th xpssion and pogssion of disas. DAMP, damag-associatd molcula pattns. had mo fquntly undtakn flights and/o jounys to gions >2,000 m abov sa lvl within 4 wks of th fla up whn compad with patints in mission (21 of 52 (40.4%) vsus 8 of 51 (15.7%, P = 0.005)). Ths sults suggst that jounys and/o flights to highaltitud gions a isk factos fo IBD fla-ups occuing within 4 wks of tavl, and th ol of hypoxia in IBD waants futh invstigation. Basd on th hypothsis that hypoxia lads to intstinal inflammation, sval studis hav xamind whth hypbaic oxygn thapy can b usd to tat Cohn s disas o ulcativ colitis. A systmatic viw of 17 studis (mostly small cas studis) includd 631 patints and dmonstatd that hypbaic oxygn thapy was associatd with an 86% ovall spons at with an 88% spons fo pinal Cohn s disas 81. A small pilot poof of concpt andomizd tial that includd 18 patints dmonstatd hypbaic oxygn thapy to b bnficial in modat-to sv ulcativ colitis 82. Systms biology appoach Th nxt stp in unavlling th pathognsis of such complx disass such as IBD quis bidging of th gap btwn clinical and pidmiological obsvations gading vaious nvionmntal influncs and mchanistic ffcts on intstinal inflammation, tanslating into nw thaputic tagts. Owing to th lag numb and vaity of nvionmntal factos, th invstigation into ths factos and thi tagts quis mathmatical modlling, nabling infomation to b oganizd into ntwoks a systms-biology-basd appoach that sks to undstand th complxity of disas pathognsis. A systms appoach in which biodata (that is, blood, stool and biopsy sampls) a inputtd and intgatd via high-thoughput tchnologis is idal to study complx conditions such as ulcativ colitis and Cohn s disas. An xampl of this appoach is th us of th iclust algoithm fo idntification of IBD molcula subtyps by pfoming pattn discovy that intgats divs data typs: binay (somatic mutation), catgoical (copy numb gain, nomal, loss), and continuous (gn xpssion) valus 83. Anoth xampl is th us of intgativ Baysian analysis (ibag) fo intgation of IBD mchanistic data with clinical data 84. Th basic constuction of th ibag modl consists of two componnts: a mchanistic modl to captu infomation by patitioning th gn xpssion into componnts xplaind by diffnt upstam platfoms and a clinical modl that subsquntly incopoats ths componnts to modl th ffcts on a clinical outcom of intst. Combind, ths appoachs nabl th idntification of gns latd to clinical outcom and gnat insight into th biological mchanisms undlying ths ffcts 85. Although it cannot b dscibd as xamining th ffct of an nvionmntal facto, th idntification of th micorna mir 214 as a potntial thaputic tagt in ulcativ colitis ntwok is a good xampl of how ffctiv an IBD systms appoach can b in idntifying nw thaputic tagts and poviding mchanistic vidnc in suppot of its fficacy. Spcifically, it was th intgation of tansciptomic and pignomic data dictly divd fom colonic tissus fom mous modls with colitis that idntifid mir 214 as a cntal gulato of an inflammatoy molcula ntwok 86. Th tansciption facto signal tansduc and activato of tansciption 3 (STAT3) is upgulatd in adult ulcativ colitis, which ld to th subsqunt invstigation of th IL 6 STAT3 pathway in padiatic disas 87. Th lvls of phosphoylatd STAT3 and th gns it gulats w upgulatd in tissu sampls fom patints with padiatic ulcativ colitis compad with contols. MiR 124 is a known pigntic facto that gulats th xpssion of STAT3 (REF. 88). Th ducd lvls of mir 124 in colon tissus of childn with ulcativ colitis sult in incasd xpssion and activity of STAT3, which pomots inflammation 88. Consistnt with this finding, a high-thoughput functional scn of th full st of human micornas found that mir 214 gulatd th activity of nucla facto κb. Intgation