The Dox-pDC - A murine conditionally immortalized plasmacytoid dendritic cell line with native immune profile.

Plasmacytoid dendritic cells (pDC) constitute a very rare blood cell population and play a significant role in immune response and immune-mediated disorders. Investigations on primary pDCs are hindered not only due to their rarity but also because they represent a heterogeneous cell population which is difficult to culture ex vivo. We generated a conditionally immortalized pDC line (Dox-pDC) from mice with Doxycycline-inducible SV40 Large T Antigen with a comparable immune profile to primary pDCs. The Dox-pDC secrete pro- and anti-inflammatory cytokines upon Toll-like receptor 9 stimulation and upregulate their MHCI, MHCII and costimulatory molecules. Further, the Dox-pDC activate and polarize naïve T cells in vivo and in vitro in response to the model antigen Ovalbumin. Due to their long-term culture stability and their robust proliferation Dox-pDC represent a reliable alternative to primary mouse pDC.

UTX - moonlighting in the cytoplasm?

The X-linked histone demethylase UTX has a pivotal role in cellular and developmental processes including embryogenesis, hematopoiesis and cancer. UTX removes di- and trimethyl groups on histone H3 lysine 27, thereby regulating gene expression. But there is growing evidence that UTX displays biological functions independent of its histone demethylase activity. To elucidate these novel functions, it is of great interest to define subcellular localizations of UTX. Here we show for the first time that native UTX is primarily localized in the cytoplasm whereas ectopic GFP and Flag-tagged UTX display nuclear and cytoplasmic localization. While its epigenetic function is exerted in the nucleus, its cytoplasmic localization points to a novel function.

Neurotrophin Receptor p75NTR Regulates Immune Function of Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Notch signaling enhances osteogenic differentiation while inhibiting adipogenesis in primary human bone marrow stromal cells.

OBJECTIVE: The Notch signaling pathway has been shown to play a role in bone marrow-derived stromal cell differentiation, however, the precise outcome of Notch activation remains controversial. The aim of this study was to evaluate the effect of Notch signaling in primary human bone marrow-derived stromal cells (hBMSCs). MATERIALS AND METHODS: hBMSCs were transduced to >90% with lentiviral vectors containing either human notch1 intracellular domain (NICD), jagged1, or dominant negative mastermind1. Cells were exposed to adipogenic and osteogenic differentiation stimuli and differentiation was quantified by oil red or alizarin red staining, alkaline phosphatase liver/bone/kidney (ALPL) activity and expression of adipogenic or osteogenic marker genes. RESULTS: NICD and jagged1 transgene-expressing hBMSCs demonstrated enhanced mineralization, nodule formation, and ALPL activity in osteogenic differentiation media. These findings correlated with increased gene expression of bone morphogenetic protein 2 and ALPL. In contrast, NICD or jagged1 transgene expression strongly inhibited adipocyte formation and reduced peroxisome proliferator-activated receptor-gamma, fatty acid binding protein 4, and adiponectin precursor gene expression. Co-overexpression of dominant negative mastermind1 and NICD or jagged1 led to a partial rescue of the differentiation phenotypes. In addition, high endogenous jagged1 expression levels were observed in hBMSCs samples with strong ALPL activity compared to a group of samples with low ALPL activity. CONCLUSION: In summary, our data suggest that induction of Notch signaling enhances the osteogenic differentiation of hBMSCs while inhibiting the adipogenic fate.

Stromal cell-derived factor-1alpha-directed chemoattraction of transiently CXCR4-overexpressing bone marrow stromal cells into functionalized three-dimensional biomimetic scaffolds.

Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface into deeper structures of 3D porous bone substitute scaffolds. Here we show that transient overexpression of CXCR4 in human BMSCs induced by mRNA transfection enhances stromal cell-derived factor-1alpha (SDF-1alpha)-directed chemotactic capacity to invade internal compartments of porous 3D bone substitute scaffolds in vitro and in vivo. In vitro native BMCSs invaded up to 500 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSCs invaded up to 800 mum within 5 days. In addition, 60% downregulation of endogenous SDF-1 transcription in BMSCs by endoribonuclease-prepared siRNA before CXCR4 mRNA transfection enhanced SDF-1alpha-directed migration of human BMSCs by 50%. Implantation of SDF-1alpha-releasing scaffolds seeded with transiently CXCR4-overexpressing BMSCs resulted in an increase of invasion into internal compartments of the scaffolds in a mouse model. In vivo native BMCS invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo.