The DPP-4 inhibitor vildagliptin impacts the gut microbiota and prevents disruption of intestinal homeostasis induced by a Western diet in mice

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1 Dietologi (18) 61: ARTICLE The DPP-4 inhiitor vildgliptin impcts the gut microiot nd prevents disruption of intestinl homeostsis induced y Western diet in mice Mrt Olivres 1 & Audrey M Neyrinck 1 & Srh A Pötgens 1 & Mrtin Beumont 1 & Nuri Slzr & Ptrice D Cni 1,3 & Lure B Bindels 1 & Nthlie M Delzenne 1 Received: 8 Ferury 18 /Accepted: 5 April 18 /Pulished online: 5 My 18 # The Author(s) 18 Astrct Aims/hypothesis Dipeptidyl peptidse 4 (DPP-4) inhiitors re gents designed to increse the hlf-life of incretins Although they re dministered orlly, little is known out their effects on the gut microiot nd functions, despite the fct tht some cteri present in the gut microiot exhiit DPP-4-like ctivity Our ojective ws to study the impct of the DPP-4 inhiitor vildgliptin on gut functions nd the intestinl ecosystem in murine model of oesity induced y Western diet () Methods Twenty seven mle C57BL/6J mice were rndomised to receive control diet, (45% kj from ft nd 17% kj from sucrose) or + vildgliptin (6 mg/ml in drinking wter) for 8 weeks Results Vildgliptin significntly reduced DPP-4 ctivity in the cecl content nd feces Vildgliptin impcted on the composition of the gut microiot nd its metolic ctivity It minly decresed Oscillicter spp ( direct effect independent of DPP-4 ctivity ws shown on cultured O vlericigenes), incresed Lctocillus spp nd propionte, nd reduced the lignds of Tolllike receptors nd 4 Vildgliptin protected ginst the reductions in crypt depth nd ilel expression of ntimicroil peptides induced y the In the liver, the expression of immune cell popultions (Cd3g nd Cd11c [lso known s Itgx]) nd cytokines ws decresed in the + vildgliptin-fed mice compred with the -fed group Ex vivo exposure of precisioncut liver slices to vildgliptin showed tht this response ws not relted to direct effect of the drug on the liver tissue Conclusions/interprettion Our study is the first to consider the DPP-4-like ctivity of the gut microiot s trget of DPP-4 inhiition We propose tht vildgliptin exerts eneficil effects t the intestinl level in ssocition with modultion of gut microiot, with consequences for heptic immunity If relevnt in humns, this could open new therpeutic uses of DPP-4 inhiition to tckle gut dysfunctions in different pthophysiologicl contexts Dt vilility The sequences used for nlysis cn e found in the MG-RAST dtse under the project nme MYNEWGUT3 Keywords Antimicroil peptides DPP-4 ctivity Gut microiot Gut liver xis Vildgliptin Western diet Electronic supplementry mteril The online version of this rticle ( contins peer-reviewed ut unedited supplementry mteril, which is ville to uthorised users Nthlie M Delzenne nthliedelzenne@uclouvine 1 3 Metolism nd Nutrition Reserch Group, Louvin Drug Reserch Institute, Université ctholique de Louvin, 73 v E Mounier, Box B17311, 1 Brussels, Belgium Deprtment of Microiology nd Biochemistry, Instituto de Productos Lácteos de Asturis (IPLA-CSIC), Villvicios, Spin Wlloon Excellence in Life sciences nd BIOtechnology (WELBIO), Louvin Drug Reserch Institute, Université ctholique de Louvin, Brussels, Belgium Arevitions AMP Antimicroil peptide DPP-4 Dipeptidyl peptidse 4 GIP Glucose-dependent insulinotropic peptide GLP Glucgon-like peptide OTU Opertionl txonomic unit PCLS Precision-cut liver slices PCoA Principl coordinte nlysis PNA Pr-nitronilide qpcr Quntittive rel-time PCR SCFA Short-chin ftty cids TLR Toll-like receptor Western diet

2 Dietologi (18) 61: Introduction Enteroendocrine cells relese incretins in response to nutrient ingestion The two min incretins glucgon-like peptide (GLP) 1 nd glucose-dependent insulinotropic peptide (GIP) regulte the postprndil secretion of insulin [1] One limittion of GLP-1 nd GIP ctivities is their short hlf-life, ecuse they re rpidly cleved nd inctivted y dipeptidyl peptidse 4 (DPP-4) DPP-4 exists s memrnenchored cell surfce peptidse nd s solule form in the circultion [] The expression nd circultion levels of DPP-4 in people with type dietes re higher thn in those without dietes [3] Additionlly, DPP-4 is considered n dipokine positively correlted to ody weight, dipose tissue inflmmtion nd insulin resistnce in oese individuls [4, 5] Sitgliptin, ngliptin nd vildgliptin re new type of DPP-4 inhiitors which re dministered orlly nd re well tolerted [6] Although first pproved in 6, their effects on essentil intestinl functions remin poorly studied In 11, Wget