Ursodeoxycholic acid and its taurine/glycine conjugated species reduce colitogenic dysbiosis and equally suppress experimental colitis in mice.

1 Ursodeoxycholic acid and its taurine/glycine conjugated species reduce colitogenic dysbiosis and 1 equally suppress experimental colitis in mice 2 3 4 Lien Van den Bossche1, Pieter Hindryckx1, Lindsey Devisscher1, Sarah Devriese1, Sophie Van 5 Welden1, Tom Holvoet1, Ramiro Vilchez-Vargas2, Marius Vital3, Dietmar H. Pieper3, Julie 6 Vanden Bussche4, Lynn Vanhaecke4, Tom Van de Wiele2, Martine De Vos1 and Debby 7 Laukens1# 8 9 1Department of Gastroenterology, Ghent University, Ghent, Belgium; 2Center for Microbial 10 Ecology and Technology, Ghent University, Ghent, Belgium; 3Microbial Interactions and 11 Processes Research Group, Department of Medical Microbiology, Helmholtz Centre for Infection 12 Research (HZI), Braunschweig, Germany; 4Laboratory of Chemical Analysis, Department of 13 Veterinary Public Health and Food Safety, Faculty of Veterinary Medicine, Ghent University, 14 Ghent, Belgium 15 16 17 Running title: UDCA and its conjugates reduce colitogenic dysbiosis 18 19 20 #Address correspondence to: Debby Laukens, [email protected]. 21 22 AEM Accepted Manuscript Posted Online 23 January 2017 Appl. Environ. Microbiol. doi:10.1128/AEM.02766-16 Copyright © 2017 American Society for Microbiology. All Rights Reserved. on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 2 ABSTRACT 23 The promising results with secondary bile acids in experimental colitis suggest that they may 24 represent an attractive and safe class of drugs for the treatment of inflammatory bowel diseases 25 (IBD). However, the exact mechanism by which bile acid therapy confers protection from 26 colitogenesis is currently unknown. Since the gut microbiota plays a crucial role in the 27 pathogenesis of IBD, and exogenous bile acid administration may affect the community structure 28 of the microbiota, we examined the impact of the secondary bile acid ursodeoxycholic acid 29 (UDCA) and its taurine/glycine conjugates on the fecal microbial community structure during 30 experimental colitis. Daily oral administration of UDCA, tauroursodeoxycholic acid (TUDCA) or 31 glycoursodeoxycholic acid (GUDCA) equally lowered the severity of dextran sodium sulfate-32 induced colitis in mice, as evidenced by reduced body weight loss, colonic shortening and 33 expression of inflammatory cytokines. Illumina sequencing demonstrated that bile acid therapy 34 during colitis did not restore fecal bacterial richness and diversity. However, bile acid therapy 35 normalized the colitis-associated increased ratio of Firmicutes to Bacteroidetes. Interestingly, 36 administration of bile acids prevented the loss of Clostridium cluster XIVa and increased the 37 abundance of Akkermansia muciniphila, bacterial species known to be particularly decreased in 38 IBD patients. We conclude that UDCA, which is an FDA-approved drug for cholestatic liver 39 disorders, could be an attractive treatment option to reduce dysbiosis and improve inflammation 40 in human IBD. 41 42 IMPORTANCE 43 Secondary bile acids are emerging as attractive candidates for the treatment of inflammatory 44 bowel disease. Although bile acids may affect the intestinal microbial community structure, 45 which significantly contributes to the course of these inflammatory disorders, the impact of bile 46 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 3 acid therapy on the fecal microbiota during colitis has not yet been considered. Here, we studied 47 the alterations in the fecal microbial abundance in colitic mice following the administration of 48 secondary bile acids. Our results show that secondary bile acids reduce the severity of colitis and 49 improve colitis-associated fecal dysbiosis at the phylum level. This study indicates that secondary 50 bile acids might act as a safe and effective drug for inflammatory bowel disease. 51 52 INTRODUCTION 53 Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal 54 tract characterized by intestinal dysbiosis. Restricted bacterial diversity and underrepresentation 55 of anti-inflammatory microorganisms such as Clostridium cluster XIVa and Akkermansia 56 muciniphila represent typical dysbiotic features in IBD (1–4). Since the intestinal microbial 57 community performs a wide range of bile acid modifications including deconjugation, 58 dehydroxylation, oxidation and epimerization (5), shifts in the composition of the gut microbiota 59 are associated with perturbations of the fecal bile acid profile (6, 7). Of particular interest, Duboc 60 and colleagues demonstrated that the conversion of primary bile acids (synthesized in the liver 61 from cholesterol) to secondary bile acids (generated by bacterial modifications) is impaired in 62 IBD patients (7). Because secondary bile acids exhibit immunomodulatory functions (7–10), 63 increasing secondary bile acid levels in the intestinal lumen could be an efficient therapeutic 64 approach for IBD. In line with this hypothesis, the administration of the secondary hydrophilic 65 bile acid ursodeoxycholic acid (UDCA) ameliorates experimental colitis but the exact mechanism 66 protecting from colitogenesis is not fully understood (11). 