Dendritic Differentiation Research Articles

RIG-I-like Receptor Triggering by Dengue Virus Drives Dendritic Cell Immune Activation and TH1 Differentiation.

Dengue virus (DENV) causes 400 million infections annually and is one of several viruses that can cause viral hemorrhagic fever, which is characterized by uncontrolled immune activation resulting in high fever and internal bleeding. Although the underlying mechanisms are unknown, massive cytokine secretion is thought to be involved. Dendritic cells (DCs) are the main target cells of DENV, and we investigated their role in DENV-induced cytokine production and adaptive immune responses. DENV infection induced DC maturation and secretion of IL-1β, IL-6, and TNF. Inhibition of DENV RNA replication abrogated these responses. Notably, silencing of RNA sensors RIG-I or MDA5 abrogated DC maturation, as well as cytokine responses by DENV-infected DCs. DC maturation was induced by type I IFN responses because inhibition of IFN-α/β receptor signaling abrogated DENV-induced DC maturation. Moreover, DENV infection of DCs resulted in CCL2, CCL3, and CCL4 expression, which was abrogated after RIG-I and MDA5 silencing. DCs play an essential role in TH cell differentiation, and we show that RIG-I and MDA5 triggering by DENV leads to TH1 polarization, which is characterized by high levels of IFN-γ. Notably, cytokines IL-6, TNF, and IFN-γ and chemokines CCL2, CCL3, and CCL4 have been associated with disease severity, endothelial dysfunction, and vasodilation. Therefore, we identified RIG-I and MDA5 as critical players in innate and adaptive immune responses against DENV, and targeting these receptors has the potential to decrease hemorrhagic fever in patients.

Lenalidomide increases human dendritic cell maturation in multiple myeloma patients targeting monocyte differentiation and modulating mesenchymal stromal cell inhibitory properties.

The use of Lenalidomide (LEN), to reverse tumor-mediated immune suppression and amplify multiple myeloma-specific immunity is currently being explored. Particularly, LEN effects on dendritic cells (DCs) are still unclear. In this study, we investigated the potential effect of LEN on DC differentiation and activity. DCs were differentiated either from CD14+ cells obtained from patients with multiple myeloma or from a human monocytic cell line.LEN, at the concentration range reached in vivo, significantly increased the median intensity expression of HLA-DR, CD86 and CD209 by DCs derived from both bone marrow and peripheral myeloma monocytes and enhanced the production of Interleukin-8, C-C motif chemokine ligand (CCL) 2, CCL5 and tumor necrosis factor-α. Consistently, LEN pre-treated DCs showed an increased ability to stimulate autologous CD3+ cell proliferation. LEN effect on dendritic differentiation was associated with the degradation of the Cereblon-related factors Ikaros and Aiolos. Moreover, we showed that LEN also blunted mesenchymal stromal cell inhibitory effect on dendritic differentiation, inhibiting Casein Kinase-1α levels. Finally, in vitro data were confirmed in ex vivo cultures obtained from relapsed myeloma patients treated with LEN, showing a significant increase of DC differentiation from peripheral blood monocytes.In conclusion, LEN increased the expression of mature dendritic markers both directly and indirectly and enhanced DC ability to stimulate T cell proliferation and to release chemokines. This suggests a new possible mechanism by which LEN could exert its anti-myeloma activity.

Abstract 17: Apolipoprotein C-III Promotes Anti-Inflammatory Regulatory T Cell Phenotype Through Modulation of Intestinal Dendritic Cell Activation