et l descried how inhiition of DPP-4 ctivity in the intestine ws involved in regulting glycemi [7] Recently, Mulvihill et l demonstrted tht glucose tolernce depended on inhiition of DPP-4 ctivity in hemtopoietic nd endothelil cells, ut there ws no involvement of the DPP-4 expressed y enterocytes [8] DPP-4 ctivity modultes the functionlity of more thn 4 potentil sustrtes, including cytokines, chemokines nd growth fctors, some of which re prticulrly relevnt for gut homeostsis [9] For exmple, the preservtion of GLP- with DPP-4 inhiitors (vline pyrrolidide) hs een proposed to stimulte intestinl epithelil growth [1, 11] Besides cleving gut peptides, DPP-4 plys role in the immune response Solule DPP-4 increses the expression of Toll-like receptors (TLRs) nd ctivtes NFκB signlling, resulting in the secretion of proinflmmtory cytokines [1, 13] These effects cn e reduced y DPP-4 inhiitors [14 16] For instnce, in mcrophges vildgliptin countercts the production of cytokines nd expression of TLR- nd TLR-4 induced y solule DPP-4 nd lipopolyscchride [13] As TLRs re prt of the innte immune response nd ply crucil role in the mintennce of gut homeostsis, investigting the effect of vildgliptin in the context of intestinl helth my produce prticulrly relevnt results [17] Vildgliptin is principlly sored in the smll, nd to lesser extent in the lrge, intestine [18] During its pssge through the intestinl trct, eukryotic cells s well s the gut microiot re exposed to vildgliptin It hs een widely descried tht intestinl microes respond to environmentl fctors such s drugs nd food, generting protective or detrimentl effects [19, ] Interestingly, some gener of cteri in the gut microiot, such s Prevotell nd Lctocillus, hve een reported to exert DPP-4-like ctivity [1 3] Furthermore, some strins of Bifidocterium nd Lctocillus cn produce DPP-4 inhiitors [4, 5] To our knowledge, the DPP-4-like ctivity of the gut microiot hs never een studied One study (without ssessment of DPP-4

3 184 Dietologi (18) 61: ctivity) reported tht vildgliptin cused chnges in the gut microiot nd tht the chnges were more significnt in rts fed high-ft diet thn in those fed control diet [6] Furthermore, recent report showed tht high-ft, highsugr diets ltered the ctivtion of enteric neurons y GLP- 1 regulting insulin secretion, n effect tht seemed to e lso dependent on the gut microiot [7] We therefore ssessed the effect of vildgliptin on the gut microiot nd intestinl functions of murine model of oesity induced y Western diet (), model tht induces gut dysfunction nd relted heptic inflmmtion Methods Animls nd tretments Two niml experiments were performed In experiment 1, 7 mle 9-week-old C57BL/6J mice (Jnvier Ls, Sint-Berthevin, Frnce) were purchsed Three mice were housed in one individully ventilted cge In experiment, four mle 1-week-old C57BL/6J mice (Jnvier Ls) were housed in one individully ventilted cge Mice were kept in pthogen-free environment with 1 h dylight cycle nd free ccess to food nd wter The cclimtistion period lsted 1 week on stndrd diet (AIN-93M; ssniff, Soest, Germny) The experiments were pproved y nd performed in ccordnce with the guidelines of the locl ethics committee of Université ctholique de Louvin Housing conditions were s specified y the Belgin Lw of 9 My 13 regrding the protection of lortory nimls (Agreement no LA 13314) In experiment 1, mice were rndomised sed on ody composition ssessed y NMR (LF5 minispec; Bruker, Rheinstetten, Germny) to minimise differences (initil men ody weight ± SEM ws 446 ± 3 g) No linding procedure ws followed The groups (n = 9) were: (1) group-fed control diet (D145K; Reserch Diets, New Brunswick, NJ, USA) contining 1% kj ft; () group-fed (D1451; Reserch Diets) contining 45% kj ft nd 17% kj sucrose; nd (3) group-fed plus vildgliptin (Cymn, Ann Aror, MI, USA [supplied y Snio, Uden, the Netherlnds]) in the drinking wter (6 mg/ml ccording to previous studies [13], corresponding to pproximtely 5 mg kg 1 dy 1 ) To discriminte the effect of vildgliptin from the effect of the on DPP-4 ctivity, we pretreted with vildgliptin for weeks In the third week, the ws introduced A scheme of the experimentl design is shown in electronic supplementry mteril (ESM) Fig 1 After 8 weeks nd 6 h of fsting, glycemi ws mesured using glucometer (Roche Dignostics, Bsel, Switzerlnd) on lood from the til Mice were nesthetised with isoflurne gs (Aot, Lke Bluff, IL, USA) Portl lood ws collected Mice were necropsied fter cervicl disloction Liver, dipose tissue nd cecl content nd tissue were weighed Blood, liver, cecl content nd tissue, ileum nd