67 When administered orally, unconjugated UDCA is rapidly conjugated with glycine in humans, 68 and to a lesser extent with taurine, on its first pass through the liver (12, 13). Based on the 69 observation that fecal bile acid hydrophobicity correlates with the severity of colitis (14), it is 70 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 4 reasonable to assume that conjugates of UDCA, which are more hydrophilic than unconjugated 71 UDCA, might be more favorable therapeutic agents for intestinal inflammation. In this regard, we 72 and others have shown that tauroursodeoxycholic acid (TUDCA) alleviates dextran sodium 73 sulfate (DSS)-induced colitis in mice (15, 16). The potential beneficial effect of 74 glycoursodeoxycholic acid (GUDCA) in colitis, however, has not been addressed so far and 75 studies comparing the therapeutic effectiveness of these different bile acid species are lacking. 76 While the composition of the luminal bile acid pool is controlled by intestinal bacteria, it is well 77 established that bile acids, in turn, also shape the gut microbiota. Bile acids restrict bacterial 78 proliferation and overgrowth directly by causing membrane damage, which is positively 79 correlated with bile acid hydrophobicity (17–19). Thus, the bactericidal activity of bile acids 80 decreases with increasing numbers of hydroxyl groups and by conjugation of the bile acid side 81 chain with taurine or glycine (19). In addition to their role as antimicrobial agents, bile acids also 82 stimulate the growth of selected bacterial species (5). Similarly, these properties are determined 83 both by the hydroxylation pattern and the conjugation status of the bile acid steroid nucleus. For 84 example, increased intestinal levels of bile acids carrying a hydroxyl group at position C7 of the 85 steroid core favor the growth of 7α-dehydroxylating bacteria, such as Clostridium cluster XIVa 86 members (20, 21). Furthermore, the amino acids in conjugated bile acids act as microbial 87 substrates for distinct bacterial groups; glycine is metabolized by Clostridium species (22, 23), 88 while taurine is a source of sulphite from which Bilophila wadsworthia derives energy for its 89 growth (24, 25). Interestingly, a diet high in saturated fat promotes taurine-conjugation of hepatic 90 bile acids, resulting in the outgrowth of B. wadsworthia and exacerbation of colitis (26). 91 Considering that the gut microbial architecture and metabolism contribute to the course of IBD 92 (27, 28), we compared the therapeutic effectiveness of UDCA and its taurine/glycine conjugates 93 in DSS-induced colitis in mice and investigated their impact on the fecal microbial community. 94 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 5 95 MATERIALS AND METHODS 96 Animals. Male 8-week-old C57Bl/6J mice were obtained from Harlan (Harlan Laboratories, 97 Horst, The Netherlands) and maintained under standard laboratory conditions with ad libitum 98 access to food (mice maintenance chow, Carfil Labofood, Pavan Service, Belgium) and water. 99 Prior to the experiment, mice were co-housed to homogenize gut microbiota between 100 experimental groups. After a one-week acclimatization, mice were assigned to the treatment 101 groups based on body weights. In order to avoid bacterial cross-contamination between groups, 102 mice of different treatment groups were housed in separate cages. The study was approved by the 103 Institutional Review Board of the Faculty of Medicine and Health Science of Ghent University 104 (ECD 2014-25). 105 Bile acid treatment. Mice were divided into five groups (n = 8 in each group). Three of them 106 received bile acid treatment: UDCA (Tokyo Chemical Industry Co. Ltd, Toshima-Ku, Tokyo, 107 Japan), TUDCA (Calbiochem, Darmstadt, Germany) or GUDCA (Sigma-Aldrich, Diegem, 108 Belgium). Bile acids were dissolved in phosphate-buffered saline (PBS) or Labrafil® M1944 109 (Gattefosse, Saint-Priest Cedex, France) and administered daily by oral gavage (500 mg/kg/day). 110 Treatments started at day 0 of DSS exposure. A non-DSS control group and DSS control group 111 (referred to as placebo-treated group) received the vehicle (PBS or Labrafil®) alone. 112 Induction and assessment of colitis. Acute colitis was established by adding 4% (w/v) DSS 113 (molecular weight 36,000–50,000; MP Biomedicals, Illkirch, France) to the drinking water for 7 114 days, followed by normal water for 3 days. A non-DSS control group received normal drinking 115 water throughout the experiment. Body weight and disease activity were recorded daily. A 116 disease activity index (DAI) was calculated as the combined score of body weight loss (0, none; 117 1, 0-10%; 2, 10-20%; 3, >20%), stool consistency (0, normal droppings; 1, loose droppings; 2, 118 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 6 diarrhea) and fecal blood loss (0, none; 1, hemoccult positive; 2, gross bleeding). Occult blood 119 was detected using the Coloscreen Hemoccult kit (Helena Laboratories Inc., Beaumont, Texas, 120 USA). Ten days after initiation of the experiment, mice were anesthetized and blood was 121 collected from the retro-orbital sinus. The mice were then sacrificed by cervical dislocation, the 122 colons were removed and their lengths were measured. Segments of distal colon were cut, rinsed 123 with PBS and frozen in liquid nitrogen. The blood was centrifuged (10.000 rpm for 10 min at 124 4°C) and serum was collected. All samples were stored at -80°C until further processing. 