Modulation of the gut microbiota impacts nonalcoholic fatty liver disease: a potential role for bile acids

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids. Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids. The worldwide epidemic of obesity is the primary driver for the global increase in the prevalence of type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) (1Finucane M.M. Stevens G.A. Cowan M.J. Danaei G. Lin J.K. Paciorek C.J. Singh G.M. Gutierrez H.R. Lu Y. Bahalim A.N. National, regional, and global trends in body-mass index since 1980: systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants.Lancet. 2011; 377: 557-567Abstract Full Text Full Text PDF PubMed Scopus (3129) Google Scholar). While caloric overconsumption and the associated positive energy balance are the sine qua non of obesity development, emerging evidence implicates gut microbes in the promotion of obesity and related metabolic disturbances (2Ridaura V.K. Faith J.J. Rey F.E. Cheng J. Duncan A.E. Kau A.L. Griffin N.W. Lombard V. Henrissat B. Bain J.R. Gut microbiota from twins discordant for obesity modulate metabolism in mice.Science. 2013; 341: 1241214Crossref PubMed Scopus (2435) Google Scholar, 3Vrieze A. Van Nood E. Holleman F. Salojärvi J. Kootte R.S. Bartelsman J.F.W.M. Dallinga-Thie G.M. Ackermans M.T. Serlie M.J. Oozeer R. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome.Gastroenterology. 2012; 143: 913-916Abstract Full Text Full Text PDF PubMed Scopus (1926) Google Scholar, 4Lam Y.Y. Ha C.W.Y. Campbell C.R. Mitchell A.J. Dinudom A. Oscarsson J. Cook D.I. Hunt N.H. Caterson I.D. Holmes A.J. Increased gut permeability and microbiota change associate with mesenteric fat inflammation and metabolic dysfunction in diet-induced obese mice.PLoS One. 2012; 7: e34233Crossref PubMed Scopus (413) Google Scholar, 5Janssen A.W.F. Kersten S. The role of the gut microbiota in metabolic health.FASEB J. 2015; 29: 3111-3123Crossref PubMed Scopus (132) Google Scholar). The human intestine contains a variety of microbiota, mainly consisting of bacteria and complemented by other microorganisms, such as fungi, protozoa, and viruses. The gut microbiota form a mutualistic relationship with the host and have an important role in host health. Besides protecting the host against invading pathogenic microorganisms, intestinal bacteria facilitate the digestion of otherwise indigestible carbohydrates, produce essential vitamins, stimulate the development of the immune system, and maintain tissue homeostasis (6Sommer F. Bäckhed F. The gut microbiota–masters of host development and physiology.Nat. Rev. Microbiol. 2013; 11: 227-238Crossref PubMed Scopus (2102) Google Scholar, 7Clemente J.C. Ursell L.K. Parfrey L.W. Knight R. The impact of the gut microbiota on human health: an integrative view.Cell. 2012; 148: 1258-1270Abstract Full Text Full Text PDF PubMed Scopus (2286) Google Scholar). In recent years, the gut microbiota have increasingly been connected with a number of diseases, including irritable bowel syndrome, Crohn's disease, obesity, type 2 diabetes, atherosclerosis, and NAFLD (5Janssen A.W.F. Kersten S. The role of the gut microbiota in metabolic health.FASEB J. 2015; 29: 3111-3123Crossref PubMed Scopus (132) Google Scholar, 8Biedermann L. Rogler G. The intestinal microbiota: its role in health and disease.Eur. J. Pediatr. 2015; 174: 151-167Crossref PubMed Scopus (126) Google Scholar, 9Wang Z. Klipfell E. Bennett B.J. Koeth R. Levison B.S. Dugar B. Feldstein A.E. Britt E.B. Fu X. Chung Y.-M. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease.Nature. 2011; 472: 57-63Crossref PubMed Scopus (3502) Google Scholar, 10Janssen A.W.F. Kersten S. Potential mediators linking gut bacteria to metabolic health: a critical view.J. Physiol. 