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U sodeoxycholic acid and i s au ine/glycine conjuga ed species educe coli ogenic dysbiosis and 1
equally supp ess expe imen al coli is in mice 2
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Lien Van den Bossche1, Pie e Hind yckx1, Lindsey De issche 1, Sa ah De iese1, Sophie Van 5
Welden1, Tom Hol oe 1, Rami o Vilchez-Va gas2, Ma ius Vi al3, Die ma H. Piepe 3, Julie 6
Vanden Bussche4, Lynn Vanhaecke4, Tom Van de Wiele2, Ma ine De Vos1 and Debby 7
Laukens1# 8
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1Depa men o Gas oen e ology, Ghen Uni e si y, Ghen , Belgium; 2Cen e o Mic obial 10
Ecology and Technology, Ghen Uni e si y, Ghen , Belgium; 3Mic obial In e ac ions and 11
P ocesses Resea ch G oup, Depa men o Medical Mic obiology, Helmhol z Cen e o In ec ion 12
Resea ch (HZI), B aunschweig, Ge many; 4Labo a o y o Chemical Analysis, Depa men o 13
Ve e ina y Public Heal h and Food Sa e y, Facul y o Ve e ina y Medicine, Ghen Uni e si y, 14
Ghen , Belgium 15
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Running i le: UDCA and i s conjuga es educe coli ogenic dysbiosis 18
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#Add ess co espondence o: Debby Laukens, debby.laukens@ugen .be. 21
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AEM Accep ed Manusc ip Pos ed Online 23 Janua y 2017
Appl. En i on. Mic obiol. doi:10.1128/AEM.02766-16
Copy igh © 2017 Ame ican Socie y o Mic obiology. All Righ s Rese ed.
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ABSTRACT 23
The p omising esul s wi h seconda y bile acids in expe imen al coli is sugges ha hey may 24
ep esen an a ac i e and sa e class o d ugs o he ea men o in lamma o y bowel diseases 25
(IBD). Howe e , he exac mechanism by which bile acid he apy con e s p o ec ion om 26
coli ogenesis is cu en ly unknown. Since he gu mic obio a plays a c ucial ole in he 27
pa hogenesis o IBD, and exogenous bile acid adminis a ion may a ec he communi y s uc u e 28
o he mic obio a, we examined he impac o he seconda y bile acid u sodeoxycholic acid 29
(UDCA) and i s au ine/glycine conjuga es on he ecal mic obial communi y s uc u e du ing 30
expe imen al coli is. Daily o al adminis a ion o UDCA, au ou sodeoxycholic acid (TUDCA) o 31
glycou sodeoxycholic acid (GUDCA) equally lowe ed he se e i y o dex an sodium sul a e-32
induced coli is in mice, as e idenced by educed body weigh loss, colonic sho ening and 33
exp ession o in lamma o y cy okines. Illumina sequencing demons a ed ha bile acid he apy 34
du ing coli is did no es o e ecal bac e ial ichness and di e si y. Howe e , bile acid he apy 35
no malized he coli is-associa ed inc eased a io o Fi micu es o Bac e oide es. In e es ingly, 36
adminis a ion o bile acids p e en ed he loss o Clos idium clus e XIVa and inc eased he 37
abundance o Akke mansia muciniphila, bac e ial species known o be pa icula ly dec eased in 38
IBD pa ien s. We conclude ha UDCA, which is an FDA-app o ed d ug o choles a ic li e 39
diso de s, could be an a ac i e ea men op ion o educe dysbiosis and imp o e in lamma ion 40
in human IBD. 41
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IMPORTANCE 43
Seconda y bile acids a e eme ging as a ac i e candida es o he ea men o in lamma o y 44
bowel disease. Al hough bile acids may a ec he in es inal mic obial communi y s uc u e, 45
which signi ican ly con ibu es o he cou se o hese in lamma o y diso de s, he impac o bile 46
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acid he apy on he ecal mic obio a du ing coli is has no ye been conside ed. He e, we s udied 47
he al e a ions in he ecal mic obial abundance in coli ic mice ollowing he adminis a ion o 48
seconda y bile acids. Ou esul s show ha seconda y bile acids educe he se e i y o coli is and 49