125 Luminex. Colonic tissues were homogenized in PBS containing protease and phosphatase 126 inhibitors and total protein concentration was measured using the Bradford method (Bio-Rad, 127 Nazareth, Belgium). Protein levels of chemokine (C-X-C motif) ligand 1 (CXCL1), granulocyte 128 colony-stimulating factor (G-CSF) and interleukin (IL)-6 were determined in colon homogenates 129 and serum using the Bio-Plex Pro Mouse Cytokine Group I multiplex kit (Bio-Rad), according to 130 the manufacturer’s instructions. Measurements were performed with the Bio-Plex MAGPIX 131 Multiplex Reader and data were analyzed using the Bio-Plex Manager 6.1 software (Bio-Rad). 132 DNA extraction from fecal samples. Fresh fecal pellets were collected at day 9 of colitis and 133 immediately stored at -80°C. Total DNA was extracted from the fecal samples using the QIAamp 134 DNA Stool Mini Kit (Qiagen Benelux, Venlo, The Netherlands). First, 180 to 220 mg of stool 135 was resuspended in 1.4 ml buffer ASL. Then, 0.5 g 0.1 mm Zirconia beads (Biospec Products, 136 Bartlesville, Oklahoma) and 4 glass beads (Biospec Products) were added and samples were 137 homogenized by vortexing. The suspension was then heated at 95°C for 15 min and the 138 manufacturer’s instructions were followed. 139 Illumina sequencing. The V1-2 region of the 16S rRNA gene was amplified as previously 140 described (29). Briefly, in a first 20 cycle polymerase chain reaction (PCR) reaction, the 16S 141 rRNA gene target was enriched using the well-documented 27F and 338R primers (30, 31) as 142 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 7 previously specified (32). This reaction mixture was used as template in a second 15 cycle PCR 143 reaction with primers comprising sequences complementary to the Illumina specific adaptors to 144 the 5’-ends (29). The latter reaction mixture was then used as template in a third 10 cycle PCR 145 reaction with primers designed to integrate both the sequence of the specific Illumina 146 multiplexing sequencing primers and the index primers. Libraries prepared by pooling equimolar 147 ratios of amplicons were finally sequenced on a MiSeq (Illumina, Hayward, CA, USA). 148 Afterwards, reads were annotated as described by Verstraelen et al. (33). 149 Illumina data analysis. Data-analysis was performed as previously described (33). After 150 resampling to the minimum sequencing depth using the phyloseq package (34) from the R 151 program (35), a total of 8,911 reads were obtained. Rarefaction curves were generated using the 152 vegan package from R (36). All phylotypes were assigned a taxonomic affiliation based on the 153 naive Bayesian classification (RDP classifier) (37) with a threshold of 80%. Relative abundances 154 of all phylotypes were then compared between different experimental groups. 155 Quantitative real-time PCR (qRT-PCR). Total fecal DNA was diluted 1:2 in water and 3 µl 156 was used in qRT-PCR with SYBR Green (SensiMix™ SYBR No-ROX Kit, Bioline Reagents, 157 UK) and 250 nM of each primer (BioLegio, Nijmegen, The Netherlands). Primer sequences used 158 for amplification of A. muciniphila were 5’-CAGCACGTGAAGGTGGGGAC-3’ and 5’-159 CCTTGCGGTTGGCTTCAGAT-3’ (38). A two-step program was performed on the LightCycler 160 480 (Roche). Cycling conditions were 95°C for 10 min, 45 cycles of 95°C for 10 s and 60°C for 1 161 min. The amount of A. muciniphila 16S rRNA gene in each sample was normalized to the total 162 amount of bacterial 16S rRNA gene. For the quantification of total 16S rRNA gene copies, fecal 163 DNA was diluted 1:10 in water and the universal bacterial 16S rRNA gene primers PRBA338f 164 5’-ACTCCTACGGGAGGCAGCAG-3’ and PRUN518r 5’-ATTACCGCGGCTGCTGG-3’ were 165 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 8 used (39). Total bacterial load was calculated using the formula 2deltaCt / [total DNA 166 concentration]. 167 Bile acid quantification. 168 Sample preparation. Fresh fecal pellets were collected at day 4 of colitis and immediately stored 169 at -80°C. Before bile acids were extracted from fecal samples, 20 µl of an internal standard 170 solution (TUDCA-d5 (Santa-Cruz Biotechnology, Heidelberg, Germany) at 25 ng/µl in methanol 171 (VWR International, Merck Millipore, Darmstadt, Germany)) was added to 25 mg feces. The 172 extraction protocol started with the addition of 5 ml ice-cold acetonitrile (VWR International) 173 containing 5% ammonium hydroxide (Merck Millipore, Darmstadt, Germany). The solution was 174 homogenized with an Ultra-Turrax homogenizer, thoroughly mixed by vortexing for 1 min and 175 then placed in an ultrasonic bath for 30 min. The resultant mixture was centrifuged at 9,000 x g 176 for 10 min and supernatant was