Impact of the Transcription Factor HoxA9 on Toll-like Receptor-Mediated Innate Immune Responses and Development of Dendritic Cells and Macrophages

Purpose of this work: Toll-like receptors (TLRs) are sensors of the innate immune system that perceive evolutionary conserved microbial structures and act as first line defense mechanisms against bacteria, viruses, fungi, and parasites. As a subset of the TLR family, intracellular TLRs reside in end...

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Bibliographic Details
Main Author: Bleyl, Tobias Mischa
Contributors: Bauer, Stefan (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2018
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Summary:Purpose of this work: Toll-like receptors (TLRs) are sensors of the innate immune system that perceive evolutionary conserved microbial structures and act as first line defense mechanisms against bacteria, viruses, fungi, and parasites. As a subset of the TLR family, intracellular TLRs reside in endosomal compartments to encounter their ligand comprising different types of nucleic acids. Important members are TLR3, 7, 8, and 9 recognizing double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), again ssRNA, and unmethylated CpG-motif containing DNA, respectively. Innate immune cells including dendritic cells (DCs) and macrophages (MΦs) show different expression profiles and varying functions of intracellular TLRs. In mice, conventional dendritic cells (cDCs) and MΦs express TLR3, 7, and 9, which predominantly induce secretion of proinflammatory cytokines such as IL-6 after activation, whereas type I interferons (IFNs) are only upregulated upon ligation of TLR3 in these cell types. A rare subset of DCs referred to as plasmacytoid dendritic cells (pDCs) uniquely possesses the ability to massively induce Interferon-α and -β (IFN-α/-β; type I IFNs) upon stimulation of TLR7 and 9. Furthermore, murine pDCs have been shown to sense DNA via TLR9 in a CpGmotif independent way. However, the molecular mechanisms of cell type-dependent functional variations of intracellular TLRs are not fully understood and mostly remain elusive. Besides involvement in embryonic tissue patterning, the homeodomaincontaining transcription factor HoxA9 is known to play essential roles in normal and malignant hematopoietic processes such as maintaining the stem cell status, leukemogenesis, and the generation of B cell progenitors. The latter circumstance has been partly linked to transcriptional regulation of the cytokine receptor fms-like tyrosine kinase 3 (Flt3) in hematopoietic progenitors. However, Flt3 and its ligand Flt3L are also crucial signals for the development and homeostasis of DCs. Moreover, prominent mRNA expression levels of hoxa9 upon stimulation of TLR9 in murine pDCs (unpublished data) suggest participation of this transcription factor in TLR9 biology particularly in pDCs. The impact of HoxA9 on the function of intracellular TLRs as well as development of DCs and MΦs was therefore investigated in this study using HoxA9 knockout (KO) mice. Hypothesis: Upregulation of HoxA9 upon activation of TLR9 in pDCs and involvement in B cell differentiation via Flt3 suggests a prominent role of this gene in TLR-mediated immune responses as well as development of DCs and MΦs. Results: HoxA9-/- mice displayed an insignificant hypocellularity of total nucleated bone marrow (BM) cells, which was reported before by several other groups. FACS analyses of total BM cells ex vivo revealed significantly reduced B cell counts but normal quantities of mature DC subsets in HoxA9-deficient mice, again confirming previous studies. TLR stimulation experiments of HoxA9-/- total BM cells as well as FACS-sorted pDC fractions ex vivo exhibited statistically significant impaired IFN-α responses upon TLR7 and 9 activation, whereas FACS-sorted cDCs did not show significant alterations in TLR function. Moreover, in vitro generated HoxA9-deficient Flt3L-induced DC cultures, which contain pDCs and cDCs, displayed almost completely abolished IFN-α levels and clearly reduced IL-6 levels upon TLR7/9 stimulation compared to wild type (WT). Importantly, Flt3L-induced DC cultures generated from BM cells of HoxA9deficient animals presented notably reduced amounts of differentiated cells after the maturation period, a higher rate of dead cells, and a shifted pDC/cDC ratio in comparison to WT littermates. Conversely, in vitro generated GM-CSF-induced cDCs and M-CSF-induced MΦs demonstrated no significant differences in surface marker expression and TLR-mediated cytokine responses in the KO cultures. Primary splenocytes of HoxA9deficient and WT animals showed equal quantities of both DC subsets regarding surface markers measured by FACS. Nonetheless, TLR7/9-mediated IFN-α levels of HoxA9 splenic pDCs displayed reductions similarly to those found in the BM. Finally, genomewide microarray expression profiling of FACS-sorted BM-derived pDCs revealed differential expression of several genes between KO and WT including the HoxA9 cofactor Meis1. Conclusions: Collectively, these results implicate a pivotal role for HoxA9 in TLR7/9-mediated pDC-specific immunity especially impacting on IFN-α responses. TLR biology of cDCs and MΦs is apparently not affected by the HoxA9 knockout. DC development among HoxA9-/- BM cells is partly impaired in vitro. However, this effect is compensated in vivo. The emerging evidence that pDCs play central roles in the pathogenesis of numerous human disorders such as viral infections, autoimmune diseases, and the tumorigenesis of various types of cancer underlines the importance of these cells and emphasizes the fact that HoxA9 could serve as a potential therapeutic target.
Physical Description:199 Pages