Evaluation of inter-individual differences in gut bacterial isoflavone bioactivation in humans by PCR-based targeting of genes involved in equol formation.

AIM: To identify human subjects harbouring intestinal bacteria that bioactivate daidzein to equol using a targeted PCR-based approach. METHODS AND RESULTS: In a pilot study including 17 human subjects, equol formation was determined in faecal slurries. In parallel, faecal DNA was amplified by PCR using degenerate primers that target highly conserved regions of dihydrodaidzein reductase and tetrahydrodaidzein reductase genes. PCR products of the expected size were observed for six of the eight subjects identified as equol producers. Analysis of clone libraries revealed the amplification of sequences exclusively related to Adlercreutzia equolifaciens in four of the subjects tested positive for equol formation, whereas in three of the equol producers, only sequences related to Slackia isoflavoniconvertens were observed. No amplicons were obtained for one equol-forming subject, thus suggesting the presence of nontargeted alternative genes. Amplicons were only sporadically observed in the nonequol producers. CONCLUSION: The majority of human subjects who produced equol were also detected with the developed PCR-based approach. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results shed light on the distribution and the diversity of known equol-forming bacterial species in the study group and indicate the presence of as yet unknown equol-forming bacteria.

Intestinal alkaline phosphatase at the crossroad of intestinal health and disease - a putative role in type 1 diabetes.

BACKGROUND: Patients with type 1 diabetes have shown an increase in circulating cytokines, altered lipoprotein metabolism and signs of vascular dysfunction in response to high-fat meals. Intestinal alkaline phosphatase (IAP) regulates lipid transport and inflammatory responses in the gastrointestinal tract. We therefore hypothesized that changes in IAP activity could have profound effects on gut metabolic homeostasis in patients with type 1 diabetes. METHODS: Faecal samples of 41 nondiabetic controls and 46 patients with type 1 diabetes were analysed for IAP activity, calprotectin, immunoglobulins and short-chain fatty acids (SCFAs). The impact of oral IAP supplementation on intestinal immunoglobulin levels was evaluated in C57BL/6 mice exposed to high-fat diet for 11 weeks. RESULTS: Patients with type 1 diabetes exhibited signs of intestinal inflammation. Compared to controls, patients with diabetes had higher faecal calprotectin levels, lower faecal IAP activities accompanied by lower propionate and butyrate concentrations. Moreover, the amount of faecal IgA and the level of antibodies binding to oxidized LDL were decreased in patients with type 1 diabetes. In mice, oral IAP supplementation increased intestinal IgA levels markedly. CONCLUSION: Deprivation of protective intestinal factors may increase the risk of inflammation in the gut - a phenomenon that seems to be present already in patients with uncomplicated type 1 diabetes. Low levels of intestinal IgA and antibodies to oxidized lipid epitopes may predispose such patients to inflammation-driven complications such as cardiovascular disease and diabetic nephropathy. Importantly, oral IAP supplementation could have beneficial therapeutic effects on gut metabolic homeostasis, possibly through stimulation of intestinal IgA secretion.

Isolation of catechin-converting human intestinal bacteria.

AIMS: To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet. METHODS AND RESULTS: Two bacterial strains, rK3 and aK2, were isolated from an epicatechin-converting human faecal suspension. The isolates catalysed individual steps in the degradation of ⁻-epicatechin and ⁺-catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both ⁻-epicatechin and ⁺-catechin giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol to 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific. CONCLUSIONS: The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability. SIGNIFICANCE AND IMPACT OF THE STUDY: The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin-converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.

Reduced coenzyme F420: heterodisulfide oxidoreductase, a proton- translocating redox system in methanogenic bacteria.

