L-threonine supplementation during colitis onset delays disease recovery

phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 1 BRIEF RESEARCH REPORT published: 05 Sep embe 2018 doi: 10.3389/ phys.2018.01247 Edi ed by: Ma ia Te esa C uz, Uni e si y o Coimb a, Po ugal Re iewed by: Vicen e Lahe a, Complu ense Uni e si y o Mad id, Spain Diana Ju ado Se a, Uni e si y o Coimb a, Po ugal *Co espondence: Rica do Sil es e [email p o ec ed] Special y sec ion: This a icle was submi ed o In eg a i e Physiology, a sec ion o he jou nal F on ie s in Physiology Recei ed: 09 Ma ch 2018 Accep ed: 17 Augus 2018 Published: 05 Sep embe 2018 Ci a ion: Gai em J, Gonçal es LG, Dinis-Oli ei a RJ, Cunha C, Ca alho A, To ado E, Rod igues F, Sa ai a M, Cas o AG and Sil es e R (2018) L-Th eonine Supplemen a ion Du ing Coli is Onse Delays Disease Reco e y. F on . Physiol. 9:1247. doi: 10.3389/ phys.2018.01247 L-Th eonine Supplemen a ion Du ing Coli is Onse Delays Disease Reco e y Joana Gai em1,2, Luís G. Gonçal es3, Rica do J. Dinis-Oli ei a4,5,6, C is ina Cunha1,2, Agos inho Ca alho1,2, Egídio To ado1,2, Fe nando Rod igues1,2, Ma ga ida Sa ai a7,8, An ónio G. Cas o1,2 and Rica do Sil es e1,2* 1Li e and Heal h Sciences Resea ch Ins i u e, School o Heal h Sciences, Uni e si y o Minho, B aga, Po ugal, 2ICVS/3B’s – PT Go e nmen Associa e Labo a o y, Guima ães, Po ugal, 3Ins i u o de Tecnologia Química e Biológica An ónio Xa ie , Uni e sidade NOVA de Lisboa, Oei as, Po ugal, 4IINFACTS – Ins i u e o Resea ch and Ad anced T aining in Heal h Sciences and Technologies, Depa men o Sciences, CESPU, CRL, Uni e si y Ins i u e o Heal h Sciences, Gand a, Po ugal, 5UCIBIO, REQUIMTE, Labo a o y o Toxicology, Depa men o Biological Sciences, Facul y o Pha macy, Uni e si y o Po o, Po o, Po ugal, 6Depa men o Public Heal h and Fo ensic Sciences, and Medical Educa ion, Facul y o Medicine, Uni e si y o Po o, Po o, Po ugal, 7Ins i u o de In es igação e Ino ação em Saúde, Uni e sidade do Po o, Po o, Po ugal, 8Ins i u o de Biologia Molecula e Celula , Uni e sidade do Po o, Po o, Po ugal Die a y nu ien s ha e eme ged as po en ial he apeu ic adjunc s o in lamma o y bowel disease (IBD) gi en hei impac on in es inal homeos asis h ough he modula ion o immune esponse, gu mic obio a composi ion and epi helial ba ie s abili y. Se e al nu ien s ha e al eady been associa ed wi h a p o ec i e pheno ype. Ye , he e is a lack o knowledge owa d he mos p omising ones as well as he mos adequa e phase o ac ion. To un eil he mos p ominen he apy candida es we cha ac e ized he colon me abolic p o ile du ing coli is de elopmen . We ha e obse ed a wo old dec ease in h eonine le els in mice subjec ed o DSS-induced coli is. We hen assessed he e ec o h eonine supplemen a ion in he beginning o he in lamma o y p ocess (DSS +Th ) o when in lamma ion is al eady es ablished (DSS +Th D8). Coli is p og ession was simila be ween he ea ed g oups and con ol coli ic mice, ye h eonine had a su p isingly de imen al e ec when adminis e ed in he beginning o he disease, wi h mice displaying a delayed eco e y when compa ed o con ol mice and mice supplemen ed wi h h eonine a e day 8. Al hough no majo changes we e ound in hei me abolic p o ile, DSS +Th mice displayed al e ed exp ession in mucin-encoding genes, as well as in goble cell coun s, un eiling an impai ed abili y o p oduce mucus. Mo eo e , IL-22 sec e ion was dec eased in DSS +Th mice when compa ed o DSS +Th D8 mice. O e all, hese esul s sugges ha supplemen a ion wi h h eonine du ing coli is induc ion impac goble cell numbe and delays he eco e y pe iod. This ein o ces he impo ance o a deepe unde s anding ega ding h eonine supplemen a ion in IBD. Keywo ds: IBD, h eonine, DSS-induced coli is, goble cells, me abolomics, IL-22, mucin F on ie s in Physiology | www. on ie sin.o g 1Sep embe 2018 | Volume 9 | A icle 1247 phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 2 Gai em e al. Th eonine Supplemen a ion E ec in Coli is INTRODUCTION In lamma o y bowel disease (IBD) is a complex debili a ing diso de o he gas oin es inal ac which comp ises bo h C ohn’s disease and ulce a i e coli is. Despi e he unclea e iology o IBD, se e al ac o s ha e been accoun ed as key o he de elopmen o he disease, such as gene ics, immune sys em and en i onmen al ac o s, namely die and gu mic obio a composi ion (Kho e al., 2011). Die a y supplemen a ion has eme ged as a p omising he apeu ic p ac ice in he p e en ion and ea men o IBD (Du chschein e al., 2016). Recen e idence has e ealed ha ibe -en iched die s p omo e p o ec ion agains IBD de elopmen , since die a y ibe is mainly e men ed by in es inal mic obio a in o sho -chain a y acids (SCFAs), such as bu y a e, ace a e and p opiona e (den Bes en e al., 2013). The p o ec i e p ope ies o hese me aboli es a e widely desc ibed by hei impac on immune cell ac i a ion and epi helial ba ie s abili y (Kelly e al., 2015;Macia e al., 2015), wi h dec eased le els o SCFAs being ound in colon samples om IBD pa ien s (Huda- Faujan e al., 2010). O he s udies ha e also poin ed ou se e al speci ic amino acids ha can imp o e in es inal homeos asis, mainly by boos ing mucosal healing and egene a ion. Fo ins ance, glu amine is known o p omo e p o ec ion in dex an sul a e sodium (DSS)- and 2,4,6- ini obenzenesul onic acid (TBNS)-induced in es inal in lamma ion, ac ing ia NF-κB down egula ion (K e zmann e al., 2008). Coli ic mice o ally adminis e ed wi h glu amine displayed supp essi e Th1/Th17 immune esponses and subsequen ly dec eased in lamma ion when compa ed o mice ed wi h egula die (Hsiung e al., 2014). O he amino acids ha e been also associa ed wi h a p o ec i e pheno ype agains coli is. Using dis inc animal models o coli is, die s en iched in h eonine, se ine, p oline and cys eine, gi en be o e and h oughou disease de elopmen , ha e been shown o es o e mucin syn hesis and s abiliza ion o gu mic obio a (Fau e e al., 2006). Simila indings we e obse ed wi h he adminis a ion o a mix u e o h eonine, me hionine and monosodium glu ama e a e coli is induc ion (Liu e al., 2013). Se e al s udies ha e so a add essed whe he adminis a ion o speci ic nu ien s may a ise as a p ophylac ic and/o he apeu ic app oach. Howe e , he e is a lack o knowledge owa d he mos p omising and adequa e phase o ac ion. Thus, we in es iga ed he me abolic p o ile o mice de eloping coli is, aiming o iden i y a ia ion o me aboli es du ing in lamma ion. The iden i ica ion o he mos a ac i e po en ial a ge s o he apy and he de ini ion o a ime ange mo e p one o po en ia e he e ec s o hei supplemen a ion may be ele an o u u e applica ions in IBD p ophylaxis and he apy. MATERIALS AND METHODS Animals Se en o nine-week old C57BL/6J male mice we e pu chased om Cha les Ri e Labo a o ies and housed in i3S animal acili ies, unde pa hogen ee condi ions, wi h ood and wa e ad libi um. All expe imen al p ocedu es we e app o ed by he i3S Animal E hics Commi ee and licensed by he Po uguese Na ional Au ho i y o Animal Heal h (DGAV) wi h e e ence 014811/2016-07-13. Coli is Induc ion Dex an sul a e sodium (DSS; TdB Consul ancy; 2% (w/ ), molecula weigh app oxima ely 40000 Da) was adminis e ed in d inking wa e ad libi um o 5 days. Clinical signs o coli is we e moni o ed daily and sco ed as a disease ac i i y index (DAI; Supplemen a y Table S1). L-Th eonine Adminis a ion Mice we e di ided in o DSS (con ol), DSS wi h L-Th eonine (DSS +Th ) and DSS ollowed by L-Th eonine adminis a ion a day 8 (DSS +Th D8), as shown in Figu e 2A.L-Th eonine [Th ; Sigma–Ald ich; 0.166% (w/ ) co esponding o 250 mg/Kg/day] was gi en in he d inking wa e ad libi um. The dose was chosen acco ding o he daily in ake in p e ious s udies wi h oden s (Fau e e al., 2006;Liu