| Monocytes are the key components of innate and adaptive immunity, and are important in the mononuclear phagocytic system. Many studies have shown that bone marrow monocytes are common precursors for dendritic cells and macrophages, with build up the defense system effectively against the invasion of foreign micro-organisms. Meanwhile, these cells play an indispensable role in the removal of seniscent and apoptosis cells to maintain the homeostasis of bodies under physiological conditions. Dendritic cells are known to be the most powerful antigen -presenting cells and can activate T cells to initiate the immune responses in vivo. Macrophages, on the orther hand, can take up virus, bacteria, dead cells for apoptosis and antigen-antibody complex by phagocytosis and endocytosis, and remove them after degradation lysosomes. They also have strong antigen processing and presenting capacity and can play a variety of immune regulation roles and mediate inflammatory responses through secretion of cytokines. Abnormal development of monocytes, dendritic cells or macrophages will inevitably lead to disorders in the immune system and the internal environment, ultimately inducing various diseases of the body.Cell differetiation is a complex process, which requires a broad range of the crosstalk among signal transduction pathways. The Notch signaling pathway is highly conserved in evolution, and plays an important role in a variety of organisms from nematodes to humans, from embryonic development to adulthood. Notch receptor and ligand families belong to type I transmembrane proteins. When Notch receptor is triggered by direct interaction with its ligands, the intracellular domain of the Notch receptor (NICD) is released from the membrane after receptor cleavage executed by aγ-secretase-like protease. NICD translocates to nucleus and associates with RBP-J through its N-terminal RAM (RBP-J association molecule) domain, and transactivates promoters harboring RBP-J-binding sites, leading to the expression of genes associated cell differentiation. The Notch signaling pathway plays a decisive role in cell fate differentiation in many different systems, such as the nervous system, vascular system, hematopoietic system, etc. During the development of the immune system, the Notch signaling pathway is involved in various aspects of the differentiation of immune cells, including T versus B cell fate commitment and the marginal zone B versus follicular B cell fate commitment. Meanwhile, Notch signaling pathway can influence the differetiation of T cells in Th1 versus Th2 immune responses. However, the role of Notch signaling pathway in other types of cells such as DC, macrophages and NK cells is still not clear. Although there are reports that blocking Notch signaling can inhibit the development of DC, the molecular mechanism of the process has been poorly understood.In order to clarify the role of Notch signaling during the development of the immune system more clearly, using gene conditional knockout technology, we disrupted the key transcription factor RBP-J of Notch signaling pathway in mouse hematopoietic stem cells. In this study, we investigaed the changes of development and function of monocytes, DCs and macrophages after the deletion of RBP-J. The related molecular mechanisms are also analyzed using cellular and molecular technologies.The main finds are as follows:1. RBP-Jflox mice were crossed with Mx-Cre transgenic mice and to get Mx-Cre×RBP-Jflox/flox mice. We got RBP-J knockout mice after induction for 8-12 injecion of poly:I-C. Southern blot analysis of DNA isolated from bone marrow cells indicated about nearly 100% deletion efficiency.Bone marrow transplantation experiments showed that the differentiation of monocytes to DC was inhibited, but the differentiation to macrophages are strengthened upon RBP-J deletion. Cell differentiation by induction in vitro confirmed the experimental results in vivo. The expression of M-CSFR detected by molecular biology methods was significantly increased in RBP-J-/- macrophages, which indicated that the Notch signaling pathway might regulate the differentiation of monocytes through directly or indirectly controlling M-CSFR expression.2. We analyzed the changes of function and differentiation of DC after that RBP-J was removed. The results showed that LPS failed to stimulate DCs maturation, and DC development remained at an immature stage with low dendrites and low MHCII expression. At the same time their migration and antigen-presenting function were damaged. Further studies have shown that the chemokine receptor CXCR4 was an indirect downstream target of the Notch signaling pathway, which could promote out-growth of dendrites and the expression of MHCII during the development of DC phenotypic defects RBP-J-/- DC could be rescued by the over-expression of CXCR4 using lenti-virus. Our results showed that there was a signaling pathway that CXCR4 regulated by Notch could promote DC maturation.3. RBP-J-/- macrophages could not be polarized to the M1-type macrophages after LPS stimulation, but underwent for the M2-type differetiation. Their migration, phagocytosis, antigen processing and presentation abilities were all decreased. In tumors, RBP-J-/- macrophages had lower anti-tumor function, as shown by that tumor weight and size were suppressed by the existence of macrophages. Immunohistochemistry results showed that RBP-J-/- macrophages could promote tumor angiogenesis, indicating that it had certain characteristics of TAM. Meanwhile, activation of the Notch signaling pathway in macrophages could enhance their capacity of antigen-presenting and stimulate a strong Th1-type immune response. These results suggested that Notch signaling pathway should be activated in macrophages when these cells were used in treatment.In summary, we first provide the evidence that the development regulation of bone marrow monocyte-derived DC and macrophages was required by Notch/RBP-J signaling. Our results show that, Notch signals involved in regulating the fate decision of monocyte development, could promote the maturation of DC by regulating the expression of CXCR4, and was also necessary for the M1 macrophage polarization. Our results reveal understanding of the function and mechanisms of the Notch signaling pathway in the development of immune system. |
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