Washed everted vesicles of the methanogenic bacterium strain Go1 were found to couple the F420H2-dependent heterodisulfide reduction with the transfer of protons across the membrane into the lumen of the everted vesicles. The transmembrane electrochemical potential of protons thereby generated was shown to be competent in driving ATP synthesis from ADP + Pi, exhibiting a stoichiometry of 2 H+ translocated or 0.4 ATP synthesized per F420H2 oxidized. This enzyme system exhibits the phenomenon of coupling and uncoupling and represents a different kind of electron transport chain with the heterodisulfide of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate as terminal electron acceptor. The heterodisulfide and methane are formed in the methyl coenzyme M reductase reaction. The reducing equivalents are derived from reduced coenzyme F420, which represents an analogue of NADH + H+ in other respiratory chains. It is assumed that the proton-translocating oxidoreductase discovered in strain Go1 is of principal importance to all methanogenic bacteria not utilizing H2.

An improved method for the automated enumeration of fluorescently labelled bacteria in human faeces.

The study aimed to improve microscopy-based automated recognition of faecal bacterial cells labelled with 16S rRNA-targeted oligonucleotides and 4',6-diamidino-2-phenylindole (DAPI). Based on the software KS400 (Carl Zeiss Vision, Hallbergmoos, Germany), designed for automising microscopy-based image capture and image analysis, a routine was developed that affords the recognition of doubly stained bacteria and the rejection of artefacts. The accuracy of the automated enumeration was investigated by comparing the resulting data with those obtained by manual counting. The newly developed method was subsequently used to compare the total bacterial counts in human faecal samples using the domain specific probe Eub338 alone and a mixture of 5 domain-specific probes, respectively. Faecal samples from 90 healthy volunteers were analysed. The cell counts obtained with Eub338 were 10% lower than those obtained with the probe mixture. Since the cells detected with the probe mixture covered a wide range of signal intensities, a dynamic analysis routine was developed to effectively detect the whole range of bright to weak signals within the same image, while at the same time reliably rejecting artefacts.

Bacterial response to eukaryotic cells. Analysis of differentially expressed proteins using nano liquid chromatography-electrospray ionization tandem mass spectrometry.

Host-bacteria interactions have mostly been investigated with regard to the host response or to activities of pathogenic bacteria. In contrast, we aim to identify reactions of non-pathogenic bacteria that result from their contact with host cells of the gastrointestinal tract. In a proteomic approach, the response of non-pathogenic human Escherichia coli bacteria on gut epithelial cells (rat IEC-6) was investigated in an in vitro co-culture model. For this purpose, a sensitive analytical procedure was developed based on the identification of two-dimensional polyacrylamide gel electrophoresis separated proteins by online nanoLC-electrospray ionization MS/MS using a quadrupole time-of-flight tandem mass spectrometer for accurate mass determination. We demonstrate here the efficiency of this technique by the identification of a total of 43 differentially expressed proteins, out of which 25 were up-regulated and 18 were down-regulated. They represent a wide range of molecular weight and different metabolic and physiological functions.

A methyl-CoM methylreductase system from methanogenic bacterium strain Gö 1 not requiring ATP for activity.

Crude inside-out vesicles from the methanogenic strain Gö1 were prepared via protoplasts. These vesicles catalyzed methane formation from methyl-CoM and H2 at a maximal rate of 35 nmol/min.mg protein. Methane formation by the vesicles did not depend on the addition of ATP. This was in contrast to conventionally prepared crude extracts from the same organism or from Methanosarcina barkeri which exhibited strict ATP dependence of methanogenesis. ATP analogues inhibited methanogenesis by extracts to a much higher extent than that by vesicles. Both, particulate and soluble components prepared from the crude vesicles by ultracentrifugation were necessary for ATP-independent methane formation from methyl-CoM and H2. Hydrogenase activity was mainly associated with the particulate fraction whereas methyl-CoM methylreductase could be assigned to the soluble fraction. The detergent sulfobetaine inhibited methane formation from methyl-CoM without affecting hydrogenase or titanium citrate-dependent methylreductase activities, indicating that an additional membraneous component is involved in methanogenesis for methyl-CoM and H2.

Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons.

