Naturalizing laboratory mice by housing in a farmyard-type habitat confers protection against colorectal carcinogenesis.

Living in a farm environment in proximity to animals is associated with reduced risk of developing allergies and asthma, and has been suggested to protect against other diseases, such as inflammatory bowel disease and cancer. Despite epidemiological evidence, experimental disease models that recapitulate such environments are needed to understand the underlying mechanisms. In this study, we show that feralizing conventional inbred mice by continuous exposure to a livestock farmyard-type environment conferred protection toward colorectal carcinogenesis. Two independent experimental approaches for colorectal cancer induction were used; spontaneous (Apc Min/+ mice on an A/J background) or chemical (AOM/DSS). In contrast to conventionally reared laboratory mice, the feralized mouse gut microbiota structure remained stable and resistant to mutagen- and colitis-induced neoplasia. Moreover, the feralized mice exhibited signs of a more mature immunophenotype, indicated by increased expression of NK and T-cell maturation markers, and a more potent IFN-γ response to stimuli. In our study, hygienically born and raised mice subsequently feralized post-weaning were protected to a similar level as life-long exposed mice, although the greatest effect was seen upon neonatal exposure. Collectively, we show protective implications of a farmyard-type environment on colorectal cancer development and demonstrate the utility of a novel animal modeling approach that recapitulates realistic disease responses in a naturalized mammal.

High microbiota reactivity of adult human intestinal IgA requires somatic mutations.

The gut is home to the body's largest population of plasma cells. In healthy individuals, IgA is the dominating isotype, whereas patients with inflammatory bowel disease also produce high concentrations of IgG. In the gut lumen, secretory IgA binds pathogens and toxins but also the microbiota. However, the antigen specificity of IgA and IgG for the microbiota and underlying mechanisms of antibody binding to bacteria are largely unknown. Here we show that microbiota binding is a defining property of human intestinal antibodies in both healthy and inflamed gut. Some bacterial taxa were commonly targeted by different monoclonal antibodies, whereas others selectively bound single antibodies. Interestingly, individual human monoclonal antibodies from both healthy and inflamed intestines bound phylogenetically unrelated bacterial species. This microbiota cross-species reactivity did not correlate with antibody polyreactivity but was crucially dependent on the accumulation of somatic mutations. Therefore, our data suggest that a system of affinity-matured, microbiota cross-species-reactive IgA is a common aspect of SIgA-microbiota interactions in the gut.

Activated ATF6 Induces Intestinal Dysbiosis and Innate Immune Response to Promote Colorectal Tumorigenesis.

BACKGROUND & AIMS: Activating transcription factor 6 (ATF6) regulates endoplasmic reticulum stress. We studied whether ATF6 contributes to the development of colorectal cancer (CRC) using tissue from patients and transgenic mice. METHODS: We analyzed data from 541 patients with CRC in The Cancer Genome Atlas database for genetic variants and aberrant expression levels of unfolded protein response genes. Findings were validated in a cohort of 83 patients with CRC in Germany. We generated mice with intestinal epithelial cell-specific expression of the active form of Atf6 (nATF6IEC) from 2 alleles (homozygous), mice with expression of nATF6IEC from 1 allele (heterozygous), and nATF6IECfl/fl mice (controls). All nATF6IEC mice were housed under either specific-pathogen-free or germ-free conditions. Cecal microbiota from homozygous nATF6IEC mice or control mice was transferred into homozygous nATF6IEC mice or control mice. nATF6IEC mice were crossed with mice with disruptions in the myeloid differentiation primary response gene 88 and toll-like receptor adaptor molecule 1 gene (Myd88/Trif-knockout mice). Intestinal tissues were collected from mice and analyzed by histology, immunohistochemistry, immunoblots, gene expression profiling of unfolded protein response and inflammatory genes, array-based comparative genome hybridization, and 16S ribosomal RNA gene sequencing. RESULTS: Increased expression of ATF6 was associated with reduced disease-free survival times of patients with CRC. Homozygous nATF6IEC mice developed spontaneous colon adenomas at 12 weeks of age. Compared with controls, homozygous nATF6IEC mice had changes in the profile of their cecal microbiota, increased proliferation of intestinal epithelial cells, and loss of the mucus barrier-all preceding tumor formation. These mice had increased penetration of bacteria into the inner mucus layer and activation of signal transducer and activator of transcription 3, yet inflammation was not observed at the pretumor or tumor stages. Administration of antibiotics to homozygous nATF6IEC mice greatly reduced tumor incidence, and germ-free housing completely prevented tumorigenesis. Analysis of nATF6IEC MyD88/TRIF-knockout mice showed that tumor initiation and growth required MyD88/TRIF-dependent activation of signal transducer and activator of transcription 3. Transplantation of cecal microbiota from nATF6IEC mice and control mice, collected before tumor formation, caused tumor formation in ex-germ-free nATF6IEC mice. CONCLUSIONS: In patients with CRC, ATF6 was associated with reduced time of disease-free survival. In studies of nATF6IEC mice, we found sustained intestinal activation of ATF6 in the colon to promote dysbiosis and microbiota-dependent tumorigenesis.