e al., 2013) and o a high- h eonine human supplemen a ion s udy (Pencha z e al., 2008). The sa e y o ou p o ocol measu ing bioma ke s o enal and li e damage was e alua ed o con i m he absence o oxici y (Supplemen a y Figu e S1). Simila luid in ake was ound among all g oups. Me abolomic Analysis by Nuclea Magne ic Resonance (NMR) Me hanol/wa e ex ac s o colon we e analyzed a an Ul ashiedTM 800 Plus (B uke ) spec ome e as desc ibed in G aça e al. (2017). Me aboli e concen a ions we e pe o med by in eg a ion o 1H-NMR esonances using TSP as e e ence. Quan i a i e Real-Time PCR (qPCR) To al RNA was isola ed om colonic samples (T ipleX ac o , G isp). As DSS inhibi s bo h polyme ase and e e se ansc ip ase ac i i ies, RNA was pu i ied wi h li hium chlo ide, as in Viennois e al. (2013). qPCR was pe o med as desc ibed in Co eia e al. (2017). The lis o p ime s used is in Supplemen a y Table S2. His ology and Goble Cell Coun Colons we e ixed in 10% bu e ed o malin (Sigma–Ald ich) and embedded in pa a in. Sec ions o 5 µm we e s ained wi h hema oxylin/eosin and Alcian Blue/Pe iodic acid-Schi . Goble cell numbe was assessed o each expe imen al condi ion in a blinded ashion. Only c yp s cu longi udinally om c yp opening o bo om we e analyzed. Cy okine Quan i ica ion Colonic explan cul u es we e pe o med as p e iously desc ibed (McNamee e al., 2011). Cy okine quan i ica ion was pe o med in supe na an s by ELISA (Biolegend). Tissue explan s we e homogenized and o al p o ein was measu ed using B ad o d assay. The concen a ion o sec e ed cy okines in he supe na an was no malized o o al issue p o ein and exp essed as picog am o cy okine pe µg o o al issue p o ein. F on ie s in Physiology | www. on ie sin.o g 2Sep embe 2018 | Volume 9 | A icle 1247 phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 3 Gai em e al. Th eonine Supplemen a ion E ec in Coli is FIGURE 1 | Th eonine le els in he colon dec ease wi h in es inal in lamma ion. (A) Mice we e ea ed wi h DSS o induce coli is and colon me aboli es we e measu ed a day 0 and day 5 by NMR. (B) Pa ial leas squa es – disc iminan analysis (PLS-DA) was ob ained o samples o bo h days. (C) Top- i e ea u es iden i ied by he combina ion o he p- alue om - es analysis and magni ude o he change ( old change om day 5 e sus day 0). (D) Impo an me aboli es selec ed by olcano plo wi h old change h eshold (x) 1.8 and - es h eshold (y) o 0.05. The pu ple ci cles ep esen ea u es abo e he h eshold. Bo h old changes and p- alues a e log ans o med. (E) Th eonine le els in he colon a day 0 and day 5. Da a is shown as mean ±SD; n= 5 mice/g oup. One ep esen a i e expe imen is shown ou o wo. ∗p<0.05. S a is ical Analysis Me aboli e di e ences we e e alua ed by ANOVA in R s a is ical so wa e. Pa ial leas squa es – disc iminan analysis (PLS-DA) models we e pe o med in SIMCA so wa e. O he s a is ical analyses we e ca ied ou wi h G aphPad P ism ( e sion 6.01). Fo mul iple g oup compa isons one-way ANOVA es wi h a Tukey mul iple-compa ison pos es was pe o med, while o mul iple g oup compa isons wi h epea ed measu es wo- way ANOVA es wi h a Tukey mul iple-compa ison pos es was applied. Da a a e p esen ed as mean ±s anda d de ia ion (SD). S a is ically signi ican alues a e: ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001. RESULTS AND DISCUSSION To cha ac e ize he colon me abolic p o ile du ing coli is de elopmen , me aboli es p esen on colonic ex ac s o mice p io (day 0) and a e 5 days o DSS-induced coli is we e analyzed by NMR (Figu e 1A). These ime poin s we e selec ed since i allows he compa ison o a homeos a ic p o ile (day 0) agains a pe iod wi h es ablished in lamma ion and lesion, ye e e sible and ea able (day 5). Mul i a ia e analyses o he me abolomic da a only demons a ed mino al e a ions be ween p o iles o day 0 and day 5 o coli is de elopmen (Figu e 1B). Howe e , uni a ia e analyses pe o med on he me aboli es allow o disc imina e ADP and pa icula ly h eonine as signi ican ly al e ed om day 0 o day 5 (Figu es 1C,D). Among essen ial amino acids, h eonine has a p ominen ole in main aining a heal hy gu . Th eonine is able o gene a e he main h ee SCFAs, namely ace a e, bu y a e and p opiona e (Neis e al., 2015). In ac , i has been p e iously iden i ied se e al biosyn he ic genes o h eonine me abolism in he human gu mic obio a, sugges ing he ele ance o his amino acid o mic obio a biology (Abubucke e al., 2012). SCFAs a e desc ibed as impo an modula o s o immune esponse, since hey a e ligands o G-p o ein-coupled ecep o 43 (GPR43) ha is exp essed by immune cells on he lamina p op ia, such as egula o y T cells, egula ing he p oin lamma o y esponses in he in es ine (Boll a h and Pow ie, 2013). Mo eo e , h eonine is as ly me abolized in he in es ine o mucin syn hesis (Fau e e al., 2005). These p o eins a e pa amoun in in es inal s abili y, since he mucus laye in he colonic ou e laye p e en s he di ec con ac o luminal mic oo ganisms wi h he epi helium (Johansson e al., 2014). I has also been sugges ed ha h eonine equi emen s a e inc eased unde pa hological se ings o main ain p ope in es inal unc ion, such as p oduc ion and o ma ion o he mucus laye (Remond e al., 2009). The e o e, by pa icipa ing in he mucus laye syn hesis and p oduc ion o an i-in lamma o y SCFAs, h eonine me abolism by gu mic obio a p o es o be essen ial o gu ba ie in eg i y and unc ion. Ou da a show ha h eonine le els d op 2- old du ing coli is de elopmen un il day 5 (Figu es 1C–E). The e o e, we hypo hesized ha h eonine supplemen a ion du ing ac i e in lamma o y disease could help o es o e he in es inal homeos asis and hus p esen some he apeu ic po en ial. P e ious s udies ha e in es iga ed h eonine in combina ion wi h o he amino acids as a po en ial candida e o he apy agains coli is (Fau e e al., 2006;Liu e al., 2013). Ne e heless, F on ie s in Physiology | www. on ie sin.o g 3Sep embe 2018 | Volume 9 | A icle 1247 phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 4 Gai em e al. Th eonine Supplemen a ion E ec in Coli is FIGURE 2 | Th eonine supplemen a ion on he onse o coli is de elopmen delays eco e y and dec eases goble cell numbe . (A) Mice we e supplemen ed wi h h eonine in he beginning o coli is induc ion (DSS + Th ) o a e es ablished in lamma ion (DSS + Th D8). Mice ea ed only o coli is induc ion we e used as con ol (DSS). (B) Disease p og ession was assessed by sco ing he disease ac i i y index (DAI) h oughou he expe imen . (C) Quan i ica ion o sho -chain a y acids (ace a e, bu y a e, p opiona e) and succina e by NMR was pe o med a day 13 o expe imen . (D) Exp ession o mucin-encoding genes Muc1 and Muc2 was pe o med by qPCR a day 13. (E) Colonic issue sec ions we e s ained wi h Alcian Blue/pe iodic acid-Schi . (F) Quan i ica ion o goble cell numbe pe c yp . Scale ba = 50 µm. (G) IL-22 quan i ica ion on colonic explan cul u es by ELISA. Da a is shown as mean ±SD. n= 5 mice/g oup. One ep esen a i e expe imen is shown ou o wo. The symbol ∗co esponds o s a is ical di e ences be ween DSS + Th and DSS; while # is ela ed o s a is ical di e ences be ween DSS + Th and DSS + Th D8. ∗p<0.05; ∗∗p<0.01; ∗∗∗p<0.001. no only single h eonine supplemen a ion was no e alua ed be o e, bu also he e is sca ce e idence ega ding he mos adequa e ime ame o i s supplemen a ion in coli is ea men . Acco dingly, we add essed h eonine supplemen a ion in wo dis inc phases: (1) in pa allel wi h he ini ia ion o he in lamma o y p ocess, i.e., simul aneously wi h DSS adminis a ion (DSS +Th ), and (2) on day 8, when in lamma ion is al eady es ablished (DSS +Th D8). Mice subjec ed o DSS-induced coli is bu wi hou h eonine supply we e used as con ol (DSS) (Figu e 2A). We obse ed ha he h ee g oups de eloped