of bioinfomatic and gnom-wid pofil analyss showd that mir 214 activats an inflammatoy spons that is amplifid though a fdback loop cicuit mdiatd by phosphatas and tnsin homologu (PTEN) and PDZ and LIM domain potin 2 (PDLIM2) 86. Thus, basd on ths computational and molcula data, mir 214 is a pomising dug tagt fo ulcativ colitis. In fact, a mir 214 inhibito blockd th inflammatoy ntwok and ducd th svity of colitis in mic inducd by dxtan sulfat sodium 86. This xampl dmonstats th pow of th systms biology appoach to idntify ky molculs involvd in IBD pathognsis and dvlop novl thapis. Intgation of clinical data and high-thoughput molcula data, including pigntic data, can thfo povid th most fficint and comp hnsiv appoach to tating complx disods such as IBD. An intgatd appoach fo nw thapis Th cuntly accptd componnts of IBD patho gnsis includ th xposom, th gnom, pignom, th gut micobiom and th immun systm (FIG. 3). Ths componnts all intact with ach anoth, and ach psnts a potntial thaputic tagt. NATURE REVIEWS GASTROENTEROLOGY & HEPATOLOGY ADVANCE ONLINE PUBLICATION 7

8 Gnom. A numb of xposom-divd pigntic factos dtmin th xpssion of an IBD pignotyp o clinically vidnt IBD. Th combination of IBDassociatd gn vaiants, matnal factos, pinatal micobial xposus, antibiotics, dit and xnobiotics, among oths, may dtmin th isk of dvloping IBD and th typ and svity of th associatd clinical manifstation. Dltion o placmnt of gn vaiants lading to disas is tchnically fasibl (in xpimntal animals) but might b associatd with unknown isks, and ffctivnss in humans is unctain. Box 1 Diffnt ntwok intactions can sult in diffnt disass Diffnt intactions btwn hypothtical biological ntwoks that contain th sam numb and typ of biological componnts can sult in diffnt disass. Th fou hypothtical scnaios (ntwoks) a ach composd of th sam numb of biological componnts: fou classs of T clls (T hlp (Th)1, 2, 17 and gulatoy (T g ) clls), fou cytokins (IL 1, IL 6, IL 17 and TNF), two cptos (IL 23R and ICAM 1) and two signalling molculs (TRAF1 and STAT3). Each biological componnt can only connct to two oth componnts, but th connctions vay dastically btwn th scnaios, vn though th numb of componnts (12) mains th sam: in th upp lft scnaio, th 12 biological componnts connct in a way that sults in fou tiangls; in th upp ight scnaio, th 12 componnts connct in a way that sult in th squas; in th low lft scnaio, th 12 biological componnts fom two hxagons; and in th low ight scnaio, thy fom on cicl. Each of th fou diffnt aangmnts (ntwoks) has th sam numb of componnts but yilds a diffnt biological outcom that, if th outcom is a disas pocss, sults in fou diffnt disass. As a puly hypothtical xampl, th outcom of th aangmnt composd of th tiangls could yild Cohn s disas; th th tiangls could yild micoscopic colitis; th two hxagons could yild ncotizing ntocolitis; and th cicl could yild pancolitis. Each of ths distinct clinical ntitis would thn qui diffnt foms of tatmnt, vn though ach ntity divs fom xactly th sam numb and typ of biological componnts that only diff in th way thy connct to ach oth. Cohn s ilitis IL-1 1 IL-17 2 TNF T g STAT3 ICAM-1 Ncotizing ntocolitis 2 17 ICAM-1 IL-17 IL-6 T g TRAF1 STAT3 IL-6 17 TRAF1 IL-23R IL-23R 1 IL-1 TNF Micoscopic colitis STAT3 IL-6 Pancolitis IL-23R STAT3 TNF TRAF1 Ntwoks concpt adaptd fom REF. 103, Macmillan Publishs Limitd. 