Proteome and Phosphoproteome Analysis of Commensally Induced Dendritic Cell Maturation States

Abstract

Dendritic cells are integral components of the mammalian immune system, which take part in orchestrating and regulating the delicate balance of immune response. Dendritic cells are potent activators of destructive responses of the immune system, at the same time, dendritic cells also take part in activating regulatory T-cells and dampening overly destructive immune responses, as well as mediating immune tolerance. This multifaceted and at times contradictory functions of dendritic cells are, at least in part, brought about by the phenotypical differences that regulate the respective immune response. As an example of the importance of the differences in dendritic cell phenotypes, we have previously reported that feeding of B. vulgatus to IL-2-/- mice leads to production of semi-mature dendritic cells and prevents colitis, whereas feeding of E. coli to IL-2-/- mice leads to fully mature dendritic cells and severe intestinal inflammation (Waidmann et al., 2003). Therefore, we believe that these maturation differences in dendritic cells have an important effect on disease manifestation and progression in colitis. However, the intracellular factors and processes taking part in regulating dendritic cell maturation are not fully understood. In our project we aimed to provide a closer look at the proteome profiles of DCs and intracellular signalling pathways/processes that underlie dendritic cell maturation. Using dendritic cells generated in vitro from cultured mouse bone marrow, we induced semi-maturation by B. vulgatus stimulation and complete maturation by E. coli stimulation. The resulting cells were harvested and lysed for proteomic analysis. We performed comparative proteomics to analyse proteins that differ in their expression levels, and shotgun phosphoproteomics to detect proteins that are differentially phosphorylated. Thereby we aimed to catalogue proteins, processes, and signalling pathways that lead to the observed phenotypical differences between the semi-mature and fully-mature dendritic cells. To this end, subsequent to the mass spectrometry runs we have employed bioinformatical tools i.e. pathway analysis, upstream regulator analysis and kinase analysis. We have determined various pathways and regulators, many of which have key roles in immunity and inflammation. We hope that our results provide a more systematic and comprehensive information on factors governing different states of dendritic cell maturation, as well as the effects of commensal and pathogenic bacteria on dendritic cell mediated immune regulation.