Apolipoprotein C-III (apoC-III) is a cardio-metabolic modulator in multiple tissues. Circulating apoC-III in humans is an independent risk factor for cardiovascular disease (CVD) because apoC-III increases circulating triglyceride (TG) through inhibition of the LDLR in the liver and lipoprotein lipase on the capillary endothelium. In the liver, apoC-III overexpression results in increased synthesis and secretion of more TG-rich VLDL. These actions increase the resident time and amount of atherogenic remnants in circulation. Paradoxically, apoC-III acts in the intestine to inhibit dietary fat absorption, a potentially beneficial action. Using human-apoC-III transgenic mice (hu-apoCIII tg mice), which have increased circulating TG compared to C57Bl6/J littermate controls (WT), we have identified an increase in circulating IL-10 at basal conditions. Regulatory T cells (Tregs), a T cell subset that is known to delay atheroprogression, secrete the anti-inflammatory cytokine IL-10. Knowing that the immune system plays a dynamic role in atherogenesis, we asked whether apoC-III is a potential modulator of Tregs during a western diet (WD) challenge. We subjected hu-apoCIII tg and WT mice to 12 weeks of a WD and through flow cytometry, found significantly increased circulating and intestinal Tregs in hu-apoCIII tg mice compared to WT. Hu-apoCIII tg intestinal gene expression showed increases in TGF-β and maintenance of IL-10 and FOXP3, key Treg-related genes, after the WD compared to WT. We hypothesized that apoC-III may act in the intestine to induce tolerance to dietary antigens through dendritic cell (DC) activation and subsequent differentiation of naïve T cells to the Treg phenotype. Using flow cytometry of the lamina propria, we saw increased activation of intestinal DCs, in hu-apoCIII tg mice at basal and WD-challenged conditions suggesting that intestinal DCs are influenced by apoC-III overexpression. These results suggest that apoC-III may influence Treg differentiation or proliferation through alteration of intestinal DC activation. The phenotype we have uncovered in hu-apoC-III tg mice suggests a link between apoC-III overexpression, dietary fat absorption, and the immune system, and that this may be protective against inflammation.

PLGA nanoparticles modified with a TNFα mimicking peptide, soluble Leishmania antigens and MPLA induce T cell priming in vitro via dendritic cell functional differentiation

Poly(lactide-co-glycolide) nanoparticles (PLGA NPs) represent a new approach for vaccine delivery due to their ability to be taken up by phagocytes and to activate immune responses. In the present study PLGA NPs were surface-modified with a TNFα mimicking peptide, and encapsulated soluble Leishmania antigens (sLiAg) and MPLA adjuvant. The synthesized PLGA NPs exhibited low cytotoxicity levels, while surface-modified NPs were more efficiently taken up by dendritic cells (DCs). The prepared nanoformulations induced maturation and functional differentiation of DCs by elevating co-stimulatory molecule levels and stimulating IL-12 and IL-10 production. Sensitized DCs promoted T cell priming, characterized by the development of mixed T cell subsets differentiation expressing Th lineage-specific transcriptional factors and cytokine genes. Moreover, PLGA NPs were biocompatible, while they were located in lymphoid organs and taken up by phagocytic cells. Our results suggest that surface-modified PLGA NPs encapsulating sLiAg and MPLA could be considered as an effective vaccine candidate against leishmaniasis.

Lung cancer-derived galectin-1 contributes to cancer associated fibroblast-mediated cancer progression and immune suppression through TDO2/kynurenine axis.

Communication between cancer cells and their microenvironment plays an important role in cancer development, but the precise mechanisms by which cancer-associated fibroblasts (CAF) impact anti-cancer immunity and cancer progression in lung cancer are poorly understood. Here, we report that lung fibroblasts when activated by lung cancer cells produce tryptophan metabolite kynurenine (Kyn) that inhibits dendritic cells' differentiation and induces cancer growth as well as migration. We identified TDO2 (tryptophan 2,3-dioxygenase) as the main enzyme expressed in fibroblasts capable of tryptophan metabolism. Mechanistically, condition medium of CAF or exogenous kynurenine stimulated AKT, with no lysine 1 (WNK1) and cAMP response element-bindingprotein (CREB) phosphorylation in lung cancer cells. Inhibition of the AKT/CREB pathway prevents cancer proliferation, while inhibition of the AKT/ WNK1 reverted epithelial-to-mesenchymal transition and cancer migration induced by kynurenine. Moreover, we also demonstrate that lung cancer-derived galectin-1 contributes to the upregulation of TDO2 in CAF through an AKT-dependent pathway. Immunohistochemical analysis of lung cancer surgical specimens revealed increased TDO2 expression in the fibroblasts adjacent to the cancer. Furthermore, in vivo studies showed that administration of TDO2 inhibitor significantly improves DCs function and T cell response, and decreases tumor metastasis in mice. Taken together, our data identify the feedback loop, consisting of cancer-derived galectin-1 and CAF-producing kynurenine, that sustains lung cancer progression. These findings suggest that targeting this pathway may be a promising therapeutic strategy.