Nlrp3-dependent IL-1ß inhibits CD103+ dendritic cell differentiation in the gut.

Inflammatory bowel disease (IBD) is associated with enhanced levels of the IL-1 family cytokines IL-1ß and IL-18, which are activated by the Nlrp3 inflammasome. Here, we investigated the role of inflammasome-driven cytokine release on T cell polarization and DC differentiation in steady state and T cell transfer colitis. In vitro and in vivo data showed that IL-1ß induces Th17 polarization and increases GM­CSF production by T cells. Reduced IL-1ß levels in Nlrp3-/- mice correlated with enhanced FLT3L levels and increased frequency of tolerogenic CD103+ DC. In the T cell transfer colitis model, Nlrp3 deficiency resulted in lower IL­1ß levels, reduced Th17 immunity, and less severe colitis. Unaltered IL-18 levels in both mouse strains pointed toward Nlrp3-independent processing. Importantly, cohousing revealed that the gut microbiome had no impact on the observed Nlrp3-/- phenotype. This study demonstrates that NLRP3 acts as a molecular switch of intestinal homeostasis by shifting local immune cells toward an inflammatory phenotype via IL-1ß.

Determinants of postprandial plasma bile acid kinetics in human volunteers.

Bile acids (BA) are signaling molecules with a wide range of biological effects, also identified among the most responsive plasma metabolites in the postprandial state. We here describe this response to different dietary challenges and report on key determinants linked to its interindividual variability. Healthy men and women (n = 72, 62 ± 8 yr, mean ± SE) were enrolled into a 12-wk weight loss intervention. All subjects underwent an oral glucose tolerance test and a mixed-meal tolerance test before and after the intervention. BA were quantified in plasma by liquid chromatography-tandem mass spectrometry combined with whole genome exome sequencing and fecal microbiota profiling. Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma BA profiles. Fasting and postprandial BA profiles revealed high interindividual variability, and three main patterns in postprandial BA response were identified using multivariate analysis. Although the women enrolled were postmenopausal, effects of sex difference in BA response were evident. Exome data revealed the contribution of preselected genes to the observed interindividual variability. In particular, a variant in the SLCO1A2 gene, encoding the small intestinal BA transporter organic anion-transporting polypeptide-1A2 (OATP1A2), was associated with delayed postprandial BA increases. Fecal microbiota analysis did not reveal evidence for a significant influence of bacterial diversity and/or composition on plasma BA profiles. The analysis of plasma BA profiles in response to two different dietary challenges revealed a high interindividual variability, which was mainly determined by genetics and sex difference of host with minimal effects of the microbiota.NEW & NOTEWORTHY Considering the average response of all 72 subjects, no effect of the successful weight loss intervention was found on plasma bile acid (BA) profiles. Despite high interindividual variability, three main patterns in postprandial BA response were identified using multivariate analysis. A variant in the SLCO1A2 gene, encoding the small intestinal BA transporter organic anion-transporting polypeptide-1A2 (OATP1A2), was associated with delayed postprandial BA increases in response to both the oral glucose tolerance test and the mixed-meal tolerance test.

Oral versus intravenous iron replacement therapy distinctly alters the gut microbiota and metabolome in patients with IBD.

