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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.

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 human microbiota consists of bacteria, archaea, viruses, and fungi that build a highly complex network of interactions between each other and the host. While there are many examples for commensal bacterial influence on host health and immune modulation, little is known about the role of commensal fungi inside the gut community. Up until now, fungal research was concentrating on opportunistic diseases caused by fungal species, leaving the possible role of fungi as part of the microbiota largely unclear. Interestingly, fungal and bacterial abundance in the gut appear to be negatively correlated and disruption of the bacterial microbiota is a prerequisite for fungal overgrowth. The mechanisms behind bacterial colonization resistance are likely diverse, including direct antagonism as well as bacterial stimulation of host defense mechanisms. In this work, we will review the current knowledge of the development of the intestinal bacterial and fungal community, the influence of the microbiota on human health and disease, and the role of the opportunistic yeast C. albicans. We will furthermore discuss the possible benefits of commensal fungal colonization. Finally, we will summarize the recent findings on bacterial-fungal interactions.

The human microbiota consists of bacteria, archaea, viruses, and fungi that build a highly complex network of interactions between each other and the host. While there are many examples for commensal bacterial influence on host health and immune modulation, little is known about the role of commensal fungi inside the gut community. Up until now, fungal research was concentrating on opportunistic diseases caused by fungal species, leaving the possible role of fungi as part of the microbiota largely unclear. Interestingly, fungal and bacterial abundance in the gut appear to be negatively correlated and disruption of the bacterial microbiota is a prerequisite for fungal overgrowth. The mechanisms behind bacterial colonization resistance are likely diverse, including direct antagonism as well as bacterial stimulation of host defense mechanisms. In this work, we will review the current knowledge of the development of the intestinal bacterial and fungal community, the influence of the microbiota on human health and disease, and the role of the opportunistic yeast C. albicans. We will furthermore discuss the possible benefits of commensal fungal colonization. Finally, we will summarize the recent findings on bacterial-fungal interactions.

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1β processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1β maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1β secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate–induced colitis model resulted in a strong increase in intestinal IL-1β, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1β cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1β by a novel, caspase-8–dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1β, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.

An increasing body of evidence suggests that probiotic bacteria are effective in the treatment of enteric infections, although the molecular basis of this activity remains elusive. To identify putative probiotics, we tested commensal bacteria in terms of their toxicity, invasiveness, inhibition of Yersinia -induced inflammation in vitro and in vivo, and modulation of dextran sodium sulfate (DSS)-induced colitis in mice. The commensal bacteria Escherichia coli , Bifidobacterium adolescentis , Bacteroides vulgatus , Bacteroides distasonis , and Streptococcus salivarius were screened for adhesion to, invasion of, and toxicity for host epithelial cells (EC), and the strains were tested for their ability to inhibit Y. enterocolitica -induced NF-κB activation. Additionally, B. adolescentis was administered to mice orally infected with Y. enterocolitica and to mice with mucosae impaired by DSS treatment. None of the commensal bacteria tested was toxic for or invaded the EC. B. adolescentis , B. distasonis , B. vulgatus , and S. salivarius inhibited the Y. enterocolitica -induced NF-κB activation and interleukin-8 production in EC. In line with these findings, B. adolescentis -fed mice had significantly lower results for mean pathogen burden in the visceral organs, intestinal tumor necrosis factor alpha mRNA expression, and loss of body weight upon oral infection with Y. enterocolitica . In addition, the administration of B. adolescentis decelerated inflammation upon DSS treatment in mice. We suggest that our approach might help to identify new probiotics to be used for the treatment of inflammatory and infectious gastrointestinal disorders.

During acute infection and inflammation, dramatic shifts in tissue metabolism are typical, thereby resulting in profound tissue hypoxia (1). Tissue hypoxia influences the innate immune response of the involved tissue (2). Therefore we pursued the hypothesis, that tissue hypoxia may influence innate immune responses by transcriptional modulation of toll-like receptor (TLR) expression and function. Murine dendritic cells were exposed to hypoxia (2% O2, 98% N2 24h) and transcriptional profiling of all mammalian TLRs was performed using real-time RT-PCR. Additional studies in human cell lines including dendritic cells, monocytic cells (MM6), endothelia (HMEC-1) and intestinal epithelia (Caco-2) were performed to evaluate TLR transcripts, protein and function during hypoxia. Cell lines characterized by stable loss and gain of hypoxia inducible factor (HIF)-1α were exposed to hypoxia. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate binding of HIF-1α within the putative promoter regions of the TLRs. Wildtype (WT) and animals with conditional HIF-1α knockout (cHIF- 1α) were exposed to normobaric hypoxia (8% O2, 92% N2, 6h) and TLR expression determined. Transcriptional levels of TLR2 (2.36±0.7-fold; P