Essential Roles for ARID1B in Dendritic Arborization and Spine Morphology of Developing Pyramidal Neurons.

Haploinsufficiency of ARID1B, a component of chromatin remodeling complex, causes intellectual disability. However, the role of ARID1B in brain development is unknown. Here, we demonstrate that ARID1B is required for neuronal differentiation in the developing brain, such as in dendritic arborization and synapse formation. Our findings suggest that ARID1B plays a critical role in the establishment of cognitive circuitry by regulating dendritic complexity. Thus, ARID1B deficiency may cause intellectual disability via abnormal brain wiring induced by the defective differentiation of cortical neurons.

Quantitative and Qualitative Analysis of Transient Fetal Compartments during Prenatal Human Brain Development.

The cerebral wall of the human fetal brain is composed of transient cellular compartments, which show characteristic spatiotemporal relationships with intensity of major neurogenic events (cell proliferation, migration, axonal growth, dendritic differentiation, synaptogenesis, cell death, and myelination). The aim of the present study was to obtain new quantitative data describing volume, surface area, and thickness of transient compartments in the human fetal cerebrum. Forty-four postmortem fetal brains aged 13–40 postconceptional weeks (PCW) were included in this study. High-resolution T1 weighted MR images were acquired on 19 fetal brain hemispheres. MR images were processed using in-house software (MNI-ACE toolbox). Delineation of fetal compartments was performed semi-automatically by co-registration of MRI with histological sections of the same brains, or with the age-matched brains from Zagreb Neuroembryological Collection. Growth trajectories of transient fetal compartments were reconstructed. The composition of telencephalic wall was quantitatively assessed. Between 13 and 25 PCW, when the intensity of neuronal proliferation decreases drastically, the relative volume of proliferative (ventricular and subventricular) compartments showed pronounced decline. In contrast, synapse- and extracellular matrix-rich subplate compartment continued to grow during the first two trimesters, occupying up to 45% of telencephalon and reaching its maximum volume and thickness around 30 PCW. This developmental maximum coincides with a period of intensive growth of long cortico-cortical fibers, which enter and wait in subplate before approaching the cortical plate. Although we did not find significant age related changes in mean thickness of the cortical plate, the volume, gyrification index, and surface area of the cortical plate continued to exponentially grow during the last phases of prenatal development. This cortical expansion coincides developmentally with the transformation of embryonic cortical columns, dendritic differentiation, and ingrowth of axons. These results provide a quantitative description of transient human fetal brain compartments observable with MRI. Moreover, they will improve understanding of structural-functional relationships during brain development, will enable correlation between in vitro/in vivo imaging and fine structural histological studies, and will serve as a reference for study of perinatal brain injuries.

Deletion of the GluRδ2 Receptor in the Hotfoot Mouse Mutant Causes Granule Cell Loss, Delayed Purkinje Cell Death, and Reductions in Purkinje Cell Dendritic Tree Area