OBJECTIVE: Iron deficiency is a common complication in patients with IBD and oral iron therapy is suggested to exacerbate IBD symptoms. We performed an open-labelled clinical trial to compare the effects of per oral (PO) versus intravenous (IV) iron replacement therapy (IRT). DESIGN: The study population included patients with Crohn's disease (CD; N=31), UC (N=22) and control subjects with iron deficiency (non-inflamed, NI=19). After randomisation, participants received iron sulfate (PO) or iron sucrose (IV) over 3 months. Clinical parameters, faecal bacterial communities and metabolomes were assessed before and after intervention. RESULTS: Both PO and IV treatments ameliorated iron deficiency, but higher ferritin levels were observed with IV. Changes in disease activity were independent of iron treatment types. Faecal samples in IBD were characterised by marked interindividual differences, lower phylotype richness and proportions of Clostridiales. Metabolite analysis also showed separation of both UC and CD from control anaemic participants. Major shifts in bacterial diversity occurred in approximately half of all participants after IRT, but patients with CD were most susceptible. Despite individual-specific changes in phylotypes due to IRT, PO treatment was associated with decreased abundances of operational taxonomic units assigned to the species Faecalibacterium prausnitzii, Ruminococcus bromii, Dorea sp. and Collinsella aerofaciens. Clear IV-specific and PO-specific fingerprints were evident at the level of metabolomes, with changes affecting cholesterol-derived host substrates. CONCLUSIONS: Shifts in gut bacterial diversity and composition associated with iron treatment are pronounced in IBD participants. Despite similar clinical outcome, oral administration differentially affects bacterial phylotypes and faecal metabolites compared with IV therapy. TRIAL REGISTRATION NUMBER: clinicaltrial.gov (NCT01067547).

Dietary fat and gut microbiota interactions determine diet-induced obesity in mice.

OBJECTIVE: Gut microbiota may promote positive energy balance; however, germfree mice can be either resistant or susceptible to diet-induced obesity (DIO) depending on the type of dietary intervention. We here sought to identify the dietary constituents that determine the susceptibility to body fat accretion in germfree (GF) mice. METHODS: GF and specific pathogen free (SPF) male C57BL/6N mice were fed high-fat diets either based on lard or palm oil for 4 wks. Mice were metabolically characterized at the end of the feeding trial. FT-ICR-MS and UPLC-TOF-MS were used for cecal as well as hepatic metabolite profiling and cecal bile acids quantification, respectively. Hepatic gene expression was examined by qRT-PCR and cecal gut microbiota of SPF mice was analyzed by high-throughput 16S rRNA gene sequencing. RESULTS: GF mice, but not SPF mice, were completely DIO resistant when fed a cholesterol-rich lard-based high-fat diet, whereas on a cholesterol-free palm oil-based high-fat diet, DIO was independent of gut microbiota. In GF lard-fed mice, DIO resistance was conveyed by increased energy expenditure, preferential carbohydrate oxidation, and increased fecal fat and energy excretion. Cecal metabolite profiling revealed a shift in bile acid and steroid metabolites in these lean mice, with a significant rise in 17ß-estradiol, which is known to stimulate energy expenditure and interfere with bile acid metabolism. Decreased cecal bile acid levels were associated with decreased hepatic expression of genes involved in bile acid synthesis. These metabolic adaptations were largely attenuated in GF mice fed the palm-oil based high-fat diet. We propose that an interaction of gut microbiota and cholesterol metabolism is essential for fat accretion in normal SPF mice fed cholesterol-rich lard as the main dietary fat source. This is supported by a positive correlation between bile acid levels and specific bacteria of the order Clostridiales (phylum Firmicutes) as a characteristic feature of normal SPF mice fed lard. CONCLUSIONS: In conclusion, our study identified dietary cholesterol as a candidate ingredient affecting the crosstalk between gut microbiota and host metabolism.

Dual Role of the Adaptive Immune System in Liver Injury and Hepatocellular Carcinoma Development.