imp o e coli is-associa ed ecal dysbiosis a he phylum le el. This s udy indica es ha seconda y 50
bile acids migh ac as a sa e and e ec i e d ug o in lamma o y bowel disease. 51
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INTRODUCTION 53
In lamma o y bowel diseases (IBD) a e ch onic in lamma o y diso de s o he gas oin es inal 54
ac cha ac e ized by in es inal dysbiosis. Res ic ed bac e ial di e si y and unde ep esen a ion 55
o an i-in lamma o y mic oo ganisms such as Clos idium clus e XIVa and Akke mansia 56
muciniphila ep esen ypical dysbio ic ea u es in IBD (1–4). Since he in es inal mic obial 57
communi y pe o ms a wide ange o bile acid modi ica ions including deconjuga ion, 58
dehyd oxyla ion, oxida ion and epime iza ion (5), shi s in he composi ion o he gu mic obio a 59
a e associa ed wi h pe u ba ions o he ecal bile acid p o ile (6, 7). O pa icula in e es , Duboc 60
and colleagues demons a ed ha he con e sion o p ima y bile acids (syn hesized in he li e 61
om choles e ol) o seconda y bile acids (gene a ed by bac e ial modi ica ions) is impai ed in 62
IBD pa ien s (7). Because seconda y bile acids exhibi immunomodula o y unc ions (7–10), 63
inc easing seconda y bile acid le els in he in es inal lumen could be an e icien he apeu ic 64
app oach o IBD. In line wi h his hypo hesis, he adminis a ion o he seconda y hyd ophilic 65
bile acid u sodeoxycholic acid (UDCA) amelio a es expe imen al coli is bu he exac mechanism 66
p o ec ing om coli ogenesis is no ully unde s ood (11). 67
When adminis e ed o ally, unconjuga ed UDCA is apidly conjuga ed wi h glycine in humans, 68
and o a lesse ex en wi h au ine, on i s i s pass h ough he li e (12, 13). Based on he 69
obse a ion ha ecal bile acid hyd ophobici y co ela es wi h he se e i y o coli is (14), i is 70
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easonable o assume ha conjuga es o UDCA, which a e mo e hyd ophilic han unconjuga ed 71
UDCA, migh be mo e a o able he apeu ic agen s o in es inal in lamma ion. In his ega d, we 72
and o he s ha e shown ha au ou sodeoxycholic acid (TUDCA) alle ia es dex an sodium 73
sul a e (DSS)-induced coli is in mice (15, 16). The po en ial bene icial e ec o 74
glycou sodeoxycholic acid (GUDCA) in coli is, howe e , has no been add essed so a and 75
s udies compa ing he he apeu ic e ec i eness o hese di e en bile acid species a e lacking. 76
While he composi ion o he luminal bile acid pool is con olled by in es inal bac e ia, i is well 77
es ablished ha bile acids, in u n, also shape he gu mic obio a. Bile acids es ic bac e ial 78
p oli e a ion and o e g ow h di ec ly by causing memb ane damage, which is posi i ely 79
co ela ed wi h bile acid hyd ophobici y (17–19). Thus, he bac e icidal ac i i y o bile acids 80
dec eases wi h inc easing numbe s o hyd oxyl g oups and by conjuga ion o he bile acid side 81
chain wi h au ine o glycine (19). In addi ion o hei ole as an imic obial agen s, bile acids also 82
s imula e he g ow h o selec ed bac e ial species (5). Simila ly, hese p ope ies a e de e mined 83
bo h by he hyd oxyla ion pa e n and he conjuga ion s a us o he bile acid s e oid nucleus. Fo 84
example, inc eased in es inal le els o bile acids ca ying a hyd oxyl g oup a posi ion C7 o he 85
s e oid co e a o he g ow h o 7α-dehyd oxyla ing bac e ia, such as Clos idium clus e XIVa 86
membe s (20, 21). Fu he mo e, he amino acids in conjuga ed bile acids ac as mic obial 87
subs a es o dis inc bac e ial g oups; glycine is me abolized by Clos idium species (22, 23), 88
while au ine is a sou ce o sulphi e om which Bilophila wadswo hia de i es ene gy o i s 89
g ow h (24, 25). In e es ingly, a die high in sa u a ed a p omo es au ine-conjuga ion o hepa ic 90
