Flagellin hypervariable region determines symbiotic properties of commensal Escherichia coli strains.

Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22-mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow-chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.

Weak Agonistic LPS Restores Intestinal Immune Homeostasis.

Generated by gram-negative bacteria, lipopolysaccharides (LPSs) are one of the most abundant and potent immunomodulatory substances present in the intestinal lumen. Interaction of agonistic LPS with the host myeloid-differentiation-2/Toll-like receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor κB (NF-κB) activation, followed by the robust induction of pro-inflammatory immune responses. Here we have isolated LPS from a common gut commensal, Bacteroides vulgatus mpk (BVMPK), which provides only weak agonistic activity. This weak agonistic activity leads to the amelioration of inflammatory immune responses in a mouse model for experimental colitis, and it was in sharp contrast to strong agonists and antagonists. In this context, the administration of BVMPK LPS into mice with severe intestinal inflammation re-established intestinal immune homeostasis within only 2 weeks, resulting in the clearance of all symptoms of inflammation. These inflammation-reducing properties of weak agonistic LPS are grounded in the induction of a special type of endotoxin tolerance via the MD-2/TLR4 receptor complex axis in intestinal lamina propria CD11c+ cells. Thus, weak agonistic LPS represents a promising agent to treat diseases involving pathological overactivation of the intestinal immune system, e.g., in inflammatory bowel diseases.

The complex methylome of the human gastric pathogen Helicobacter pylori.

The genome of Helicobacter pylori is remarkable for its large number of restriction-modification (R-M) systems, and strain-specific diversity in R-M systems has been suggested to limit natural transformation, the major driving force of genetic diversification in H. pylori. We have determined the comprehensive methylomes of two H. pylori strains at single base resolution, using Single Molecule Real-Time (SMRT®) sequencing. For strains 26695 and J99-R3, 17 and 22 methylated sequence motifs were identified, respectively. For most motifs, almost all sites occurring in the genome were detected as methylated. Twelve novel methylation patterns corresponding to nine recognition sequences were detected (26695, 3; J99-R3, 6). Functional inactivation, correction of frameshifts as well as cloning and expression of candidate methyltransferases (MTases) permitted not only the functional characterization of multiple, yet undescribed, MTases, but also revealed novel features of both Type I and Type II R-M systems, including frameshift-mediated changes of sequence specificity and the interaction of one MTase with two alternative specificity subunits resulting in different methylation patterns. The methylomes of these well-characterized H. pylori strains will provide a valuable resource for future studies investigating the role of H. pylori R-M systems in limiting transformation as well as in gene regulation and host interaction.

Bacterial Immunogenicity Is Critical for the Induction of Regulatory B Cells in Suppressing Inflammatory Immune Responses.

B cells fulfill multifaceted functions that influence immune responses during health and disease. In autoimmune diseases, such as inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis, depletion of functional B cells results in an aggravation of disease in humans and respective mouse models. This could be due to a lack of a pivotal B cell subpopulation: regulatory B cells (Bregs). Although Bregs represent only a small proportion of all immune cells, they exhibit critical properties in regulating immune responses, thus contributing to the maintenance of immune homeostasis in healthy individuals. In this study, we report that the induction of Bregs is differentially triggered by the immunogenicity of the host microbiota. In comparative experiments with low immunogenic Bacteroides vulgatus and strong immunogenic Escherichia coli, we found that the induction and longevity of Bregs depend on strong Toll-like receptor activation mediated by antigens of strong immunogenic commensals. The potent B cell stimulation via E. coli led to a pronounced expression of suppressive molecules on the B cell surface and an increased production of anti-inflammatory cytokines like interleukin-10. These bacteria-primed Bregs were capable of efficiently inhibiting the maturation and function of dendritic cells (DCs), preventing the proliferation and polarization of T helper (Th)1 and Th17 cells while simultaneously promoting Th2 cell differentiation in vitro. In addition, Bregs facilitated the development of regulatory T cells (Tregs) resulting in a possible feedback cooperation to establish immune homeostasis. Moreover, the colonization of germfree wild type mice with E. coli but not B. vulgatus significantly reduced intestinal inflammatory processes in dextran sulfate sodium (DSS)-induced colitis associated with an increase induction of immune suppressive Bregs. The quantity of Bregs directly correlated with the severity of inflammation. These findings may provide new insights and therapeutic approaches for B cell-controlled treatments of microbiota-driven autoimmune disease.

Galleria mellonella: A Novel Invertebrate Model to Distinguish Intestinal Symbionts From Pathobionts.

Insects and mammals share evolutionary conserved innate immune responses to maintain intestinal homeostasis. We investigated whether the larvae of the greater wax moth Galleria mellonella may be used as an experimental organism to distinguish between symbiotic Bacteroides vulgatus and pathobiotic Escherichia coli, which are mammalian intestinal commensals. Oral application of the symbiont or pathobiont to G. mellonella resulted in clearly distinguishable innate immune responses that could be verified by analyzing similar innate immune components in mice in vivo and in vitro. The differential innate immune responses were initiated by the recognition of bacterial components via pattern recognition receptors. The pathobiont detection resulted in increased expression of reactive oxygen and nitrogen species related genes as well as antimicrobial peptide gene expression. In contrast, the treatment/application with symbiotic bacteria led to weakened immune responses in both mammalian and insect models. As symbionts and pathobionts play a crucial role in development of inflammatory bowel diseases, we hence suggest G. mellonella as a future replacement organism in inflammatory bowel disease research.

Genome Sequence of Galleria mellonella (Greater Wax Moth).

The larvae of the greater wax moth, Galleria mellonella, are pests of active beehives. In infection biology, these larvae are playing a more and more attractive role as an invertebrate host model. Here, we report on the first genome sequence of Galleria mellonella.

TLR signaling-induced CD103-expressing cells protect against intestinal inflammation.

BACKGROUND: Toll-like receptor (TLR) expression in patients with inflammatory bowel disease is increased when compared with healthy controls. However, the impact of TLR signaling during inflammatory bowel disease is not fully understood. METHODS: In this study, we used a murine model of acute phase inflammation in bone marrow chimeric mice to investigate in which cell type TLR2/4 signal induction is important in preventing intestinal inflammation and how intestinal dendritic cells are influenced. Mice were either fed with wild-type bacteria, able to initiate the TLR2/4 signaling cascade, or with mutant strains with impaired signal induction capacity. RESULTS: The induction of the TLR2/4 signal cascade in epithelial cells resulted in inflammation in bone marrow chimeric mice, whereas induction in hematopoietic cells had an opposed function. Furthermore, feeding of wild-type bacteria prevented disease; however, differing signal induction of bacteria had no effect on lamina propria dendritic cell activation. In contrast, functional TLR2/4 signals resulted in increased frequencies of CD103-expressing lamina propria and mesenteric lymph node dendritic cells, which were able to ameliorate disease. CONCLUSIONS: The TLR-mediated amelioration of disease, the increase in CD103-expressing cells, and the beneficial function of TLR signal induction in hematopoietic cells indicate that the increased expression of TLRs in patients with inflammatory bowel disease might result in counterregulation of the host and serve in preventing disease.