Hepatocellular carcinoma (HCC) represents a classic example of inflammation-linked cancer. To characterize the role of the immune system in hepatic injury and tumor development, we comparatively studied the extent of liver disease and hepatocarcinogenesis in immunocompromised versus immunocompetent Fah-deficient mice. Strikingly, chronic liver injury and tumor development were markedly suppressed in alymphoid Fah(-/-) mice despite an overall increased mortality. Mechanistically, we show that CD8(+) T cells and lymphotoxin ß are central mediators of HCC formation. Antibody-mediated depletion of CD8(+) T cells as well as pharmacological inhibition of the lymphotoxin-ß receptor markedly delays tumor development in mice with chronic liver injury. Thus, our study unveils distinct functions of the immune system, which are required for liver regeneration, survival, and hepatocarcinogenesis.

The mouse gut microbiome revisited: From complex diversity to model ecosystems.

Laboratory mice are the most commonly used animal model in translational medical research. In recent years, the impact of the gut microbiota (i.e. communities of microorganisms in the intestine) on host physiology and the onset of diseases, including metabolic and neuronal disorders, cancers, gastrointestinal infections and chronic inflammation, became a focal point of interest. There is abundant evidence that mouse phenotypes in disease models vary greatly between animal facilities or commercial providers, and that this variation is associated with differences in the microbiota. Hence, there is a clear discrepancy between the widespread use of mouse models in research and the patchwork knowledge on the mouse gut microbiome. In the present manuscript, we summarize data pertaining to the diversity and functions of the mouse gut microbiota, review existing work on gnotobiotic mouse models, and discuss challenges and opportunities for current and future research in the field.

Dysbiotic gut microbiota causes transmissible Crohn's disease-like ileitis independent of failure in antimicrobial defence.

OBJECTIVES: Dysbiosis of the intestinal microbiota is associated with Crohn's disease (CD). Functional evidence for a causal role of bacteria in the development of chronic small intestinal inflammation is lacking. Similar to human pathology, TNF(deltaARE) mice develop a tumour necrosis factor (TNF)-driven CD-like transmural inflammation with predominant ileal involvement. DESIGN: Heterozygous TNF(deltaARE) mice and wildtype (WT) littermates were housed under conventional (CONV), specific pathogen-free (SPF) and germ-free (GF) conditions. Microbial communities were analysed by high-throughput 16S ribosomal RNA gene sequencing. Metaproteomes were measured using LC-MS. Temporal and spatial resolution of disease development was followed after antibiotic treatment and transfer of microbial communities into GF mice. Granulocyte infiltration and Paneth cell function was assessed by immunofluorescence and gene expression analysis. RESULTS: GF-TNF(deltaARE) mice were free of inflammation in the gut and antibiotic treatment of CONV-TNF(deltaARE) mice attenuated ileitis but not colitis, demonstrating that disease severity and location are microbiota-dependent. SPF-TNF(deltaARE) mice developed distinct ileitis-phenotypes associated with gradual loss of antimicrobial defence. 16S analysis and metaproteomics revealed specific compositional and functional alterations of bacterial communities in inflamed mice. Transplantation of disease-associated but not healthy microbiota transmitted CD-like ileitis to GF-TNF(deltaARE) recipients and triggered loss of lysozyme and cryptdin-2 expression. Monoassociation of GF-TNF(deltaARE) mice with the human CD-related Escherichia coli LF82 did not induce ileitis. CONCLUSIONS: We provide clear experimental evidence for the causal role of gut bacterial dysbiosis in the development of chronic ileal inflammation with subsequent failure of Paneth cell function.

Orally administered allyl sulfides from garlic ameliorate murine colitis.

SCOPE: Inflammatory bowel disease (IBD) is an incurable disease which affects millions of people. Garlic (Allium sativum) preparations have been traditionally employed for the treatment of diseases affecting the digestive tract. Here, we have investigated the effect of diallyl sulfide (DAS) and diallyl disulfide (DADS), two garlic-derived sulfur compounds, on intestinal inflammation in vivo as well as in intestinal isolated cells. METHODS AND RESULTS: Colitis was induced in mice by intracolonic administration of dinitrobenzenesulfonic acid. Intestinal damage was assessed by evaluating colon weight/colon length ratio and by histology. Murine intestinal epithelial cells stimulated with IFN-γ were used to evaluate the possible in vitro DAS and DADS anti-inflammatory effects. DAS and DADS, given for two consecutive days after DNBS administration, reduced inflammation and damage. In IFN-γ-stimulated intestinal epithelial cells, DADS reduced IP-10 and IL-6 levels, while DAS inhibited nitric oxide production and STAT-1 expression. CONCLUSION: DAS and DADS exert therapeutic effects in the DNBS model of colitis. The actions of these compounds on the production of IP-10, IL-6, hydrogen sulfide or nitric oxide and on the expression of STAT-1 observed in intestinal cells stimulated with IFN-γ, might explain the protective action of DAS and DADS in experimental IBD.

Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes.

Hepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8(+) T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8(+) T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8(+) and NKT cells cooperatively induce liver damage. Hepatocellular LTßR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development.

Fetal exposure to maternal inflammation does not affect postnatal development of genetically-driven ileitis and colitis.

BACKGROUND: Chronic inflammatory disorders have been increasing in incidence over the past decades following geographical patterns of industrialization. Fetal exposure to maternal inflammation may alter organ functions and the offspring's disease risk. We studied the development of genetically-driven ileitis and colitis in response to maternal inflammation using mouse models. METHODS: Disease susceptible (TnfΔARE/+ and IL10-/-) and disease-free (Tnf+/+ and IL10-/+) offspring were raised in inflamed and non-inflamed dams. Ileal, caecal and colonic pathology was evaluated in the offspring at 8 or 12 weeks of age. Ly6G-positive cells in inflamed sections from the distal ileum and distal colon were analysed by immunofluorescence microscopy. Gene expression of pro-inflammatory cytokines was measured in whole tissue specimens by quantitative PCR. Microarray analyses were performed on laser microdissected intestinal epithelium. Caecal bacterial communities were assessed by Illumina sequencing of 16S rRNA amplicons. RESULTS: Disease severity, the number of infiltrated neutrophils as well as Tnf and Il12p40 mRNA expression were independent of maternal inflammation in the offspring of mouse models for ileitis (TnfΔARE/+) and colitis (IL10-/-). Although TNF-driven maternal inflammation regulated 2,174 (wild type) and 3,345 (TnfΔARE/+) genes in the fetal epithelium, prenatal gene expression patterns were completely overwritten after birth. In addition, co-housing experiments revealed no change in phylogenetic diversity of the offspring's caecal microbiota in response to maternal inflammation. This is independent of the offspring's genotype before and after the onset of tissue pathology. CONCLUSIONS: Disease risk and activity in mouse models of chronic ileitis and colitis was independent of the fetal exposure to maternal inflammation. Likewise, maternal inflammation did not alter the diversity and composition of offspring's caecal microbiota, clearly demonstrating that changes of the gene expression program in the fetal gut epithelium were not relevant for the development of chronic inflammatory disorders in the gut.

Lignan transformation by gut bacteria lowers tumor burden in a gnotobiotic rat model of breast cancer.

High dietary lignan exposure is implicated in a reduced breast cancer risk in women. The bacterial transformation of plant lignans to enterolignans is thought to be essential for this effect. To provide evidence for this assumption, gnotobiotic rats were colonized with the lignan-converting bacteria Clostridium saccharogumia, Eggerthella lenta, Blautia producta and Lactonifactor longoviformis (LCC rats). Germ-free rats were used as the control. All animals were fed a lignan-rich flaxseed diet and breast cancer was induced with 7,12-dimethylbenz(a)anthracene. The lignan secoisolariciresinol diglucoside was converted into the enterolignans enterodiol and enterolactone in the LCC but not in the germ-free rats. This transformation did not influence cancer incidence at the end of the 13 weeks experimental period but significantly decreased tumor numbers per tumor-bearing rat, tumor size, tumor cell proliferation and increased tumor cell apoptosis in LCC rats. No differences between LCC and control rats were observed in the expression of the genes encoding the estrogen receptors (ERs) α, ERß and G-coupled protein 30. The same was true for IGF-1 and EGFR involved in tumor growth. The activity of selected enzymes involved in the degradation of oxidants in plasma and liver was significantly increased in the LCC rats. However, plasma and liver concentrations of reduced glutathione and malondialdehyde, considered as oxidative stress markers, did not differ between the groups. In conclusion, our results show that the bacterial conversion of plant lignans to enterolignans beneficially influences their anticancer effects.