Constipation is an ailment encountered often in elderly people. A study was initiated to test the effects of lactose or inulin on the bowel habits of constipated elderly patients and to correlate these effects with several variables measured in feces such as microflora composition, concentration of lactate and short-chain fatty acids (SCFAs), pH, and the activities of beta-glucosidase and beta-glucuronidase, Groups of 15 and 10 patients received lactose and inulin, respectively, for a period of 19 d. The dose, 20 g/d from days 1 to 8, was gradually increased to 40 g/d from days 9 to 11 and was kept at this dose from days 12 to 19. There was considerable interindividual variations with this kind of dietary intervention. Inulin increased bifidobacteria significantly from 7.9 to 9.2 log10/g dry feces, but decreased enterococci in number and enterobacteria in frequency. In individuals consuming lactose, a noticeable increase in fecal counts of enterococci and a decrease in lactobacilli and clostridia was detected. Total bacterial counts remained unchanged. No changes in the concentrations of fecal SCFAs and lactate were observed. SCFAs showed a slight trend toward higher molar ratios of acetate to butyrate in response to the intake of lactose or inulin. The fecal pH and the beta-glucosidase and beta-glucuronidase activities were not influenced by sugar intake. Inulin showed a better laxative effect than lactose and reduced functional constipation with only mild discomfort.

Bifidobacterium adolescentis modulates the specific immune response to another human gut bacterium, Bacteroides thetaiotaomicron, in gnotobiotic rats.

In order to investigate the capability of an autochthonous bacterium to modulate the host's immune response against the indigenous microfiora, the immunogenicity of two selected bacterial species of the human gut was investigated in a gnotobiotic rat model. Germ-free (GF) rats were monoassociated with either Bifidobacterium (B.) adolescentis or Bacteroides (B.) thetaiotaomicron and the development of bacteria-specific IgG and IgA in serum and specific secretory IgA (sIgA) in feces of the animals were measured. Knowing the antibody levels in gnotobiotic rats induced by monoassociation, we subsequently diassociated two groups of rats in order to investigate the impact of B. adolescentis on the immune reaction against B. thetaiotaomicron. One group was diassociated simultaneously with B. adolescentis and B. thetaiotaomicron, the second group was diassociated with these bacteria in sequence. In contrast to B. thetaiotaomicron, B. adolescentis was not able to induce a systemic immune response in monoassociated animals as evident from serum IgG and IgA. However, both bacterial species challenged the mucosal immune system as indicated by an increase in sIgA in the feces. The specific immune response to B. thetaiotaomicron was significantly lower in diassociated animals than in animals monoassociated with B. thetaiotaomicron. This effect was more pronounced in the rats, that had been associated sequentially. The presence of B. adolescentis down-regulated the humoral immunity to B. thetaiotaomicron.

Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria.

Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE). Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor. Resting cells of S. ovata reductively dechlorinated PCE at a rate of 9.8 nmol h-1 (mg protein)-1 to trichloroethylene (TCE) as the sole product. The dechlorination activity depended on concomitant acetogenesis from methanol and CO2. Cell-free extracts of S. ovata, Clostridium formicoaceticum, Acetobacterium woodii, and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors. Corrinoids were shown in S. ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide. Rates of dechlorination followed a pseudo-first-order kinetic.

A fluorescence quenching test for the detection of flavonoid transformation.

A novel fluorescence quenching test for the detection of flavonoid degradation by microorganisms was developed. The test is based on the ability of the flavonoids to quench the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Several members of the anthocyanidins, flavones, isoflavones, flavonols, flavanones, dihydroflavanones, chalcones, dihydrochalcones and catechins were tested with regard to their quenching properties. The anthocyanidins were the most potent quenchers of DPH fluorescence, while the flavanones, dihydroflavanones and dihydrochalcones, quenched the fluorescence only weakly. The catechins had no visible impact on DPH fluorescence. The developed test allows a quick and easy differentiation between flavonoid-degrading and flavonoid-non-degrading bacteria. The investigation of individual reactions of flavonoid transformation with the developed test system is also possible.

The F420H2-dehydrogenase from Methanolobus tindarius: cloning of the ffd operon and expression of the genes in Escherichia coli.

