The disruption of the protective intestinal barrier—the ‘leaky gut’—is a common complication of the inflammatory bowel disease. There is limited data on the mechanisms of the intestinal barrier disruption upon low-grade inflammation characteristic of patients with inflammatory bowel disease in clinical remission. Thus, animal models that recapitulate the complexity of chronic intestinal inflammation in vivo are of particular interest. In this study, we used Mucin-2 (Muc2) knockout mice predisposed to colitis to study intestinal barrier upon chronic inflammation. We used 4-kDa FITC-Dextran assay and transmission electron microscopy to demonstrate the increased intestinal permeability and morphological defects in intercellular junctions in Muc2 knockout mice. Confocal microscopy revealed the disruption of the apical F-actin cytoskeleton and delocalization of tight junction protein Claudin-3 from the membrane. We further demonstrate mitochondrial damage, impaired oxygen consumption and the reduction of the intestinal ATP content in Muc2 knockout mice. Finally, we show that chemically induced mitochondrial uncoupling in the wild type mice mimics the intestinal barrier disruption in vivo and causes partial loss of F-actin and membrane localization of Claudin-3. We propose that mitochondrial damage and metabolic shifts during chronic inflammation contribute to the leaky gut syndrome in Muc2 knockout animal model of colitis.

Growing evidence suggests that intestinal mucosa homeostasis impacts immunity, metabolism, the Central Nervous System (CNS), and behavior. Here, we investigated the effect of the monosaccharide fucose on inflammation, metabolism, intestinal microbiota, and social behavior in the Dextran Sulfate Sodium (DSS)-induced chronic colitis mouse model. Our data show that chronic colitis is accompanied by the decrease of the serum tryptophan level and the depletion of the intestinal microbiota, specifically tryptophan-producing E. coli and Bifidobacterium. These changes are associated with defects in the male mouse social behavior such as a lack of preference towards female bedding in an odor preference test. The addition of fucose to the test animals' diet altered the bacterial community, increased the abundance of tryptophan-producing E. coli, normalized blood tryptophan levels, and ameliorated social behavior deficits. At the same time, we observed no ameliorating effect of fucose on colon morphology and colitis. Our results suggest a possible mechanism by which intestinal inflammation affects social behavior in male mice. We propose fucose as a promising prebiotic, since it creates a favorable environment for the beneficial bacteria that promote normalization of serum tryptophan level and amelioration of the behavioral abnormalities in the odor preference test.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory disorders of the gastrointestinal tract with complex etiology and no strategies for complete cure. IBD are often complicated by mental disorders like anxiety and depression, indicating substantial shifts in the microbiota gut-brain axis. However, the mechanisms connecting IBD to mental diseases are still under debate. Here we use Muc2 knockout mouse model of chronic colitis to uncouple the effects of the intestinal microbiota on host behavior from chronic inflammation in the gut. Muc2 knockout male mice exhibit high exploratory activity, reduced anxiety-related behaviors, impaired sensorimotor gating, and altered social preference towards males and females. Microbial transfer to wild-type mice via littermate co-housing shows that colitis-associated microbiota rather than inflammation per se defines behavioral features in Muc2 colitis model. Metagenomic profiling and combination of antibiotic treatments revealed that bacterial species Akkermansia muciniphila is associated with the behavioral phenotype in mutants, and that its intestinal abundance correlates with social preference towards males. Metabolomic analysis together with pharmacological inhibition of Gly and NMDA receptors helped us to determine that brain glycine is responsible for the behavioral phenotype in Muc2 mice. Blood and brain metabolic profiles suggest that microbiota-dependent changes in choline metabolism might be involved in regulation of central glycine neurotransmission. Taken together, our data demonstrates that colitis-associated microbiota controls anxiety, sensorimotor gating and social behavior via metabolic regulation of the brain glycinergic system, providing new venues to combat neurological complications of IBD.