Catechols in caffeic acid phenethyl ester are essential for inhibition of TNF-mediated IP-10 expression through NF-κB-dependent but HO-1- and p38-independent mechanisms in mouse intestinal epithelial cells.

SCOPE: Caffeic acid phenethyl ester (CAPE) is an active constituent of honeybee propolis inhibiting nuclear factor (NF)-κB. The aims of our study were to provide new data on the functional relevance and mechanisms underlying the role of CAPE in regulating inflammatory processes at the epithelial interface in the gut and to determine the structure/activity relationship of CAPE. METHODS AND RESULTS: CAPE significantly inhibited TNF-induced IP-10 expression in intestinal epithelial cells. Using various analogues, we demonstrated that substitution of catechol hydroxyl groups and addition of one extra hydroxyl group on ring B reversed the functional activity of CAPE to inhibit IP-10 production. The anti-inflammatory potential of CAPE was confirmed in ileal tissue explants and embryonic fibroblasts derived from TNF(ΔARE/+) mice. Interestingly, CAPE inhibited both TNF- and LPS-induced IP-10 production in a dose-dependent manner, independently of p38 MAPK, HO-1 and Nrf2 signaling pathways. We found that CAPE did not inhibit TNF-induced IκB phosphorylation/degradation or nuclear translocation of RelA/p65, but targeted downstream signaling events at the level of transcription factor recruitment to the gene promoter. CONCLUSION: This study reveals the structure-activity effects and anti-inflammatory potential of CAPE in the intestinal epithelium.

Depletion of luminal iron alters the gut microbiota and prevents Crohn's disease-like ileitis.

BACKGROUND: Iron replacement therapy is a common treatment in patients with anaemia and Crohn's disease, but oral iron supplements are less tolerated. The pathogenesis of Crohn's disease is attributed to intestinal bacteria and environmental factors that trigger disease in a genetically predisposed host. The aim of this study was to characterise the interrelationship between luminal iron sulfate, systemic iron, the gut microbiota and the development of chronic ileitis in a murine model of Crohn's disease. METHODS: Wild type (WT) and heterozygous TNF(ΔARE/WT) mice were fed with an iron sulfate containing or iron sulfate free diet in combination with intraperitoneal control injections or iron injections for 11 weeks. RESULTS: TNF(ΔARE/WT) mice develop severe inflammation of the distal ileum but remained completely healthy when transferred to an iron sulfate free diet, even if iron was systemically repleted. Absence of luminal iron sulfate reduced cellular markers of endoplasmic reticulum (ER) stress responses and pro-apoptotic mechanisms in the ileal epithelium. Phenotype or reactivity of major effector intraepithelial CD8αß(+) T cells were not altered in the absence of luminal iron. Interestingly, ER stress mechanisms sensitised the small intestinal epithelial cell (IEC) line Mode-K to cytotoxic function of effector T cells from TNF(ARE/WT) mice. Pyrosequencing of 16S rRNA tags of the caecal microbiota revealed that depletion of luminal iron sulfate induced significant compositional alterations, while total microbial diversity (Shannon's diversity index) and number of total operational taxonomic units were not affected. CONCLUSION: This study showed that an iron sulfate free diet in combination with systemic iron repletion prevents the development of chronic ileitis in a murine model of Crohn's disease. Luminal iron may directly affect IEC function or generate a pathological milieu in the intestine that triggers epithelial cell stress-associated apoptosis through changes in microbial homeostasis. These results suggest that oral replacement therapy with iron sulfate may trigger inflammatory processes associated with progression of Crohn's disease-like ileitis.

Posttranslational inhibition of proinflammatory chemokine secretion in intestinal epithelial cells: implications for specific IBD indications.