The membrane-bound F420H2-dehydrogenase from the methylotrophic methanogen Methanolobus tindarius oxidizes reduced coenzyme F420 and feeds the electrons into an energy-conserving electron transport chain. Based on the N-terminal amino acid sequence of the 40-kDa subunit of F420H2-dehydrogenase the corresponding gene ffdB was detected in chromosomal DNA of M. tindarius. Sequence analysis, primer extension, and RT-PCR experiments indicated that ffdB is part of an operon harboring three additional open reading frames (ffdA, ffdC, ffdD). The corresponding mRNA transcript and transcription start sites were determined. All four genes could be heterologously expressed in Escherichia coli.

Feeding resistant starch affects fecal and cecal microflora and short-chain fatty acids in rats.

The effects of different forms of resistant potato starch (RS) on the major microbial population groups and short-chain fatty acids (SCFA) in the cecum and feces of rats were studied over a 5-mo feeding period. Thirty 8-wk-old male Wistar rats, averaging 210 g initial body weight, were adapted for 7 d to a balanced basal diet containing 60% waxy maize starch devoid of any RS. On d 8, three groups of 10 rats each were fed diets containing the following forms of starch: 1) rapidly digestible waxy maize starch (basal diet), 2) a mixture of 83.3% waxy maize starch and 16.7% native granular potato starch (RS 1), or 3) a mixture of 33.3% waxy maize starch and 66.7% modified potato starch (RS 2). The final RS content in RS 1 and RS 2 was 10%. Fecal samples were collected at d 8 and 1, 3, and 5 mo after the start of the experiment. Cecal contents were taken after 5 mo. The colony counts of microbial groups did not vary with time in the control or the RS 1 group (P > .05). Only the number of Bacteroides/fusobacteria decreased between mo 1 and 5 in rats fed RS 1 (P < .05). The RS 2 diet led to a significant increase in total culturable bacteria, lactobacilli, streptococci, and enterobacteria between mo 1 and 5. The RS 1 and RS 2 diets stimulated the growth of bifidobacteria. Cecal numbers of lactobacilli, streptococci, and enterobacteria were higher in rats fed RS 2 than in rats fed RS 1 or control diet (P < .05). Lactobacillus cellobiosus occurred only in rats fed RS 1 or RS 2. Acetate increased in mo 3 compared with d 8 in all groups (P < .05). The fecal and cecal SCFA displayed higher concentrations of acetate and propionate and a higher molar proportion of propionate in RS 2 than in RS 1 or control rats (P < .05). Stimulation of bifidobacteria, lactobacilli, and SCFA may be useful for the suppression of pathogenic organisms in the colon.

A new technique to determine hydrogen excreted by gnotobiotic rats.

A new system, that allowed the monitoring of hydrogen (H2) excretion by gnotobiotic rats without affecting their defined microbial status, was developed. The system consists of an isolator containing a chamber for an experimental animal, and a life-support system (LSS), with a sampling port outside the isolator connected to it. H2 accumulation in the system was measured by analysing a defined volume of gas after removal. H2 concentrations were determined with an electrochemical cell or by gas chromatography. To validate this technique, H2 excretion by germ-free (GF) and mono-associated rats fed a chemically defined diet was measured after oral application of lactulose. Mono-associated rats had been obtained by colonizing GF rats with a H2-producing Clostridium perfringens type A strain isolated from human faeces of a healthy volunteer. Application of 50 mg lactulose to the mono-associated rats resulted in a significant increase in H2 excretion. The net H2 excretion was 7.82+/-1.28 ml H2 in 12 h corresponding to a net maximal rate of 1.1+/-0.3 ml H2/h. In contrast, in experiments with GF rats, less than 0.13 ml H2 were detectable within 12 h. The technique presented is a useful tool for studying bacterial H2 metabolism in vivo under gnotobiotic conditions.

Evolution of the gut microbiota and the influence of diet.