collected. The extraction procedure was repeated once more and 177 the combined supernatants were subsequently evaporated under nitrogen at 40°C. Each dry 178 extract was then resuspended in 200 µl of a 40:60 mixture of solvent A (7.5 mM ammonium 179 acetate (Merck Millipore) in ultrapure water, pH 4.0) and solvent B (5% acetonitrile in 180 methanol), centrifuged at 9,000 x g for 10 min and supernatant was collected. To compensate for 181 matrix effects, the standard addition method was applied for bile acid quantification (40). Briefly, 182 supernatant was divided into two equal aliquots and transferred to liquid chromatography-mass 183 spectrometry vials. One aliquot was spiked with 20 µl of the 40:60 mixture of solvent A and B. 184 The other aliquot was spiked with 20 µl of bile acid solution (a 40:60 mixture of solvent A and B, 185 supplemented with 16.5 ng/µl lithocholic acid (LCA; Sigma-Aldrich), 89.4 ng/µl UDCA (Simga-186 Aldrich), 0.75 ng/µl TUDCA (Calbiochem) and 0.75 ng/µl GUDCA (Sigma-Aldrich)). A 10 μl 187 aliquot of each sample was injected into the ultra-high performance liquid chromatography with 188 high resolution mass spectrometry (UHPLC-HRMS) system. 189 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 9 UHPLC-HRMS analysis. Chromatographic separation of bile acids was carried out on an Accela 190 UHPLC system of Thermo Fisher Scientific (San José, CA, USA), with an Acquity UPLC HSS 191 C18 column (1.8 μm, 50 mm × 2.1 mm, Waters). The binary solvent system consisting of two 192 solvents A and B was set at a constant flow rate of 300 µl/min at 35°C. For elution, a gradient 193 profile was applied with the following proportions (v/v) of solvent A: 0 – 1.0 min at 40%, 1.0 – 194 6.0 min from 40% to 1%, 6.0 – 8.0 min at 1%, 8.0 – 8.1 min from 1% to 40%, followed by 3.9 195 min of re-equilibration. 196 HRMS analysis was performed on an ExactiveTM stand-alone benchtop mass spectrometer 197 (Thermo Fisher Scientific), equipped with a heated electrospray ionization source (HESI-II), 198 operating in the negative ionization mode. Ionization source working parameters were optimized 199 and were set to a sheath, auxiliary and sweep gas of 40, 5 and 1 arbitrary units (au), respectively, 200 heater and capillary temperature of 120°C and 375°C and tube lens, skimmer, capillary and spray 201 voltage of 123 V, 22 V, 43.5 V and 4 kV (+/-), respectively. A scan range of m/z 300-550 was 202 selected and the resolution was set at 100,000 FWHM at 1 Hz (1 scan per second). The automatic 203 gain control (AGC) target was set at high dynamic range (3 × E6 ions) and the maximum 204 injection time was 100 ms. 205 Data processing. HRMS data processing was performed with Xcalibur™ 3.0 (Thermo Fisher 206 Scientific). The concentration of a selected bile acid was calculated using the following formula 207 (40): 208 Cunk= CSA × ARunk ARSA – ARunk with Cunk being the unknown concentration of the bile acid in the original fecal sample, CSA being 209 the spiked concentration of the bile acid in the fecal sample after standard addition, ARunk being 210 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 16 degree of dysbiosis in IBD (55–57). Of note, the phylum-level population shifts from 354 Bacteroidetes to Firmicutes induced by DSS in our study resemble those observed in obese 355 individuals and in animals on a high-fat diet, and have been associated with low-grade intestinal 356 and systemic inflammation in obesity (58, 59). In this context, fecal calprotectin and plasma C-357 reactive protein levels showed a positive correlation with bacteria belonging to the Firmicutes, 358 whereas a negative correlation was found between C-reactive protein levels and specific groups 359 within the Bacteroidetes (58). It is therefore likely that bile acid therapy counteracts the 360 development of a “pro-inflammatory” microbiota during colitis. This is speculative since it 361 remains unknown if the changes seen in the Firmicutes/Bacteroidetes ratio following bile acid 362 treatment are a cause, rather than a consequence, of the bile acid anti-inflammatory effect. 363 However, bile acid therapy did not prevent the DSS-induced decrease in unclassified members of 364 the phylum Bacteroidetes but increased the relative abundance of Bacteroidia, which was not 365 affected by DSS. Thus, we can speculate that bile acid therapy directly interferes with an 366 imbalanced microbial environment. 