bile acids, esul ing in he ou g ow h o B. wadswo hia and exace ba ion o coli is (26). 91
Conside ing ha he gu mic obial a chi ec u e and me abolism con ibu e o he cou se o IBD 92
(27, 28), we compa ed he he apeu ic e ec i eness o UDCA and i s au ine/glycine conjuga es 93
in DSS-induced coli is in mice and in es iga ed hei impac on he ecal mic obial communi y. 94
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MATERIALS AND METHODS 96
Animals. Male 8-week-old C57Bl/6J mice we e ob ained om Ha lan (Ha lan Labo a o ies, 97
Ho s , The Ne he lands) and main ained unde s anda d labo a o y condi ions wi h ad libi um 98
access o ood (mice main enance chow, Ca il Labo ood, Pa an Se ice, Belgium) and wa e . 99
P io o he expe imen , mice we e co-housed o homogenize gu mic obio a be ween 100
expe imen al g oups. A e a one-week acclima iza ion, mice we e assigned o he ea men 101
g oups based on body weigh s. In o de o a oid bac e ial c oss-con amina ion be ween g oups, 102
mice o di e en ea men g oups we e housed in sepa a e cages. The s udy was app o ed by he 103
Ins i u ional Re iew Boa d o he Facul y o Medicine and Heal h Science o Ghen Uni e si y 104
(ECD 2014-25). 105
Bile acid ea men . Mice we e di ided in o i e g oups (n = 8 in each g oup). Th ee o hem 106
ecei ed bile acid ea men : UDCA (Tokyo Chemical Indus y Co. L d, Toshima-Ku, Tokyo, 107
Japan), TUDCA (Calbiochem, Da ms ad , Ge many) o GUDCA (Sigma-Ald ich, Diegem, 108
Belgium). Bile acids we e dissol ed in phospha e-bu e ed saline (PBS) o Lab a il® M1944 109
(Ga e osse, Sain -P ies Cedex, F ance) and adminis e ed daily by o al ga age (500 mg/kg/day). 110
T ea men s s a ed a day 0 o DSS exposu e. A non-DSS con ol g oup and DSS con ol g oup 111
( e e ed o as placebo- ea ed g oup) ecei ed he ehicle (PBS o Lab a il®) alone. 112
Induc ion and assessmen o coli is. Acu e coli is was es ablished by adding 4% (w/ ) DSS 113
(molecula weigh 36,000–50,000; MP Biomedicals, Illki ch, F ance) o he d inking wa e o 7 114
days, ollowed by no mal wa e o 3 days. A non-DSS con ol g oup ecei ed no mal d inking 115
wa e h oughou he expe imen . Body weigh and disease ac i i y we e eco ded daily. A 116
disease ac i i y index (DAI) was calcula ed as he combined sco e o body weigh loss (0, none; 117
1, 0-10%; 2, 10-20%; 3, >20%), s ool consis ency (0, no mal d oppings; 1, loose d oppings; 2, 118
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dia hea) and ecal blood loss (0, none; 1, hemoccul posi i e; 2, g oss bleeding). Occul blood 119
was de ec ed using he Colosc een Hemoccul ki (Helena Labo a o ies Inc., Beaumon , Texas, 120
USA). Ten days a e ini ia ion o he expe imen , mice we e anes he ized and blood was 121
collec ed om he e o-o bi al sinus. The mice we e hen sac ificed by ce ical disloca ion, he 122
colons we e emo ed and hei leng hs we e measu ed. Segmen s o dis al colon we e cu , insed 123
wi h PBS and ozen in liquid ni ogen. The blood was cen i uged (10.000 pm o 10 min a 124
4°C) and se um was collec ed. All samples we e s o ed a -80°C un il u he p ocessing. 125
Luminex. Colonic issues we e homogenized in PBS con aining p o ease and phospha ase 126
inhibi o s and o al p o ein concen a ion was measu ed using he B ad o d me hod (Bio-Rad, 127
Naza e h, Belgium). P o ein le els o chemokine (C-X-C mo i ) ligand 1 (CXCL1), g anulocy e 128
colony-s imula ing ac o (G-CSF) and in e leukin (IL)-6 we e de e mined in colon homogena es 129
and se um using he Bio-Plex P o Mouse Cy okine G oup I mul iplex ki (Bio-Rad), acco ding o 130
he manu ac u e ’s ins uc ions. Measu emen s we e pe o med wi h he Bio-Plex MAGPIX 131
Mul iplex Reade and da a we e analyzed using he Bio-Plex Manage 6.1 so wa e (Bio-Rad). 132
DNA ex ac ion om ecal samples. F esh ecal pelle s we e collec ed a day 9 o coli is and 133