colon were collected, frozen in liquid nitrogen nd stored t 8 C until nlysed Detils of experiment re given elow in the section Precision-cut liver slices Biochemicl nlysis Portl ctive GLP-1 (7-36 mide nd 7-37), insulin nd totl GIP were determined in plsm collected in n EDTA tue with DPP-4 inhiitor (DDP4-1; Millipore, Drmstdt, Germny) using multiplex immunossy kit (Bio-Plex; Bio-Rd, Nzreth, Belgium) Anlysis of gut microiot composition Genomic DNA ws extrcted from the cecl content using QIAmp DNA Stool Mini Kit (Qigen, Hildren, Germny), including ed-eting step The composition of the gut microiot ws nlysed y Illumin sequencing of the 16S rrna gene (Illumin, Sn Diego, CA, USA) Illumin sequencing ws performed s previously descried [8, 9] The V5 V6 region of the 16S rrna gene ws mplified y PCR using modified primers The mplicons were purified, quntified nd sequenced using the Illumin MiSeq system to produce 3 p sequencing products, t the University of Minnesot Genomics Center Susequent ioinformtics nd iosttistics nlyses were performed s previously descried [8] The full protocol nd sttisticl nlyses re descried in ESM Methods In ddition, in the cecl content, some components of the gut microiot were nlysed y quntittive rel-time PCR (qpcr) s previously descried [8] Primers re presented in ESM Tle 1 Gene expression nlyses Totl RNA ws isolted from tissues nd qpcr ws performed using the StepOne System (Applied Biosystems, Wlthm, MA, USA) to nlyse expression of the following genes: Cd3g, Cd11c (lso known s Itgx), Cd68, Cd163, Cludin (lsoknownscldn), DefA, F4/8 (lso known s Adgre1), Il1, Il6, Il1, Ki67 (lso known s Mki67), Lyz1, Mcp1 (lso known s Ccl), Muc, Ocln, Plg, Reg3g, Tcf4, Tnfα (lso known s Tnf) nd Zo1 (lso known s Tjp1) Dt were nlysed using the -ΔΔCT method nd expression ws normlised to Rpl19 (see ESM Methods nd ESM Tle for further detils) Short-chin ftty cids Cecl content ws diluted 1:6 in LAL regent wter (Lonz, Wlkersville, MD, USA) nd homogenised with tissue lyser (Qigen) for 4 min without eds to void cteri disruption Smples were centrifuged (8 g, min) Cell-free superntnts were used for the quntifiction of cette, utyrte nd propionte s previously descried [3] nd for the nlyses of TLR gonists s descried elow TLR- nd TLR-4 gonists TLR- nd TLR-4 gonists were mesured using HEK-Blue reporter cell lines ccording to

4 Dietologi (18) 61: the mnufcturer s instructions (InvivoGen, Sn Diego, CA, USA) Cells were exposed to the superntnts of cecl content nd the protocol previously descried ws followed [31] Histologicl nlysis of the intestine Crypt depth nd villus length were mesured fter H&E stining Sections were digitised (Leic SCN4; Leic, Wetzlr, Germny) nd imges were cptured using Leic Imge Viewer softwre, version 44 At lest ten mesurements per mouse were mde Precision-cut liver slices After 1 week of cclimtistion, mice in experiment were nesthetised with ketmine (1 mg/kg ody weight) (Nimtek; Eurovet, Bldel, Netherlnds) nd xylzine (1 mg/kg ody weight) (Rompun; Byer, Leverkusen, Germny) Precision-cut liver slices (PCLS) were prepred s previously descried [3, 33] PCLS were incuted without (control medium) or with vildgliptin (6 mg/ml) in oxygented nd supplemented Wymouth s medium for 4 h t 37 C under gittion PCLS were frozen in dry ice until nlysed Bcteril growth conditions Growth curves of Oscillicter vlericigenes DSM186 nd L reuteri 1-3 in the presence of vildgliptin (6 mg/ml) or sence of vildgliptin (control roth) were determined (ESM Methods) To mesure DPP-4 ctivity 5 ml of overnight cultures were hrvested nd processed s descried elow DPP-4 ctivity DPP-4 ctivity ws mesured y the clevge of pr-nitronilide (PNA) from Gly-Pro-PNA (Sigm, St Louis, MO, USA) Smples ( 5 mg) were suspended in TRIS-se uffer (5 mmol/l, 1% N-octylglucoside, ph 83), homogenised forminwithtissuelysernd centrifuged (3 g,min) The superntnt ( μl) ws incuted with Gly-Pro-PNA The enzymtic ctivity ws mesured in kinetic nlysis of 3 min t 37 C (38 nm) (SpectrMx M; Moleculr Devices, Sn Jose,CA,USA)ndquntifiedwithstndrdcurveoffree PNA In tissues nd cteril smples, the vlues were normlised y the mount of protein (Brdford method) Sttisticl nlyses The numer of mice per group ws sed on previous experiments to mesure the primry outcomes (the incresed the ody weight) with the minimum numer of nimls [34] Anlyses were performed y one-wy ANOVA followed y post hoc Tukey s tests (GrphPd Prism, version 5; GrphPd, L Joll, CA, USA) For the growth curves, twowy ANOVA followed y Bonferroni s post hoc test ws performed A χ test ws used for ctegoricl dt (ctive GLP-1 nd TLR-4 gonist) (Sttgrphics Plus, version 51; Sttgrphics, The Plins, VA, USA) Dt of microiot composition were nlysed using Welch s t test nd the flse discovery rte ws pplied for p vlue correction (q vlue) ccording to the Benjmini Hocherg procedure Ecologicl descriptors nd dt from the PCLS experiment were nlysed using Welch s t test Multiple correltion nlyses were performed in R version 31 (wwwr-projectorg), with djustment of p vlues ccording to the Benjmini Hocherg procedure Results were considered sttisticlly significnt t p < 5 Any exclusion decision ws supported y Grus test Plots were generted using GrphPd showing the men ± SEM Ech dot represents one iologicl replicte Results Vildgliptin decresed systemic nd intestinl DPP-4 ctivity Vildgliptin reduced DPP-4 ctivity in the portl vein nd incresed the concentrtion of ctive GLP-1 (Fig 1, ) In ddition, vildgliptin significntly reduced DPP-4 ctivity in the ileum, the cecum nd the colon (Fig 1c e) Orgn weights nd food intke re presented in ESM Tle 3 -fed mice showed significnt increse in ody weight nd diposity Body weight evolution, glycemi, insulin nd GIP re shown in ESM Fig Neither nor vildgliptin modified the mrkers relted to glucose homeostsis Vildgliptin olished DPP-4 ctivity in feces nd cecl content DPP-4 ctivity in the feces did not differ etween groups efore the introduction of the tretments The dministrtion of vildgliptin for weeks completely olished DPP-4 ctivity in the feces (Fig ) The effect persisted fter the introduction of the, s shown y the mesurement of DPP-4 ctivity in the cecl content t the end of the experiment (Fig ) Vildgliptin modified gut microiot composition nd ctivity in vivo nd inhiited growth of O vlericigenes in vitro, independently of DPP-4 ctivity Vildgliptin incresed the weight of the cecl content nd cecl tissue compred with its control group (the -fed mice) (Fig 3, ) To ssess whether these oservtions were linked to cteril fermenttion, we quntified the production of short-chin ftty cids (SCFA) in the cecl content Vildgliptin significntly incresed the mount of propionte compred with the group (Fig 3e) There were no differences in totl mount of SCFA, cette or utyrte (Fig 3c, d, f) We sequenced the 16S rrna gene of the two -fed groups with or without ddition of vildgliptin Principl coordinte nlysis (PCoA) of et diversity sed on Morisit Horn distnce showed tht vildgliptin ccounted for 38% of the vrition in the dtset (Adonis method, 1 permuttions; p < 1) The mjority of the vrition explined y the tretment ws further confirmed y PCoA using inry Jccrd nd Bry Curtis indexes (14% nd 6%, respectively; p <1)(Fig4 c) No differences were oserved in ny of the six indexes of lph diversity nlysed (ESM Fig 3) A totl of 93 opertionl txonomic units (OTUs) were

5 184 Dietologi (18) 61: DPP-4 ctivity (pkt/ml) V Active GLP-1 (pg/ml) identified, of which 67 differed significntly etween the two groups t the p < 5 threshold (ESM Tle 4) When the flse discovery rte ws pplied for p vlue correction, only two OTUs corresponding to the genus Oscillicter spp (OTU 4, p <1, q = 16) (Fig 4d) nd unclssified Ruminococccee (OTU 41, p < 1, q = 16)(Fig 4e) remined significntly reduced y vildgliptin Furthermore, some specific cteri were quntified using qpcr to confirm the reductions in Oscillicter spp nd to quntify the cteri initilly hypothesised to e ffected y DPP-4 inhiition This included cteri with DPP-4-like ctivity (Prevotell nd Lctocillus)[1 3] nd those tht produce DPP-4 inhiitors (Bifidocterium nd Lctocillus)[4, 5] Vildgliptin cused significnt reduction in Oscillicter/ Oscillospir ndnincreseinlctocillus spp (Fig 5) The ltter oservtion is consistent with the sequencing dt of L johnsonii tht tends to increse with vildgliptin (OTU 177, p = 16, q =155)(ESMTle4) To study whether vildgliptin explined these chnges, we exposed O vlericigenes to vildgliptin in vitro nd oserved tht the drug inhiited its growth (Fig 6) In prllel, we performed the sme test with L reuteri, which is crrier of +V c 15 1 Fig 1 () DPP-4 ctivity in the portl vein, () concentrtion of ctive GLP-1 in the portl vein nd (c) DPP-4 ctivity in the ileum, (d) cecum nd (e) colon Mice were fed control diet, or + vildgliptin DPP-4 ctivity (pkt/mg protein) V +V Feces dy Feces dy 14 DPP-4 ctivity (pkt/mg protein) 3 1 DPP-4 ctivity (pkt/ml) 5 + V Fig DPP-4 ctivity () in the feces efore nd fter dministrtion of vildgliptin for weeks (14 dys) nd () in the cecl content (8 weeks) Mice were fed control diet, or + vildgliptin (+V) Significnt differences etween conditions re shown s p < 1 nd p < 1 ccording to one-wy ANOVA