Untersuchungen zur post-transkriptionalen Kontrolle der Genexpression in Maus-Makrophagen

Macrophages are the first line of defense against invading pathogenic organisms. Pathogenic components like lipopolysaccharides (LPS) of gram negative bacteria trigger the intracellular immune response. To avoid damage to the organism this immune reaction must be tightly controlled. A regulation is possible at different steps: at kinase activation, degradation of signaling proteins, transcriptional and post-transcriptional activation of gene expression. Post-transcriptional control is mediated for instance through modulation of mRNA stability and mRNA translation by RNA binding proteins or microRNAs. The aim of this work was to identify and characterize RNA binding proteins and microRNAs, which operate during the LPS induced macrophage activation. Thus the specific function of the heterogeneous nuclear ribonucleoprotein (hnRNP) K, which is already described as a post-transcriptional regulator, was studied initially. Two mRNAs could be identified, which are translationally regulated by hnRNP K during inflammation. The mRNA of transforming growth factor-beta activated kinase 1 (TAK1) is translationally inhibited by hnRNP K in untreated macrophages. During LPS induced macrophage activation the interaction between the mRNA and hnRNP K is abrogated and the translation of TAK1 mRNA is induced. A possible c-Src dependent tyrosine phosphorylation could play a role in this process. Further it was shown that the reduction of hnRNP K expression leads to an increased production of cytokines - the mediators of inflammation. HnRNP K binds the mRNA of protein kinase B-beta (AKT2) and activates its translation in untreated as well as in LPS-stimulated macrophages. The mechanism and the consequences of this regulation are yet unknown. There are other proteins and microRNAs which are involved in the post-transcriptional regulation of the immune response. By enrichment and identification of RNA binding proteins in macrophages an "mRNA interactome" was created. The interactome serves as a basis to study additional regulatory mechanisms during inflammation. Furthermore, by identification of LPS-regulated microRNAs a foundation was established for the analysis of microRNA mediated post-transcriptional control

Untersuchungen zur post-transkriptionalen Kontrolle der Genexpression in Maus-Makrophagen

Macrophages are the first line of defense against invading pathogenic organisms. Pathogenic components like lipopolysaccharides (LPS) of gram negative bacteria trigger the intracellular immune response. To avoid damage to the organism this immune reaction must be tightly controlled. A regulation is possible at different steps: at kinase activation, degradation of signaling proteins, transcriptional and post-transcriptional activation of gene expression. Post-transcriptional control is mediated for instance through modulation of mRNA stability and mRNA translation by RNA binding proteins or microRNAs. The aim of this work was to identify and characterize RNA binding proteins and microRNAs, which operate during the LPS induced macrophage activation. Thus the specific function of the heterogeneous nuclear ribonucleoprotein (hnRNP) K, which is already described as a post-transcriptional regulator, was studied initially. Two mRNAs could be identified, which are translationally regulated by hnRNP K during inflammation. The mRNA of transforming growth factor-beta activated kinase 1 (TAK1) is translationally inhibited by hnRNP K in untreated macrophages. During LPS induced macrophage activation the interaction between the mRNA and hnRNP K is abrogated and the translation of TAK1 mRNA is induced. A possible c-Src dependent tyrosine phosphorylation could play a role in this process. Further it was shown that the reduction of hnRNP K expression leads to an increased production of cytokines - the mediators of inflammation. HnRNP K binds the mRNA of protein kinase B-beta (AKT2) and activates its translation in untreated as well as in LPS-stimulated macrophages. The mechanism and the consequences of this regulation are yet unknown. There are other proteins and microRNAs which are involved in the post-transcriptional regulation of the immune response. By enrichment and identification of RNA binding proteins in macrophages an "mRNA interactome" was created. The interactome serves as a basis to study additional regulatory mechanisms during inflammation. Furthermore, by identification of LPS-regulated microRNAs a foundation was established for the analysis of microRNA mediated post-transcriptional control

CX3CR1 Mediates the Development of Monocyte-Derived Dendritic Cells during Hepatic Inflammation

Recent evidence suggests that hepatic dendritic cells (HDCs) contribute to the evolution of chronic liver diseases. However, the HDC subsets involved and the mechanisms driving these responses are still poorly understood. In this study, we have investigated the role of the fractalkine receptor CX3CR1 in modulating monocyte-derived dendritic cell (moDC) differentiation during liver inflammation. The phenotype of HDC and functional relevance of CX3CR1 was assessed in mice following necro-inflammatory liver injury induced by the hepatotoxic agent carbon tetrachloride (CCl4) and in steatohepatitis caused by a methionine/choline-deficient (MCD) diet. In both the experimental models, hepatic inflammation was associated with a massive expansion of CD11c+/MHCIIhigh/CD11b+ myeloid HDCs. These cells also expressed the monocyte markers Ly6C, chemokine (C-C Motif) receptor 2 (CCR2), F4/80 and CD88, along with CX3CR1, allowing their tentative identification as moDCs. Mice defective in CX3CR1 showed a reduction in liver-moDC recruitment following CCl4 poisoning in parallel with a defective maturation of monocytes into moDCs. The lack of CX3CR1 also affected moDC differentiation from bone marrow myeloid cells induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) in vitro. In wild-type mice, treatment with the CX3CR1 antagonist CX3-AT (150 \ub5g, i.p.) 24 h after CCl4 administration reduced liver moDCS and significantly ameliorated hepatic injury and inflammation. Altogether, these results highlight the possible involvement of moDCs in promoting hepatic inflammation following liver injury and indicated a novel role of CX3CL1/CX3CR1 dyad in driving the differentiation of hepatic moDCs