Diet is a major force that shapes the composition and activity of the gut microbiota. This is evident from alterations in gut microbiota composition after weaning or drastic dietary changes. Owing to the complexity of the microbiota, interactions of intestinal bacteria with the host are difficult to study. Gnotobiotic animal models offer the opportunity to reduce the complexity and the interindividual variability of the intestinal microbiota. Germ-free animals were associated with a simplified microbial community consisting of eight bacterial species, that are found in the human gut. These microbes were selected because their genome sequences are available, and they mimic to some extent the metabolic activity of the human gut microbiota. The microbiota responded to dietary modifications by changes in the relative proportions of the community members. This model offers the chance to better define the role of intestinal bacteria in obesity development, but little is known on how diet affects intestinal bacteria at the cellular level. Mice monoassociated with Escherichia coli were used as a simplified model to investigate the influence of dietary factors on bacterial protein expression in the intestine. The mice were fed three different diets: a carbohydrate (lactose)-rich diet, a protein-rich diet and a diet rich in starch. The lactose-rich diet led to an induction of proteins involved in E. coli's oxidative stress response (Fur, AhpF, Dps). The corresponding genes are under control of the OxyR transcriptional regulator which is activated by oxidative stress. Further experiments demonstrated that osmotic stress exerted by various carbohydrates leads to an upregulation of proteins belonging to the oxyR regulon. The data suggest that the upregulated proteins enable intestinal E. coli to better cope with diet-induced osmotic stress. These examples demonstrate that gnotobiotic animal models are a valuable tool for studying diet-induced changes at the community and the cell level.

Increased bacterial putrescine has no impact on gut morphology and physiology in gnotobiotic adolescent mice.

Gut bacteria influence host anatomy and physiology. It has been proposed that bacterial metabolites including polyamines are responsible for intestinal maturation and mucosal growth. We have hypothesised that bacterially produced polyamines act as trophic factors and thereby influence large intestinal crypt depth and thickness of the different gut layers. For that purpose, germ-free mice were associated with two different microbial consortia. One group was colonised with a simplified human microbiota (SIHUMI). The second group was associated with SIHUMI + Fusobacterium varium (SIHUMI + Fv), which is known to produce high amounts of polyamines. Polyamine concentrations were measured by HPLC and morphological parameters were determined microscopically. Germ-free and conventional mice served as controls. The caecal putrescine concentration of the SIHUMI + Fv was 61.8 µM (47.6-75.5 µM), whereas that of conventional and SIHUMI mice was 28.8 µM (1.3-41.7 µM) and 24.5 µM (16.8-29.1 µM), respectively. The caecal putrescine concentration of germ-free mice was only 0.6 µM (0-1.0 µM). Caecal crypt depth and thickness of the different caecal layers revealed no significant differences between SIHUMI and SIHUMI + Fv mice. However, the crypt depth in the caeca of conventional, SIHUMI and SIHUMI + Fv mice was increased by 48.6% (P<0.001), 39.7% (P<0.001) and 28.5% (P<0.05), respectively, compared to germ-free mice. These findings indicate that increased intestinal putrescine concentrations do not influence gut morphology in our gnotobiotic adolescent mice.

Enterococcus faecium NCIMB 10415 does not protect interleukin-10 knock-out mice from chronic gut inflammation.

Enterococcus faecium NCIMB 10415 reduces diarrhoea incidence and duration in animals and human study subjects. We tested whether the strain is also capable of reducing chronic gut inflammation and aimed to identify mechanisms that are involved in possible probiotic effects. To identify health-promoting mechanisms of the strain, we used interleukin-10-deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB 10415 for 3, 8 and 24 weeks, respectively. Control mice were fed a diet which was identically composed but did not contain the strain. After 3 weeks of intervention the experimental animals were less inflamed in the caecum than the control animals. This effect was not observed in the colon and there were no differences between experimental and control mice at any other time point. The application of the strain was associated with higher expression levels of interferon gamma and interferon gamma-induced protein 10 after 3 and 24 but not after 8 weeks of feeding. No differences between the animals were observed in intestinal barrier function or intestinal microbiota composition. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB 10415 for 8 weeks. These low cell numbers were associated with a significantly lower caecal inflammation score and improved paracellular permeability as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB 10415 is not capable of reducing gut inflammation in the IL-10-/- mouse model. The exact role of A. muciniphila and of a possible interaction between this bacterium, NCIMB 10415 and the host in gut inflammation requires further investigation.

Expression of heparan sulfate proteoglycans in murine models of experimental colitis.