coli is wi h a simila p og ession p o ile. Ne e heless, a e his ime poin , he eco e y p o ile o coli ic mice supplemen ed wi h h eonine in he d inking wa e (DSS +Th ) was slowe han ha o he o he wo g oups, showing s a is ically signi ican di e ences om day 10 o he inal day o expe imen when compa ed o con ol g oup (DSS). Besides, DSS +Th mice had also a dis inc i e DAI sco e a day 11 and 12 when compa ed wi h mice ha only ecei ed h eonine a e day 8 (DSS +Th D8) (Figu e 2B). Despi e he di e gen F on ie s in Physiology | www. on ie sin.o g 4Sep embe 2018 | Volume 9 | A icle 1247 phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 5 Gai em e al. Th eonine Supplemen a ion E ec in Coli is pheno ype, no majo di e ences we e ound in colon leng h nei he in he in es inal pe meabili y (Supplemen a y Figu e S1). Th eonine adminis a ion was su p isingly de imen al o disease eco e y when gi en a he se ing o he in lamma o y p ocess. To unde s and his pheno ype, we i s e alua ed he po en ial al e a ions in he colon me abolic p o ile among he di e en g oups. No signi ican di e ences we e ound be ween mos o he me aboli es. Pa icula ly, he le els o he majo SCFAs (i.e., ace a e, bu y a e, and p opiona e), no mally associa ed wi h a p o ec i e pheno ype, we e simila among g oups. Only succina e le els, which is as an impo an ma ke o in lamma ion p omo ing IL-1βinduc ion in in lamma o y con ex s (Tannahill e al., 2013), we e ound o be ma kedly dec eased in DSS +Th D8 mice (Figu e 2C). No majo al e a ions we e obse ed in he in lamma o y in il a e p o ile, issue o ganiza ion and hepa ic and enal oxici y se um bioma ke s (Supplemen a y Figu e S1). Th eonine plays a majo ole in mucin syn hesis and consequen ly in he o ma ion o he mucus laye . Indeed, lack o h eonine is also known o impai in es inal pa acellula pe meabili y and is associa ed wi h ewe goble cells and mucus syn hesis (Fau e e al., 2005;Mao e al., 2011). The mucus laye se es as a ba ie agains mic obial ansloca ion o he lamina p op ia and he e o e i s in eg i y is pa amoun o in es inal homeos asis. When we analyzed he exp ession o mucin-encoding genes, we ound ha Muc2 exp ession is dec eased in DSS +Th mice when compa ed o mice ha only ecei ed h eonine a day 8 (DSS +Th D8) (Figu e 2D). Muc2 encodes o he oligome ic mucus gel- o ming mucin 2 p o ein ha is he majo esponsible o mucus syn hesis. Indeed, impai men o o al absence o he mucus laye is associa ed wi h se e e coli is, as obse ed in Muc2-de icien mice (Van de Sluis e al., 2006). We also ound ha DSS +Th mice display highe exp ession o Muc1, which has shown o con ibu e o in es inal in lamma ion and colon cance p og ession (Baldus e al., 2004; Takahashi e al., 2015). We nex quan i ied he numbe o goble cells o he colonic mucosa. These a e a sec e o y epi helial cell lineage ound in bo h he small and he la ge in es ines, whose majo unc ion is he p oduc ion o mucus. By analyzing colon slices s ained wi h Alcian Blue/Pe iodic acid-Schi , we obse ed ha DSS +Th mice displayed signi ican ly ewe goble cells when compa ed o bo h DSS and DSS +Th D8 (Figu es 2E,F). The e o e, ou esul s sugges ha an al e a ion in mucus syn hesis due o h eonine adminis a ion du ing he onse o disease may impac in es inal in eg i y, by delaying he eco e y o disease. Fau e e al. (2006) ha e demons a ed ha supplemen a ion wi h die con aining highe doses o amino acids, including L- h eonine, lead o an inc ease in goble cell numbe , egula ed mucin p oduc ion in he colon and es o ed mic obio a composi ion a e DSS ea men in a s. No wi hs anding, no only he animal model is di e en , bu also L- h eonine was gi en be o e coli is induc ion, which may be unde lying he dis inc i e ou come. P e ious s udies ha e linked se e al cy okines o mucus p oduc ion in he in es ine (Pa ks e al., 2015). To examine he immunological p o ile o he h ee