Notch signaling in differentiation and function of dendritic cells

Hematopoietic stem cells give rise to multiple lineages of cells. This process is governed by a tightly controlled signaling network regulated by cytokines and a direct cell-cell contact. Notch signaling represents one of the major pathways activated during direct interaction between hematopoietic progenitor cells and bone marrow stroma. A critical role of Notch signaling in differentiation of T- and B-lymphocytes has now been established. Until recently, the role of Notch signaling in the development of myeloid cells and particular dendritic cells remained unclear. In this review, we discuss recent exciting findings that shed light on the critical role of Notch in differentiation and the function of dendritic cells and its impact on immune responses.     

Notch signaling controls the generation and differentiation of early T lineage progenitors

Signaling by the transmembrane receptor Notch is critical for T lineage development, but progenitor subsets that first receive Notch signals have not been defined. Here we identify an immature subset of early T lineage progenitors (ETPs) in the thymus that expressed the tyrosine kinase receptor Flt3 and had preserved B lineage potential at low progenitor frequency. Notch signaling was active in ETPs and was required for generation of the ETP population. Additionally, Notch signals contributed to the subsequent differentiation of ETPs. In contrast, multipotent hematopoietic progenitors circulated in the blood even in the absence of Notch signaling, suggesting that critical Notch signals during early T lineage development are delivered early after thymic entry.     

Notch signaling regulates the differentiation of post-mitotic intestinal epithelial cells

The intestinal epithelium comprises differentiated cells of four lineages maintained by precursor cells. As the Notch pathway controls the fate of proliferating cells in many systems, we investigated the effect of conditional expression of an activated Notch mutant in intestinal epithelium. An increase in the number of goblet cells occurs within 8 h of induction, due to an effect of Notch on post-mitotic cells, not on precursors. This observation broadens the role of Notch into controlling postmitotic differentiation and indicates that the composition of the epithelium is not solely determined by progenitor cells.     

Histological and functional differentiation of non-lymphoid cells in the chicken spleen.

The phenotypes and functions of various populations of non-lymphoid cells in the chicken spleen were investigated with monoclonal antibodies and after in vivo administration of antigens. Monoclonal antibody CVI-ChNL-68.1 was used to detect red pulp macrophages, interdigitating cells, and monocytes, whereas CVI-ChNL-68.2 was used to detect ellipsoid-associated non-lymphoid cells (EANC). Two new monoclonal antibodies were developed: CVI-ChNL-74.2, which specifically recognized red pulp macrophages and a ring of macrophages surrounding the peri-ellipsoid lymphocyte sheath; and CVI-ChNL-74.3, which specifically recognized follicular dendritic cells (FDC) in germinal centres and small clusters of stromal cells in T-cell areas. After intravenous injection of lipopolysaccharide (LPS), the number of 68.1+ and 74.2+ macrophages decreased dramatically, whereas 68.2+ EANC and 74.3+ FDC were unaffected. After intravenous injection of heat-inactivated Brucella abortus, the numbers of both macrophages and EANC decreased. In contrast, a significant increase of 74.3+ cells was observed in the T-cell areas outside the germinal centres. As expected, intravenous injection of non-mitogenic antigens, such as keyhole limpet haemocyanin and Ficoll, and carrageenan did not affect the non-lymphoid cell populations. At least six subpopulations of non-lymphoid cells in the chicken spleen can now be discriminated with monoclonal antibodies. Our results show that mononuclear phagocytes are sensitive for mitogenic stimulators such as LPS and Brucella abortus. In contrast, stromal non-lymphoid cells are only sensitive to the particulate mitogen Brucella abortus. We conclude that the complex formed by ellipsoid cells, the peri-ellipsoid B-cell sheath, and the surrounding macrophages, is the functional equivalent of the mammalian marginal zone.     

骨髓间充质干细胞体外增殖及分化过程中Notch信号的表达变化

目的研究Notch信号系统对骨髓间充质干细胞的增殖与分化的调控作用。方法用密度梯度离心法分离培养犬骨髓间充质干细胞,观察其增殖及传代情况,并进行成骨、成软骨及成心肌细胞诱导,Western blot检测细胞增殖及分化过程中Notch信号蛋白的表达。结果骨髓间充质干细胞呈梭形、旋涡样生长,增殖及传代能力强,可在体外形成钙结节,分化为软骨细胞,免疫组化示心肌细胞标志物的表达。Western blot显示在增殖过程中可见Notch信号蛋白的表达,为1.00±0.13,当分化为骨细胞及心肌细胞时,其表达上调,分别为1.20±0.14及1.70±0.17,与增殖的干细胞相比有明显差异(P<0.05)。结论 Notch信号系统对骨髓间充质干细胞的增殖及分化起重要的调控作用。     

Notch ligand-bearing thymic epithelial cells initiate and sustain Notch signaling in thymocytes independently of T cell receptor signaling.