367 We demonstrated that Clostridium cluster XIVa species were significantly underrepresented upon 368 DSS challenge, confirming previous observations in both human and experimental IBD (2, 60–369 62). However, oral administration of UDCA or its taurine/glycine conjugates was able to provoke 370 an enrichment of these species compared with placebo-treated mice. It has been shown that 371 selected members within the clostridial cluster XIVa possess 7α-dehydroxylation activity (20), 372 which is involved in a multistep biochemical pathway converting UDCA to LCA (63, 64). In our 373 experiment, orally administered TUDCA and GUDCA were rapidly deconjugated to UDCA, so 374 either bile acid treatment created a substrate-rich environment for these species. This may explain 375 the bloom of Clostridium cluster XIVa that was observed in colitic mice that were treated with 376 bile acids. Clostridium spp. belonging to cluster XIVa are important inducers of regulatory T 377 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 17 cells in the colon (65). In addition, 80% of the butyrate-producing strains isolated from human 378 fecal samples belong to the clostridial cluster XIVa (66). Butyrate is a short-chain fatty acid with 379 distinctive anti-inflammatory properties that has already proven its efficacy in Crohn’s disease 380 (67). However, butyrate-producing bacteria are depleted in the fecal microbiota of IBD patients 381 (61, 62, 68). Thus, our observation that UDCA or its taurine/glycine conjugates increased the 382 abundance of Clostridium cluster XIVa during colonic inflammation is of particular interest and 383 may suggest an immunomodulatory role of these bile acids. 384 Another finding of this study was the overrepresentation of Bacteroidaceae, Prevotellaceae and 385 Akkermansia in fecal samples of bile acid-treated mice following DSS exposure. These results 386 might be related to the stimulatory effect of bile acids on mucin secretion as a defense 387 mechanism to protect the gastrointestinal epithelium against potential bile acid toxicity (69–71). 388 Bacterial species belonging to the genera Bacteroides, Prevotella and Akkermansia produce one 389 or more enzymes required for mucin degradation (72), which is enhanced during the acute phase 390 of DSS-induced colitis (46). Therefore, it is reasonable to assume that these bacteria can grow 391 better in an environment that is, resulting from exogenous bile acid administration, enriched with 392 mucins. A. muciniphila is a commensal bacterium residing in the mucus layer of the intestinal 393 tract and has been shown to be reduced in IBD patients (3, 4). Although conflicting results were 394 obtained in studies assessing the role of Akkermansia in colonic inflammation, these species are 395 thought to play a key role in the regulation of gut barrier function and mucosal immune responses 396 toward the commensal microbiota (73, 74). 397 The molecular structure of a bile acid determines its metabolism, physicochemical properties and 398 biological effects (75). In the present study, we used three bile acids sharing the same steroidal 399 hydroxylation pattern but differing in their amino acid conjugation pattern. Neither bile acid 400 species tested proved to be more or less efficacious than the other in reducing colonic 401 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 18 inflammation. Likewise, administration of UDCA induced similar changes in the bacterial 402 community compared with its taurine/glycine conjugated species. These observations can be 403 explained by the rapid in vivo biotransformation of orally administered bile acids by the liver and 404 by the intestinal microbiota. With the exception of fecal GUDCA concentrations, which were 405 only increased following GUDCA therapy, there were no differences in fecal concentrations of 406 UDCA, TUDCA or LCA between mice that were administered UDCA or its conjugates. This is 407 in contrast with data from previous studies in patients with primary biliary cirrhosis (12) and rats 408 (43) showing that, compared with UDCA, orally administered TUDCA undergoes reduced 7-409 dehydroxylation to LCA. It is conceivable that interspecies differences in intestinal microbiota 410 account for these discrepancies. For example, deconjugation of TUDCA or GUDCA is a 411 prerequisite for further 7-dehydroxylation and is catalyzed by bile salt hydrolases (43). Because 412 Lactobacilli, which express bile salt hydrolases, are more abundant in the mouse gut microbiota 413 as compared to the human gut microbiota (76), it is likely that biotransformation of these 414 conjugated bile acids occurs to a larger extent in mice. 415 In summary, we report that UDCA and its taurine/glycine conjugated species ameliorate colonic 416 inflammation in mice without differing in therapeutic effectiveness, and reduce DSS-induced 417 fecal dysbiosis at the phylum level, irrespective of the bile acid conjugation status. As we 418 demonstrated no advantage of using either the taurine or glycine conjugate of UDCA, we suggest 419 that UDCA could be a safe and readily available treatment option for IBD. This conclusion is 420 further supported by the current therapeutic use of UDCA in cholestatic patients (77) and by its 421 preventive effects on IBD-associated colorectal carcinogenesis (78–80). 422 423 ACKNOWLEDGEMENTS 424 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 19 The authors are grateful to Dr. Falk Pharmaceuticals, who kindly provided TUDCA for research 425 purposes. We would also like to thank Hilde Devlies, Griet Driesschaert and Petra Van 426 Wassenhove for providing technical assistance. There are no conflicts of interest to declare. 