g oups, cy okine le els in he colon we e quan i ied. No majo changes we e obse ed be ween he g oups o in e leukin (IL)-1β, IL-12p70, IL-10, IL-17A/F and g anulocy e-mac ophage colony-s imula ing ac o (GM-CSF) le els (Supplemen a y Figu e S2). No wi hs anding, he amoun o IL-22 was signi ican ly dec eased in DSS +Th mice when compa ed o DSS +Th D8 mice (Figu e 2G). IL-22 is a membe o he IL-10 amily o cy okines and has been as ly s udied in he con ex o in es inal homeos asis. I can be p oduced by se e al cell ypes, such as T helpe (Th1) 1, Th17, Th22 and inna e lymphoid cells (ILCs), and p esen se e al oles in he gas oin es inal ac , such as issue egene a ion and main enance o he in es inal epi helial ba ie (Ru z e al., 2013). Thus, he dec eased IL-22 le els may be associa ed wi h delayed eco e y o he in es inal balance. O e all, ou da a demons a e ha supplemen a ion o h eonine du ing coli is induc ion impai s goble cell numbe , wi h concomi an dec eased Muc2 exp ession and IL-22 p oduc ion. These a ia ions a e likely o be he cause o delayed eco e y obse ed in his si ua ion. In e es ingly, hese e ec s a e no seen when h eonine is adminis e ed once coli is is es ablished. Acu e DSS-induced coli is is known o p omo e gu mic obial dysbiosis (Munyaka e al., 2016). Th eonine is me abolized by some in es inal commensal bac e ia, leading o he p oduc ion o se e al me aboli es used o in es inal main enance and o media e immune esponses (Neis e al., 2015). Thus, h eonine supplemen a ion du ing induc ion o coli is may impac di e en ly he colonic mic obio a popula ions p esen du ing he onse and upon he es ablishmen o in lamma ion, ha ing ul ima ely dis inc e ec s in in es inal unc ion. Fu he unde s anding o he mechanisms unde lying h eonine supplemen a ion may gi e new insigh s on how die a y nu ien s modula e he dynamic balance be ween mic obiome, immune esponse and ba ie unc ion. ETHICS STATEMENT This s udy was ca ied ou in acco dance wi h he ecommenda ions o Eu opean Council Di ec i e (2010/63/EU) guidelines ha whe e ansposed in o Po uguese law (Dec ee- Law n.◦113/2013, Augus 7 h), i3S Animal E hics Commi ee and licensed by he Po uguese Na ional Au ho i y o Animal Heal h (DGAV). The p o ocol was app o ed by he i3S Animal E hics Commi ee and licensed by he Po uguese Na ional Au ho i y o Animal Heal h (DGAV) wi h e e ence 014811/2016-07-13. AUTHOR CONTRIBUTIONS JG, ET, CC, AgC, FR, MS, AnC, and RS designed he expe imen s. JG, LG, RD-O, and RS pe o med he expe imen s. JG, LG, RD-O, and RS analyzed he da a. JG, LG, RD-O, and RS in e p e ed he esul s. JG and RS d a ed he manusc ip and p epa ed he ables and igu es. JG, LG, RD-O, ET, CC, AgC, FR, MS, AnC, and RS e ised he pape and app o ed he inal e sion o he manusc ip . F on ie s in Physiology | www. on ie sin.o g 5Sep embe 2018 | Volume 9 | A icle 1247 phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 6 Gai em e al. Th eonine Supplemen a ion E ec in Coli is FUNDING This wo k was suppo ed by he No he n Po ugal Regional Ope a ional P og amme (NORTE 2020), unde he Po ugal 2020 Pa ne ship Ag eemen , h ough he Eu opean Regional De elopmen Fund (FEDER) (NORTE-01-0145-FEDER- 000013) and he Fundação pa a a Ciência e Tecnologia (FCT) (con ac s PD/BD/106053/2015 o JG ia In e - Uni e si y Doc o al P og amme in Ageing and Ch onic Disease – PhDOC, IF/00021/2014 o RS, IF/01390/2014 o ET, IF/01147/2013 o RD-O, IF/00735/2014 o AgC, SFRH/BPD/96176/2013 o CC, and SFRH/BPD/111100/2015 o LG). MS is a FCT in es iga o . The NMR da a was acqui ed a CERMAX (Cen o de Ressonância Magné ica An ónio Xa ie ) which is a membe o he Na ional NMR ne wo k wi h he suppo o P ojec LISBOA-01-0145-FEDER- 007660. SUPPLEMENTARY MATERIAL The Supplemen a y Ma e ial o his a icle can be ound online a : h ps://www. on ie sin.o g/a icles/10.3389/ phys. 2018.01247/ ull#supplemen a y-ma e ial