followed y Tukey s post hoc test +V d DPP-4 ctivity (pkt/ml) V DPP-4-like ctivity in the gut microiot L reuteri grew similrly in oth conditions (Fig 6) No DPP-4 ctivity ws detected in the cell-free extrcts of O vlericigenes Therefore, the effect of vildgliptin on cteril growth is not truly dependent on the inhiition of cteril DPP-4 ctivity (ESM Fig 4) Vildgliptin reduced TLR lignds in cecl content nd restored expression of ntimicroil peptides nd crypt depth in ileum The cecl extrcts of mice treted with vildgliptin presented lower TLR- nd TLR-4 lignds compred with those of -fed mice (Fig 7, ) In the ileum, the expression of ntimicroil peptides (AMPs) such s C-type lectins (Reg3g), phospholipses (Plg) ndα-defensin (DefA) were downregulted in -fed mice (Fig 8) Vildgliptin countercted the induced downregultion of Reg3g, Plg nd DefA We studied whether vildgliptin could lso hve impcted the morphology nd some prolifertion mrkers ssocited with Pneth cells Vildgliptin significntly restored crypt depth (Fig 8) without chnging villus length (Fig 8c) The histologicl imges re shown in ESM Fig 5 No differences were oserved in Ki67 or Tcf4 expression (Fig 8d, e) No mjor chnges due to vildgliptin were oserved in the expression of mrkers relted to gut rrier function or cytokines; vildgliptin only incresed Cludin nd Mcp1 (ESM Fig 6) In the colon, we nlysed some vriles previously shown to e ffected y vildgliptin in the ileum Only significnt increse in crypt depth ws oserved, indicting tht the mjor chnges induced y vildgliptin occur in the ileum (ESM Fig 7) Finlly, we performed Spermn correltion nlysis to evlute the ssocitions etween gut microes nd host vriles (ESM Fig 8) We oserved positive correltions etween TLR-4 gonists nd three OTUs (OTU 4 [Oscillicter spp], OTU 41 [unclssified Ruminococccee] nd OTU 4 [Ctcter spp]) nd negtive correltions etween TLR-4 gonists nd two OTUs (OTU 117 [unclssified Lchnospircee] nd OTU 7 [Prcteroides goldsteinii]) Also positive ssocition ws found etween Reg3g nd OTU 16 (unclssified Porphyromondcee) e DPP-4 ctivity (pkt/ml) V (+V) Significnt differences etween conditions re shown s p < 5, p < 1 nd p < 1 ccording to one-wy ANOVA followed y Tukey s post hoc test

6 Dietologi (18) 61: Fig 3 Weight of () cecl tissue nd () ceclcontent(c f) Amount of SCFA in the cecl content (CC): (c) totlscfa,(d) cette, (e) propionte nd (f) utyrte Mice were fed control diet, or + vildgliptin (+V) Significnt differences etween conditions re shown s p <5ndp <1 ccording to one-wy ANOVA followed y Tukey s post hoc test c Cecl tissue (g) V Cecl content (g) V Totl SCFA (µg)/totl CC V d e f Acette (µg)/totl CC 6 4 Propionte (µg)/totl CC V +V Butyrte (µg)/totl CC V Vildgliptin indirectly reduced gene expression of proinflmmtory cytokines in liver Vildgliptin reduced DPP-4 ctivity in the portl vein (Fig 1), suggesting tht the liver is exposed to n efficient dose Consistent with this, we show tht vildgliptin decresed DPP-4 ctivity in the liver (ESM Fig 9) Vildgliptin ws lso ssocited with significnt reduction in the expression of Cd3g, Cd11c nd the proinflmmtory mrkers Mcp1, Tnfα, Il1 nd Il6 (Fig 9, ) To ssess whether the chnges responded to direct effect, we performed n ex vivo experiment in which PCLS were exposed to vildgliptin In contrst to our oservtions in vivo, no chnges in the expression of ny cytokines were oserved (Fig 9c) c PCo (19%) PCo (14%) PCo (14%) PCo1 (38%) PCo1 (1%) PCo1 (3%) PCo3 (14%) PCo3 (1%) PCo3 (1%) d OTU 4 Oscillicter spp Rel undnce (%) V Fig 4 Principl coordinte (PCo) plots of et diversity sed on () Morisit Horn, () inry Jccrd nd(c) Bry Curtis indexes (d, e) Reltive (Rel) undnce of OTUs modified y vildgliptin ssessed y Illumin sequencing in DNA extrcted from cecl content Mice were e OTU 41 Unclssified Ruminococccee Rel undnce (%) V fed (lck symols) or + vildgliptin (+V; grey symols) Dt were nlysed using Welch s t test nd p vlues were corrected using the Benjmini Hocherg method Significnt differences re shown s q <5

