Yao-Chun Wang

Notch Signaling Determines the M1 versus M2 Polarization of Macrophages in Antitumor Immune Responses

Macrophages are important tumor-infiltrating cells and play pivotal roles in tumor growth and metastasis. Macrophages participate in immune responses to tumors in a polarized manner: classic M1 macrophages produce interleukin (IL) 12 to promote tumoricidal responses, whereas M2 macrophages produce IL10 and help tumor progression. The mechanisms governing macrophage polarization are unclear. Here, we show that the M2-like tumor-associated macrophages (TAM) have a lower level of Notch pathway activation in mouse tumor models. Forced activation of Notch signaling increased M1 macrophages which produce IL12, no matter whether M1 or M2 inducers were applied. When Notch signaling was blocked, the M1 inducers induced M2 response in the expense of M1. Macrophages deficient in canonical Notch signaling showed TAM phenotypes. Forced activation of Notch signaling in macrophages enhanced their antitumor capacity. We further show that RBP-J-mediated Notch signaling regulates the M1 versus M2 polarization through SOCS3. Therefore, Notch signaling plays critical roles in the determination of M1 versus M2 polarization of macrophages, and compromised Notch pathway activation will lead to the M2-like TAMs. These results provide new insights into the molecular mechanisms of macrophage polarization and shed light on new therapies for cancers through the modulation of macrophage polarization through the Notch signaling.

RBP-J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice

In adults, angiogenic abnormalities are involved in not only tumor growth but several human inherited diseases as well. It is unclear, however, concerning how the normal vascular structure is maintained and how angiogenesis is initiated in normal adults. Using the Cre‐LoxP‐mediated conditional gene deletion, we show in the present study that in adult mice disruption of the transcription factor recombination signal‐binding protein Jκ (RBP‐J) in endothelial cells strikingly induced spontaneous angiogenesis in multiple tissues, including retina and cornea, as well as in internal organs, such as liver and lung. In a choroidal neovascularization model, which mimics the angiogenic process in tumor growth and age‐related macular degeneration, RBP‐J deficiency induced a more intensive angiogenic response to injury. This could be transmitted by bone marrow, indicating that RBP‐J could modulate bone marrow‐derived endothelial progenitor cells in adult angiogenesis. In addition, in the absence of RBP‐J, proliferation of endothelial cells increased significantly, leading to accumulative vessel outgrowth. These findings suggest that in adults RBP‐J‐mediated Notch signaling may play an essential role in the maintenance of vascular homeostasis by repressing endothelial cell proliferation.—Dou, G.‐R., Wang, Y.‐C., Hu, X.‐B., Hou, L.‐H., Wang, C.‐M., Xu, J.‐F., Wang, Y.‐S., Liang, Y.‐M., Yao, L.‐B., Yang, A.‐G., Han, H. RBP‐J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice. FASEB J. 22, 1606–1617 (2008)

Lipopolysaccharide-induced Maturation of Bone Marrow-derived Dendritic Cells Is Regulated by Notch Signaling through the Up-regulation of CXCR4

Dendritic cells (DCs) are professional antigen presenting cells to initiate immune response against pathogens, but mechanisms controlling the maturation of DCs are unclear. Here we report that, in the absence of recombination signal binding protein-Jκ (RBP-J, the transcription factor mediating Notch signaling), lipopolysaccharide-stimulated monocyte-derived DCs are arrested at a developmental stage with few dendrites, low major histocompatibility complex II (MHC II) expression, and reduced motility and antigen presentation ability. RBP-J null DCs had lower expression of CXCR4. Transduction with a CXCR4-expressing lentivirus rescued developmental arrest of RBP-J-deficient DCs. Activation of Notch signaling in DCs up-regulated CXCR4 expression and increased the outgrowth of dendrites and the expression of MHC II. These effects were abrogated by a CXCR4 inhibitor. Therefore, Notch signaling is essential for DCs to transit from a dendritelowMHC IIlow immature state into a dendritehighMHC IIhigh mature state, during the lipopolysaccharide-induced DC maturation, most likely through the up-regulation of CXCR4.