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are considered important in maintaining physiological homeostasis in many systems. Their expression is altered greatly in several pathophysiological conditions. Herein, we assess the expression and cellular localization of HSPGs in two murine models of human inflammatory bowel disease (IBD). METHODS: Expression and localization of HSPGs, syndecans, and HS epitopes were examined in the colon of 129SvEv interleukin 10 knockout (IL10(-/-)), C3Bir IL10(-/-), and their genetic control (IL10(+/+)) counterparts (129SvEv; C3H/HeJ). mRNA expression of syndecans and heparan sulfate biosynthesis enzymes were evaluated by real-time polymerase chain reaction (PCR). Localization of HSPGs was determined by immunofluorescence. RESULTS: mRNA for all syndecans was detected and expression in colonic tissues altered in IL10(-/-) mice. Syndecan-1 protein was expressed in the intestinal epithelium and on lamina propria cells of IL10(-/-) and control mice but was significantly reduced on the intestinal epithelial cells of IL10(-/-), mice particularly with severe colitis. Syndecan-2 was not detected, whereas syndecan-3 immunoreactivity was localized in the lamina propria but did not differ between control and IL10(-/-) mice. Syndecan-4 was present on epithelial cells of all mice but was significantly reduced in IL10(-/-) mice. Differences in the expression of HS epitopes between control and IL10(-/-) mice were also confirmed. CONCLUSIONS: The study has revealed altered expression of syndecan-1 and -4 and HS epitopes in the gut of mice with an IBD-like gut disorder. The IL10(-/-) mouse is a useful model for further study of the functional role of HSPGs in chronic inflammation and in maintaining healthy gut barrier.

Balancing inflammatory, lipid, and xenobiotic signaling pathways by VSL#3, a biotherapeutic agent, in the treatment of inflammatory bowel disease.

BACKGROUND: The interleukin 10 knockout mouse (IL10-KO) is a model of human inflammatory bowel disease (IBD) used to study host microbial interactions and the action of potential therapeutics. Using Affymetrix data analysis, important signaling pathways and transcription factors relevant to gut inflammation and antiinflammatory probiotics were identified. METHODS: Affymetrix microarray analysis on both wildtype (WT) and IL10-KO mice orally administered with and without the probiotic VSL#3 was performed and the results validated by real-time polymerase chain reaction (PCR), immunocytochemistry, proteomics, and histopathology. Changes in metabolically active bacteria were assessed with denaturing gradient gel electrophoresis (DGGE). RESULTS: Inflammation in IL10-KO mice was characterized by differential regulation of inflammatory, nuclear receptor, lipid, and xenobiotic signaling pathways. Probiotic intervention resulted in downregulation of CXCL9 (fold change [FC] = -3.98, false discovery rate [FDR] = 0.019), CXCL10 (FC = -4.83, FDR = 0.0008), CCL5 (FC = -3.47, FDR = 0.017), T-cell activation (Itgal [FC = -4.72, FDR = 0.00009], Itgae [FC = -2.54 FDR = 0.0044]) and the autophagy gene IRGM (FC = -1.94, FDR = 0.01), a recently identified susceptibility gene in human IBD. Consistent with a marked reduction in integrins, probiotic treatment decreased the number of CCL5+ CD3+ double-positive T cells and upregulated galectin2, which triggers apoptosis of activated T cells. Importantly, genes associated with lipid and PPAR signaling (PPARalpha [FC = 2.36, FDR = 0.043], PPARGC1alpha [FC = 2.58, FDR = 0.016], Nr1d2 [FC = 3.11, FDR = 0.0067]) were also upregulated. Altered microbial diversity was noted in probiotic-treated mice. CONCLUSIONS: Bioinformatics analysis revealed important immune response, phagocytic and inflammatory pathways dominated by elevation of T-helper cell 1 type (TH1) transcription factors in IL10-KO mice. Probiotic intervention resulted in a site-specific reduction of these pathways but importantly upregulated PPAR, xenobiotic, and lipid signaling genes, potential antagonists of NF-kappaB inflammatory pathways.