Dominique Rainteau

Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases

Objective Gut microbiota metabolises bile acids (BA). As dysbiosis has been reported in inflammatory bowel diseases (IBD), we aim to investigate the impact of IBD-associated dysbiosis on BA metabolism and its influence on the epithelial cell inflammation response. Design Faecal and serum BA rates, expressed as a proportion of total BA, were assessed by high-performance liquid chromatography tandem mass spectrometry in colonic IBD patients (42) and healthy subjects (29). The faecal microbiota composition was assessed by quantitative real-time PCR. Using BA profiles and microbiota composition, cluster formation between groups was generated by ranking models. The faecal BA profiles in germ-free and conventional mice were compared. Direct enzymatic activities of BA biotransformation were measured in faeces. The impact of BA on the inflammatory response was investigated in vitro using Caco-2 cells stimulated by IL-1β. Results IBD-associated dysbiosis was characterised by a decrease in the ratio between Faecalibacterium prausntizii and Escherichia coli. Faecal-conjugated BA rates were significantly higher in active IBD, whereas, secondary BA rates were significantly lower. Interestingly, active IBD patients exhibited higher levels of faecal 3-OH-sulphated BA. The deconjugation, transformation and desulphation activities of the microbiota were impaired in IBD patients. In vitro, secondary BA exerted anti-inflammatory effects, but sulphation of secondary BAs abolished their anti-inflammatory properties. Conclusions Impaired microbiota enzymatic activity observed in IBD-associated dysbiosis leads to modifications in the luminal BA pool composition. Altered BA transformation in the gut lumen can erase the anti-inflammatory effects of some BA species on gut epithelial cells and could participate in the chronic inflammation loop of IBD.

Identification of an anti-inflammatory protein from Faecalibacterium prausnitzii, a commensal bacterium deficient in Crohn’s disease

Background Crohn’s disease (CD)-associated dysbiosis is characterised by a loss of Faecalibacterium prausnitzii, whose culture supernatant exerts an anti-inflammatory effect both in vitro and in vivo. However, the chemical nature of the anti-inflammatory compounds has not yet been determined. Methods Peptidomic analysis using mass spectrometry was applied to F. prausnitzii supernatant. Anti-inflammatory effects of identified peptides were tested in vitro directly on intestinal epithelial cell lines and on cell lines transfected with a plasmid construction coding for the candidate protein encompassing these peptides. In vivo, the cDNA of the candidate protein was delivered to the gut by recombinant lactic acid bacteria to prevent dinitrobenzene sulfonic acid (DNBS)-colitis in mice. Results The seven peptides, identified in the F. prausnitzii culture supernatants, derived from a single microbial anti-inflammatory molecule (MAM), a protein of 15 kDa, and comprising 53% of non-polar residues. This last feature prevented the direct characterisation of the putative anti-inflammatory activity of MAM-derived peptides. Transfection of MAM cDNA in epithelial cells led to a significant decrease in the activation of the nuclear factor (NF)-κB pathway with a dose-dependent effect. Finally, the use of a food-grade bacterium, Lactococcus lactis, delivering a plasmid encoding MAM was able to alleviate DNBS-induced colitis in mice. Conclusions A 15 kDa protein with anti-inflammatory properties is produced by F. prausnitzii, a commensal bacterium involved in CD pathogenesis. This protein is able to inhibit the NF-κB pathway in intestinal epithelial cells and to prevent colitis in an animal model.

Increase in fecal primary bile acids and dysbiosis in patients with diarrhea‐predominant irritable bowel syndrome

Background  Irritable bowel syndrome (IBS) is a multifactorial disease for which a dysbiosis of the gut microbiota has been described. Bile acids (BA) could play a role as they are endogenous laxatives and are metabolized by gut microbiota. We compared fecal BA profiles and microbiota in healthy subjects (HS) and patients with diarrhea‐predominant IBS (IBS‐D), and we searched for an association with symptoms.