BACKGROUND AND AIM: Inflammatory bowel diseases (IBD) are immune-mediated chronic diseases that are characterized by an overreaction of the intestinal immune system to the intestinal microbiota. VSL#3, a mixture of 8 different lactic acid bacteria, is a clinically relevant probiotic compound in the context of IBD, but the bacterial structures and molecular mechanisms underlying the observed protective effects are largely unknown. The intestinal epithelium plays a very important role in the maintenance of the intestinal homeostasis, as the intestinal epithelial cells (IEC) are capable of sensing, processing, and reacting upon signals from the luminal microbiota and the intestinal immune system. This immune regulatory function of the IEC is lost in IBD owing to dysregulated activation of the IEC. Thus, the aim of this study was to reveal protective mechanisms of VSL#3 on IEC function. RESULTS: In vitro, VSL#3 was found to selectively inhibit activation-induced secretion of the T-cell chemokine interferon-inducible protein (IP)-10 in IEC. Cell wall-associated proteins of VSL#3-derived Lactobacillus casei (L. casei) were identified to be the active anti-inflammatory component of VSL#3. Mechanistically, L. casei did not impair initial IP-10 protein production, but induced posttranslational degradation of IP-10 in IEC. Feeding studies in tumor necrosis factor (TNF)Delta ARE/+ mice, a mouse model for experimental ileitis, revealed that neither VSL#3 nor L. casei is capable of reducing ileal inflammation. Even preweaning feeding of VSL#3 did not prevent the development of severe ileitis in TNF Delta ARE/+ mice. In contrast, VSL#3 feeding studies in IL-10-/- mice, a model for experimental colitis, revealed that VSL#3 has local, intestinal compartment-specific protective effects on the development of inflammation. Reduced histopathologic inflammation in the cecum of IL-10-/- mice after VSL#3 treatment was found to correlate with reduced levels of IP-10 protein in primary cecal epithelial cells. CONCLUSION AND OUTLOOK: These results suggest that the inhibitory effect of VSL#3-derived L. casei on IP-10 secretion in IEC is an important probiotic mechanism that contributes to the anti-inflammatory effects of VSL#3 in specific subsets of patients with IBD. An important future aim is the identification of the active probiotic protein, which could serve as a basis for the development of new efficient therapies in the context of IBD.

Isolation of bacteria from the ileal mucosa of TNFdeltaARE mice and description of Enterorhabdus mucosicola gen. nov., sp. nov.

The diversity of bacteria associated with inflamed mucosa was investigated by culturing ileal samples from TNF(deltaARE) mice on a selective medium containing mucin. Among eight isolates, two strains (Mt1B3 and Mt1B8(T)) belonged to bacterial groups not yet cultured from the mouse intestine. Whereas strain Mt1B3 was identified as a member of the family Planococcaceae and is closely related to Sporosarcina species and Filibacter limicola DSM 13886(T), strain Mt1B8(T) was a novel bacterium. Based on phylogenetic analysis, strain Mt1B8(T) is a member of the family Coriobacteriaceae. The closest relatives with validly published names were Asaccharobacter celatus, Adlercreutzia equolifaciens (<96 % similarity) and Eggerthella species (<92 %). With respect to Asaccharobacter celatus and Eggerthella, the phylogenetic position of strain Mt1B8(T) was confirmed at the chemotaxonomic level by Fourier-transform infrared spectroscopic analysis. The major fatty acid of strain Mt1B8(T) is C(16 : 0) (23.9 %). Menaquinones were monomethylated. DNA-DNA relatedness between strain Mt1B8(T) and Asaccharobacter celatus DSM 18785(T) was 28 %. Strain Mt1B8(T) is a Gram-positive-staining rod that does not form spores and has a high DNA G+C content (64.2 mol%). Cells are aerotolerant but grow only under strictly anoxic conditions. They are sensitive to cefotaxime, clarithromycin, erythromycin, metronidazole, tetracycline, tobramycin and vancomycin. API and VITEK analysis showed the ability of strain Mt1B8(T) to convert a variety of amino acid derivatives. According to these findings, it is proposed to create a novel genus and species, Enterorhabdus mucosicola gen. nov., sp. nov., to accommodate strain Mt1B8(T). The type strain of Enterorhabdus mucosicola is Mt1B8(T) (=DSM 19490(T) =CCUG 54980(T)).

Post-translational inhibition of IP-10 secretion in IEC by probiotic bacteria: impact on chronic inflammation.

BACKGROUND: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC). METHODOLOGY/PRINCIPAL FINDINGS: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF(DeltaARE) and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells. CONCLUSIONS/SIGNIFICANCE: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.