2017; 595: 477-487Crossref PubMed Scopus (48) Google Scholar). NAFLD describes a spectrum of related liver diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis (11Schuppan D. Schattenberg J.M. Non-alcoholic steatohepatitis: pathogenesis and novel therapeutic approaches.J. Gastroenterol. Hepatol. 2013; 28: 68-76Crossref PubMed Scopus (194) Google Scholar). Although the exact pathogenesis of NAFLD is unknown, multiple factors likely contribute to the progression of NAFLD, including genetic predisposition, lipid overload, and inflammatory insults. Indeed, it is believed that inflammatory mediators released locally or derived from tissues, such as the intestine and adipose tissue, play an important role in the progression of steatosis to NASH (12Tilg H. Moschen A.R. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis.Hepatology. 2010; 52: 1836-1846Crossref PubMed Scopus (1619) Google Scholar, 13Nassir F. Ibdah J. Role of mitochondria in nonalcoholic fatty liver disease.Int. J. Mol. Sci. 2014; 15: 8713-8742Crossref PubMed Scopus (218) Google Scholar). Recent studies have raised the possibility that NAFLD may be related to disturbances in the gut microbial composition. In particular, it was found that the gut microbial composition was different between healthy individuals and patients with NAFLD (14Jiang W. Wu N. Wang X. Chi Y. Zhang Y. Qiu X. Hu Y. Li J. Liu Y. Dysbiosis gut microbiota associated with inflammation and impaired mucosal immune function in intestine of humans with non-alcoholic fatty liver disease.Sci. Rep. 2015; 5: 8096Crossref PubMed Scopus (375) Google Scholar, 15Mouzaki M. Comelli E.M. Arendt B.M. Bonengel J. Fung S.K. Fischer S.E. Mcgilvray I.D. Allard J.P. Intestinal microbiota in patients with nonalcoholic fatty liver disease.Hepatology. 2013; 58: 120-127Crossref PubMed Scopus (521) Google Scholar). Furthermore, mouse studies suggest that changes in the gut microbial community may impact the development of hepatic steatosis (16Jiang C. Xie C. Li F. Zhang L. Nichols R.G. Krausz K.W. Cai J. Qi Y. Fang Z. Takahashi S. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease.J. Clin. Invest. 2015; 125: 386-402Crossref PubMed Scopus (449) Google Scholar), inflammation (17Henao-Mejia J. Elinav E. Jin C. Hao L. Mehal W.Z. Strowig T. Thaiss C.A. Kau A.L. Eisenbarth S.C. Jurczak M.J. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity.Nature. 2012; 482: 179-185Crossref PubMed Scopus (1699) Google Scholar, 18De Minicis S. Rychlicki C. Agostinelli L. Saccomanno S. Candelaresi C. Trozzi L. Mingarelli E. Facinelli B. Magi G. Palmieri C. Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice.Hepatology. 2014; 59: 1738-1749Crossref PubMed Scopus (202) Google Scholar), and fibrosis (18De Minicis S. Rychlicki C. Agostinelli L. Saccomanno S. Candelaresi C. Trozzi L. Mingarelli E. Facinelli B. Magi G. Palmieri C. Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice.Hepatology. 2014; 59: 1738-1749Crossref PubMed Scopus (202) Google Scholar). To further elaborate the concept that the gut microbiota may affect the development of NAFLD, we studied the influence of modulation of the gut microbiota on NAFLD. To that end, in a mouse model of NAFLD, we stimulated the gut bacteria by feeding mice the highly fermentable dietary fiber, guar gum (GG), using the poorly fermentable dietary fiber, resistant starch (RS), as control (CTRL). Inasmuch as GG escapes digestion in the small intestine and is thought to act exclusively via bacterial fermentation and concomitant alterations in gut microbiota, feeding GG is an attractive strategy to study the role of the gut microbes in NAFLD. Additionally, in the same mouse model of NAFLD, we studied the effect on NAFLD of suppressing the gut bacteria using a mixture of antibiotics. Overall, our data indicate that the gut microbiota have a marked impact on NAFLD. Specifically, specific modulation of the gut microbiota by feeding GG worsened features of