Lipopolysaccharide | EPFL Graph Search

vignette|460x460px| Structure d'un lipopolysaccharide (LPS) Les lipopolysaccharides (LPS), également appelés lipoglycanes ou endotoxines, sont de grosses molécules constituées d’un lipide et d’un polysaccharide composé d’un antigène O, d’un noyau externe et d’un noyau interne reliés par une liaison covalente. Ils se trouvent dans la membrane externe des bactéries à Gram négatif. Le lipopolysaccharide (LPS) se lie au récepteur TLR4 et promeut la libération de cytokines pro-inflammatoires. C'est une endotoxine pyrogène. Il induit chez l'homme des signes cliniques (fièvre, agrégation des hématies, choc septique, diminution de la pression artérielle). C'est la partie lipide A qui est considérée comme la plus toxique (action sur les granulocytes et les cellules épithéliales). En recherche fondamentale, le LPS est souvent utilisé pour provoquer l'inflammation tel que l'induction de la cyclooxygénase-2 sur des cellules ou des organes en culture. Généralement il est utilisé à une concentr

Detection of environmental glucocorticoids and investigation of their effects using the zebrafish embryo as a model

Anita Orsolya Hidasi

Synthetic glucocorticoids (GCs) are widely used anti-inflammatory drugs. They mimic cortisol, the natural stress hormone in humans and in fish. Cortisol binds and activates the glucocorticoid receptor (GR), which regulates genes governing development, immune response, osmoregulation, and behavior. Therefore, the presence of GCs in the aquatic environment may cause endocrine disruption through impairing these essential physiological processes in fish. The aim of this thesis was to assess the effects of environmentally relevant concentrations of GCs in teleost fish, focusing on the inflammatory response and molecular mechanisms of GC action on different levels, from mRNA to proteins to metabolites. To confirm the environmental presence of GCs, GC activity was determined along a Swiss freshwater stream impacted by a wastewater treatment plant, using effect-directed analysis. The effluent samples showed the highest potency to activate the GR, as determined by the GR-CALUX® assay. The chemical analysis found clobetasol propionate (CP), a highly potent GC, to be responsible for 63% of the total GR activity in the sample. CP was selected as a model chemical to study GC effects in fish. Embryos of zebrafish (Danio rerio) aged until 5 days post fertilization (dpf) were used as a model organism; working with embryonic stages is a refinement of animal experiments. We demonstrated that CP is readily taken up by the embryos. Bacterial lipopolysaccharides (LPS) induce one of the main inflammatory cascades. As observed in LPS challenge assays, the inflammatory response of the embryos was significantly reduced after exposure to ¿0.1 nM CP. We showed that mRNA expression of several LPS mechanism- and GC action-related genes was regulated by CP. Among them, Annexin a1b (anxa1b) was significantly down-regulated after exposure to ¿0.05 nM CP. Sensitive regulation of anxa1b by the non-steroidal anti-inflammatory drug diclofenac (DCF) suggested that this gene product is a potential biomarker of steroidal and non-steroidal immunosuppressive effects. An LC-MS/MS-based targeted proteomics technique was developed to measure proteins, as it is these molecules that actually carry out cellular functions. 12 out of 40 GC action-related protein targets were detectable in control embryo digests and were subsequently monitored after CP, DCF and grab water sample exposures. We found that the muscle protein Myhz2 was regulated only after DCF exposure, while I¿B¿, an anti-inflammatory protein, was regulated only by CP, in agreement with mRNA level data. Global and targeted metabolomics analyses were performed in zebrafish embryos exposed to CP from 4 to 5 dpf, in collaboration with M. Lamoree (VU Amsterdam). Several of the detected compounds showed significant response to CP: lysine, nicotinamide, choline, hypoxanthine, tyrosine and tryptophan. To conclude, this thesis demonstrates that GCs are present in the aquatic environment and may suppress the inflammatory response of fish at environmentally relevant concentrations.

EPFL2016

Modulation of Dendritic Cells Function by H. polygyrus Products

Blaise Dayer

Dendritic cells (DCs) are crucial antigen-presenting cells that can drastically change the development of an immune response by polarising T helper cells toward a Th1 or a Th2 phenotype. Heligmosomoides polygyrus, a murine model of human helminth, has been shown to strongly down-regulate its host immune response. At the DC level, its excretory/secretory products (HES) modulate the cytokine response and co-stimulatory marker expression to bacterial stimulation in favour of an antiinflammatory environment. A regulatory T cell-inducing TGF[beta]-like activity was also discovered in HES. In this work, we further characterised the immunomodulatory properties of HES and heat-inactivated HES both in vitro, using LPS-pulsed GM-CSF-grown bone marrowderived DCs, and in in vivo adoptive transfer of HES-, bacterial extract- or co-pulsed DCs. To determine if the TGF[beta], C-type lectin receptors (CLRs) or toll-like receptors (TLRs) were implicated in this immunomodulation, we treated DCs with blocking antibodies, chemical kinase inhibitors and grew DCs from knockout mice. These studies revealed that HES immunomodulation of DCs was independent of the TGF[beta] receptor, the two CLRs, Dectin-1 and 2, as well as any TLR. Spleen tyrosine kinase (Syk), which is crucial for the signalling of many CLRs, and phosphoinositide 3- kinase seemed not to be needed as well. In an attempt to reduce the number of potential immunomodulators in HES, HES size fractions were tested for their ability to reduce LPS-induced inflammatory cytokine production of DCs. The activity was limited to few fractions, fraction 14 being the most potent. Finally, physical interactions between DCs and biotinylated HES were measured by flow cytometry and revealed that CD24, a molecule required for HES interactions with B cells, was implicated, but not crucial. HES binding to CD24-/- DCs was reduced, but the LPS-induced cytokines and co-stimulatory markers levels were still down-regulated upon HES co-treatment.

2011

Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner

Kristina Schoonjans

Stringent control of immune responses in the intestinal mucosa is critical for the maintenance of immune homeostasis and prevention of tissue damage, such as observed during inflammatory bowel disease. Intestinal epithelial cells, primarily thought to form a simple physical barrier, critically regulate intestinal immune cell functions by producing immunoregulatory glucocorticoids on T-cell activation. In this study we investigated whether stimulation of cells of the innate immune system results in the induction of intestinal glucocorticoids synthesis and what role TNF-alpha plays in this process. Stimulation of the innate immune system with lipopolysaccharide (LPS) led to an up-regulation of colonic steroidogenic enzymes and the induction of intestinal glucocorticoid synthesis. The observed induction was dependent on macrophage effector functions, as depletion of macrophages using clodronate-containing liposomes, but not absence of T and B cells, inhibited intestinal glucocorticoid synthesis. LPS-induced glucocorticoid synthesis was critically dependent on TNF-alpha as it was significantly decreased in TNF-alpha-deficient animals. Both TNF receptor-1 and -2 were found to be equally involved in LPS- and T-cell-induced intestinal GC synthesis. These results describe a novel and critical role of TNF-alpha in immune cell-induced intestinal glucocorticoid synthesis.

2010