Project description:Analysis of MyD88 as a putative mediator in host-microbe-interactions MyD88-Hairpin was micro-injected in Hydra AEP embryos. The resulting Hatchling D3 showed a patchy distribution of the transgene observed by GFP. By asexual reproduction via budding and constant selection for the marker gene GFP the transgene could be driven into different cell ines. Therefore two different cultures were established. Polyps that showed no GFP-Signal anymore (control) and Polyps with both, endodermal and ectodermal transgenic cell-lines (knockdown).
Project description:Host pathways mediating changes in immune states elicited by intestinal microbial colonization are incompletely characterized. Here we describe alterations of the host immune state induced by colonization of germ-free zebrafish larvae with an intestinal microbial community or single bacterial species. We show that microbiota-induced changes in intestinal leukocyte subsets and whole-body host gene expression are dependent on the innate immune adaptor gene myd88. Similar patterns of gene expression are elicited by colonization with conventional microbiome, as well as mono-colonization with two different zebrafish commensal bacterial strains. By studying loss-of-function myd88 mutants, we find that colonization suppresses Myd88 at the mRNA level. Tlr2 is essential for microbiota-induced effects on myd88 transcription and intestinal immune cell composition.
Project description:Transcriptional profiling of Murine Embryonic Fibroblasts (MEFs) infected with Ad-MyD88 vs. Ad-GFP or mock infected. Three-condition experiment, Ad-MyD88 vs. Ad-GFP vs. Mock infected cells. Biological replicates: 3 Ad-MyD88, 3 Ad-GFP, 3 mock, independently grown and harvested. One replicate per array.
Project description:Transcriptional profiling of Bone-Marrow derived mouse Dendritic Cells (bmDCs) infected with Ad-MyD88 vs. Ad-GFP or mock infected Three-condition experiment, Ad-MyD88 vs. Ad-GFP vs. Mock infected cells. Biological replicates: 3 Ad-MyD88, 3 Ad-GFP, 3 mock, independently grown and harvested. One replicate per array.
Project description:The MYD88 L265P mutation, activator of NF-kappa B (NF-kB), is found in 80% to 90% of WM. So far, there is no existing animal model for WM to evaluate the role of MYD88 activation, and the only published mouse model harboring the mouse ortholog MYD88 L252P mutation develops aggressive B-cell lymphomas in aged mice. The MYD88 L252P coding sequence was attached to YFP cDNA with an IRES sequence. This construct was flanked with loxP sites and inserted in the Rosa26 locus. The expression of MYD88 L252P protein is induced only in B cells after crossing with CD19_Cre mice.
Project description:To elucidate the differences occurring in the B cell composition between WM patients, we performed single cell RNA sequencing on CD19 + sorted cells from patients with MYD88 L265P versus MYD88 WT phenotype and compared them with two healthy controls.
Project description:We compared in vitro mTECs-high stimulated with TLR9 ligand CpG ODN (1826) or non stimulated from MyD88 -/- and MyD88 +/+ mice: Thymi were enzymaticaly digested, cells were MACS enriched for CD45- fraction, and FACS sorted using BD Influx sorter. mTECs-high were gated as EpCAM+CD11c-Ly51-MHCII+CD80+. Cell from MyD88 -/- and MyD88 +/+ mice were incubated in RPMI with or without CpG ODN (1826) for 24 hours. Total RNA was isolated using RNeasy Plus Micro Kit (Qiagen). 4 samples per condition were used.
Project description:Transcriptome analysis of submandibular glands in female MyD88+/+ and MyD88−/− NOD mice. Sjögren's syndrome (SS) is an autoimmune disease characterized by dysfunction of salivary glands (SGs) and lacrimal glands, which is caused by chronic inflammation associated with autoantibody and autoreactive lymphocyte infiltration. The pathogenic mechanism of SS has not been fully elucidated. Infiltrated lymphocytes form regularized structures similar to lymphoid follicles of secondary lymphoid organs, such as T/B cell compartments, high endothelial venules (HEVs), lymphatic vessels, and germinal centers, therefore being believed as an ectopic lymphoid tissue called tertiary lymphoid organs (TLO). We previously found that deletion of the Toll-like receptor/IL-1 receptor (TLR/IL-1R) adaptor molecule gene Myd88 in SS model mice NOD reduced the frequency of lymphocyte infiltration and HEV formation in SGs. In this study, we analyzed the effect of MyD88 deficiency on lymphoid follicle formation in SGs of NOD mice. Microarray analysis showed decreased expression of genes related to TLO, such as Cxcl13 and Cxcr5, in Myd88-deficient SGs. These results indicate that deficiency of TLR/IL-1R signaling decrease gene expression ot chemokines in SGs, suggesting MyD88-dependent signaling is directly involved in formation of lymphoid follicles in SS.