NAFLD, whereas suppression of the gut bacteria using oral antibiotics protected against NAFLD, possibly via changes in the portal delivery of bile acids. Animal studies were performed using pure-bred male C57Bl/6 mice. Mice were individually housed in temperature- and humidity-controlled specific pathogen-free conditions. Mice had ad libitum access to food and water. In study 1, 11-week-old mice received a low-fat diet or a high-fat/high-cholesterol/high-fructose diet (HFCFD) for 18 weeks, providing 10% or 45% energy as triglycerides (formula D12450B or D12451 from Research Diets, Inc., manufactured by Research Diet Services, Wijk bij Duurstede, The Netherlands). The fat source of this HFCFD was replaced by safflower oil and supplemented with 1% cholesterol (Dishman, Veenendaal, The Netherlands). Mice receiving the HFCFD were provided with 20% fructose water (w/v) to promote development of NAFLD (19Nomura K. Yamanouchi T. The role of fructose-enriched diets in mechanisms of nonalcoholic fatty liver disease.J. Nutr. Biochem. 2012; 23: 203-208Crossref PubMed Scopus (167) Google Scholar). The fiber-enriched diets were identical to the HFCFD (CTRL) except that corn starch and part of the maltodextrin 10 were replaced by 10% (w/w) RS or GG. The composition of the diets is shown in supplemental Table S1. RS, a retrograded tapioca starch classified as RS type 3 (brand name C*Actistar 11700) and GG (brand name Viscogum) were obtained from Cargill R&D Centre Europe (Vilvoorde, Belgium). Bodyweight and food intake were assessed weekly. One mouse fed the GG-enriched diet died during the study for reasons unrelated to the intervention. In study 2, 10-week-old mice received a HFCFD (CTRL) for 18 weeks, providing 45% energy as triglycerides (formula 58V8 manufactured by TestDiet, St. Louis, MO). This diet was supplemented with 1% cholesterol (Dishman). The mice received 20% fructose (w/v) in the drinking water. The trimethylamine N-oxide (TMAO)-enriched diet was identical to the CTRL diet except that 0.296% of sucrose (w/w) was replaced by 0.296% TMAO-dihydrate (w/w) (Sigma, Zwijndrecht, The Netherlands) to obtain a final concentration of 0.2% TMAO (w/w). Bodyweight and food intake were assessed weekly. One mouse fed the TMAO-enriched diet died during the study for reasons unrelated to the intervention. One mouse in the CTRL group and one mouse fed the TMAO-enriched diet failed to thrive and were excluded from further analysis. In study 3, 4-month-old male mice received either chow (CTRL) or chow supplemented with 0.5% (w/w) taurocholic acid (Calbiochem) for 7 days (20Song P. Zhang Y. Klaassen C.D. Dose-response of five bile acids on serum and liver bile acid concentrations and hepatotoxicty in mice.Toxicol. Sci. 2011; 123: 359-367Crossref PubMed Scopus (101) Google Scholar). Bodyweight and food intake were assessed daily. In study 4, 10-week-old mice received a HFCFD (CTRL) for 18 weeks, providing 45% energy as triglycerides (formula 58V8 manufactured by TestDiet). The diet was supplemented with 1% cholesterol (Dishman). The mice received 20% fructose (w/v) in the drinking water. Mice that were given antibiotics received the same CTRL diet with the addition of 1 g/l ampicillin, 1 g/l neomycin sulfate, 1 g/l metronidazole, and 0.5 g/l vancomycin in the drinking water for 22 weeks. This antibiotic cocktail has previously been shown to deplete all detectable commensal bacteria (21Rakoff-Nahoum S. Paglino J. Eslami-Varzaneh F. Edberg S. Medzhitov R. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis.Cell. 2004; 118: 229-241Abstract Full Text Full Text PDF PubMed Scopus (3299) Google Scholar). Bodyweight and food intake were assessed weekly. One mouse in the CTRL group and one mouse in the antibiotics group failed to thrive and were excluded from further analysis. At the end of each study, mice were anesthetized using isoflurane and blood was collected by orbital puncture. Mice were euthanized via cervical dislocation after which tissues were excised and weighed and intestinal content was sampled. The mice were not fasted prior to euthanasia (e.g., ad libitum fed state). The animal experiments were approved by the local animal ethics committee of Wageningen University. In study 1, mice were fasted for 5 h prior to the intraperitoneal glucose tolerance test. During this period, fructose water was replaced by tap water. Blood samples were collected from the tail vein immediately before (t = 0 min) and at selected time points after intraperitoneal injection with glucose (0.8 g/kg bodyweight). Glucose was measured using Accu-chek Compact. Plasma concentrations of insulin were quantified after 5 h fasting according to manufacturer's instructions (Crystal Chem, Downers Grove, IL). Total RNA was extracted from epididymal white adipose tissue, liver, and scrapings of the distal small intestine using TRIzol reagent (Life Technologies, Bleiswijk, The Netherlands). Subsequently, 500 ng RNA was used to synthesize cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories, Veenendaal, The Netherlands). Changes in gene expression were determined by real-time PCR on a CFX384 real-time PCR detection system (Bio-Rad Laboratories) by using SensiMix (Bioline; GC Biotech, Alphen aan den Rijn, The Netherlands). The housekeeping gene, 36b4 or β-actin, was used for normalization. Sequences of the used primers are listed in supplemental Table S2. H&E staining of sections was performed using standard protocols. Paraffin-embedded liver sections (5 μm) were stained for collagen using fast green FCF/Sirius red F3B. Staining of neutral lipids was performed on frozen liver sections using Oil Red O according to standard protocols. Visualization of hepatic stellate cells was performed on paraffin-embedded liver sections with an antibody against α smooth muscle actin (αSMA) (M0851; Dako, Heverlee, Belgium). To detect macrophages, 5 μm frozen liver sections were stained for Cd68 Kupffer cells (Cd68 marker, FA11) as described previously (22Bieghs V. Wouters K. van Gorp P.J. Gijbels M.J.J. de Winther M.P.J. Binder C.J. Lütjohann D. Febbraio M. Moore K.J. van Bilsen M. Role of scavenger receptor A and CD36 in diet-induced nonalcoholic steatohepatitis in hyperlipidemic mice.Gastroenterology. 2010; 138: 2477-2486Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). In studies 1 and 3, blood was collected in EDTA-coated tubes and centrifuged for 15 min at 1,000 g to obtain plasma. In studies 2 and 4, blood was collected, allowed to clot for 45 min, and centrifuged for 15 min at 1,000 g to obtain serum. The mice were not fasted prior to blood collection. Plasma and serum alanine aminotransferase (ALT) activity was measured with a kit from Abcam (Cambridge, UK). The commercially available Limulus Amebocyte Lysate assay (Lonza, Walkersville, MD) was used to quantify plasma and portal serum endotoxin levels. Plasma concentration of total bile acids was determined using a colorimetric assay kit (Diazyme Laboratories, Poway, CA). Free and conjugated bile acid subspecies were quantified by LC-MS/MS using a SHIMADZU LC system (SHIMADZU, Kyoto, Japan) and tandem AB SCIEX API-3,200 triple quadrupole mass spectrometers (AB SCIEX, Framingham, MA), as previously described (23Nagy R.A. van Montfoort A.P.A. Dikkers A. van Echten-Arends J. Homminga I. Land J.A. Hoek A. Tietge U.J.F. Presence of bile acids in human follicular fluid and their relation with embryo development in modified natural cycle IVF.Hum. Reprod. 2015; 30: 1102-1109Crossref PubMed Scopus (25) Google Scholar). Trimethylamine (TMA) and TMAO plasma levels and TMAO serum levels were quantified by stable isotope dilution LC-MS/MS, as previously described (24Wang Z. Levison B.S. Hazen J.E. Donahue L. Li X.M. Hazen S.L. Measurement of trimethylamine-N-oxide by stable isotope dilution liquid chromatography tandem mass spectrometry.Anal. Biochem. 2014; 455: 35-40Crossref PubMed Scopus (221) Google Scholar, 25Koeth R.A. Levison B.S. Culley M.K. Buffa J.A. Wang Z. Gregory J.C. Org E. Wu Y. Li L. Smith J.D. γ-Butyrobetaine Is a proatherogenic intermediate in gut microbial metabolism of L–carnitine to TMAO.Cell Metab. 2014; 20: 799-812Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). In study 1, during the sixth week of dietary intervention, feces were collected over a period of 48 h. Total lipids and free fatty acids were measured in the fecal samples as mentioned by Govers and Van der Meet (26Govers M.J. Van der Meet R. Effects of dietary calcium and phosphate on the intestinal interactions between calcium, phosphate, fatty acids, and bile acids.Gut. 1993; 34: 365-370Crossref PubMed Scopus (109) Google Scholar). Briefly, 100 mg of fecal samples were weighed, dried, and acidified using HCl. Lipids were then extracted using petroleum and diethyl ether. The ether fraction was collected and evaporated and the total lipids were weighed. Free and conjugated bile acid subspecies in the feces, collected at the end of the dietary intervention in studies 1 and 4, were determined by capillary gas chromatography (Agilent 6890; Amstelveen, The Netherlands) as described previously (27van Meer H. Boehm G. Stellaard F. Vriesema A. Knol J. Havinga R. Sauer P.J. Verkade H.J. Prebiotic oligosaccharides and the enterohepatic circulation of bile salts in rats.Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 294: G540-G547Crossref PubMed Scopus (23) Google Scholar). Microarray analysis was performed on liver samples from eight mice of the CTRL group and eight mice of the GG group. RNA was isolated as described above and subsequently purified using the RNeasy Micro kit from Qiagen (Venlo, The Netherlands). RNA integrity was verified with RNA 6000 Nano chips on an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, The Netherlands). Purified RNA (100 ng) was labeled with the Ambion WT expression kit (Invitrogen, Carlsbad, CA) and hybridized to an Affymetrix Mouse Gene 1.1 ST array plate (Affymetrix, Santa Clara, CA). Hybridization, washing, and scanning were carried out on an Affymetrix GeneTitan platform according to the manufacturer's instructions. Arrays were normalized using the Robust Multi-array Average method (28Bolstad B.M. Irizarry R.A. Astrand M. Speed T.P. A comparison of normalization metholds for high density oligonucleotide array data based on variance and bias.Bioinformatics. 2003; 19: 185-193Crossref PubMed Scopus (6420) Google Scholar, 29Irizarry R.A. Bolstad B.M. Collin F. Cope L.M. Hobbs B. Speed T.P. Summaries of affymetrix genechip probe level data.Nucleic Acids Res. 2003; 31: e15Crossref PubMed Scopus (4023) Google Scholar). Probe sets were defined according to Dai et al. (30Dai M. Wang P. Boyd A.D. Kostov G. Athey B. Jones E.G. Bunney W.E. Myers R.M. Speed T.P. Akil H. Evolving gene/transcript definitions significantly alter the interpretation of GeneChip data.Nucleic Acids Res. 2005; 33: e175Crossref PubMed Scopus (1398) Google Scholar). In this method, probes are assigned to Entrez IDs as a unique gene identifier. The P values were calculated using an intensity-based moderated t-statistic (31Sartor M.A. Tomlinson C.R. Wesselkamper S.C. Sivaganesan S. Leikauf G.D. Medvedovic M. Intensity-based hierarchical Bayes method improves testing for differentially expressed genes in microarray experiments.BMC Bioinformatics. 