immedia ely s o ed a -80°C. To al DNA was ex ac ed om he ecal samples using he QIAamp 134
DNA S ool Mini Ki (Qiagen Benelux, Venlo, The Ne he lands). Fi s , 180 o 220 mg o s ool 135
was esuspended in 1.4 ml bu e ASL. Then, 0.5 g 0.1 mm Zi conia beads (Biospec P oduc s, 136
Ba les ille, Oklahoma) and 4 glass beads (Biospec P oduc s) we e added and samples we e 137
homogenized by o exing. The suspension was hen hea ed a 95°C o 15 min and he 138
manu ac u e ’s ins uc ions we e ollowed. 139
Illumina sequencing. The V1-2 egion o he 16S RNA gene was ampli ied as p e iously 140
desc ibed (29). B ie ly, in a i s 20 cycle polyme ase chain eac ion (PCR) eac ion, he 16S 141
RNA gene a ge was en iched using he well-documen ed 27F and 338R p ime s (30, 31) as 142
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p e iously speci ied (32). This eac ion mix u e was used as empla e in a second 15 cycle PCR 143
eac ion wi h p ime s comp ising sequences complemen a y o he Illumina speci ic adap o s o 144
he 5’-ends (29). The la e eac ion mix u e was hen used as empla e in a hi d 10 cycle PCR 145
eac ion wi h p ime s designed o in eg a e bo h he sequence o he speci ic Illumina 146
mul iplexing sequencing p ime s and he index p ime s. Lib a ies p epa ed by pooling equimola 147
a ios o amplicons we e inally sequenced on a MiSeq (Illumina, Haywa d, CA, USA). 148
A e wa ds, eads we e anno a ed as desc ibed by Ve s aelen e al. (33). 149
Illumina da a analysis. Da a-analysis was pe o med as p e iously desc ibed (33). A e 150
esampling o he minimum sequencing dep h using he phyloseq package (34) om he R 151
p og am (35), a o al o 8,911 eads we e ob ained. Ra e ac ion cu es we e gene a ed using he 152
egan package om R (36). All phylo ypes we e assigned a axonomic a ilia ion based on he 153
nai e Bayesian classi ica ion (RDP classi ie ) (37) wi h a h eshold o 80%. Rela i e abundances 154
o all phylo ypes we e hen compa ed be ween di e en expe imen al g oups. 155
Quan i a i e eal- ime PCR (qRT-PCR). To al ecal DNA was dilu ed 1:2 in wa e and 3 µl 156
was used in qRT-PCR wi h SYBR G een (SensiMix™ SYBR No-ROX Ki , Bioline Reagen s, 157
UK) and 250 nM o each p ime (BioLegio, Nijmegen, The Ne he lands). P ime sequences used 158
o ampli ica ion o A. muciniphila we e 5’-CAGCACGTGAAGGTGGGGAC-3’ and 5’-159
CCTTGCGGTTGGCTTCAGAT-3’ (38). A wo-s ep p og am was pe o med on he Ligh Cycle 160
480 (Roche). Cycling condi ions we e 95°C o 10 min, 45 cycles o 95°C o 10 s and 60°C o 1 161
min. The amoun o A. muciniphila 16S RNA gene in each sample was no malized o he o al 162
amoun o bac e ial 16S RNA gene. Fo he quan i ica ion o o al 16S RNA gene copies, ecal 163
DNA was dilu ed 1:10 in wa e and he uni e sal bac e ial 16S RNA gene p ime s PRBA338 164
5’-ACTCCTACGGGAGGCAGCAG-3’ and PRUN518 5’-ATTACCGCGGCTGCTGG-3’ we e 165
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used (39). To al bac e ial load was calcula ed using he o mula 2del aC / [ o al DNA 166
concen a ion]. 167
Bile acid quan i ica ion. 168
Sample p epa a ion. F esh ecal pelle s we e collec ed a day 4 o coli is and immedia ely s o ed 169
a -80°C. Be o e bile acids we e ex ac ed om ecal samples, 20 µl o an in e nal s anda d 170
solu ion (TUDCA-d5 (San a-C uz Bio echnology, Heidelbe g, Ge many) a 25 ng/µl in me hanol 171
(VWR In e na ional, Me ck Millipo e, Da ms ad , Ge many)) was added o 25 mg eces. The 172
ex ac ion p o ocol s a ed wi h he addi ion o 5 ml ice-cold ace oni ile (VWR In e na ional) 173
con aining 5% ammonium hyd oxide (Me ck Millipo e, Da ms ad , Ge many). The solu ion was 174
homogenized wi h an Ul a-Tu ax homogenize , ho oughly mixed by o exing o 1 min and 175
hen placed in an ul asonic ba h o 30 min. The esul an mix u e was cen i uged a 9,000 x g 176
