Rbpj expression in regulatory T cells is critical for restraining TH2 responses.

The transcriptional regulator Rbpj is involved in T-helper (TH) subset polarization, but its function in Treg cells remains unclear. Here we show that Treg-specific Rbpj deletion leads to splenomegaly and lymphadenopathy despite increased numbers of Treg cells with a polyclonal TCR repertoire. A specific defect of Rbpj-deficient Treg cells in controlling TH2 polarization and B cell responses is observed, leading to the spontaneous formation of germinal centers and a TH2-associated immunoglobulin class switch. The observed phenotype is environment-dependent and can be induced by infection with parasitic nematodes. Rbpj-deficient Treg cells adopt open chromatin landscapes and gene expression profiles reminiscent of tissue-derived TH2-polarized Treg cells, with a prevailing signature of the transcription factor Gata-3. Taken together, our study suggests that Treg cells require Rbpj to specifically restrain TH2 responses, including their own excessive TH2-like differentiation potential.

Molecular cloning and functional characterization of a homolog of the transcriptional regulator CSL in Litopenaeus vannamei.

The Notch signaling pathway transcriptional regulator, CSL (also called as CBF1, Suppressor of Hairless or Lag-1 in different species, generally designated as CSL1), is not only associated with cell proliferation and differentiation but also involved in tumorigenesis, inflammation and immune regulation in vertebrates. We recently showed that Notch signaling was involved in the immune response of Litopenaeus vannamei shrimp. However, as an important transcriptional regulator of this pathway, whether or not shrimp CSL was also involved in immune response had not been explored. Here, we cloned and characterized the CSL gene in L. vannamei (LvCSL), which has a 2271 bp open reading frame (ORF) encoding a putative protein of 756 amino acids, and contains two conserved Lag1-DNA bind as well as beta trefoil domains (BTD). LvCSL clustered with invertebrates in the phylogenetic tree and closely related to the RBP Jk X1 of Parasteatoda tepidariorum. The transcript level of LvCSL analyzed by quantitative polymerase chain reaction (qPCR) showed that LvCSL was widely expressed in all tissues tested, with induced levels observed in the hepatopancreas and hemocytes following immune challenge with Vibrio parahaemolyticus, Streptoccocus iniae, lipopolysaccharide (LPS), and white spot syndrome virus (WSSV), therefore, suggesting LvCSL involvement in shrimp immune response to pathogens. Besides, LvCSL knockdown decreased the expression of proliferation-related genes (LvHey2 and LvAstakine), and attenuated the expression of immune-related genes L. vannamei hypoxia inducible factor alpha (LvHIF-α), LvLectin and L. vannamei small subunit hemocyanin (LvHMCS) in shrimp hemocytes, as well as significantly decreased total hemocyte count. Moreover, high cumulative mortality was observed in LvCSL depleted shrimp challenged with V. parahaemoliticus. In conclusion, our present data strongly suggest that LvCSL is an important factor in shrimp, vital for shrimp survival and contributing to immune resistance to pathogens.

IL-17 induced NOTCH1 activation in oligodendrocyte progenitor cells enhances proliferation and inflammatory gene expression.

NOTCH1 signalling contributes to defective remyelination by impairing differentiation of oligodendrocyte progenitor cells (OPCs). Here we report that IL-17 stimulation induces NOTCH1 activation in OPCs, contributing to Th17-mediated demyelinating disease. Mechanistically, IL-17R interacts with NOTCH1 via the extracellular domain, which facilitates the cleavage of NOTHC1 intracellular domain (NICD1). IL-17-induced NOTCH1 activation results in the interaction of IL-17R adaptor Act1 with NICD1, followed by the translocation of the Act1-NICD1 complex into the nucleus. Act1-NICD1 are recruited to the promoters of several NOTCH1 target genes (including STEAP4, a metalloreductase important for inflammation and cell proliferation) that are specifically induced in the spinal cord by Th17 cells. A decoy peptide disrupting the IL-17RA-NOTCH1 interaction inhibits IL-17-induced NOTCH1 activation and attenuates Th17-mediated experimental autoimmune encephalitis (EAE). Taken together, these findings demonstrate critical crosstalk between the IL-17 and NOTCH1 pathway, regulating Th17-induced inflammatory and proliferative genes to promote demyelinating disease.

