ABSTRACT: Interferon regulatory factor (IRF) 5 has a major role in defining inflammatory macrophage polarization, but the molecular mechanisms of its function as a transcriptional regulator of inflammatory genes are not fully understood. Here, we characterise the sites of IRF5 recruitment in inflammatory macrophages in response to LPS.
Project description:Interferon regulatory factor (IRF) 5 has a major role in defining inflammatory macrophage polarization, but the molecular mechanisms of its function as a transcriptional regulator of inflammatory genes are not fully understood. Here, we characterise the sites of IRF5 recruitment in inflammatory macrophages in response to LPS.
Project description:Interferon regulatory factor (IRF) 5 has a major role in defining inflammatory macrophage polarization, but the molecular mechanisms of its function as a transcriptional regulator of nflammatory genes are not fully understood. Here, we characterise the sites of IRF5 recruitment in inflammatory macrophages in response to lipopolysaccharide (LPS). Samples in this experiment have also been used in Illumina BeadChip expression profling assay (see ArrayExpress experiment E-MTAB-2032, https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2032).
Project description:To ditinguish different immune response and investigate the rule of proper recognition of fungi, a transcriptional analysis on moDCs exposed to a pathogenic and a harmless fungi was performed. Monocyte-derived dendritic cells were treated with Candida albicans, two different forms of Saccaromyces cerevisiae (whole organism and spore ascum) or untreated.
Project description:Samples of monocyte derived dendritic cells (moDCs) from three healthy volunteers untreated and treated with LPS [100ng/ml] or R848 [2.5µg/ml] for different time points (0h,3h,6h,12h and 24h).
Project description:Monocyte-derived dendritic cells obtained from healthy donors were collected and either left untreated, or treated for three hours with heat-killed Candida albicans. Three distinct donors were used. Related data sets have been deposited in ArrayExpress under accessions E-MTAB-750 and E-MTAB-1213
Project description:In order to dissect the response from different fungal cell wall components, monocyte-derived dendritic cells obtained from healthy donors were collected and either left untreated, or treated for four hours with either mannan, zymosan, curdlan, whole yeast, or yeast spores. This experiment is related to E-MTAB-1213.
Project description:All experiments are performed on human dendritic cells (DCs) differentiated in GM-CSF and IL-4 from CD14+ cells and bone marrow mesenchimal stem cells (MSCs). We found that MSCs impair active immune synapse formation and we evidenced at electron microscopy two types of contact between MSCs and DCs: gap and adherent junctions. In the same experiments we show that MSC contacts induce a reorganization of DC cytoskeleton by the formation of actin podosomes, structures typical of an immature, tolerogenic state of DCs. These results suggest that MSCs exert a tolerogenic effect on DCs by mechanism mediated by cell-cell contact. This induces DC cytoskeleton reorganization with formation of actin podosomes.
Project description:Interferon regulatory factor 5 (IRF5) plays a distinct role in the regulation of immune responses in health and disease and is a genetic risk factor several autoimmune conditions, including systemic lupus erythematosus, diabetes, arthritis and inflammatory bowel disease. IRF5 function as a transcription factor requires post-translational modifications of the protein such as phosphorylation. Though several upstream signalling regulators of IRF5 have been identified, there is as yet little understanding of how they control IRF5 activity or whether other unknown kinases are involved. In this study, we systematically searched for IRF5 kinases using a reporter-based method for screening a library of 365 inhibitors of protein kinases. Based on the pool of the identified IRF5-inhibiting compounds we have compiled a list of putative IRF5 kinases, which we tested in subsequent in vitro assays. Protein-tyrosine kinase 2-beta (Ptk2b or PYK2) was one of the top hits, capable of binding to and phosphorylating mouse IRF5 at tyrosine (Y) 171. We have shown that IRF5 recruitment to target genes and IRF5-dependent transcription was impaired in cells treated with highly selective PYK2 inhibitors or in PYK2-deficient cells. Moreover, expression of pro-inflammatory cytokines was blocked in the supernatants of human biopsies from inflamed colon of patients with ulcerative colitis and treated ex vivo with a PYK2 inhibitor. Thus, we have identified a major role for PYK2 in regulating the inflammatory response and mapped its activity to the IRF5 innate sensing pathways.
Project description:Inflammatory immune disorders such as inflammatory bowel disease (IBD) and multiple sclerosis (MS) are major health problems. Currently, the intestinal whipworm Trichuris suis is being explored in clinical trials to reduce inflammation in these diseases, however, the mechanisms by which the parasite affects the host immune system are not known. Here we determined the effects of T. suis soluble products (SPs) on toll-like receptor-4 (TLR4)-stimulated human dendritic cells (DCs) using Illumina bead chip gene arrays. Pathway analysis of lipopolysaccharide (LPS)-stimulated DCs with or without T. suis treatment showed that costimulation with T. suis SPs resulted in a down-regulation of both the myeloid differentiation primary response gene 88 (MyD88)-dependent and the TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent signalling pathways triggered by TLR4. These data were verified using quantitative real-time PCR of several key genes within these pathways and/or defining their protein levels. In addition, T. suis SPs induce Rab7b, a negative regulator of TLR4 signalling that interferes with its trafficking, which coincided with a reduced surface expression of TLR4. These data indicate that the mechanism by which T. suis SPs reduce inflammatory responses is through suppression of both TLR4 signalling and surface expression on DCs.
Project description:To discover the key determinants of IRF-specific enhancer selection, we identified and characterized IRF binding regions. Using chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq), we mapped the binding regions of IRF3, IRF5, and IRF9 in murine dendritic cells (DCs) stimulated with TLR3 and TLR9 agonists, as well as IFNβ, respectively. We comprehensively analysed the IRF cistromes to identify characteristic features, including DNA motifs for IRFs and other transcription factors, and chromatin status.