7 1844 Dietologi (18) 61: Fig 5 Gut microiot composition nlysed y qpcr in DNA extrcted from cecl content () Totl cteri, () Oscillicter/Oscillospir, (c) Lctocillus spp, (d) Bifidocterium spp nd (e) Bcteroides/Prevotell Mice were fed or + vildgliptin (+V) Significnt differences re shown s p < 5 ccording to Welch s t test c Totl cteri log 1 (cells/g) V d 9 Oscillicter/Oscillospir log 1 (cells/g) e +V 11 Lctocillus spp log 1 (cells/g) V Bifidocterium spp log 1 (cells/g) V Bcteroides/Prevotell log 1 (cells/g) V Discussion Inhiition of DPP-4 ctivity to tckle metolic disorders through the preservtion of incretins hs een widely descried However, the dditionl effects of DPP-4 inhiitors on the intestinl ecosystem remin poorly studied In the present study, we evluted whether the DPP-4 inhiitor vildgliptin could impct intestinl homeostsis including the composition nd ctivity of gut microiot using in vivo, ex vivo nd in vitro pproches Our work is the first to consider the DPP-4-like ctivity of the gut microiot s potentil trget of DPP-4 inhiitors nd evlute, in n niml model of diet-induced oesity, the effect of vildgliptin on gut rrier function, innte immune response nd liver Overll, our results show tht vildgliptin improved gstrointestinl function judged y restortion of the expression of AMPs nd the depth of the crypts in the ileum In the liver, vildgliptin ws ssocited with reduction of cytokine expression However, the ex vivo experiment with PCLS showed tht reductions were not due to direct effect of the O vlericigenes Asornce (6 nm) Time (h) L reuteri Asornce (6 nm) Time (h) Fig 6 Growth curves of () O vlericigenes nd () L reuteri in control roth (lck line) nd roth with vildgliptin (grey line) Significnt differences etween conditions re shown s p < 1 ccording to twowy ANOVA followed y Bonferroni s post hoc test OD, opticl density drug, reinforcing the ide tht reduced heptic inflmmtory tone might e relted to chnges t the intestinl level Furthermore, vildgliptin ws ssocited with chnges in et diversity indexes, reductions in Oscillicter spp nd increses in Lctocillus spp nd cecl propionte levels Among the 67 OTUs chnged y vildgliptin, we cn pinpoint some unclssified Lchnospircee nd Ruminococccee, s well s P goldsteinii, s potentil contriutors to the increse in propionte levels [35], even if no sttisticlly significnt correltion cked up this fct DPP-4 is multifunctionl protein nd therefore it is plusile tht its inhiition y vildgliptin hs induced the rod chnges tht we oserved through severl mechnisms In this regrd, we demonstrted three effects of vildgliptin on the intestine, nmely: (1) restortion of the ctive form of gut peptides such s GLP-1; () modultion of the innte immune response; nd (3) TLR- gonist equivlent (pg FSL-1/mg CC) V TLR-4 gonist equivlent (pg LPS/mg CC) V Fig 7 Lignds of () TLR- nd() TLR-4 in cecl content (CC) Mice were fed control diet, or + vildgliptin (+V) Dt for TLR- were nlysed using one-wy ANOVA followed y Tukey s post hoc test Ctegoricl dt (positive/totl) for TLR-4 were nlysed using the χ test Significnt differences etween conditions re shown s p < 5 nd p < 1 FSL-1, firolst-stimulting lipopeptide; LPS, lipopolyscchride

8 Dietologi (18) 61: AMP mrna (reltive expression) 3 1 +V +V Reg3g Plg DefA Lyz1 +V +V Immune cell mrna (reltive expression) V +V +V +V +V Cd3g F4/8 Cd11c Cd68 Cd163 Crypt depth (µm) d Ki67 mrna (reltive expression) V +V modultion of the gut microiot There could e link etween these effects, s illustrted for instnce in study performed in oese preiotic-fed mice which reported tht the gut microiot influenced the relese of sustrtes of DPP-4 such s GLP-1 nd GLP- [36] Furthermore, high-ft diet cn impir enteroendocrine cell function nd the relese of GLP-1, s confirmed here [37] We propose tht the increse in ctive GLP-1 in mice treted with vildgliptin cn lso e prtly explined y the increses in propionte in the cecl content [38] The commuttive reltionship etween microiot nd host is lso supported y the fct tht AMPs shpe the composition of the gut microiot, nd, conversely, the gut microiot influences host immunity [39] Since we know tht some cteri exert DPP-4-like ctivity, one of our outcome mesures ws to ssess whether vildgliptin trgets DPP-4 ctivity of the gut microiot [1 3] We hve shown for the first time tht vildgliptin completely olished DPP-4 ctivity in the cecl content nd feces Anlyses of the composition of the gut microiot c Villus length (µm) e Tcf4 mrna (reltive expression) V +V Fig 8 () Gene expression of AMPs Reg3g, Plg, DefA nd Lyz1() Crypt depth, (c) villus length, (d) prolifertion mrker Ki67 nd (e) Pneth cell differentition mrker Tcf4 in the ileum Mice were fed control diet, or + vildgliptin (+V) Significnt differences etween conditions re shown s p < 5 nd p < 1 ccording to one-wy ANOVA followed y Tukey s post hoc test Cytokine mrna (reltive expression) c Cytokine mrna (reltive expression) V +V +V +V +V Mcp1 Tnfα Il1 Il6 Il1 Vildgliptin Vildgliptin Vildgliptin Vildgliptin Vildgliptin Mcp1 Tnfα Il1 Il6 Il1 Fig 9 In n in vivo experiment, mice were fed control diet, or + vildgliptin (+V) In n ex vivo experiment, PCLS were exposed to control medium or medium with