Thymic epithelial cells are specialized to play essential roles at multiple stages of T cell development in the thymus, yet the molecular basis of this specialization is largely unknown. Recently, the Notch family of transmembrane proteins has been implicated in thymocyte development. Such proteins interact with cell surface proteins of the Delta-like and Jagged families. It is known that Notch ligands are expressed intrathymically, and that Notch signaling is regulated by Notch ligands expressed on either the same or third-party cells. However, functional analysis of Notch ligand expression, and elucidation of the mechanism of Notch ligand signaling in thymocyte development, are unclear. Here, we find that Notch ligand expression in the thymus is compartmentalized, with MHC class II(+) thymic epithelium, but not thymocytes nor dendritic cells, expressing Jagged-1, Jagged-2 and Delta-like-1. We also provide evidence that contact with Notch ligands on thymic epithelium is necessary to activate and sustain Notch signaling in thymocytes, and that this can occur independently of positive selection induction. Our data suggest that Notch ligand expression by thymic epithelium may partly explain the specialization of these cells in supporting thymocyte development, by regulating Notch activation via an inductive signaling mechanism independently of signaling leading to positive selection.     

Nuclear Notch1 signaling and the regulation of dendritic development

To understand the function of Notch in the mammalian brain, we examined Notch1 signaling and its cellular consequences in developing cortical neurons. We found that the cytoplasmic domain of endogenous Notch1 translocated to the nucleus during neuronal differentiation. Notch1 cytoplasmic-domain constructs transfected into cortical neurons were present in multiple phosphorylated forms, localized to the nucleus and could induce CBF1-mediated transactivation. Molecular perturbation experiments suggested that Notch1 signaling in cortical neurons promoted dendritic branching and inhibited dendritic growth. These observations show that Notch1 signaling to the nucleus exerts an important regulatory influence on the specification of dendritic morphology in neurons.     

Notch信号调节外周T细胞的活化、增殖与分化

初始T细胞分化为效应T和记忆T细胞受到多种因素调节.最近在Notch信号途径的研究进展显示它也参于T细胞的活化与分化.大量研究已经表明Notch信号途径可以影响T细胞在中枢免疫器官的发育,现在关于它调节外周T细胞的分化状态也积累不少证据,Notch信号活化之后能够改变CD4+和CD8+T细胞分泌细胞因子的特点.以下着重介绍Notch信号参于调节外周T细胞的活化、增殖和分化的最新资料,尽管不同的研究者所得实验结果有冲突之处,但已经提示Notch信号在T细胞外周发育中的重要意义,特别重要的是抗原递呈细胞(APC)可以通过Notch信号途径调节T细胞的分化.     

Notch1 signalling controls the differentiation and function of myeloid-derived suppressor cells in systemic lupus erythematosus

Emerging studies have reported the expansion of myeloid-derived suppressor cells (MDSCs) in some autoimmune disorders, such as systemic lupus erythematosus (SLE), but the detailed molecular mechanisms of the aberrant expansion in SLE are still unclear. In the present study, we confirmed that the increased MDSCs positively correlated with disease activity in SLE patients. The suppressive capacity of MDSCs from patients with high activity was lower than that of MDSCs from patients with low activity. Moreover, the potential precursors for MDSCs, common myeloid progenitors (CMPs) and granulocyte–monocyte progenitors (GMPs), were markedly increased in the bone marrow (BM) aspirates of SLE patients. As an important regulator of cell fate decisions, aberrant activation of Notch signalling was reported to participate in the pathogenesis of SLE. We found that the expression of Notch1 and its downstream target gene hairy and enhancer of split 1 (Hes-1) increased markedly in GMPs from SLE patients. Moreover, the Notch1 signalling inhibitor DAPT profoundly relieved disease progression and decreased the proportion of MDSCs in pristane-induced lupus mice. The frequency of GMPs was also decreased significantly in lupus mice after DAPT treatment. Furthermore, the inhibition of Notch1 signalling could limit the differentiation of MDSCs in vitro. The therapeutic effect of DAPT was also verified in Toll-like receptor 7 (TLR7) agonist-induced lupus mice. Taken together, our results demonstrated that Notch1 signalling played a crucial role in MDSC differentiation in SLE. These findings will provide a promising therapy for the treatment of SLE.     

GM-CSF controls nonlymphoid tissue dendritic cell homeostasis but is dispensable for the differentiation of inflammatory dendritic cells

GM-CSF (Csf-2) is a critical cytokine for the in?vitro generation of dendritic cells (DCs) and is thought to control the development of inflammatory DCs and resident CD103(+) DCs in some tissues. Here we showed that in contrast to the current understanding, Csf-2 receptor acts in the steady state to promote the survival and homeostasis of nonlymphoid tissue-resident CD103(+) and CD11b(+) DCs. Absence of Csf-2 receptor on lung DCs abrogated the induction of CD8(+) T?cell immunity after immunization with particulate antigens. In contrast, Csf-2 receptor was dispensable for the differentiation and innate function of inflammatory DCs during acute injuries. Instead, inflammatory DCs required Csf-1 receptor for their development. Thus, Csf-2 is important in vaccine-induced CD8(+) T?cell immunity through the regulation of nonlymphoid tissue DC homeostasis rather than control of inflammatory DCs in?vivo.     