REFERENCES Abubucke , S., Sega a, N., Goll, J., Schube , A. M., Iza d, J., Can a el, B. L., e al. (2012). Me abolic econs uc ion o me agenomic da a and i s applica ion o he human mic obiome. PLoS Compu . Biol. 8:e1002358. doi: 10.1371/jou nal. pcbi.1002358 Baldus, S. E., Monig, S. P., Huxel, S., Landsbe g, S., Hanisch, F. G., Engelmann, K., e al. (2004). MUC1 and nuclea be a-ca enin a e coexp essed a he in asion on o colo ec al ca cinomas and a e bo h co ela ed wi h umo p ognosis. Clin. Cance Res. 10, 2790–2796. doi: 10.1158/1078-0432.CCR-0 3-0163 Boll a h, J., and Pow ie, F. (2013). Immunology. Feed you T egs mo e ibe . Science 341, 463–464. doi: 10.1126/science.1242674 Co eia, C. R., Gai em, J., Oli ei a, M. B., Sil es e, R., and Mano, J. F. (2017). The in luence o su ace modi ied poly(l-lac ic acid) ilms on he di e en ia ion o human monocy es in o mac ophages. Bioma e . Sci. 5, 551–560. doi: 10.1039/ c6bm00920d den Bes en, G., an Eunen, K., G oen, A. K., Venema, K., Reijngoud, D. J., and Bakke , B. M. (2013). The ole o sho -chain a y acids in he in e play be ween die , gu mic obio a, and hos ene gy me abolism. J. Lipid Res. 54, 2325–2340. doi: 10.1194/jl .R036012 Du chschein, F., Pe i sch, W., and Hamme , H. F. (2016). Die he apy o in lamma o y bowel diseases: he es ablished and he new. Wo ld J. Gas oen e ol. 22, 2179–2194. doi: 10.3748/wjg. 22.i7.2179 Fau e, M., Me aux, C., Moennoz, D., Godin, J. P., Vuichoud, J., Rocha , F., e al. (2006). Speci ic amino acids inc ease mucin syn hesis and mic obio a in dex an sul a e sodium- ea ed a s. J. Nu . 136, 1558–1564. doi: 10.1093/jn/136.6. 1558 Fau e, M., Moennoz, D., Mon igon, F., Me aux, C., B euille, D., and Balle e, O. (2005). Die a y h eonine es ic ion speci ically educes in es inal mucin syn hesis in a s. J. Nu . 135, 486–491. doi: 10.1093/jn/135. 3.486 G aça, G., Des e o, J., Sousa, J., Fonseca, C., Sil ei a, M., Se pa, J., e al. (2017). Iden i ica ion o pu a i e bioma ke s o lep omeningeal in asion in B-cell non-Hodgkin lymphoma by NMR me abolomics. Me abolomics 13:136. doi: 10.1007/s11306-017-1269-9 Hsiung, Y. C., Liu, J. J., Hou, Y. C., Yeh, C. L., and Yeh, S. L. (2014). E ec s o die a y glu amine on he homeos asis o CD4+T cells in mice wi h dex an sul a e sodium-induced acu e coli is. PLoS One 9:e84410. doi: 10.1371/jou nal. pone.0084410 Huda-Faujan, N., Abdulami , A. S., Fa imah, A. B., Anas, O. M., Shuhaimi, M., Yazid, A. M., e al. (2010). The impac o he le el o he in es inal sho chain Fa y acids in in lamma o y bowel disease pa ien s e sus heal hy subjec s. Open Biochem. J. 4, 53–58. doi: 10.2174/1874091x010040 10053 Johansson, M. E., Gus a sson, J. K., Holmen-La sson, J., Jabba , K. S., Xia, L., Xu, H., e al. (2014). Bac e ia pene a e he no mally impene able inne colon mucus laye in bo h mu ine coli is models and pa ien s wi h ulce a i e coli is. Gu 63, 281–291. doi: 10.1136/gu jnl-2012-303207 Kelly, C. J., Zheng, L., Campbell, E. L., Saeedi, B., Scholz, C. C., Bayless, A. J., e al. (2015). C oss alk be ween mic obio a-de i ed sho -chain a y acids and in es inal epi helial HIF augmen s issue ba ie unc ion. Cell Hos Mic obe 17, 662–671. doi: 10.1016/j.chom.2015.03.005 Kho , B., Ga de , A., and Xa ie , R. J. (2011). Gene ics and pa hogenesis o in lamma o y bowel disease. Na u e 474, 307–317. doi: 10.1038/na u e10209 K e zmann, N. A., Fillmann, H., Mau iz, J. L., Ma oni, C. A., Ma oni, N., Gonzalez-Gallego, J., e al. (2008). E ec s o glu amine on p oin lamma o y gene exp ession and ac i a ion o nuclea ac o kappa B and signal ansduce s and ac i a o s o ansc ip ion in TNBS-induced coli is. In lamm. Bowel Dis. 14, 1504–1513. doi: 10.1002/ibd.20543 Liu, X., Beaumon , M., Walke , F., Chaumon e , C., And iamihaja, M., Ma sumo o, H., e al. (2013). Bene icial e ec s o an amino acid mix u e on colonic mucosal healing in a s. In lamm. Bowel Dis. 19, 2895–2905. doi: 10. 