o 10 min and supe na an was collec ed. The ex ac ion p ocedu e was epea ed once mo e and 177
he combined supe na an s we e subsequen ly e apo a ed unde ni ogen a 40°C. Each d y 178
ex ac was hen esuspended in 200 µl o a 40:60 mix u e o sol en A (7.5 mM ammonium 179
ace a e (Me ck Millipo e) in ul apu e wa e , pH 4.0) and sol en B (5% ace oni ile in 180
me hanol), cen i uged a 9,000 x g o 10 min and supe na an was collec ed. To compensa e o 181
ma ix e ec s, he s anda d addi ion me hod was applied o bile acid quan i ica ion (40). B ie ly, 182
supe na an was di ided in o wo equal aliquo s and ans e ed o liquid ch oma og aphy-mass 183
spec ome y ials. One aliquo was spiked wi h 20 µl o he 40:60 mix u e o sol en A and B. 184
The o he aliquo was spiked wi h 20 µl o bile acid solu ion (a 40:60 mix u e o sol en A and B, 185
supplemen ed wi h 16.5 ng/µl li hocholic acid (LCA; Sigma-Ald ich), 89.4 ng/µl UDCA (Simga-186
Ald ich), 0.75 ng/µl TUDCA (Calbiochem) and 0.75 ng/µl GUDCA (Sigma-Ald ich)). A 10 μl 187
aliquo o each sample was injec ed in o he ul a-high pe o mance liquid ch oma og aphy wi h 188
high esolu ion mass spec ome y (UHPLC-HRMS) sys em. 189
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UHPLC-HRMS analysis. Ch oma og aphic sepa a ion o bile acids was ca ied ou on an Accela 190
UHPLC sys em o The mo Fishe Scien i ic (San José, CA, USA), wi h an Acqui y UPLC HSS 191
C18 column (1.8 μm, 50 mm × 2.1 mm, Wa e s). The bina y sol en sys em consis ing o wo 192
sol en s A and B was se a a cons an low a e o 300 µl/min a 35°C. Fo elu ion, a g adien 193
p o ile was applied wi h he ollowing p opo ions ( / ) o sol en A: 0 – 1.0 min a 40%, 1.0 – 194
6.0 min om 40% o 1%, 6.0 – 8.0 min a 1%, 8.0 – 8.1 min om 1% o 40%, ollowed by 3.9 195
min o e-equilib a ion. 196
HRMS analysis was pe o med on an Exac i eTM s and-alone bench op mass spec ome e 197
(The mo Fishe Scien i ic), equipped wi h a hea ed elec osp ay ioniza ion sou ce (HESI-II), 198
ope a ing in he nega i e ioniza ion mode. Ioniza ion sou ce wo king pa ame e s we e op imized 199
and we e se o a shea h, auxilia y and sweep gas o 40, 5 and 1 a bi a y uni s (au), espec i ely, 200
hea e and capilla y empe a u e o 120°C and 375°C and ube lens, skimme , capilla y and sp ay 201
ol age o 123 V, 22 V, 43.5 V and 4 kV (+/-), espec i ely. A scan ange o m/z 300-550 was 202
selec ed and he esolu ion was se a 100,000 FWHM a 1 Hz (1 scan pe second). The au oma ic 203
gain con ol (AGC) a ge was se a high dynamic ange (3 × E6 ions) and he maximum 204
injec ion ime was 100 ms. 205
Da a p ocessing. HRMS da a p ocessing was pe o med wi h Xcalibu ™ 3.0 (The mo Fishe 206
Scien i ic). The concen a ion o a selec ed bile acid was calcula ed using he ollowing o mula 207
(40): 208
Cunk=
CSA × ARunk
ARSA – ARunk
wi h Cunk being he unknown concen a ion o he bile acid in he o iginal ecal sample, CSA being 209
he spiked concen a ion o he bile acid in he ecal sample a e s anda d addi ion, ARunk being 210
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deg ee o dysbiosis in IBD (55–57). O no e, he phylum-le el popula ion shi s om 354
Bac e oide es o Fi micu es induced by DSS in ou s udy esemble hose obse ed in obese 355
indi iduals and in animals on a high- a die , and ha e been associa ed wi h low-g ade in es inal 356
and sys emic in lamma ion in obesi y (58, 59). In his con ex , ecal calp o ec in and plasma C-357
eac i e p o ein le els showed a posi i e co ela ion wi h bac e ia belonging o he Fi micu es, 358
whe eas a nega i e co ela ion was ound be ween C- eac i e p o ein le els and speci ic g oups 359
wi hin he Bac e oide es (58). I is he e o e likely ha bile acid he apy coun e ac s he 360
de elopmen o a “p o-in lamma o y” mic obio a du ing coli is. This is specula i e since i 361