β-mannosyl linkages inhibit CAWS arteritis by negatively regulating dectin-2-dependent signaling in spleen and dendritic cells

Abstract

Aims: CAWS, Candida albicans water-soluble fraction, is an extracellular mannoprotein produced by C. albicans NBRC1385. It is a ligand of dectin-2, the C-type lectin receptor for innate immunity, and has strong potency for induction of vasculitis in DBA/2 mice. The structure of this mannoprotein is known to be modulated by the culture conditions. To clarify the structure required for vasculitis, CAWSs were prepared in the two culture conditions with or without pH control, and biological properties were compared.

Methods: CAWSs prepared by the standard protocol and pH controlled at 7.0 were designated as CAWS and CAWS727, respectively. The antigenicity was detected by the anti-Candida mannan IgG. These chemical structures were assessed by nuclear magnetic resonance analysis and the lectin array system. The in vitro activity of CAWSs was tested by tumor necrosis factor-α (TNF-α) induction using bone marrow-derived dendritic cells and spleen cell cultures.

Results: The antigenicity of CAWS727 was similar to CAWS but the nuclear magnetic resonance analysis showed a higher ratio of β-mannosyl linkages were detected in CAWS727. The lectin array showed relative affinities of CAWS727 to α-mannosyl specific lectins were weaker than those of CAWS. CAWS induced severe vasculitis in DBA/2 mice while CAWS727 did not. CAWS significantly induced TNF-α but CAWS727 did slightly. In addition, CAWS-induced TNF-α production was inhibited by mixing with CAWS727 in a concentration dependent manner.

Conclusion: The α-mannosyl linkages of Candida mannan is a key molecule for the immunotoxicity. CAWS727, which conatins β-mannosyl linkages, competitively bound to lectin receptors, and resulted in reductions in the inflammatory response.     

The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways

Most dendritic cell (DC) responses to Toll-like receptor (TLR) ligands depend on the activation of mitogen-activated protein kinases (MAPKs), but the contributions of the many MAPK-activated kinases (MKs) that act 'downstream' of the MAPKs Erk and p38 are not known. Here we sought to determine which MKs are required for acute TLR-driven, MAPK-dependent DC endocytic responses. Two specific and structurally different inhibitors of the MK Rsk suppressed TLR-induced endocytosis, thus defining in DCs a specific requirement for MKs in TLR responses. In addition, we identify in DCs a previously unknown configuration of the MAPK system whereby Rsk is activated not only by Erk but also by p38 through the intermediates MK2 and MK3. Thus, in DCs, p38 contributes to the activation of all known MK families.     

Notch信号系统调控骨髓间充质干细胞向心肌样细胞的分化

背景：Notch信号系统在调控骨髓间充质干细胞的定向分化中起关键作用,但尚无涉及干细胞分化为心肌细胞及其分化机制的报道。 目的：分析Notch信号系统在骨髓间充质干细胞分化为心肌细胞过程中的调控作用。 方法：将分离培养的骨髓间充质干细胞与心肌细胞共培养,Jagged1组加入Notch激活剂Jagged1,DAPT组加入Notch激活剂Jagged1和抑制剂DAPT,对照组加入PBS缓冲液。用反转录-聚合酶链反应、免疫组化等方法检测干细胞分化为心肌细胞的情况及Notch信号系统的表达。 结果与结论：骨髓间充质干细胞在体外可分化为心肌细胞,与DAPT组及对照组相比,Jagged1组的干细胞分化为心肌细胞的比率提高,心肌标志物表达增多,并且Notch1和Jagged1表达增强。证实Notch信号系统对骨髓间充质干细胞分化为心肌细胞起正向调控作用。     

Notch signaling and the emergence of hematopoietic stem cells

The hematopoietic stem cell (HSC) is able to give rise to all blood cell lineages in vertebrates. HSCs are generated in the early embryo after two precedent waves of primitive hematopoiesis. Canonical Notch signaling is at the center of the complex mechanism that controls the development of the definitive HSC. The successful in vitro generation of hematopoietic cells from pluripotent stem cells with the capacity for multilineage hematopoietic reconstitution after transplantation requires the recapitulation of the most important process that takes place in the hemogenic endothelium during definitive hematopoiesis, that is the endothelial-to-hematopoietic transition (EHT). To meet this challenge, it is necessary to thoroughly understand the molecular mechanisms that modulate Notch signaling during the HSC differentiation process considering different temporal and spatial dimensions. In recent years, there have been important advances in this field. Here, we review relevant contributions describing different genes, factors, environmental cues, and signaling cascades that regulate the EHT through Notch interactions at multiple levels. The evolutionary conservation of the hematopoietic program has made possible the use of diverse model systems. We describe the contributions of the zebrafish model and the most relevant ones from transgenic mouse studies and from in vitro differentiated pluripotent cells.