Recent studies have found that in the cerebellum, the δ2 glutamate receptor (GluRδ2) plays a key role in regulating the differentiation of parallel fiber-Purkinje synapses and mediating key physiological functions in the granule cell-Purkinje cell circuit. In the hotfoot mutant or GluRδ2 knockout mice, the absence of GluRδ2 expression results in impaired motor-related tasks, ataxia, and disruption of long-term depression at parallel fiber-Purkinje cell synapses. The goal of this study was to determine the long-term consequences of deletion of GluRδ2 expression in the hotfoot mutant (GluRδ2 ho/ho ) on Purkinje and granule cell survival and Purkinje cell dendritic differentiation. Quantitative estimates of Purkinje and granule cell numbers in 3-, 12-, and 20-month-old hotfoot mutants and wild-type controls showed that Purkinje cell numbers are within control values at 3 and 12months in the hotfoot mutant but reduced by 20% at 20months compared with controls. In contrast, the number of granule cells is significantly reduced from 3months onwards in GluRδ2 ho/ho mutant mice compared to wild-type controls. Although the overall structure of Purkinje cell dendrites does not appear to be altered, there is a significant 27% reduction in the cross-sectional area of Purkinje cell dendritic trees in the 20-month-old GluRδ2 ho/ho mutants. The interpretation of the results is that the GluRδ2 receptor plays an important role in the long-term organization of the granule-Purkinje cell circuit through its involvement in the regulation of parallel fiber-Purkinje cell synaptogenesis and in the normal functioning of this critical cerebellar circuit.

Microtubule-Actin Crosslinking Factor 1 Is Required for Dendritic Arborization and Axon Outgrowth in the Developing Brain.

Dendritic arborization and axon outgrowth are critical steps in the establishment of neural connectivity in the developing brain. Changes in the connectivity underlie cognitive dysfunction in neurodevelopmental disorders. However, molecules and associated mechanisms that play important roles in dendritic and axon outgrowth in the brain are only partially understood. Here, we show that microtubule-actin crosslinking factor 1 (MACF1) regulates dendritic arborization and axon outgrowth of developing pyramidal neurons by arranging cytoskeleton components and mediating GSK-3 signaling. MACF1 deletion using conditional mutant mice and in utero gene transfer in the developing brain markedly decreased dendritic branching of cortical and hippocampal pyramidal neurons. MACF1-deficient neurons showed reduced density and aberrant morphology of dendritic spines. Also, loss of MACF1 impaired the elongation of callosal axons in the brain. Actin and microtubule arrangement appeared abnormal in MACF1-deficient neurites. Finally, we found that GSK-3 is associated with MACF1-controlled dendritic differentiation. Our findings demonstrate a novel role for MACF1 in neurite differentiation that is critical to the creation of neuronal connectivity in the developing brain.

Amniotic mesenchymal cells from pre-eclamptic placentae maintain immunomodulatory features as healthy controls.

Pre-eclampsia (PE) is one of the most severe syndromes in human pregnancy, and the underlying mechanisms of PE have yet to be determined. Pre-eclampsia is characterized by the alteration of the immune system's activation status, an increase in inflammatory Th1/Th17/APC cells, and a decrease in Th2/Treg subsets/cytokines. Moreover, inflammatory infiltrates have been detected in the amniotic membranes of pre-eclamptic placentae, and to this date limited data are available regarding the role of amniotic membrane cells in PE. Interestingly, we and others have previously shown that human amniotic mesenchymal stromal cells (hAMSC) possess anti-inflammatory properties towards almost all immune cells described to be altered in PE. In this study we investigated whether the immunomodulatory properties of hAMSC were altered in PE. We performed a comprehensive study of cell phenotype and investigated the in vitro immunomodulatory properties of hAMSC isolated from pre-eclamptic pregnancies (PE-hAMSC), comparing them to hAMSC from normal pregnancies (N-hAMSC). We demonstrate that PE-hAMSC inhibit CD4/CD8 T-cell proliferation, suppress Th1/Th2/Th17 polarization, induce Treg and block dendritic cells and M1 differentiation switching them to M2 cells. Notably, PE-hAMSC generated a more prominent induction of Treg and higher suppression of interferon-γ when compared to N-hAMSC, and this was associated with higher transforming growth factor-β1 secretion and PD-L2/PD-L1 expression in PE-hAMSC. In conclusion, for the first time we demonstrate that there is no intrinsic impairment of the immunomodulatory features of PE-hAMSC. Our results suggest that amniotic mesenchymal stromal cells do not contribute to the disease, but conversely, could participate in offsetting the inflammatory environment which characterizes PE.

TPX2 regulates neuronal morphology through kinesin-5 interaction.