Outer membrane vesicles (OMVs) are produced by many pathogenic and non-pathogenic bacteria through ubiquitous blebbing of the outer membrane. These vesicles consist of lipids, proteins, lipopolysaccharides and other luminal molecules. Supplementary to the roles in pathogenesis, cell-to-cell communication and stress response, recent work has suggested that OMVs play important roles in immunomodulation and the establishment of a balanced gut microbiota for maintaining the intestinal homeostasis. We focus on the influences of OMVs derived from the symbiotic gram-negative B. vulgatus mpk on the innate immune system. These vesicles show anti-inflammatory functions resulting in inhibition of T cell proliferation. Responsible for this effect is the induction of semi-mature dendritic cells in the host, characterised by an intermediate MHC-class-II surface expression, low secretion of the proinflammatory cytokines TNFα, IL-1s and IL-12 with simultaneous remarkable production of IL-6. Stimulation of wt, TLR2−/−, TLR4−/− and TLR2−/−x TLR4−/− DCs indicate the crucial role of different Pathogen-associated molecular patterns (PAMPs) of the bacterial membrane for the development of this non-responsive semi-mature phenotype. In addition to bacterial LPS, binding to host TLR4, a yet unknown compound to host TLR2 is important for the production of this phenotypical cytokine profile. We propose that outer membrane vesicles derived from commensal B. vulgatus mpk are promising drugs for therapeutical treatment of systemic diseases like e.g. colitis, type-I-diabetes or multiple sclerosis. By the induction of semi-mature dendritic cells, OMVs have the ability to modulate the cytokine secretion in the gastrointestinal tract in order to maintaining permanent anti-inflammatory conditions.

We investigated whether commensal bacteria modulate activation and maturation of bone marrow-derived DC and their ability to prime CD4(+) T cells. We used Escherichia coli mpk, which induces colitis in gnotobiotic IL-2-deficient (IL-2(-/-)) mice, and Bacteroides vulgatus mpk, which prevents E. coli-induced colitis. Stimulation of DC with E. coli induced TNF-alpha, IL-12 and IL-6 secretion and expression of activation markers. Moreover, stimulation of DC with E. coli increased T cell activation and led to Th1 polarization. Stimulation with B. vulgatus led only to secretion of IL-6, and DC were driven into a semi-mature state with low expression of activation markers and did not promote Th1 responses. B. vulgatus-induced semi-mature DC were non-responsive to stimulation by E. coli in terms of maturation, T cell priming and TNF-alpha but not IL-6 production. The non-responsiveness of B. vulgatus-stimulated DC was abrogated by addition of anti-IL-6 mAb or mimicked with rIL-6. These data suggest that B. vulgatus-induced IL-6 drives DC into a semi-mature state in which they are non-responsive to proinflammatory activation by E. coli. This in vitro mechanism might contribute to the prevention of E. coli-triggered colitis development by B. vulgatus in vivo; high IL-6 mRNA expression was consistently found in B. vulgatus-colonized or B. vulgatus/E. coli co-colonized IL-2(-/-) mice and was associated with absence of colitis.

Background IL-2 deficient (IL-2(-/-)) mice mono-colonized with E. coli mpk develop colitis whereas IL-2(-/-)-mice mono-colonized with B. vulgatus mpk do not and are even protected from E. coli mpk induced colitis. Methodology/principal findings We investigated if mono-colonization with E. coli mpk or B. vulgatus mpk differentially modulates distribution, activation and maturation of intestinal lamina propria (LP) dendritic cells (DC). LP DC in mice mono-colonized with protective B. vulgatus mpk or co-colonized with E. coli mpk/B. vulgatus mpk featured a semi-mature LP DC phenotype (CD40(lo)CD80(lo)MHC-II(hi)) whereas mono-colonization with colitogenic E. coli mpk induced LP DC activation and maturation prior to onset of colitis. Accordingly, chemokine receptor (CCR) 7 surface expression was more strikingly enhanced in mesenteric lymph node DC from E. coli mpk than B. vulgatus mpk mono- or co-colonized mice. Mature but not semi-mature LP DC promoted Th1 polarization. As B. vulgatus mpk promotes differentiation of semi-mature DC presumably by IL-6, mRNA and protein expression of IL-6 was investigated in LP DC. The data demonstrated that IL-6 mRNA and protein was increased in LP DC of B. vulgatus mpk as compared to E. coli mpk mono-colonized IL-2(-/-)-mice. The B. vulgatus mpk mediated suppression of CCR7 expression and DC migration was abolished in IL-6(-/-)-DC in vitro. Conclusions/significance From this data we conclude that the B. vulgatus triggered IL-6 secretion by LP DC in absence of proinflammatory cytokines such as IL-12 or TNF-alpha induces a semi-mature LP DC phenotype, which might prevent T-cell activation and thereby the induction of colitis in IL-2(-/-)-mice. The data provide new evidence that IL-6 might act as an immune regulatory cytokine in the mucosa by targeting intestinal DC.

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.