2 T g 1 IL-17 TRAF1 ICAM-1 IL-6 T g TNF 17 IL-17 ICAM-1 17 IL-1 IL-1 IL-23R 2 1 Exposom. Exposom-divd pigntic factos abl to modulat th micobiom and immun systm potins a innumabl. Among thm a changs in ditay miconutints o maconutints, salt intak, atificial swtns, mulsifis, smoking and titani um dioxid nanopaticls, all of which can induc chonic inflammation. Studis in shp hav shown that matnal obsity inducs intstinal inflammation in th offsping, which colatd with incasd xpssion of tansfoming gowth facto-β and IL 17, suggsting that matnal obsity pdisposs to IBD 89. Compaisons of th distal gut micobiota of gntically obs mic and thi lan littmats, as wll as thos of obs and lan human volunts, hav vald that obsity is associatd with changs in th lativ abundanc of two domin ant bactial phyla, Bactoidts and Fimicuts 90. Impotant obstacls xist whn considing IBD thaputic intvntions dictd towads nvionmntal factos: tagts a mostly unknown, intvntions only hav slctiv fficacy (fo xampl, smoking cssation) and bhavioual modifications must b liflong, lading to complianc and ffctivnss issus. Gut micobiom. Vaious micobial statgis could b usd to maintain gut micobial homostasis: antibiotics hav a ol in th moval o suppssion of undsiabl micooganisms; pobiotics can intoduc missing dsiabl micooganisms, whas pbiotics facilitat th polifation of bnficial micooganisms; dfnsins might bcom availabl to plnish antimicobial pptids to balanc th gut micobiota. Clinical studis dmonstating a bnficial ffct of antibiotics o pobiotics on disas activity in IBD hav bn caid out only with mtonidazol o onidazol in pvnting postopativ cunc 91, ifaximin in mild ilal Cohn s disas 92, and th pobiotic VSL#3 in pvnting cunt pouchitis 93 and in mild to modat ulc ativ colitis 94. Facal micobiota tansplantation (FMT) is a nw statgy involving th infusion of a facal suspnsion of stools fom a halthy dono into th gastointstinal tact of a patint to cu a spci fic disas 95,96. To dat, in patints with IBD, FMT only offs slctiv, tansint, dono dpndnt and cipint-dpndnt modification of th gut micobiota with unpdictabl clinical bnfits 96,97. Two andomizd clinical tials of FMT in ulcativ colitis achd divging conclusions gading fficacy, with on dmonstating a bnfit and th oth showing no ffct of FMT in ulcativ colitis 96,97. Howv, mo dtaild analysis of on of th tials vald a stong dono ffct in dtmining th fficacy of FMT 98,99. A thid, multicnt tial adoptd a mo aggssiv gimn of FMT via nma 5 days a wk fo 8 wks in compaison with placbo 100. At th nd of th tial, which includd 85 patints, stoid f clinical mission was achivd by 27% of patints civing FMT compad with 8% civing placbo (P = 0.021) 100. Thus, tatmnts tagting gut mico biota can occasionally b fficacious but a not tagt slctiv, can b usd only fo limitd piods of tim, and might hav unpdictabl advs ffcts. 8 ADVANCE ONLINE PUBLICATION

9 Mtabolomics Study of chmical fingpints (mtabolits) psnt within an oganism. Phnomics Masumnt of physical and biochmical taits of an oganism. Box 2 Qustions on th nvionmnt and IBD How do nvionmntal factos modify th isk o natual histoy of Cohn s disas and ulcativ colitis? Whn, duing on s liftim, a nvionmntal xposus impotant fo dvlopmnt of Cohn s disas and ulcativ colitis? How do nvionmntal xposus xt thi ffct on isk of IBD, and why do som hav divgnt ffcts on Cohn s disas compad to ulcativ colitis? A som individuals mo suscptibl to spcific nvionmntal factos, and is this govnd by gntics, thnicity, gogaphy, compting influncs o oth biological mchanisms? Can modification of nvionmntal factos b ffctiv in pimay and sconday pvntion of IBD? Can changing nvionmntal factos b usd as standalon o adjunct tatmnt fo managmnt of IBD? Will btt dfinition of nvionmntal factos impov ou undstanding of disas biology and idntify nw thaputic pathways? Immun systm. Modulating th immun systm is by fa th most common appoach to tating IBD, and it involvs using dugs to tagt cytokins, cptos, cll adhsion molculs and signalling pathways. Howv, ths tagts might chang with gut inflammation, which is an volving biological pocss contolld