TPX2 (targeting protein for Xklp2) is a multifunctional mitotic spindle assembly factor that in mammalian cells localizes and regulates mitotic motor protein kinesin-5 (also called Eg5 or kif11). We previously showed that upon depletion or inhibition of kinesin-5 in cultured neurons, microtubule movements increase, resulting in faster growing axons and thinner dendrites. Here, we show that depletion of TPX2 from cultured neurons speeds their rate of process outgrowth, similarly to kinesin-5 inhibition. The phenotype is rescued by TPX2 re-expression, but not if TPX2's kinesin-5-interacting domain is deleted. These results, together with studies showing a spike in TPX2 expression during dendritic differentiation, suggest that the levels and distribution of TPX2 are likely to be determinants of when and where kinesin-5 acts in neurons.

The role of Ikaros transcriptional factor in normal hematopoiesis and leukemogenesis: biological and clinical aspects

Investigation of the pathogenesis and factors effecting recurrence, progression and drug resistance in acute leukemia (AL) remains a major challenge for hematology and other related areas. The role of more than 50 genes and proteins in the AL pathogenesis has been shown, including the well-studied tumor suppressor (CDKN2A/CDKN2B, RB1, PTEN, p53), and classical fusion genes (BCR/ABL1, TEL/AML1, E2A/PBX, MLL translocations). In addition, high frequency of aberrations in genes responsible for lymphoid differentiation have been identified such as transcription factors (PAX5, IKZF1 and EBF1), transcriptional regulation of the genes (ETV6, ERG), and signaling pathways of antigen receptors (BTLA, CD200, TOX, BLNK, VPREB1), as well as genes involved in chemoresistance of leukemia cells (NR3C1). In recent studies, Ikaros abnormalities have been reported to be frequently associated with AL. Ikaros is a member of a Kruppel-like family of zinc finger transcription factors that also includes IKZF2 (Helios), IKZF3 (Aiolos), Eos and Pegasus, and encoded by the IKZF1 gene. In hematopoietic cells Ikaros functions as a transcription factor, a key protein controlling T-, B-, NK-, and dendritic cells early differentiation. At the early hematopoiesis stages, it represses the myeloid and erythroid lineages, and stimulates the lymphoid differentiation. Ikaros also normally modulates immune response and plays role of a tumor suppressor in lymphoid malignances. Data from numerous clinical studies confirmed an association between the presence of IKZF1 aberrations and B-cell and, to a lesser extent, T-cell acute lymphoblastic leukemia (ALL) development. Besides, loss of Ikaros function was associated with progression of myeloproliferative diseases to acute myeloid leukemia (AML) in children. From clinical point of view, particular intragenic IKZF1 deletions and a short (non-functional) protein Ikaros isoforms, which may occur as a result of intragenic deletions or aberrant splicing, are the most significant features. These mutations of IKZF1 gene and Ikaros aberrant expression play a key role in the lymphoid transformation, tumor progression, and may cause development of leukemic cells chemoresistance. Therefore, IKZF1 aberrations should be taken into account as a valuable prognostic marker for risk groups stratification, poor outcome and low survival rare. This review compiles currently available data regarding the frequency and variants of the IKZF1 (Ikaros) aberrations, and the use of them in diagnostics of all types of leukemia and minimal residual disease detection. Although Ikaros has already applied in clinical studies, a growing number of questions still remain unanswered. Molecular biology of IKZF1 expression and splicing regulation is not well understood. Clinical value of point mutations and subclonal deletion in IKZF1 locus should be elucidated. Prognostic significance of intragenic deletions and aberrant splicing is necessary to clarify for different groups of ALL patients, in connection with other genetic markers and therapy protocol. More detailed clinical analysis required for proving IKZF1 impact on probability of relapse, improving patients, risk stratification and application of minimal residual disease.

The GluN2B subunit of N-methy-D-asparate receptor regulates the radial migration of cortical neurons in vivo

The formation of layered structure of the mammalian neocortex requires a fine organized migration of post-mitotic neurons during early development. However, whether the radial migration is regulated by NMDA receptor and specific subunits remains contradictory and unknown. Here, we reported that in the developing rat cortex, migration of presumptive layer II/III neurons to their deserved destination was regulated by NMDA receptors with GluN2B but not GluN2A subunit. Using in utero electroporation of small interference RNA (siRNA) of distinct NMDA receptor subunits, we found that knockdown GluN1 and GluN2B subunits dramatically delayed the neuronal migration to proper layer II/III, while improperly stayed at lower layers or even the germinal regions, without changing the cell fate. In contrast, knockdown of GluN2A subunit did not impair the neuronal migration. Additionally, the ecotopic neurons by GluN2B RNAi developed to well dendritic differentiation, while the ones by GluN1 RNAi still kept morphology of migrating neurons. Therefore, GluN2B subunit of NMDA receptor plays an essential role in regulating proper neuronal migration and cortical lamination.

Tenascin-C aggravates autoimmune myocarditis via dendritic cell activation and Th17 cell differentiation.

BackgroundTenascin‐C (TN‐C), an extracellular matrix glycoprotein, appears at several important steps of cardiac development in the embryo, but is sparse in the normal adult heart. TN‐C re‐expresses under pathological conditions including myocarditis, and is closely associated with tissue injury and inflammation in both experimental and clinical settings. However, the pathophysiological role of TN‐C in the development of myocarditis is not clear. We examined how TN‐C affects the initiation of experimental autoimmune myocarditis, immunologically.Methods and ResultsA model of experimental autoimmune myocarditis was established in BALB/c mice by immunization with murine α‐myosin heavy chains. We found that TN‐C knockout mice were protected from severe myocarditis compared to wild‐type mice. TN‐C induced synthesis of proinflammatory cytokines, including interleukin (IL)‐6, in dendritic cells via activation of a Toll‐like receptor 4, which led to T‐helper (Th)17 cell differentiation and exacerbated the myocardial inflammation. In the transfer experiment, dendritic cells loaded with cardiac myosin peptide acquired the functional capacity to induce myocarditis when stimulated with TN‐C; however, TN‐C‐stimulated dendritic cells generated from Toll‐like receptor 4 knockout mice did not induce myocarditis in recipients.ConclusionsOur results demonstrated that TN‐C aggravates autoimmune myocarditis by driving the dendritic cell activation and Th17 differentiation via Toll‐like receptor 4. The blockade of Toll‐like receptor 4‐mediated signaling to inhibit the proinflammatory effects of TN‐C could be a promising therapeutic strategy against autoimmune myocarditis.

The Dendritic Differentiation of Purkinje Neurons: Unsolved Mystery in Formation of Unique Dendrites.

Purkinje neurons play a central role in cerebellar functions. For Purkinje neurons to play such a role, the unique morphology of their dendrites is important. Purkinje neurons have the most elaborate dendritic trees among neurons in the brain (Fig. 1). They extend long proximal dendrites towards the pial surface. These dendrites branch extensively to form numerous synapses with parallel fibers, axons of granule cells. Purkinje dendrites are formed in a plane that is oriented perpendicular to the long axis of the cerebellum. Such unique dendritic trees differentiate over several postnatal weeks in rodents [1]. To date, many studies have investigated the cellular and molecular mechanisms of the dendritic differentiation of Purkinje neurons [2, 3]. Although those studies identified a number of molecules involved in Purkinje dendrite differentiation, several important aspects concerning differentiation of the unique dendrites remain unclear. This editorial highlights these aspects of the dendritic differentiation of Purkinje neurons (Fig. 2).

Ly49 and C-type lectin receptors on dendritic cells regulate T-cell differentiation as co-stimulatory molecules