Disruption of the transcription factor recombination signal‐binding protein‐Jκ (RBP‐J) leads to veno‐occlusive disease and interfered liver regeneration in mice

Liver sinusoid (LS) endothelial cells (LSECs) support hepatocytes in resting livers and proliferate during liver regeneration to revascularize regenerated liver parenchyma. We report that recombination signal‐binding protein‐Jκ (RBP‐J), the critical transcription factor mediating Notch signaling, regulates both resting and regenerating LSECs. Conditional deletion of RBP‐J resulted in LSEC proliferation and a veno‐occlusive disease–like phenotype in the liver, as manifested by liver congestion, deposition of fibrin‐like materials in LSs, edema in the space of Disse, and increased apoptosis of hepatocytes. Regeneration of liver was remarkably impaired, with reduced LSEC proliferation and destroyed sinusoidal structure. LSEC degeneration was obvious in the regenerating liver of RBP‐J–deficient mice, with some LSECs losing cytoplasm, and organelles protruding into the remnant plasma‐membrane of LSs to hamper the microcirculation and intensify veno‐occlusive disease during liver regeneration. Hepatocytes were also degenerative, as shown by dilated endoplasmic reticulum, decreased proliferation, and increased apoptosis during liver regeneration. Molecular analyses revealed that the dynamic expression of several related molecules—such as vascular endothelial growth factor, vascular endothelial growth factor receptors 1 and 2, interleukin‐6, and hepatocyte growth factor—was disturbed. Conclusion: Notch/RBP‐J signaling may play dual roles in LSECs: in resting liver it represses proliferation, and in regenerating liver it supports proliferation and functional differentiation. (HEPATOLOGY 2009;49:268‐277.)

Notch-RBP-J Signaling Regulates the Mobilization and Function of Endothelial Progenitor Cells by Dynamic Modulation of CXCR4 Expression in Mice

Bone marrow (BM)-derived endothelial progenitor cells (EPC) have therapeutic potentials in promoting tissue regeneration, but how these cells are modulated in vivo has been elusive. Here, we report that RBP-J, the critical transcription factor mediating Notch signaling, modulates EPC through CXCR4. In a mouse partial hepatectomy (PHx) model, RBP-J deficient EPC showed attenuated capacities of homing and facilitating liver regeneration. In resting mice, the conditional deletion of RBP-J led to a decrease of BM EPC, with a concomitant increase of EPC in the peripheral blood. This was accompanied by a down-regulation of CXCR4 on EPC in BM, although CXCR4 expression on EPC in the circulation was up-regulated in the absence of RBP-J. PHx in RBP-J deficient mice induced stronger EPC mobilization. In vitro, RBP-J deficient EPC showed lowered capacities of adhering, migrating, and forming vessel-like structures in three-dimensional cultures. Over-expression of CXCR4 could at least rescue the defects in vessel formation by the RBP-J deficient EPC. These data suggested that the RBP-J-mediated Notch signaling regulated EPC mobilization and function, at least partially through dynamic modulation of CXCR4 expression. Our findings not only provide new insights into the regulation of EPC, but also have implications for clinical therapies using EPC in diseases.

The transcription factor RBP-J-mediated signaling is essential for dendritic cells to evoke efficient anti-tumor immune responses in mice

BackgroundDendritic cells (DCs) are professional antigen presenting cells that initiate specific immune responses against tumor cells. Transcription factor RBP-J-mediated Notch signaling regulates DC genesis, but whether this pathway regulates DC function in anti-tumor immunity remains unclear. In the present work we attempted to identify the role of Notch signaling in DC-mediated anti-tumor immune response.ResultsWhen DCs were co-inoculated together with tumor cells, while the control DCs repressed tumor growth, the RBP-J deficient DCs had lost tumor repression activity. This was most likely due to that DCs with the conditionally ablated RBP-J were unable to evoke anti-tumor immune responses in the solid tumors. Indeed, tumors containing the RBP-J deficient DCs had fewer infiltrating T-cells, B-cells and NK-cells. Similarly, the draining lymph nodes of the tumors with RBP-J-/- DCs were smaller in size, and contained fewer cells of the T, B and NK lineages, as compared with the controls. At the molecular level, the RBP-J deficient DCs expressed lower MHC II, CD80, CD86, and CCR7, resulting in inefficient DC migration and T-cell activation in vitro and in vivo. T-cells stimulated by the RBP-J deficient DCs did not possess efficient cytotoxicity against tumor cells, in contrast to the control DCs.ConclusionThe RBP-J-mediated Notch signaling is essential for DC-dependent anti-tumor immune responses. The deficiency of RBP-J impairs the DC-based anti-tumor immunity through affecting series of processes including maturation, migration, antigen presentation and T-cell activation. The Notch signaling pathway might be a target for the establishment of the DC-based anti-tumor immunotherapies.

Host defence against C. albicans infections in IgH transgenic mice with VH derived from a natural anti-keratin antibody

Fungal infections have been increasing and life‐threatening in recent years, but host immune responses, especially the humoral immunity, to fungi have not been fully understood. In the present study, we report that natural antibodies from unimmunized mice bind to Candida albicans. We established a monoclonal natural antibody, 3B4, which recognized a surface antigen located at germ tubes of C. albicans. The 3B4 antibody protected mice from C. albicans‐induced death in passive immunization, by mechanisms involving suppressing germ tube formation and modulating phagocytosis. Interestingly, 3B4 also bound to a self‐antigen keratin. To further study the generation and anti‐C. albicans activities of natural antibodies in vivo, we constructed a μ chain transgenic mouse (TgVH3B4) using the VH gene from 3B4. TgVH3B4 had elevated serum anti‐keratin/C. albicans IgM, and were resistant to C. albicans infections. Analyses of B cell development showed that in TgVH3B4, B cells secreting the anti‐keratin/C. albicans antibodies were enriched in the B1 B cell compartment. Our findings reveal an important role of keratin‐reactive natural antibodies in anti‐C. albicans immune responses, and suggest that keratin may function in selecting B cells into the B1 B cell compartment, where natural antibodies are made to fight fungal infections.

Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-α and NO production in macrophages

Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP–MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.

Notch-RBP-J-Independent Marginal Zone B Cell Development in IgH Transgenic Mice with VH Derived from a Natural Polyreactive Antibody

Both the B cell antigen receptor (BCR) signaling and Notch signaling pathway play important roles in marginal zone (MZ) B cell development; however, if and how these two signaling pathways engage in crosstalk with each other remain unclear. In the present study, IgH transgenic mice (TgVH3B4) were crossed with mice with Notch downstream transcription factor RBP-J floxed alleles (RBP-Jf/f) and Mx-Cre transgene. Subsequently, MZ B cell development was analyzed in 3B4/Cre/RBP-Jf/f mice that expressed the transgenic 3B4 IgH and exhibited a deficiency in Notch signaling in B cells upon poly (I:C) injection. We observed that MZ B cell numbers were severely reduced, but still detectable in 3B4/Cre/RBP-Jf/f mice, in contrast to increased numbers of MZ B cells in TgVH3B4 mice and almost no MZ B cells in Cre/RBP-Jf/f mice. The majority of the MZ B cells in the 3B4/Cre/RBP-Jf/f mice had the same antigen specificity with that of 3B4 antibody, indicating that a particular BCR specificity might direct MZ B cell development in the absence of Notch signaling. The number of MZ B precursor (MZP) cells was reduced sharply in 3B4/Cre/RBP-Jf/f mice, and the number of transitional stage 1 and transitional stage 2 cells did not change that much, indicating that the interaction between BCR and Notch signaling likely occurred during the T2-MZP stage. Based on the transgenic mouse model, our data indicate that MZ B cells with certain BCR specificity can develop in a Notch-RBP-J independent manner.