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15 p.-5 fig.-1 tab.-1 graph abst. This paper is dedicated to Prof. Jesús Jiménez-Barbero for his 60th birthday., The gut microbiota guide the development of the host immune system by setting a systemic threshold for immune activation. Lipopolysaccharides (LPSs) from gut bacteria are able to trigger systemic and local proinflammatory and immunomodulatory responses, and this capability strongly relies on their fine structures. Up to now, only a few LPS structures from gut commensals have been elucidated; therefore, the molecular motifs that may be important for LPS–mammalian cell interactions at the gut level are still obscure. Here, we report on the full structure of the LPS isolated from one of the prominent species of the genus Bacteroides, Bacteroides vulgatus. The LPS turned out to consist of a particular chemical structure based on hypoacylated and mono-phosphorylated lipid A and with a galactofuranose-containing core oligosaccharide and an O-antigen built up of mannose and rhamnose. The evaluation of the immunological properties of this LPS on human in vitro models revealed a very interesting capability to produce anti-inflammatory cytokines and to induce a synergistic action of MD-2/TLR4- and TLR2-mediated signaling pathways., F.D.L. acknowledges Progetto STAR 2018 Linea 1 grant E66C18001330003. S.M.S. acknowledges Spanish Ministry of Science (ref. CTQ2017-88353-R). A.M., F.D.L., and A.S.acknowledge H2020 Marie Skłodowska-Curie ITN 2018 “SweetCrossTalk” grant 814102. A.M. acknowledges progetto POR SATIN POR-FESR 2014−2 0 2 0 g r a n t B61C17000070007 (OR3) and Progetto POR Campania Oncoterapia 2014−2020 grant B61G18000470007. A.S. acknowledges PRIN-MIUR 2017 Glytunes project. A.S. and F.C. acknowledge COST (European Cooperation in Science and Technology) Action CA18103 (INNOGLY). F.C. was financially supported by the NWO Spinoza award of Y.K.

The Oligo-Mouse-Microbiota (OMM12) is a recently developed synthetic bacterial community for functional microbiome research in mouse models (Brugiroux et al., 2016). To date, the OMM12 model has been established in several germ-free mouse facilities world-wide and is employed to address a growing variety of research questions related to infection biology, mucosal immunology, microbial ecology and host-microbiome metabolic cross-talk. The OMM12 consists of 12 sequenced and publically available strains isolated from mice, representing five bacterial phyla that are naturally abundant in the murine gastrointestinal tract (Lagkouvardos et al., 2016). Under germ-free conditions, the OMM12 colonizes mice stably over multiple generations. Here, we investigated whether stably colonized OMM12 mouse lines could be reproducibly established in different animal facilities. Germ-free C57Bl/6J mice were inoculated with a frozen mixture of the OMM12 strains. Within 2 weeks after application, the OMM12 community reached the same stable composition in all facilities, as determined by fecal microbiome analysis. We show that a second application of the OMM12 strains after 72 h leads to a more stable community composition than a single application. The availability of such protocols for reliable de novo generation of gnotobiotic rodents will certainly contribute to increasing experimental reproducibility in biomedical research.

The proper communication between gut and brain is pivotal for maintenance of health and dysregulation of the gut-brain axis can lead to several clinical disorders. Also, in Parkinson’s disease (PD) 85% of all patients experienced constipation long before showing any signs of motor phenotypes. For differential diagnosis and when it comes to preventive treatment there is an urgent need for the identification of biomarkers indicating early disease stages long before the disease phenotype manifests. DJ-1 is a chaperon protein involved in the protection against PD and genetic mutations in this protein have been shown to cause familial PD. However, how the deficiency of DJ-1 modifies the PD risk remains incompletely understood. In the present study we provide evidence that DJ-1 is implicated in shaping the gut microbiome including their metabolite production or innate immune cells (ILCs) development. We revealed that in 4 months old mice genetic deficiency of DJ-1 leads to significantly decrease in several bacterial genera and significantly increase in two specific genera, namelyAlistipesandRikenella. DJ-1 deficient mice have a higher production of calprotectin/MCP-1 inflammatory protein - a known protein involved in colonic inflammation – and significantly higher expression of glial fibrillary acidic protein (GFAP) than control littermates. Expression of a-Synuclein, a key protein in Lewy bodies, in the colon was not significantly different between genotypes. Metabolic profiles of feces extracts analysed by H1-NMR spectroscopy showed increased short chain fatty acids (SCFAs) and decreased amino acid levels, suggesting a general switch from protein towards fibre degrading strains in DJ-1 deficient mice. We observed that Malonate - which is known to influence the immune system – has significantly higher concentrations in DJ-1 deficient mice. Moreover, DJ-1 deficient mice have high levels of the phenol derivate 3-(3-Hydroxyphenyl) propanoic acid (3-HPPA) which is a breakdown product of aromatic substrates like tyrosine, phenylalanine and polyphenols. DJ-1 deficient mice also showed significantly reduced percentage of ILCs. Thus, our data suggests that absence of DJ-1 leads to increase in gut inflammatory bacteria composition, deregulated metabolites and dysregulated innate immunity which could be a key factor in the initiation of PD disease in the gut, and potentially also in brain during disease progression.

Together with animal experiments, organotypical cell cultures are important models for analyzing cellular interactions of the mucosal epithelium and pathogenic mechanisms in the gastrointestinal tract. Here, we introduce a three-dimensional culture model from the adult mouse colon for cell biological investigations in an in vivo-like environment. These explant cultures were cultured for up to 2 weeks and maintained typical characteristics of the intestinal mucosa, including a high-prismatic epithelium with specific epithelial cell-to-cell connections, a basal lamina and various connective tissue cell types, as analyzed with immunohistological and electron microscopic methods. The function of the epithelium was tested by treating the cultures with dexamethasone, which resulted in a strong upregulation of the serum- and glucocorticoid-inducible kinase 1 similar to that found in vivo. The culture system was investigated in infection experiments with the fungal pathogen Candida albicans. Wildtype but not Delta cph1/Delta efg1-knockout Candida adhered to, penetrated and infiltrated the epithelial barrier. The results demonstrate the potential usefulness of this intestinal in vitro model for studying epithelial cell-cell interactions, cellular signaling and microbiological infections in a three-dimensional cell arrangement.