The C-type lectin receptors (CLRs) expressed on dendritic cells (DCs) participate in T-cell polarization by recognizing pathogen-associated molecular patterns and activating signaling pathways for cytokine production. In addition, some CLRs expressed on DCs function as co-stimulatory molecules via recognition of endogenous ligands on T cells and regulate proliferation and/or differentiation of T cells. We recently showed that killer cell lectin-like receptor Ly49s3 is expressed in rat thymic DCs and recognizes MHC class I molecules on T cells, for differentiation into naturally occurring regulatory T cells (nTregs). Upon binding to MHC class I molecules on T cells, Ly49s3 seems to stimulate signal transduction pathway(s) leading to up-regulation of the MHC class II genes and then functions as a co-stimulatory molecule. The signaling pathway(s) is supposed to involve Dap12, Syk/Zap70, Lat, Plc-gamma, PKC, PU.1 and C2ta proteins to attain MHC class II expression. Other than Ly49s3, Ly49Q and Ly49B have been shown to be expressed in myeloid cells including DCs and macrophages, raising the possibility that they may be involved in the regulation of T-cell differentiation through recognition of MHC class I molecules on T cells. In humans, immunoglobulin (Ig)-like receptors binding to MHC class I molecules take the place of Ly49 receptors. Among them, expression of the KIR2DL4 gene has been reported to be induced in antigen-presenting cells, although its biological significance is obscure, and immunoglobulin-like transcript 4 (ILT4) expressed in DCs has been shown to down-regulate expression of MHC class II molecules on the same cells, upon binding to MHC class I molecules. They may also be involved in regulation of T-cell differentiation. Some other CLRs are expressed on DCs and possibly function as co-stimulatory molecules. For example, dectin-1, dectin-2 and Dcal-1 have been shown to promote T-cell proliferation, Treg differentiation and IL-4 production of T cells, respectively, through binding to unidentified ligands on T cells. DC-Sign, which recognizes ICAM3 on T cells, is also suggested to be involved in T-cell differentiation. Further investigation of the functional roles of CLRs on DCs will provide insight into the regulatory mechanisms of T-cell differentiation, essential processes for regulating immune responses.

The Serine/Threonine Kinase Ndr2 Controls Integrin Trafficking and Integrin-Dependent Neurite Growth

Integrins have been implicated in various processes of nervous system development, including proliferation, migration, and differentiation of neuronal cells. In this study, we show that the serine/threonine kinase Ndr2 controls integrin-dependent dendritic and axonal growth in mouse hippocampal neurons. We further demonstrate that Ndr2 is able to induce phosphorylation at the activity- and trafficking-relevant site Thr(788/789) of β1-integrin to stimulate the PKC- and CaMKII-dependent activation of β1-integrins, as well as their exocytosis. Accordingly, Ndr2 associates with integrin-positive early and recycling endosomes in primary hippocampal neurons and the surface expression of activated β1-integrins is reduced on dendrites of Ndr2-deficient neurons. The role of Ndr2 in dendritic differentiation is also evident in vivo, because Ndr2-null mutant mice show arbor-specific alterations of dendritic complexity in the hippocampus. This indicates a role of Ndr2 in the fine regulation of dendritic growth; in fact, treatment of primary neurons with Semaphorin 3A rescues Ndr2 knock-down-induced dendritic growth deficits but fails to enhance growth beyond control level. Correspondingly, Ndr2-null mutant mice show a Semaphorin 3A(-/-)-like phenotype of premature dendritic branching in the hippocampus. The results of this study show that Ndr2-mediated integrin trafficking and activation are crucial for neurite growth and guidance signals during neuronal development.

Dlx1 transcription factor regulates dendritic growth and postsynaptic differentiation through inhibition of neuropilin‐2 and PAK3 expression

Dlx1, a member of the homeobox domain transcriptional factors, is expressed in a subset of interneurons and is involved in their differentiation. To understand the roles of Dlx1 in dendritic and postsynaptic differentiation, we manipulated Dlx1 expression in both excitatory pyramidal neurons and inhibitory interneurons in hippocampal culture. Exogenous expression of Dlx1 in pyramidal neurons, which lack endogenous Dlx1, resulted in reduced complexity of dendritic arborization. This effect was dependent on the DNA-binding motif of Dlx1. Dlx1 overexpression also induced prominent reduction of spine density, but with mild suppression in the formation of postsynaptic densities. To confirm the roles of endogenous Dlx1, we knocked down Dlx1 in interneurons and found enhanced dendritic growth. By manipulating the expression of possible downstream effectors of Dlx1, neuropilin-2 and p21-activated kinase 3, we provided evidence for the involvement of these two signaling molecules in Dlx1-dependent regulation of dendritic differentiation. Our experimental data support the idea that Dlx1 expression in developing interneurons specifically suppresses two important downstream regulators, leading to the characteristic morphology of Dlx1-expressing interneurons with less branched dendrites and few dendritic spines.