427 428 FUNDING INFORMATION 429 This work was supported by research grants from the Research Foundation Flanders (FWO; 430 11J9915N and 1298213N), a concerted action grant (GOA) from the Special Research Fund 431 (BOF; GOA 2012/01G00812) of Ghent University, and a grant from the Belgian foundation for 432 Crohn’s disease and ulcerative colitis patients (CCV vzw, research grant 2014). 433 434 REFERENCES 435 1. Nagao-Kitamoto H, Kitamoto S, Kuffa P, Kamada N. 2016. Pathogenic role of the gut 436 microbiota in gastrointestinal diseases. Intest Res 14:127–138. 437 2. Andoh A, Imaeda H, Aomatsu T, Inatomi O, Bamba S, Sasaki M, Saito Y, Tsujikawa 438 T, Fujiyama Y. 2011. Comparison of the fecal microbiota profiles between ulcerative 439 colitis and Crohn’s disease using terminal restriction fragment length polymorphism 440 analysis. J Gastroenterol 46:479–486. 441 3. Png CW, Lindén SK, Gilshenan KS, Zoetendal EG, McSweeney CS, Sly LI, McGuckin 442 MA, Florin THJ. 2010. Mucolytic bacteria with increased prevalence in IBD mucosa 443 augment in vitro utilization of mucin by other bacteria. Am J Gastroenterol 105:2420–2428. 444 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 20 4. Vigsnæs LK, Brynskov J, Steenholdt C, Wilcks A, Licht TR. 2012. Gram-negative 445 bacteria account for main differences between faecal microbiota from patients with 446 ulcerative colitis and healthy controls. Benef Microbes 3:287–297. 447 5. Wahlström A, Sayin SI, Marschall H-U, Bäckhed F. 2016. Intestinal Crosstalk between 448 Bile Acids and Microbiota and Its Impact on Host Metabolism. Cell Metab 24:41–50. 449 6. Kakiyama G, Pandak WM, Gillevet PM, Hylemon PB, Heuman DM, Daita K, Takei 450 H, Muto A, Nittono H, Ridlon JM, White MB, Noble NA, Monteith P, Fuchs M, 451 Thacker LR, Sikaroodi M, Bajaj JS. 2013. Modulation of the fecal bile acid profile by gut 452 microbiota in cirrhosis. J Hepatol 58:949–955. 453 7. Duboc H, Rajca S, Rainteau D, Benarous D, Maubert M-A, Quervain E, Thomas G, 454 Barbu V, Humbert L, Despras G, Bridonneau C, Dumetz F, Grill J-P, Masliah J, 455 Beaugerie L, Cosnes J, Chazouillères O, Poupon R, Wolf C, Mallet J-M, Langella P, 456 Trugnan G, Sokol H, Seksik P. 2013. Connecting dysbiosis, bile-acid dysmetabolism and 457 gut inflammation in inflammatory bowel diseases. Gut 62:531–539. 458 8. Calmus Y, Weill B, Ozier Y, Chéreau C, Houssin D, Poupon R. 1992. 459 Immunosuppressive properties of chenodeoxycholic and ursodeoxycholic acids in the 460 mouse. Gastroenterology 103:617–621. 461 9. Greve JW, Gouma DJ, Buurman WA. 1989. Bile acids inhibit endotoxin-induced release 462 of tumor necrosis factor by monocytes: an in vitro study. Hepatol Baltim Md 10:454–458. 463 10. Yoneno K, Hisamatsu T, Shimamura K, Kamada N, Ichikawa R, Kitazume MT, Mori 464 M, Uo M, Namikawa Y, Matsuoka K, Sato T, Koganei K, Sugita A, Kanai T, Hibi T. 465 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 21 2013. TGR5 signalling inhibits the production of pro-inflammatory cytokines by in vitro 466 differentiated inflammatory and intestinal macrophages in Crohn’s disease. Immunology 467 139:19–29. 468 11. Martínez-Moya P, Romero-Calvo I, Requena P, Hernández-Chirlaque C, Aranda CJ, 469 González R, Zarzuelo A, Suárez MD, Martínez-Augustin O, Marín JJG, de Medina 470 FS. 2013. Dose-dependent antiinflammatory effect of ursodeoxycholic acid in experimental 471 colitis. Int Immunopharmacol 15:372–380. 472 12. Invernizzi P, Setchell KD, Crosignani A, Battezzati PM, Larghi A, O’Connell NC, 473 Podda M. 1999. Differences in the metabolism and disposition of ursodeoxycholic acid and 474 of its taurine-conjugated species in patients with primary biliary cirrhosis. Hepatol Baltim 475 Md 29:320–327. 476 13. Rudolph G, Kloeters-Plachky P, Sauer P, Stiehl A. 2002. Intestinal absorption and biliary 477 secretion of ursodeoxycholic acid and its taurine conjugate. Eur J Clin Invest 32:575–580. 478 14. Stenman LK, Holma R, Forsgård R, Gylling H, Korpela R. 2013. Higher fecal bile acid 479 hydrophobicity is associated with exacerbation of dextran sodium sulfate colitis in mice. J 480 Nutr 143:1691–1697. 481 15. Cao SS, Zimmermann EM, Chuang B-M, Song B, Nwokoye A, Wilkinson JE, Eaton 482 KA, Kaufman RJ. 2013. The unfolded protein response and chemical chaperones reduce 483 protein misfolding and colitis in mice. Gastroenterology 144:989–1000.e6. 484 16. Laukens D, Devisscher L, Van den Bossche L, Hindryckx P, Vandenbroucke RE, 485 Vandewynckel Y-P, Cuvelier C, Brinkman BM, Libert C, Vandenabeele P, De Vos M. 486 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 22 2014. Tauroursodeoxycholic acid inhibits experimental colitis by preventing early intestinal 487 epithelial cell death. Lab Investig J Tech Methods Pathol 94:1419–1430. 488 17. Kurdi P, Kawanishi K, Mizutani K, Yokota A. 2006. Mechanism of Growth Inhibition by 489 Free Bile Acids in Lactobacilli and Bifidobacteria. J Bacteriol 188:1979–1986. 490 18. Taranto MP, Perez-Martinez G, Font de Valdez G. 2006. Effect of bile acid on the cell 491 membrane functionality of lactic acid bacteria for oral administration. Res Microbiol 492 157:720–725. 493 19. Sonomoto K, Yokota A. 2011. Lactic Acid Bacteria and Bifidobacteria: Current Progress 494 in Advanced Research. Horizon Scientific Press. 