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/Tolllike receptor 4 (MD-2/TLR4) receptor complex results in nuclear factor kB (NF-kB) 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 Gram negative intestinal symbiont Bacteroides vulgatus mpk is able to prevent from induction of colonic inflammation in Rag1−/− mice and promotes immune balance in Il2−/− mice. These inflammation-silencing effects are associated with B. vulgatus mpk-mediated induction of semi-mature dendritic cells, especially in the colonic lamina propria (cLP). However the beneficial interaction of bacteria with host immune cells is limited due to the existence of a large mucus layer covering the intestinal epithelium. How can intestinal bacteria overcome this physical barrier and contact the host immune system?One mechanism is the production of outer membrane vesicles (OMVs) via ubiquitous blebbing of the outer membrane. These proteoliposomes have the ability to traverse the mucus layer. Hence, OMVs play an important role in immunomodulation and the maintenance of a balanced gut microbiota. Here we demonstrate that the stimulation of bone marrow derived dendritic cells (BMDCs) with isolated OMVs originated from B. vulgatus mpk leads to the induction of a tolerant semi-mature phenotype. Thereby, microbe- associated molecular patterns (MAMPs) delivered by OMVs are crucial for the interaction and the resulting maturation of immune cells. Additional to the binding to host TLR4, a yet unknown ligand to TLR2 is indispensable for the conversion of immature BMDCs into a semi-mature state. Thus, crossing the epithelial mucus layer and directly contact host cells, OMV mediate cross-tolerance via the transport of various Toll-like receptor antigens. These features make OMVs to a key attribute of B. vulgatus mpk for a vigorous acellular prevention and treatment of systemic diseases.

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Mesenchymal stromal cells (MSCs) are multipotent cells that can be differentiated in vitro into a variety of cell types, including adipocytes or osteoblasts. Our recent studies indicated that a high expression of CD146 on MSCs from bone marrow correlates with their robust osteogenic differentiation potential. We therefore investigated if expression of CD146 on MSCs from the placenta correlates with a similar osteogenic differentiation potential. The MSCs were isolated specifically from the endometrial and fetal parts of human term placenta and expanded in separate cultures and compared with MSCs from bone marrow as controls. The expression of cell surface antigens was investigated by flow cytometry. Differentiation of MSCs was documented by cytochemistry and analysis of typical lineage marker genes. CD146-positive MSCs were separated from CD146-negative cells by magnet-assisted cell sorts (MACS). We report that the expression of CD146 is associated with a higher osteogenic differentiation potential in human placenta-derived MSCs (pMSCs) and the CD146(pos) pMSCs generated a mineralized extracellular matrix, whereas the CD146(neg) pMSCs failed to do so. In contrast, adipogenic and chondrogenic differentiation of pMSCs was not different in CD146(pos) compared with CD146(neg) pMSCs. Upon enrichment of pMSCs by MACS, the CD146(neg) and CD146(pos) populations maintained their expression levels for this antigen for several passages in vitro. We conclude that CD146(pos) pMSCs either respond to osteogenic stimuli more vividly or, alternatively, CD146(pos) pMSCs present a pMSC subset that is predetermined to differentiate into osteoblasts.

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.

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.

Success of stem cell therapies were reported in different medical disciplines, including haematology, rheumatology, orthopaedic surgery, traumatology, and others. Currently, more than 4000 clinical trials using stem cells have been completed or are underway, among which 378 investigated or are at present investigating mesenchymal stromal cells (MSCs). The majority of clinical trials using stem- or progenitor- cells, including hematopoietic stem cells and MSCs, target the immune system. However, therapies based on MSCs are increasingly implemented to treat symptoms in which failure of the resident stem cells in situ, or malfunction of tissues or structures are not associated with immune cells or inflammation, but instead are associated with mechanical or metabolic stress, ageing, developmental or acquired malformations, and other causes. To proceed further in the development of stem cell therapies as a safe and effective treatment for surgical and other medical specialities, the behaviour of MSCs implanted in preclinical models and their impact on the site of application need to be explored in detail. Depending on the pre-clinical model employed, tracking of labelled stem cells in live animals makes an enormous difference for exploration of the mechanisms and kinetics involved in MSC-mediated tissue regeneration. Here we review (pre-)clinically applicable key methods to label human MSCs for short and long-term observations in small and large animal models.

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