vildgliptin Gene expression of () iomrkers of immune cell popultions, nd () cytokines in the liver in the in vivo experiment nd (c) cytokines in PCLS in the ex vivo experiment In (, ), significnt differences etween conditions re shown s p <5 nd p < 1 ccording to one-wy ANOVA followed y Tukey s post hoc test In (c), dt were nlysed with Welch s t test showed tht Oscillicter spp ws the most striking trget of vildgliptin, ut its reduction ws not explined y DPP-4 inhiition Only one study hs considered the impct of the DPP-4 inhiitors sxgliptin, vildgliptin nd sitgliptin on cteril DPP-4-like ctivity [4] Inhiition of the DPP-4- like ctivity of Streptococcus mutns found in the orl microiot impired its iofilm formtion [4] None of the DPP-4 inhiitors tested ffected the growth of S mutns in the concentrtion rnge considered (4 48 μg/ml) [4] In our study, however, vildgliptin (6 μg/ml) inhiited the growth of O vlericigenes, even though this cterium does not exhiit DPP-4 ctivity Tken together these results demonstrte the impct of vildgliptin on different memers of the microiot (intestinl nd orl) vi inhiition of DPP-4-like ctivity

9 1846 Dietologi (18) 61: nd through mechnism still unknown A recent study reported chnges in the composition of the gut microiot y vildgliptin in Sprgue Dwley rt models of dietes [6] In greement with our results, vildgliptin cused reduction in Oscillicter tht ws interpreted s eneficil effect [6] Furthermore, other niml studies hve ssocited increses in Oscillicter with oese nd dietic phenotypes [41 43], nd with increses in intestinl permeility, condition involved in the development of metolic disorders [41] Consequently, vildgliptin induces eneficil chnges in the composition of the intestinl microiot tht might ecome therpeutic strtegy eyond the preservtion of incretins The gut is populted with multiple types of immune nd epithelil cells tht work together to mintin tolernce to the gut microiot nd food [17] The interction etween microiot nd host cells is medited y TLRs, which re sensors involved in the innte immune response In ddition, chemicl rrier consisting of the secretions of AMPs nd other cell products (cytokines nd immunogloulins) is prt of the first line of defence [44] Here, on the one hnd, we hve shown tht vildgliptin reduces levels of TLR- nd TLR-4 gonists in the cecl content Previously, it hs een reported tht vildgliptin suppresses TLR- nd TLR-4 content in mcrophges stimulted with solule DPP-4 nd lipopolyscchride [13] This oservtion is in greement with the reduction in TLR-4 gonist ctivity (such s lipopolyscchride) in the cecl content of mice treted with vildgliptin nd demonstrtes tht vildgliptin impcts the crosstlk etween the gut microiot nd the host On the other hnd, vildgliptin lso influenced the innte immune response vi restortion of AMP expression in the ileum In greement with previous studies crried out y our group, ft-enriched diet cused reduction in AMPs [34, 45] Vildgliptin completely countercted this reduction We hypothesise tht this effect might e linked to GLP- As GLP- is inctivted y DPP- 4, vildgliptin could hve preserved its functionlity [1, 11] The role of GLP- in inducing ntimicroil products hs een shown in GLP- receptor knockout mice which hd reduced AMPs nd impired mucosl ctericidl ctivity [46] Also, the restortion of crypt depth in mice tht received vildgliptin could e explined, t lest in prt, y GLP- stimultion of crypt cell prolifertion nd inhiition of poptosis [47] Unfortuntely, we could not confirm experimentlly this hypothesis due to the lck of commercilly ville methods to mesure ctive GLP- Finlly, we investigted whether the chnges oserved t the intestinl level could lso hve impcted the inflmmtory tone in the liver Mice treted with vildgliptin showed reduction in the expression of lymphocytes (Cd3g), mcrophge ctivtion (Cd11c) nd proinflmmtory mrkers The ex vivo experiment in which PCLS were exposed to vildgliptin showed no chnges in ny of the inflmmtory mrkers nlysed Even if there is is etween the in vivo nd ex vivo pproches, our oservtions indicte tht the chnges induced y vildgliptin t the intestinl level might hve lso impcted heptic homeostsis Specificlly, the reinforcement of gut immunity induced y vildgliptin could result in lower exposure of the liver to microil stimuli It would e useful to evlute how vildgliptin influences gut microil ctivity nd the gut liver xis, since microil metolites tht re prone to rech the liver through the portl vein, such s ile cids (vi the frnesoid X receptor) or SCFA (like utyrte), modulte heptic inflmmtion nd therey the occurrence