by pimay and sconday factos that diffntially modulat th immun spons in th aly and lat stags of IBD. To distinguish immun vnts in aly-stag and lat-stag Cohn s disas, mucosal T cll function was invstigatd in padiatic IBD. At disas onst, mucosal T clls can mount a typical typ 1 T hlp spons that smbls an acut infctious pocss, but this spons pattn is lost with pogssion to lat disas. This finding suggsts that immunogulation changs with th cous of human IBD, affcting th timing and fficacy of immunomodulatoy agnts 101. Th main considations in tagting th immun factos in IBD a that cunt tatmnts a asonably ffctiv, th is a limitd tagt slction, th spons is dpndnt on immun status, and fficacy can b lost ov tim. Complx disass qui omics-basd thapy. Biological malfunctions sponsibl fo disas vay fom patint to patint, vn whn th disass display simila clinical manifstations. As illustatd in this Rviw, this aspct is du to th multiplicity and complxity of th undlying molcula ntwoks that cannot b contolld by blocking individual molcula tagts. A dtaild ntwok analysis must b don to idntify spcific biological diffncs, pobabl outcoms and thaputic tagt slction 79. BOX 1 futh xplains th hypothsis on how th ffct of diffnt biological ntwoks btwn th sam componnts could yild diffnt clinical phnotyps. In IBD, th a numous omics filds contibuting to disas dvlopmnt, including gnomics (~20,000 gns), mtagnomics (~ micooganisms), tansciptomics (~ RNA tanscipts), potomics (~ potins), mtabolomics (~ mtabolits) and phnomics o xposomics (~ compounds). Thi intaction cats an almost infinit numb of pathognic outcoms that can only b undstood by adopting a systms biology appoach to idntify th cntal gulatos of th ntwoks. This multi-omics intga tion of IBD ntwoks should nabl spcific tagt idntification and is potntially cuativ, foming th basis fo psonalizd tatmnts fo IBD. Conclusions Th gut micobiom is cntal to th pathognsis of IBD. Multipl nvionmntal influncs, fom th pinatal piod to adulthood, do, howv, influnc th isk and natual histoy of ths complx, immunologically mdiatd disass. Emging vidnc has povidd pidmiological suppot and mchanistic plausibility fo sval of ths associations. Futh studis must adopt a multi-omic big data appoach, intgating sval lays of infomation on clinical data, nvionmntal xposus, gntics, pigntics, immunological function and micobial stuctu. Such an appoach offs th potntial to futh ou in dpth undstanding of th patho gnic mchanisms bhind ths disass, which, in tun, could lad to th dvlopmnt of nw avnus fo tatmnt and disas pvntion (BOX 2). 1. Ananthakishnan, A. N. Epidmiology and isk factos fo IBD. Nat. Rv. Gastontol. Hpatol. 12, (2015). 2. Kaplan, G. G. & Ng, S. C. Globalisation of inflammatoy bowl disas: pspctivs fom th volution of inflammatoy bowl disas in th UK and China. Lanct Gastontol. Hpatol. 1, (2016). 3. Kaplan, G. G. & Ng, S. C. Undstanding and pvnting th global incas of inflammatoy bowl disas. Gastontology 152, (2017). 4. Buk, K. E., Boumiti, C. & Ananthakishnan, A. N. Modifiabl nvionmntal factos in inflammatoy bowl disas. Cu. Gastontol. Rp. 19, 21 (2017). 5. Ng, S. C. t al. Gogaphical vaiability and nvionmntal isk factos in inflammatoy bowl disas. Gut 62, (2013). 6. Ng, S. C. t al. Envionmntal isk factos in inflammatoy bowl disas: a population-basd cas-contol study in Asia-Pacific. Gut 64, (2015). 7. Kostic, A. D., Xavi, R. J. & Gvs, D. Th micobiom in inflammatoy bowl disas: cunt status and th futu ahad. Gastontology 146, (2014). 8. Kahstom, C. T., Paint, N. & Wiss, U. Intstinal micobiota in halth and disas. Natu 535, 47 (2016). 9. Lynch, S. V. & Pdsn, O. Th human intstinal micobiom in halth and disas. N. Engl. J. 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