495 20. Ridlon JM, Alves JM, Hylemon PB, Bajaj JS. 2013. Cirrhosis, bile acids and gut 496 microbiota. Gut Microbes 4:382–387. 497 21. Islam KBMS, Fukiya S, Hagio M, Fujii N, Ishizuka S, Ooka T, Ogura Y, Hayashi T, 498 Yokota A. 2011. Bile acid is a host factor that regulates the composition of the cecal 499 microbiota in rats. Gastroenterology 141:1773–1781. 500 22. Dürre P, Andreesen JR. 1983. Purine and glycine metabolism by purinolytic clostridia. J 501 Bacteriol 154:192–199. 502 23. Lebertz H, Andreesen JR. 1988. Glycine fermentation by Clostridium histolyticum. Arch 503 Microbiol 150:11–14. 504 24. Laue H, Denger K, Cook AM. 1997. Taurine reduction in anaerobic respiration of 505 Bilophila wadsworthia RZATAU. Appl Environ Microbiol 63:2016–2021. 506 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 23 25. Laue H, Smits THM, Schumacher UK, Claros MC, Hartemink R, Cook AM. 2006. 507 Identification of Bilophila wadsworthia by specific PCR which targets the taurine:pyruvate 508 aminotransferase gene. FEMS Microbiol Lett 261:74–79. 509 26. Devkota S, Wang Y, Musch MW, Leone V, Fehlner-Peach H, Nadimpalli A, 510 Antonopoulos DA, Jabri B, Chang EB. 2012. Dietary-fat-induced taurocholic acid 511 promotes pathobiont expansion and colitis in Il10-/- mice. Nature 487:104–108. 512 27. Wlodarska M, Kostic AD, Xavier RJ. 2015. An integrative view of microbiome-host 513 interactions in inflammatory bowel diseases. Cell Host Microbe 17:577–591. 514 28. Brinkman BM, Becker A, Ayiseh RB, Hildebrand F, Raes J, Huys G, Vandenabeele P. 515 2013. Gut microbiota affects sensitivity to acute DSS-induced colitis independently of host 516 genotype. Inflamm Bowel Dis 19:2560–2567. 517 29. Camarinha-Silva A, Jáuregui R, Chaves-Moreno D, Oxley APA, Schaumburg F, 518 Becker K, Wos-Oxley ML, Pieper DH. 2014. Comparing the anterior nare bacterial 519 community of two discrete human populations using Illumina amplicon sequencing. 520 Environ Microbiol 16:2939–2952. 521 30. LANE DJ. 1991. 16S/23S rRNA sequencing. Nucleic Acid Tech Bact Syst 125–175. 522 31. Etchebehere C, Tiedje J. 2005. Presence of Two Different Active nirS Nitrite Reductase 523 Genes in a Denitrifying Thauera sp. from a High-Nitrate-Removal-Rate Reactor. Appl 524 Environ Microbiol 71:5642–5645. 525 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 24 32. Chaves-Moreno D, Plumeier I, Kahl S, Krismer B, Peschel A, Oxley APA, Jauregui R, 526 Pieper DH. 2015. The microbial community structure of the cotton rat nose. Environ 527 Microbiol Rep 7:929–935. 528 33. Verstraelen H, Vilchez-Vargas R, Desimpel F, Jauregui R, Vankeirsbilck N, Weyers S, 529 Verhelst R, De Sutter P, Pieper DH, Van De Wiele T. 2016. Characterisation of the 530 human uterine microbiome in non-pregnant women through deep sequencing of the V1-2 531 region of the 16S rRNA gene. PeerJ 4:e1602. 532 34. McMurdie PJ, Holmes S. 2013. phyloseq: An R Package for Reproducible Interactive 533 Analysis and Graphics of Microbiome Census Data. PLOS ONE 8:e61217. 534 35. R Core Team. 2012. R: A language and environment for statistical computing. R 535 Foundation for Statistical Computing, Vienna, Austria. 536 36. Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, Minchin PR, 537 O’Hara RB, Simpson GL, Solymos P, Stevens MHH, Szoecs E, Wagner H. 2016. vegan: 538 Community Ecology Package. 539 37. Wang Q, Garrity GM, Tiedje JM, Cole JR. 2007. Naïve Bayesian Classifier for Rapid 540 Assignment of rRNA Sequences into the New Bacterial Taxonomy. Appl Environ 541 Microbiol 73:5261–5267. 542 38. Schneeberger M, Everard A, Gómez-Valadés AG, Matamoros S, Ramírez S, Delzenne 543 NM, Gomis R, Claret M, Cani PD. 2015. Akkermansia muciniphila inversely correlates 544 with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders 545 during obesity in mice. Sci Rep 5. 546 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 25 39. Ovreås L, Lj F, Daae FL, Vl T. 1997. Distribution of bacterioplankton in Meromictic lake 547 Sælenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified 548 gene fragments coding for 16S rRNA. Appl Environ Microbiol 63:3367–73. 549 40. Cai X, Liu Y, Zhou X, Navaneethan U, Shen B, Guo B. 2012. An LC-ESI-MS method for 550 the quantitative analysis of bile acids composition in fecal materials. Biomed Chromatogr 551 BMC 26:101–108. 552 41. Alex P, Zachos NC, Nguyen T, Gonzales L, Chen T-E, Conklin LS, Centola M, Li X. 553 2009. Distinct cytokine patterns identified from multiplex profiles of murine DSS and 554 TNBS-induced colitis. Inflamm Bowel Dis 15:341–352. 555 42. Peter MR, Jerkic M, Sotov V, Douda DN, Ardelean DS, Ghamami N, Lakschevitz F, 556 Khan MA, Robertson SJ, Glogauer M, Philpott DJ, Palaniyar N, Letarte M. 2014. 557 Impaired Resolution of Inflammation in the Endoglin Heterozygous Mouse Model of 558 Chronic Colitis. Mediators Inflamm 2014. 559 43. Rodrigues CM, Kren BT, Steer CJ, Setchell KD. 1995. Tauroursodeoxycholate increases 560 rat liver ursodeoxycholate levels and limits lithocholate formation better than 561 ursodeoxycholate. Gastroenterology 109:564–572. 562 44. Alnouti Y, Csanaky IL, Klaassen CD. 2008. Quantitative-Profiling of Bile Acids and their 563 Conjugates in Mouse Liver, Bile, Plasma, and Urine Using LC-MS/MS. J Chromatogr B 564 Analyt Technol Biomed Life Sci 873:209–217. 