emains unknown i he changes seen in he Fi micu es/Bac e oide es a io ollowing bile acid 362
ea men a e a cause, a he han a consequence, o he bile acid an i-in lamma o y e ec . 363
Howe e , bile acid he apy did no p e en he DSS-induced dec ease in unclassi ied membe s o 364
he phylum Bac e oide es bu inc eased he ela i e abundance o Bac e oidia, which was no 365
a ec ed by DSS. Thus, we can specula e ha bile acid he apy di ec ly in e e es wi h an 366
imbalanced mic obial en i onmen . 367
We demons a ed ha Clos idium clus e XIVa species we e signi ican ly unde ep esen ed upon 368
DSS challenge, con i ming p e ious obse a ions in bo h human and expe imen al IBD (2, 60–369
62). Howe e , o al adminis a ion o UDCA o i s au ine/glycine conjuga es was able o p o oke 370
an en ichmen o hese species compa ed wi h placebo- ea ed mice. I has been shown ha 371
selec ed membe s wi hin he clos idial clus e XIVa possess 7α-dehyd oxyla ion ac i i y (20), 372
which is in ol ed in a mul is ep biochemical pa hway con e ing UDCA o LCA (63, 64). In ou 373
expe imen , o ally adminis e ed TUDCA and GUDCA we e apidly deconjuga ed o UDCA, so 374
ei he bile acid ea men c ea ed a subs a e- ich en i onmen o hese species. This may explain 375
he bloom o Clos idium clus e XIVa ha was obse ed in coli ic mice ha we e ea ed wi h 376
bile acids. Clos idium spp. belonging o clus e XIVa a e impo an induce s o egula o y T 377
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cells in he colon (65). In addi ion, 80% o he bu y a e-p oducing s ains isola ed om human 378
ecal samples belong o he clos idial clus e XIVa (66). Bu y a e is a sho -chain a y acid wi h 379
dis inc i e an i-in lamma o y p ope ies ha has al eady p o en i s e icacy in C ohn’s disease 380
(67). Howe e , bu y a e-p oducing bac e ia a e deple ed in he ecal mic obio a o IBD pa ien s 381
(61, 62, 68). Thus, ou obse a ion ha UDCA o i s au ine/glycine conjuga es inc eased he 382
abundance o Clos idium clus e XIVa du ing colonic in lamma ion is o pa icula in e es and 383
may sugges an immunomodula o y ole o hese bile acids. 384
Ano he inding o his s udy was he o e ep esen a ion o Bac e oidaceae, P e o ellaceae and 385
Akke mansia in ecal samples o bile acid- ea ed mice ollowing DSS exposu e. These esul s 386
migh be ela ed o he s imula o y e ec o bile acids on mucin sec e ion as a de ense 387
mechanism o p o ec he gas oin es inal epi helium agains po en ial bile acid oxici y (69–71). 388
Bac e ial species belonging o he gene a Bac e oides, P e o ella and Akke mansia p oduce one 389
o mo e enzymes equi ed o mucin deg ada ion (72), which is enhanced du ing he acu e phase 390
o DSS-induced coli is (46). The e o e, i is easonable o assume ha hese bac e ia can g ow 391
be e in an en i onmen ha is, esul ing om exogenous bile acid adminis a ion, en iched wi h 392
mucins. A. muciniphila is a commensal bac e ium esiding in he mucus laye o he in es inal 393
ac and has been shown o be educed in IBD pa ien s (3, 4). Al hough con lic ing esul s we e 394
ob ained in s udies assessing he ole o Akke mansia in colonic in lamma ion, hese species a e 395
hough o play a key ole in he egula ion o gu ba ie unc ion and mucosal immune esponses 396
owa d he commensal mic obio a (73, 74). 397
The molecula s uc u e o a bile acid de e mines i s me abolism, physicochemical p ope ies and 398
biological e ec s (75). In he p esen s udy, we used h ee bile acids sha ing he same s e oidal 399
hyd oxyla ion pa e n bu di e ing in hei amino acid conjuga ion pa e n. Nei he bile acid 400
species es ed p o ed o be mo e o less e icacious han he o he in educing colonic 401
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in lamma ion. Likewise, adminis a ion o UDCA induced simila changes in he bac e ial 402