Control Of Allograft Rejection With Mitomycin C Incubated Blood Cells

Immunosuppressive treatment in allogeneic transplantation is an effective way to control allograft rejection. But unfortunately it can cause serious side effects, such as infections or malignancies. Cell therapeutic approaches for induction of specific tolerance are on the rise and may have fewer side effects. We observed that monocytes or peripheral blood mononuclear cells (PBMC) of healthy persons after culture with dendritic cell-inducing cytokines and differentiation blockade with the chemotherapeutic drug mitomycin C (MMC) show the ability to suppress allogeneic T cell responses. Interestingly these cells resemble morphologically as well as phenotypically myeloid-derived suppressor cells (MDSC) which occur in cancer patients impairing the anti-tumor immunity. FACS- and gene expression analyses of these MMC incubated blood cells revealed downregulation of the immunostimulatory cell surface molecules CD80, CD83, CD86 and HLA-DR, and upregulated transcription of immunosuppressive and pro-apoptotic genes such as iNOS, arginase-1, IL-10, COX2, TGF-β, placental growth factor, immunoglobulin-like transcript 3, adrenomedullin, SMAD5, LRDD and of the transcription factor C/EBPβ. For use in preclinical transplantation models in vitro culture of MMC treated donor-derived blood cells with dendritic-cell inducing cytokines was not applicable and was therefore omitted. The tolerance inducing properties of donor-derived MMC incubated blood cells were investigated in two different animal models of solid organ transplantation. When injected into rats, donor-derived PBMC incubated with MMC strongly prolonged heart allograft survival. Suppression of rejection was donor-specific. Recipients showed an increased number of CD4+CD25+FoxP3+ regulatory T-cells and tolerance could be transferred to syngeneic recipients with blood or spleen cells. Donor-derived MMC incubated blood cells also prolonged kidney allograft survival in pigs. Furthermore, in a first clinical use a 6-year-old girl with relapse of acute lymphoblastic leukemia and imminent rejection of a third haploidentical stem cell transplant received donor-derived CD3/CD19-depleted peripheral blood stem cells, which have been treated in vitro with MMC, in order to prohibit graft rejection. We describe a simple method for in vitro generation of tolerance inducing donor-derived blood cells for potential use in clinical organ transplantation. Disclosures: No relevant conflicts of interest to declare.

Dendritic differentiation of cerebellar Purkinje cells is promoted by ryanodine receptors expressed by Purkinje and granule cells

Cerebellar Purkinje cells have the most elaborate dendritic trees among neurons in the brain. We examined the roles of ryanodine receptor (RyR), an intracellular Ca(2+) release channel, in the dendrite formation of Purkinje cells using cerebellar cell cultures. In the cerebellum, Purkinje cells express RyR1 and RyR2, whereas granule cells express RyR2. When ryanodine (10 µM), a blocker of RyR, was added to the culture medium, the elongation and branching of Purkinje cell dendrites were markedly inhibited. When we transferred small interfering RNA (siRNA) against RyR1 into Purkinje cells using single-cell electroporation, dendritic branching but not elongation of the electroporated Purkinje cells was inhibited. On the other hand, transfection of RyR2 siRNA into granule cells also inhibited dendritic branching of Purkinje cells. Furthermore, ryanodine reduced the levels of brain-derived neurotrophic factor (BDNF) in the culture medium. The ryanodine-induced inhibition of dendritic differentiation was partially rescued when BDNF was exogenously added to the culture medium in addition to ryanodine. Overall, these results suggest that RyRs expressed by both Purkinje and granule cells play important roles in promoting the dendritic differentiation of Purkinje cells and that RyR2 expressed by granule cells is involved in the secretion of BDNF from granule cells.