2006; 7: 538Crossref PubMed Scopus (191) Google Scholar). The q value was calculated as the measure of significance for the false discovery rate (32Storey J.D. Tibshirani R. Statistical significance for genomewide studies.Proc. Natl. Acad. Sci. USA. 2003; 100: 9440-9445Crossref PubMed Scopus (7097) Google Scholar). To identify the pathways most significantly altered in the livers of the GG group compared with the CTRL group, Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA) was performed. Input criteria were a relative fold change equal to or above 1.3 and a q value equal to or below 0.01. Array data have been submitted to the Gene Expression Omnibus under accession number GSE76087. A publicly available dataset (GSE34730) was downloaded from Gene Expression Omnibus and further processed as described above to obtain individual gene expression data. Ingenuity Pathway Analysis was performed on genes induced by deoxycholic acid by at least 2-fold. Liver homogenates were prepared by lysing liver tissue in ice-cold Pierce IP lysis buffer (ThermoScientific, Rockford, IL) containing complete protease inhibitor and PhosSTOP phosphatase inhibitor (both from Roche, Mannheim, Germany). Equal amounts of protein were loaded on a Mini Protean TGX gel, 4–15%, and subsequently transferred to a PVDF membrane (both from Bio-Rad Laboratories). The primary antibodies for tissue inhibitor of metalloproteinase (TIMP)1 (Abcam, Cambridge, UK) and HSP90 (Cell Signaling) were incubated at 4°C overnight followed by incubation with the appropriate secondary peroxidase-conjugated antibody (Sigma). Protein bands were visualized using an enhanced chemiluminescent substrate (Bio-Rad Laboratories). For liver triglyceride measurement, livers were homogenized in a buffer containing 10 mM Tris, 2 mM EDTA, and 250 mM sucrose at pH 7.5 in a Tissue Lyser II (Qiagen, Hilden, Germany) to obtain 2% homogenates. Triglycerides were subsequently quantified using a Triglyceride LiquiColormono kit from HUMAN Diagnostics (Wiesbaden, Germany). The 4-hydroxyproline content was determined spectrophotometrically in liver hydrolysates, as described previously (33Jamall I.S. Finelli V.N. Que Hee S.S. A simple method to determine nanogram levels of 4-hydroxyproline in biological tissues.Anal. Biochem. 1981; 112: 70-75Crossref PubMed Scopus (484) Google Scholar). For hepatic myeloperoxidase activity, liver homogenates were prepared and myeloperoxidase peroxidase activity was measured according to manufacturer's instructions (Abcam). For the quantification of liver total bile acid content, livers were homogenized in 75% ethanol, incubated at 50°C and centrifuged. After the collection of the supernatant, the concentration of total bile acids was determined using a colorimetric assay kit (Diazyme Laboratories). Epididymal white adipose tissue was isolated, minced and digested with 1.5 mg/ml collagenase type 2 (Sigma) in DMEM containing 0.5% fatty acid-free BSA for 45 min at 37°C by shaking. Cell suspensions were filtered through a 100 μM filter and centrifuged at 200 g for 10 min. Floating mature adipocytes were discarded and the stromal vascular cell pellet was resuspended in erythrocyte lysis buffer for 5 min. Cells were washed twice with FACS buffer (PBS + 1% BSA) and incubated with fluorescently labeled antibodies including CD45-ECD (Beckman Coulter, Fullerton, CA), F4/80-FITC and Cd11b-PE (Biolegend, San Diego, CA). Samples were analyzed using a FC500 flow cytometer (Beckman Coulter). In study 1, fecal samples derived from the second part of the colon were suspended in 10 mM Tris, 1 mM EDTA, 0.5% SDS, and 0.2 mg/ml proteinase K (ThermoScientific). After addition of 0.1–0.25 mm and 4 mm glass beads, buffered phenol (Invitrogen) was added and cells were lysed by mechanical disruption using a bead beater (MP Biomedicals, Santa Ana, CA) for 3 min. DNA was subsequently extracted using phenol:chloroform:isoamylalcohol (25:24:1) (Invitrogen), precipitated with isopropanol, and washed with 70% ethanol. In study 4, fecal samples (∼15–60 mg) derived from the second part of the colon were suspended in 500 μl S.T.A.R. buffer (Roche). After addition of 0.1 mm zirconia and 2.5 