565 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 32 euthanasia on day 10. (D,E) Cytokine levels of CXCL1, G-CSF and IL-6 in colonic tissue (D) 689 and serum (E) collected on day 10. Data are represented as the mean ± SEM (n = 8 in each 690 group). *P<0.05, **P<0.01. C = Control; P = Placebo; T = TUDCA; U = UDCA; G = GUDCA. 691 Figure 2. Oral administration of UDCA, TUDCA and GUDCA during DSS-induced colitis 692 prevents colitis-associated dysbiosis at the phylum level. Fecal samples were collected on day 693 9 of colitis and microbiota profiles were characterized by 16S rRNA Illumina MiSeq sequencing. 694 (A-C) Estimation of (A) species richness (i.e., total number of operational taxonomic units), (B) 695 species diversity (i.e., Shannon index) and (C) bacterial load in the fecal microbiota. Bacterial 696 load was calculated as 2deltaCt / [total DNA concentration]. (D) Ratio of the percentage of 16S 697 rRNA gene sequences belonging to Firmicutes and Bacteroidetes. (E) Composition of the fecal 698 microbial community at the phylum level. Data are represented as the mean ± SEM (n ≥ 6 in each 699 group). *P<0.05, **P<0.01. C = Control; P = Placebo; T = TUDCA; U = UDCA; G = GUDCA. 700 Figure 3. Oral administration of UDCA, TUDCA and GUDCA during DSS-induced colitis 701 alters the fecal microbiota at lower taxonomic levels. Fecal samples were collected on day 9 of 702 colitis and microbiota profiles were characterized by 16S rRNA Illumina MiSeq sequencing. (A) 703 Composition of the fecal microbial community at the class level. (B-F) Percentage of 16S rRNA 704 gene sequences belonging to (B) Bacteroidaceae, (C) Porphyromonadaceae, (D) Prevotellaceae, 705 (E) Clostridium cluster XIVa and (F) Akkermansia. (G) qRT-PCR results for A. muciniphila. 706 Copy numbers were normalized to the 16S rRNA gene copy number in each sample. Data are 707 represented as the mean ± SEM (n ≥ 6 in each group). *P<0.05, **P<0.01. C = Control; P = 708 Placebo; T = TUDCA; U = UDCA; G = GUDCA. 709 Figure 4. Orally administered UDCA, TUDCA and GUDCA undergo extensive 710 biotransformation. Fecal samples were collected at day 4 of colitis and bile acids were 711 quantified using UHPLC-HRMS. (A) UDCA, (B) TUDCA, (C) LCA and (D) GUDCA. Data are 712 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 33 represented as the mean ± SEM (n ≥ 7 in each group). *P<0.05, **P<0.01. C = Control; P = 713 Placebo; T = TUDCA; U = UDCA; G = GUDCA. 714 on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from 0123456789 0 1 2 3 4 5 6 Days DAI score ** * CPTUG 0 100 200 300 400 500 ColonCXCL1 (pg/mg totalprotein) ** ** DSSNo DSS D. Figure 1 A. E. CPTUG 0 1000 2000 3000 ColonG-CSF (pg/mg totalprotein) ** ** DSSNo DSS CPTUG 0 50 100 150 SerumIL-6(pg/ml) ** DSSNo DSS CPTUG 0 500 1000 1500 SerumCXCL1 (pg/ml) ** * DSSNo DSS CPTUG 0 10000 20000 30000 SerumG-CSF (pg/ml) ** ** * DSSNo DSS CPTUG 0 100 200 300 400 ColonIL-6 (pg/mg totalprotein) ** ** DSSNo DSS CPTUG 0 2 4 6 8 10 Colonlength(cm) ** * p=0.08 DSSNo DSS B. C. 12345678910 -30 -25 -20 -15 -10 -5 0 5 Days %bodyweightchange * DSS + GUDCA DSS + UDCA DSS + TUDCA DSS + Placebo No DSS control on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from Relative abundance(%) CPTUG 0 10 20 30 40 50 60 70 80 90 100 DSS No DSS Species richness CPTUG 0 200 400 600 800 1000 DSSNo DSS ** p=0.086 Speciesdiversity CPTUG 0 1 2 3 4 5 6 DSSNo DSS ** * Bacterialload CPTUG 0 10 20 30 40 50 60 DSSNo DSS ** F/Bratio CPTUG 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 DSSNo DSS ** * Figure 2 A. B. C. D. E. CPTUG 80 85 90 95 100 DSS No DSS Bacteroidetes Candidatus Saccharibacteria Deferribacteres Firmicutes Proteobacteria Unclassified Bacteria on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from Relative abundance(%) CPTUG 0 10 20 30 40 50 60 70 80 90 100 DSSNo DSS Figure 3 A. B. C. D. E. Bacteroidaceae Relative abundance(%) CPTUG 0 10 20 30 40 50 DSSNo DSS ** * ** Porphyromonadaceae Relative abundance(%) CPTUG 0 2 4 6 8*** DSSNo DSS Prevotellaceae Relative abundance(%) CPTUG 0 2 4 6 8 DSSNo DSS ** ** Akkermansia Relative abundance(%) CPTUG 0.00 0.05 0.10 0.15 0.20 0.25 DSSNo DSS * p=0.06 A. muciniphila Normalized copy number CPTUG 0 500 1000 1500 2000 2500 3000 DSSNo DSS ** p=0.086 * Clostridium cluster XIVa Relative 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 Bacteroidia Unclassified Bacteroidetes Saccharibacteria_genera_incertae_sedis Deferribacteres Bacilli Clostridia Erysipelotrichia Unclassified Firmicutes Betaproteobacteria Deltaproteobacteria Gammaproteobacteria Unclassified Proteobacteria Verrucomicrobiae Unclassified Bacteria on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from D. Figure 4 A. B. C. UDCA ng /mgfeces CPTUG 0 500 1000 1500 2000 2500 3000 ** ** DSS No DSS TUDCA ng /mgfeces CPTUG 0 50 100 150 200 * ** DSS No DSS LCA ng /mgfeces CPTUG 0 200 400 600 800 1000 1200 DSS No DSS ** ** GUDCA ng /mgfeces CPTUG 0 50 100 150 200 250 ** DSS No DSS on February 20, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aem.asm.org/Downloaded from