Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Muralikrishnan, A.
The role of C/EBPα in early dendritic cell development
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Graz Medical University; 2021. pp. 86
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Kratky Dagmar
- Strobl Herbert
- Wölfler Albert
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- Abstract:
- Steady-state dendritic cells (DCs) are crucial effector cells of the immune system, which are derived from hematopoietic stem cells and distinct progenitors. In the presence of FMS-related tyrosine kinase 3 ligand (FLT3L), multipotent progenitors (MPPs) and common myeloid progenitors (CMPs) differentiate to monocytic dendritic progenitors (MDPs) and subsequently to common dendritic precursors (CDPs). Finally, CDPs differentiate either into conventional DCs or plasmocytoid DCs. C/EBPα (CCAAT/enhancer binding protein alpha) is a basic region-leucine zipper transcription factor and indispensable for formation of mature neutrophils and eosinophils. Mice with a homozygous deletion of the Cebpa gene have normal to elevated numbers of CMPs, but completely lack downstream granulocyte/monocyte precursors (GMPs) and all subsequent granulocytic stages indicating that C/EBPα is essential for early myelopoiesis by enabling transition of CMPs to GMPs. Though the gene is well studied in myelopoiesis, little is known about its role in early steps of development of DCs. Using a CebpaCre-EYFP reporter mouse model, we identified the majority of splenic conventional DCs to be derived from Cebpa-expressing hematopoietic stem and progenitor cells (HSPCs). Detailed analysis of DC progenitor stages revealed an increase of Cebpa/EYFP+ cells in these mice from early progenitors, such as MPPs via CMPs to its maximum at the MDP stage. Since in later stages no further increase of Cebpa/EYFP+ cells was observed, these results together with data from published gene expression studies clearly indicated Cebpa expression during early DC development. To assess a functional role of C/EBP in early DC development, we next used an inducible bone marrow-specific Cebpa knockout (KO) mouse model (Mx1Cre-CebpaF/F mice), isolated HSPCs of these mice and studied their capacity to form mature DCs after in vitro culture with FLT3L. While Cebpa wildtype (WT) HSPCs produced high numbers of mature DCs after eight days of culture, HSPCs from Cebpa KO mice exhibited a profound reduction in DC numbers. In a step-wise analysis, we observed decreased formation of MDPs and a block in their transition to CDPs in KO HSPCs, whereas WT HSPCs underwent successful transition through the MDP and CDP stages towards mature DCs. Gene expression analysis of FLT3L-stimulated HSPCs from Cebpa WT and KO mice revealed a significant change in transcription factors associated with DC development, like IRF8 and PU.1, inflammatory cytokines like TNFα and IL1β and several genes related to DC development, such as Cx3cr1, as well as related to the TNFα- and NFκB-pathways. Interestingly, TNFα was also reduced in supernatants of Cebpa KO HSPCs stimulated with FLT3L. Accordingly, addition of TNFα to HSPCs cultured in vitro with FLT3L partially restored DC formation in Cebpa KO cells by upregulating Sfpi1 (the gene for PU.1) and Cx3cr1 expression. Mixed lymphocyte reactions showed that DCs generated from Cebpa KO HSPCs by TNFα addition were functionally fully active. In conclusion, we identified a critical role of C/EBPα in early DC development in this study. Expression of Cebpa was indispensable for the formation of MDPs and their transition to CDPs during FLT3L-induced DC development. The inflammatory cytokine TNFα was able to partially overcome this maturation block probably by interfering with Sfpi1 and Cx3cr1 expression. However, further studies are needed to reveal the detailed molecular mechanisms, through which C/EBPα affects early DC formation.