of non-lcoholic ftty liver disese [48, 49] For our study, we selected vildgliptin for its potentil effect on inflmmtion (reduction of TLR expression) [14] The phrmcokinetics of vildgliptin is different from tht of the other DPP-4 inhiitors (eing more lipophilic) For instnce, the percentge of vildgliptin excreted in the feces is 45%, wheres for sitgliptin nd sxgliptin it is reported to e 13% nd %, respectively [5] Therefore, the effects of other gliptins on the microiot nd relted metolic effects could e relevnt, since they re found in higher quntities in the lower intestine Humn dt my confirm this hypothesis In conclusion, we show tht in -fed mice presenting gut disorders, vildgliptin ffected the composition of the gut microiot nd improved intestinl homeostsis Due to the multifunctionl roles of DPP-4, further studies re needed to decipher the precise moleculr mechnisms If the eneficil effect of vildgliptin on the intestinl trct is confirmed in humn studies, it might represent novel mechnism of vildgliptin to improve humn helth eyond its stndrd use Acknowledgements We thnk V Alleys, I Blve, R Selleslgh nd B Es Sd (Louvin Drug Reserch Institute, Brussels, Belgium) for their skilful technicl ssistnce We thnk B Pot (Vrije Universiteit Brussel, Brussels, Belgium) for supplying the strin of L reuteri 1-3 Dt vilility The dt tht support the findings of this study re ville from the corresponding uthor upon resonle request The sequences used for nlysis cn e found in the MG-RAST dtse under the project nme MYNEWGUT3 Funding This work ws supported y EU grnt (MyNewGut) MO is eneficiry of MOVE-IN Louvin Incoming Post-Doctorl Fellowship co-funded y the Mrie Curie Actions of the Europen Commission NS enefits from Jun de l Cierv post-doctorl contrct grnted y the Spnish Ministry of Economy, Industry nd Competitiveness (MINECO) PDC is senior reserch ssocite of the Fonds de l Recherche Scientifique (FRS-FNRS) nd is supported y FRFS-WELBIO through grnt WELBIO-CGR-17, the Fonds Billet- Ltour (Grnt for Medicl Reserch 15) nd Europen Reserch Council (ERC) Strting Grnt 13 (grnt ENIGMO) LBB is the recipient of FSR susidies (Fonds Spécil de l Recherche, Université ctholique de Louvin) Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript Contriution sttement MO, AMN nd NMD conceived nd designed the experiments MO nd AMN performed the niml experiments MB

10 Dietologi (18) 61: performed the ex vivo experiment MO, AMN, SAP, MB, NS, PDC nd LBB were responsile for the cquisition of dt, or nlysis nd interprettion of dt LBB performed the correltion nlyses MO nd NMD wrote the pper AMN, SAP, MB, NS, PDC nd LBB revised the rticle nd provided importnt intellectul content NMD plnned nd supervised ll experiments nd mnuscript preprtion All uthors red the mnuscriptndpprovedtheversiontoepulishednmdisthegurntor of this work Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4 Interntionl License ( cretivecommonsorg/licenses/y/4/), which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde References 1 Seino Y, Fukushim M, Ye D (1) GIP nd GLP-1, the two incretin hormones: similrities nd differences J Dietes Investig 1:8 3 Mulvihill EE, Drucker DJ (14) Phrmcology, physiology, nd mechnisms of ction of dipeptidyl peptidse-4 inhiitors Endocr Rev 35: Lee SA, Kim YR, Yng EJ et l (13) CD6/DPP4 levels in peripherl lood nd T cells in ptients with type dietes mellitus J Clin Endocrinol Met 98: Stengel A, Goeel-Stengel M, Teuffel P et l (14) Oese ptients hve higher circulting protein levels of dipeptidyl peptidse IV Peptides 61: Sell H, Bluher M, Kloting N et l (13) Adipose dipeptidyl peptidse-4 nd oesity: correltion with insulin resistnce nd depot-specific relese from dipose tissue in vivo nd in vitro Dietes Cre 36: Kushwh RN, Hq W, Ktti SB (14) Sixteen-yers of cliniclly relevnt dipeptidyl peptidse-iv (DPP-IV) inhiitors for tretment of type- dietes: perspective Curr Med Chem 1: Wget A, Cou C, Msseoeuf M et l (11) Physiologicl nd phrmcologicl mechnisms through which the DPP-4 inhiitor sitgliptin regultes glycemi in mice Endocrinology 15: Mulvihill EE, Vrin EM, Gldnc B et l (17) Cellulr sites nd mechnisms linking reduction of dipeptidyl peptidse-4 ctivity to control of incretin hormone ction nd glucose homeostsis Cell Met 5: Mortier A, Gouwy M, Vn Dmme J, Proost P, Struyf S (16) CD6/dipeptidylpeptidse IV-chemokine interctions: douleedged regultion of inflmmtion nd tumor iology J Leukoc Biol 99: Yzeck R, Howrth GS, Aott CA (9) Dipeptidyl peptidse inhiitors, n emerging drug clss for inflmmtory disese? 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