The recessive gene ol-2 confers papillaassociated and race-non-speciWc resistance to tomato powdery mildew caused by Oidium neolycopersici. In order to facilitate marker assisted selection (MAS) in practical breeding programmes, we identiWed two simple sequence repeat (SSR) markers and one cleaved ampliWed polymorphic sequence (CAPS) marker which are linked to the resistance locus and co-dominantly inherited. Aiming to provide a base for ol-2 positional cloning, we used a large segregating F 2 population to merge these markers with all the ol-2 linked ampliWed fragment length polymorphism (AFLP ® ) markers previously identiWed in an integrated genetic map. By screening a tomato bacterial artiWcial chromosome (BAC) library, we detected two BAC clones containing two expressed sequence tags (ESTs) homologous to the gene mlo, responsible for powdery mildew resistance in barley, as well as an ol-2-linked marker. Chromosomal mapping by Fluorescence in situ Hybridization (FISH) revealed major signals of the two BAC DNAs in the pericentromeric heterochromatin of the short arm of chromosome 4, in the same region where the ol-2 gene was previously mapped. The genetic and cytogenetic co-localisation between ol-2 and tomato mlo-homologue(s), in addition to the similarity of ol-2 and mlo resistances for both genetic and phytopathological characteristics, suggests that ol-2 is likely a mlo-homologue. Thus, a homology-based cloning approach could be more suitable than positional cloning for ol-2 isolation.Keywords Fluorescence in situ hybridization · Homology-based cloning · Integrated linkage map · Map-based cloning · Oidium neolycopersici · Solanum lycopersicum

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The mucus layer in the intestine plays a critical role in regulation of host–microbe interactions and maintaining homeostasis. Disruptions of the mucus layer due to genetic, environmental, or immune factors may lead to inflammatory bowel diseases (IBD). IBD frequently are accompanied with infections, and therefore are treated with antibiotics. Hence, it is important to evaluate risks of antibiotic treatment in individuals with vulnerable gut barrier and chronic inflammation. Mice with a knockout of the Muc2 gene, encoding the main glycoprotein component of the mucus, demonstrate a close contact of the microbes with the gut epithelium which leads to chronic inflammation resembling IBD. Here we demonstrate that the Muc2−/− mice harboring a gut protozoan infection Tritrichomonas sp. are susceptible to an antibiotic-induced depletion of the bacterial microbiota. Suppression of the protozoan infection with efficient metronidazole dosage or L-fucose administration resulted in amelioration of an illness observed in antibiotic-treated Muc2−/− mice. Fucose is a monosaccharide presented abundantly in gut glycoproteins, including Mucin2, and is known to be involved in host–microbe interactions, in particular in microbe adhesion. We suppose that further investigation of the role of fucose in protozoan adhesion to host cells may be of great value.

Fucose, the terminal glycan of the intestinal glycoprotein Mucin2, was shown to have an anti-inflammatory effect in mouse colitis models and modulate immune response due to macrophage polarization changes. In this study we evaluated the effect of 0.05% L-fucose supplementation of drinking water on immune parameters in the intestine of homozygous mutant Muc2−/−, compared to Muc2+/+ mice. To get into innate and adaptive immunity mechanisms of gut inflammation, we tested PrkdcSCIDMuc2−/− strain, Muc2 knockout on SCID background, that is characterized by lack of lymphocytes, in comparison with PrkdcSCID mice. We evaluated intestinal cytokine profiling, macrophage and eosinophil infiltration, and expression of Nos2 and Arg1 markers of macrophage activation in all strains. Markers of Th1, Treg and Th17 cells (Tbx21, Foxp3, and Rorc expression) were evaluated in Muc2−/− and Muc2+/+ mice. Both Muc2−/− and PrkdcSCIDMuc2−/− mice demonstrated increased numbers of macrophages, eosinophils, elevated levels of TNFa, GM-CSF, and IL-10 cytokines. In Muc2−/− mice we observed a wide range of pro-inflammatory cytokines elevated, such as IFN-gamma, IL-1b, IL-12p70, IL-6, M-CSF, G-CSF, IL-17, MCP-1, RANTES, MIP1b, MIP2. Muc2−/− mice demonstrated increase of Nos2, Tbx21 and Foxp3 genes mRNA, while in PrkdcSCIDMuc2−/− mice Arg1 expression was increased. We found that in Muc2−/− mice L-fucose reduced macrophage infiltration and IL-1a, TNFa, IFNgamma, IL-6, MCP-1, RANTES, MIP1b levels, decreased Nos2 expression, and induced the expression of Treg marker Foxp3 gene. On the contrary, in PrkdcSCIDMuc2−/− mice L-fucose had no effect on macrophage and eosinophil numbers, but increased TNFa, GM-CSF, IL-12p70, IL-6, IL-15, IL-10, MCP1, G-CSF, IL-3 levels and Nos2 gene expression, and decreased Arg1 gene expression. We demonstrated that anti-inflammatory effect of L-fucose observed in Muc2−/− mice is not reproduced in PrkdcSCIDMuc2−/−, which lack lymphocytes. We conclude that activation of Treg cells is a key event that leads to resolution of inflammation upon L-fucose supplementation in Muc2−/− mice.

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