The receptor TGR5 protects the liver from bile acid overload during liver regeneration in mice

Many regulatory pathways are involved in liver regeneration after partial hepatectomy (PH) to initiate growth, protect liver cells, and sustain functions of the remnant liver. Bile acids (BAs), whose levels rise in the blood early after PH, stimulate both hepatocyte proliferation and protection, in part through their binding to the nuclear farnesoid X receptor (FXR). However, the effect of the BA receptor, TGR5 (G‐protein‐coupled BA receptor 1) after PH remains to be studied. Liver histology, hepatocyte proliferation, BA concentrations (plasma, bile, liver, urine, and feces), bile flow and composition, and cytokine production were studied in wild‐type (WT) and TGR5 KO (knockout) mice before and after PH. BA composition (plasma, bile, liver, urine, and feces) was more hydrophobic in TGR5 KO than in WT mice. After PH, severe hepatocyte necrosis, prolonged cholestasis, exacerbated inflammatory response, and delayed regeneration were observed in TGR5 KO mice. Although hepatocyte adaptive response to post‐PH BA overload was similar in WT and TGR5 KO mice, kidney and biliary adaptive responses were strongly impaired in TGR5 KO mice. Cholestyramine treatment, as well as Kupffer cell depletion, significantly improved the post‐PH TGR5 KO mice phenotype. After bile duct ligation or upon a cholic acid–enriched diet, TGR5 KO mice exhibited more severe liver injury than WT as well as impaired BA elimination in urine. Conclusion: TGR5 is crucial for liver protection against BA overload after PH, primarily through the control of bile hydrophobicity and cytokine secretion. In the absence of TGR5, intrahepatic stasis of abnormally hydrophobic bile and excessive inflammation, in association with impaired bile flow adaptation and deficient urinary BA efflux, lead to BA overload‐induced liver injury and delayed regeneration. (Hepatology 2013;58:1451–1460)

Bile acid profiling in human biological samples: Comparison of extraction procedures and application to normal and cholestatic patients

The role of bile acids in cell metabolism, membrane biology and cell signaling is increasingly recognized, thus making necessary a robust and versatile technique to extract, separate and quantify a large concentration range of these numerous molecular species. HPLC-MS/MS analysis provides the highest sensitivity to detect and identify bile acids. However, due to their large chemical diversity, extraction methods are critical and quite difficult to optimize, as shown by a survey of the literature. This paper compares the performances of four bile acid extraction protocols applied to either liquid (serum, urine, bile) or solid (stool) samples. Acetonitrile was found to be the best solvent for deproteinizing liquid samples and NaOH the best one for stool extraction. These optimized extraction procedures allowed us to quantitate as much as 27 distinct bile acids including sulfated species in a unique 30 min HPLC run, including both hydrophilic and hydrophobic species with a high efficiency. Tandem MS provided a non ambiguous identification of each metabolite with a good sensitivity (LOQ below 20 nmol/l except for THDCA and TLCA). After validation, these methods, successfully applied to a group of 39 control patients, detected 14 different species in serum in the range of 30-800 nmol/l, 11 species in urine in the range of 20-200 nmol/l and 25 species in stool in the range of 0.4-2000 nmol/g. The clinical interest of this method has been then validated on cholestatic patients. The proposed protocols seem suitable for profiling bile acids in routine analysis.

Improvements in Glucose Metabolism Early After Gastric Bypass Surgery Are Not Explained by Increases in Total Bile Acids and Fibroblast Growth Factor 19 Concentrations

CONTEXT Bile acids and fibroblast growth factor 19 (FGF19) have been suggested as key mediators of the improvements in glucose metabolism after Roux-en-Y gastric bypass (RYGB). OBJECTIVE To describe fasting and postprandial state total bile acid (TBA) and FGF19 concentrations before and after RYGB and relate them to parameters of glucose metabolism, glucagon-like peptide-1, cholecystokinin, and cholesterol fractions. DESIGN AND SETTING A prospective descriptive study was performed at the Department of Endocrinology, Hvidovre Hospital, Hvidovre, Denmark. PATIENTS Thirteen type 2 diabetic (T2D) patients and 12 normal glucose tolerant (NGT) subjects participated in the study. INTERVENTION A 4-hour liquid meal test was performed before and 1 week, 3 months, and 1 year after RYGB. MAIN OUTCOME MEASURES We measured fasting and postprandial TBA and FGF19 concentrations. RESULTS Fasting TBA concentrations decreased in NGT subjects (P < .001) and were unchanged in T2D patients 1 week after surgery, but then increased gradually in both groups with time from surgery (ANOVA Ptime < .001). Area under the curve (AUC) TBA was decreased in NGT subjects 1 week after RYGB (before surgery, 567 mmol * min/L [interquartile range, 481-826]; 1 wk, 419 [381-508]; P = .009) and was unchanged in T2D patients (894 [573-1002]; 695 [349-1147]; P = .97) but then increased with time from surgery in both groups (Ptime < .001). Fasting FGF19 concentrations were unchanged acutely after RYGB (NGT, 140 pg/mL [100-162], 134 [119-204], P = .42; T2D, 162 [130-196], 154 [104-164], P = .68) and remained unchanged throughout the follow-up period. AUC FGF19 increased gradually with time after surgery (Ptime < .001), resembling the changes seen with AUC TBA. One week after RYGB, glucose metabolism improved, low-density lipoprotein-cholesterol and high-density lipoprotein-cholesterol decreased, and cholecystokinin and glucagon-like peptide-1 secretion increased, whereas FFA concentrations were unchanged. CONCLUSION TBA and FGF19 do not explain acute changes in glucose metabolism, cholesterol fractions, and gut hormone secretion after RYGB.