communi y compa ed wi h i s au ine/glycine conjuga ed species. These obse a ions can be 403
explained by he apid in i o bio ans o ma ion o o ally adminis e ed bile acids by he li e and 404
by he in es inal mic obio a. Wi h he excep ion o ecal GUDCA concen a ions, which we e 405
only inc eased ollowing GUDCA he apy, he e we e no di e ences in ecal concen a ions o 406
UDCA, TUDCA o LCA be ween mice ha we e adminis e ed UDCA o i s conjuga es. This is 407
in con as wi h da a om p e ious s udies in pa ien s wi h p ima y bilia y ci hosis (12) and a s 408
(43) showing ha , compa ed wi h UDCA, o ally adminis e ed TUDCA unde goes educed 7-409
dehyd oxyla ion o LCA. I is concei able ha in e species di e ences in in es inal mic obio a 410
accoun o hese disc epancies. Fo example, deconjuga ion o TUDCA o GUDCA is a 411
p e equisi e o u he 7-dehyd oxyla ion and is ca alyzed by bile sal hyd olases (43). Because 412
Lac obacilli, which exp ess bile sal hyd olases, a e mo e abundan in he mouse gu mic obio a 413
as compa ed o he human gu mic obio a (76), i is likely ha bio ans o ma ion o hese 414
conjuga ed bile acids occu s o a la ge ex en in mice. 415
In summa y, we epo ha UDCA and i s au ine/glycine conjuga ed species amelio a e colonic 416
in lamma ion in mice wi hou di e ing in he apeu ic e ec i eness, and educe DSS-induced 417
ecal dysbiosis a he phylum le el, i espec i e o he bile acid conjuga ion s a us. As we 418
demons a ed no ad an age o using ei he he au ine o glycine conjuga e o UDCA, we sugges 419
ha UDCA could be a sa e and eadily a ailable ea men op ion o IBD. This conclusion is 420
u he suppo ed by he cu en he apeu ic use o UDCA in choles a ic pa ien s (77) and by i s 421
p e en i e e ec s on IBD-associa ed colo ec al ca cinogenesis (78–80). 422
423
ACKNOWLEDGEMENTS 424
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The au ho s a e g a e ul o D . Falk Pha maceu icals, who kindly p o ided TUDCA o esea ch 425
pu poses. We would also like o hank Hilde De lies, G ie D iesschae and Pe a Van 426
Wassenho e o p o iding echnical assis ance. The e a e no con lic s o in e es o decla e. 427
428
FUNDING INFORMATION 429
This wo k was suppo ed by esea ch g an s om he Resea ch Founda ion Flande s (FWO; 430
11J9915N and 1298213N), a conce ed ac ion g an (GOA) om he Special Resea ch Fund 431
(BOF; GOA 2012/01G00812) o Ghen Uni e si y, and a g an om he Belgian ounda ion o 432
C ohn’s disease and ulce a i e coli is pa ien s (CCV zw, esea ch g an 2014). 433
434
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eu hanasia on day 10. (D,E) Cy okine le els o CXCL1, G-CSF and IL-6 in colonic issue (D) 689
and se um (E) collec ed on day 10. Da a a e ep esen ed as he mean ± SEM (n = 8 in each 690
g oup). *P<0.05, **P<0.01. C = Con ol; P = Placebo; T = TUDCA; U = UDCA; G = GUDCA. 691
Figu e 2. O al adminis a ion o UDCA, TUDCA and GUDCA du ing DSS-induced coli is 692
p e en s coli is-associa ed dysbiosis a he phylum le el. Fecal samples we e collec ed on day 693
9 o coli is and mic obio a p o iles we e cha ac e ized by 16S RNA Illumina MiSeq sequencing. 694
(A-C) Es ima ion o (A) species ichness (i.e., o al numbe o ope a ional axonomic uni s), (B) 695
species di e si y (i.e., Shannon index) and (C) bac e ial load in he ecal mic obio a. Bac e ial 696
load was calcula ed as 2del aC / [ o al DNA concen a ion]. (D) Ra io o he pe cen age o 16S 697
RNA gene sequences belonging o Fi micu es and Bac e oide es. (E) Composi ion o he ecal 698
mic obial communi y a he phylum le el. Da a a e ep esen ed as he mean ± SEM (n ≥ 6 in each 699
g oup). *P<0.05, **P<0.01. C = Con ol; P = Placebo; T = TUDCA; U = UDCA; G = GUDCA. 700
Figu e 3. O al adminis a ion o UDCA, TUDCA and GUDCA du ing DSS-induced coli is 701