1097/01.MIB.0000435849.17263.c5 Macia, L., Tan, J., Viei a, A. T., Leach, K., S anley, D., Luong, S., e al. (2015). Me aboli e-sensing ecep o s GPR43 and GPR109A acili a e die a y ib e- induced gu homeos asis h ough egula ion o he in lammasome. Na . Commun. 6:6734. doi: 10.1038/ncomms7734 Mao, X., Zeng, X., Qiao, S., Wu, G., and Li, D. (2011). Speci ic oles o h eonine in in es inal mucosal in eg i y and ba ie unc ion. F on . Biosci. 3: 1192–1200. McNamee, E. N., Mas e son, J. C., Jedlicka, P., McManus, M., G enz, A., Collins, C. B., e al. (2011). In e leukin 37 exp ession p o ec s mice om coli is. P oc. Na l. Acad. Sci. U.S.A. 108, 16711–16716. doi: 10.1073/pnas.11119 82108 Munyaka, P. M., Rabbi, M. F., Kha ipou , E., and Ghia, J. E. (2016). Acu e dex an sul a e sodium (DSS)-induced coli is p omo es gu mic obial dysbiosis in mice. J. Basic Mic obiol. 56, 986–998. doi: 10.1002/jobm.201500726 Neis, E. P., Dejong, C. H., and Rensen, S. S. (2015). The ole o mic obial amino acid me abolism in hos me abolism. Nu ien s 7, 2930–2946. doi: 10.3390/ nu7042930 Pa ks, O. B., Pociask, D. A., Hodzic, Z., Kolls, J. K., and Good, M. (2015). In e leukin-22 signaling in he egula ion o in es inal heal h and disease. F on . Cell De . Biol. 3:85. doi: 10.3389/ cell.2015.00085 Pencha z, P. B., Elango, R., and Ball, R. O. (2008). An app oach o de ining he uppe sa e limi s o amino acid in ake. J. Nu . 138, 1996s–2002s. doi: 10.1093/ jn/138.10.1996S Remond, D., Bu ie e, C., Godin, J. P., Mi and, P. P., Obled, C., Pape , I., e al. (2009). In es inal in lamma ion inc eases gas oin es inal h eonine up ake and mucin syn hesis in en e ally ed minipigs. J. Nu . 139, 720–726. doi: 10.3945/ jn.108.101675 Ru z, S., Eidenschenk, C., and Ouyang, W. (2013). IL-22, no simply a Th17 cy okine. Immunol. Re . 252, 116–132. doi: 10.1111/im .12027 Takahashi, H., Jin, C., Rajabi, H., Pi oda, S., Alam, M., Ahmad, R., e al. (2015). MUC1-C ac i a es he TAK1 in lamma o y pa hway in colon cance . Oncogene 34, 5187–5197. doi: 10.1038/onc.2014.442 Tannahill, G. M., Cu is, A. M., Adamik, J., Palsson-McDe mo , E. M., McGe ick, A. F., Goel, G., e al. (2013). Succina e is an in lamma o y signal ha induces IL-1be a h ough HIF-1alpha. Na u e 496, 238–242. doi: 10.1038/na u e 11986 Van de Sluis, M., De Koning, B. A., De B uijn, A. C., Velcich, A., Meije ink, J. P., Van Goudoe e , J. B., e al. (2006). Muc2-de icien mice spon aneously de elop F on ie s in Physiology | www. on ie sin.o g 6Sep embe 2018 | Volume 9 | A icle 1247 phys-09-01247 Sep embe 4, 2018 Time: 9:42 # 7 Gai em e al. Th eonine Supplemen a ion E ec in Coli is coli is, indica ing ha MUC2 is c i ical o colonic p o ec ion. Gas oen e ology 131, 117–129. doi: 10.1053/j.gas o.2006.04.020 Viennois, E., Chen, F., La oui, H., Bake , M. T., and Me lin, D. (2013). Dex an sodium sul a e inhibi s he ac i i ies o bo h polyme ase and e e se ansc ip ase: li hium chlo ide pu i ica ion, a apid and e icien echnique o pu i y RNA. BMC Res. No es 6:360. doi: 10.1186/1756-0500-6-360 Con lic o In e es S a emen : The au ho s decla e ha he esea ch was conduc ed in he absence o any comme cial o inancial ela ionships ha could be cons ued as a po en ial con lic o in e es . The e iewe DS and handling Edi o decla ed hei sha ed a ilia ion a he ime o he e iew. Copy igh © 2018 Gai em, Gonçal es, Dinis-Oli ei a, Cunha, Ca alho, To ado, Rod igues, Sa ai a, Cas o and Sil es e. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums is pe mi ed, p o ided he o iginal au ho (s) and he copy igh owne (s) a e c edi ed and ha he o iginal publica ion in his jou nal is ci ed, in acco dance wi h accep ed academic p ac ice. No use, dis ibu ion o ep oduc ion is pe mi ed which does no comply wi h hese e ms. F on ie s in Physiology | www. on ie sin.o g 7Sep embe 2018 | Volume 9 | A icle 1247