Immediate neuroendocrine signaling after partial hepatectomy through acute portal hyperpressure and cholestasis

BACKGROUND & AIMS Early neuroendocrine pathways contribute to liver regeneration after partial hepatectomy (PH). We investigated one of these pathways involving acute cholestasis, immediate portal hyperpressure, and arginine vasopressin (AVP) secretion. METHODS Surgical procedure (PH, Portal vein stenosis (PVS), bile duct ligation (BDL), spinal cord lesion (SCL)) and treatments (capsaicin, bile acids (BA), oleanolic acid (OA)) were performed on rats and/or wild type or TGR5 (GPBAR1) knock-out mice. In these models, the activation of AVP-secreting supraoptic nuclei (SON) was analyzed, as well as plasma BA, AVP, and portal vein pressure (PVP). Plasma BA, AVP, and PVP were also determined in human living donors for liver transplantation. RESULTS Acute cholestasis (mimicked by BDL or BA injection) as well as portal hyperpressure (mimicked by PVS) independently activated SON and AVP secretion. BA accumulated in the brain after PH or BDL, and TGR5 was expressed in SON. SON activation was mimicked by the TGR5 agonist OA and inhibited in TGR5 KO mice after BDL. An afferent nerve pathway also contributed to post-PH AVP secretion, as capsaicin treatment or SCL resulted in a weaker SON activation after PH. CONCLUSIONS After PH in rodents, acute cholestasis and portal hypertension, via the nervous and endocrine routes, stimulate the secretion of AVP that may protect the liver against shear stress and bile acids overload. Data in living donors suggest that this pathway may also operate in humans.

High plasma level of nucleocapsid‐free envelope glycoprotein‐positive lipoproteins in hepatitis C patients

Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture–produced HCV (HCVcc) and ex vivo–characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid‐free subviral LVPs are assembled and secreted by apoB‐producing cell lines. To determine whether such subviral particles circulate in HCV‐infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low‐density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A–purified LVPs contained at least the envelope glycoprotein E2 and E2‐specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride‐rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 × 106 apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid‐free, envelope glycoprotein‐containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes. Conclusion: LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family. (HEPATOLOGY 2012;56:39–48)

Acyl Chains of Phospholipase D Transphosphatidylation Products in Arabidopsis Cells: A Study Using Multiple Reaction Monitoring Mass Spectrometry

Background Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor. Methodology/Principal findings Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. Conclusions MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.

Trade-off between bile resistance and nutritional competence drives Escherichia coli diversification in the mouse gut.

Bacterial diversification is often observed, but underlying mechanisms are difficult to disentangle and remain generally unknown. Moreover, controlled diversification experiments in ecologically relevant environments are lacking. We studied bacterial diversification in the mammalian gut, one of the most complex bacterial environments, where usually hundreds of species and thousands of bacterial strains stably coexist. Herein we show rapid genetic diversification of an Escherichia coli strain upon colonisation of previously germ-free mice. In addition to the previously described mutations in the EnvZ/OmpR operon, we describe the rapid and systematic selection of mutations in the flagellar flhDC operon and in malT, the transcriptional activator of the maltose regulon. Moreover, within each mouse, the three mutant types coexisted at different levels after one month of colonisation. By combining in vivo studies and determination of the fitness advantages of the selected mutations in controlled in vitro experiments, we provide evidence that the selective forces that drive E. coli diversification in the mouse gut are the presence of bile salts and competition for nutrients. Altogether our results indicate that a trade-off between stress resistance and nutritional competence generates sympatric diversification of the gut microbiota. These results illustrate how experimental evolution in natural environments enables identification of both the selective pressures that organisms face in their natural environment and the diversification mechanisms.