al e s he ecal mic obio a a lowe axonomic le els. Fecal samples we e collec ed on day 9 o 702
coli is and mic obio a p o iles we e cha ac e ized by 16S RNA Illumina MiSeq sequencing. (A) 703
Composi ion o he ecal mic obial communi y a he class le el. (B-F) Pe cen age o 16S RNA 704
gene sequences belonging o (B) Bac e oidaceae, (C) Po phy omonadaceae, (D) P e o ellaceae, 705
(E) Clos idium clus e XIVa and (F) Akke mansia. (G) qRT-PCR esul s o A. muciniphila. 706
Copy numbe s we e no malized o he 16S RNA gene copy numbe in each sample. Da a a e 707
ep esen ed as he mean ± SEM (n ≥ 6 in each g oup). *P<0.05, **P<0.01. C = Con ol; P = 708
Placebo; T = TUDCA; U = UDCA; G = GUDCA. 709
Figu e 4. O ally adminis e ed UDCA, TUDCA and GUDCA unde go ex ensi e 710
bio ans o ma ion. Fecal samples we e collec ed a day 4 o coli is and bile acids we e 711
quan i ied using UHPLC-HRMS. (A) UDCA, (B) TUDCA, (C) LCA and (D) GUDCA. Da a a e 712
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33
ep esen ed as he mean ± SEM (n ≥ 7 in each g oup). *P<0.05, **P<0.01. C = Con ol; P = 713
Placebo; T = TUDCA; U = UDCA; G = GUDCA. 714
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*
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Figu e 1
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E.
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*
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** **
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Colonleng h(cm)
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*
p=0.08
DSSNo
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12345678910
-30
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-20
-15
-10
-5
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%bodyweigh change
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CPTUG
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*
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Figu e 2
A. B. C.
D. E.
CPTUG
80
85
90
95
100
DSS
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Bac e oide es
Candida us Saccha ibac e ia
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Fi micu es
P o eobac e ia
Unclassi ied Bac e ia
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B. C. D.
E.
Bac e oidaceae
Rela i e abundance(%)
CPTUG
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DSSNo
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**
Po phy omonadaceae
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8***
DSSNo
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P e o ellaceae
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8
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**
Akke mansia
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0.20
0.25
DSSNo
DSS
*
p=0.06
A. muciniphila
No malized copy numbe
CPTUG
0
500
1000
1500
2000
2500
3000
DSSNo
DSS
**
p=0.086
*
Clos idium clus e XIVa
Rela i e abundance(%)
CPTUG
0.0
0.1
0.2
0.3
0.4
0.5
DSSNo
DSS
**
*
F. G.
CPTUG
70
75
80
85
90
95
100
DSS
No
DSS
Bac e oidia
Unclassi ied Bac e oide es
Saccha ibac e ia_gene a_ince ae_sedis
De e ibac e es
Bacilli
Clos idia
E ysipelo ichia
Unclassi ied Fi micu es
Be ap o eobac e ia
Del ap o eobac e ia
Gammap o eobac e ia
Unclassi ied P o eobac e ia
Ve ucomic obiae
Unclassi ied Bac e ia
on Feb ua y 20, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aem.asm.o g/Downloaded om
D.
Figu e 4
A. B.
C.
UDCA
ng /mg eces
CPTUG
0
500
1000
1500
2000
2500
3000
**
**
DSS
No
DSS
TUDCA
ng /mg eces
CPTUG
0
50
100
150
200
*
**
DSS
No
DSS
LCA
ng /mg eces
CPTUG
0
200
400
600
800
1000
1200
DSS
No
DSS
**
**
GUDCA
ng /mg eces
CPTUG
0
50
100
150
200
250
**
DSS
No
DSS
on Feb ua y 20, 2017 by Helmhol z-Zen um ue In ek ions o schung - BIBLIOTHEK-h p://aem.asm.o g/Downloaded om