mm glass beads (BioSpec, Bartlesville, OK), cells were lysed by mechanical disruption using a bead beater (MP Biomedicals) for 3 × 1 min. DNA was subsequently extracted and purified using the Maxwell 16 system (Promega). In brief, homogenates (250 μl) were transferred to a prefilled reagent cartridge (Maxwell® 16 Tissue LEV Total RNA purification kit, custom-made; Promega). Sixteen samples were processed at the same time. After 30 min, the purification process was completed and DNA was eluted in 50 μl of water (nuclease free) (Promega). For 16S rRNA gene sequencing DNA samples were sent to the Broad Institute of Massachusetts Institute of Technology and Harvard (Cambridge, MA). The microbial 16S rRNA gene was amplified targeting the hyper-variable region V4 using forward primer 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and the reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The cycling conditions consisted of an initial denaturation of 94°C for 3 min, followed by 25 cycles of denaturation at 94°C for 45 s, annealing at 50°C for 60 s, extension at 72°C for 5 min, and a final extension at 72°C for 10 min. Sequencing was performed using the Illumina MiSeq platform generating paired-end reads of 175 bp in length in each direction. Overlapping paired-end reads were subsequently aligned. The details of this protocol are as previously described (34Gevers D. Kugathasan S. Denson L.A. Vázquez-Baeza Y. Van Treuren W. Ren B. Schwager E. Knights D. Song S.J. Yassour M. The treatment-naive microbiome in new-onset Crohn's disease.Cell Host Microbe. 2014; 15: 382-392Abstract Full Text Full Text PDF PubMed Scopus (1926) Google Scholar). Raw sequence data quality was assessed using FastQC, version: 0.11.2 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads quality was checked with Sickle, version: 1.33 (https://github.com/najoshi/sickle) and low quality reads were removed. For visualizing the taxonomic composition of the fecal microbiota and further β diversity analysis, QIIME, version: 1.9.0 was used (35Caporaso J.G. Kuczynski J. Stombaugh J. Bittinger K. Bushman F.D. Costello E.K. Fierer N. Peña A.G. Goodrich J.K. Gordon J.I. QIIME allows analysis of high- throughput community sequencing data.Nat. Methods. 2010; 7: 335-336Crossref PubMed Scopus (24741) Google Scholar). In brief, closed reference OTU picking with 97% sequence similarity against GreenGenes 13.8 reference database was done. Jackknifed β-diversity of unweighted UniFrac distances with 10 jackknife replicates was measured at rarefaction depth of 22,000 reads per sample. For statistical significance, biological relevance, and visualization, we used the linear discriminant analysis (LDA) effect size (LEfSe) method (https://bitbucket.org/biobakery/biobakery/wiki/lefse) (36Segata N. Izard J. Waldron L. Gevers D. Miropolsky L. Garrett W.S. Huttenhower C. Metagenomic biomarker discovery and explanation.Genome Biol. 2011; 12: R60Crossref PubMed Scopus (7674) Google Scholar). In study 4, real-time PCR was used to quantify the 16S rRNA gene and ITS1-5.8S-ITS2 region in equal amounts of extracted DNA on a CFX384 real-time PCR detection system (Bio-Rad Laboratories) by using SensiMix (Bioline; GC Biotechnology). The 16S rRNA gene was amplified using the forward primer 1369F (5′-CGGTGAATACGTTCYCGG-3′) (37Suzuki M.T. Taylor L.T. DeLong E.F. Quantitative analysis of small-subunit rRNA genes in mixed microbial populations via 5′-nuclease assays.Appl. Environ. Microbiol. 2000; 66: 4605-4614Crossref PubMed Scopus (917) Google Scholar) and the reverse primer 1492R (5′-GGWTACCTTGTTACGACTT-3′) (38Weisburg W.G. Barns S.M. Pelletier D.A. Lane D.J. 16S ribosomal DNA amplification for phylogenetic study.J. Bacteriol. 1991; 173: 697-703Crossref PubMed Scopus (8849) Google Scholar). The cycling conditions consisted of an initial denaturation of 95°C for 5 min, follow