Transcription factor RBP-J-mediated signalling regulates basophil immunoregulatory function in mouse asthma model.

Basophils (BA) play an important role in the promotion of aberrant T helper type 2 (Th2) immune responses in asthma. It is not only the effective cell, but also modulates the initiation of Th2 immune responses. We earlier demonstrated that Notch signalling regulates the biological function of BAin vitro. However, whether this pathway plays the same role in vivo is not clear. The purpose of the present study was to investigate the effect of Notch signalling on BA function in the regulation of allergic airway inflammation in a murine model of asthma. Bone marrow BA were prepared by bone marrow cell culture in the presence of recombinant interleukin-3 (rIL-3; 300 pg/ml) for 7 days, followed by isolation of the CD49b+ microbeads. The recombination signal binding protein J (RBP-J-/- ) BA were co-cultured with T cells, and the supernatant and the T-cell subtypes were examined. The results indicated disruption of the capacity of BA for antigen presentation alongside an up-regulation of the immunoregulatory function. This was possibly due to the low expression of OX40L in the RBP-J-/- BA. Basophils were adoptively transferred to ovalbumin-sensitized recipient mice, to establish an asthma model. Lung pathology, cytokine profiles of brobchoalveolar fluid, airway hyperactivity and the absolute number of Th1/Th2 cells in lungs were determined. Overall, our results indicate that the RBP-J-mediated Notch signalling is critical for BA-dependent immunoregulation. Deficiency of RBP-J influences the immunoregulatory functions of BA, which include activation of T cells and their differentiation into T helper cell subtypes. The Notch signalling pathway is a potential therapeutic target for BA-based immunotherapy against asthma.

TNF and Bone Remodeling.

PURPOSE OF REVIEW: The mechanisms involved in the TNF-mediated deregulated bone remodeling are little appreciated. This review will discuss and summarize the impact of TNF, Notch, and RBP-J signaling on bone remodeling. RECENT FINDINGS: The integrity of the adult skeleton undergoes constant and dynamic remodeling throughout life to maintain a proper bone homeostasis, which is achieved by the essential tight control of coupling between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. The studies in this field include not only the differentiation and function of osteoblasts and osteoclasts, but also the mechanisms that simultaneously control both cell types during bone remodeling. Chronic inflammation is one of the most evident and common pathological settings that often leads to deregulated bone remodeling. The resounding success of TNF blockade therapy has demonstrated a key role for TNF in inflammation and the pathogenesis of inflammatory bone resorption associated with diseases such as rheumatoid arthritis and periodontitis. Recent studies have highlighted the function of Notch and RBP-J signaling in both physiological and TNF-mediated inflammatory bone remodeling.

The blockage of Notch signalling promoted the generation of polymorphonuclear myeloid-derived suppressor cells with lower immunosuppression.

Myeloid-derived suppressor cells (MDSCs) mostly consisting of polymorphonuclear (PMN)-MDSCs and mononuclear MDSCs have been considered to play critical roles in immunosuppression, angiogenesis, invasion and metastases of various tumours. However, it is still unclear the regulated mechanisms underlying the generation and immunosuppression of two major MDSC subsets. Here, we report Notch signalling was inhibited significantly in tumour-bearing mouse MDSCs, in which PMN-MDSCs were the major population. MDSCs without recombination signal binding protein-Jк (RBP-J), the critical transcription factor mediating signalling from all four mammalian Notch receptors, reduced their ability of inhibiting the proliferation and activation of allogenic T cells. RBP-J-deficient MDSCs could not down-regulate the expression of co-stimulation molecules on dendritic cells (DCs). The antigen presentation capacity of DCs co-cultured with RBP-J-deficient MDSCs was not impaired in contrast to controls. Moreover, we show the blockage of Notch signalling could improve the generation of PMN-MDSCs but inhibit the production of mononuclear MDSCs both in vitro and in vivo. Stat3 pathway was suppressed in MDSCs blocked Notch signalling and Stat3 activation by IL-6 could reverse the phenotype and immunosuppression of Notch signalling-deficient MDSCs. Therefore, targeting Notch signalling may be an effective therapeutic strategy in tumour therapy.

Satb1 Overexpression Drives Tumor-Promoting Activities in Cancer-Associated Dendritic Cells.

Special AT-rich sequence-binding protein 1 (Satb1) governs genome-wide transcriptional programs. Using a conditional knockout mouse, we find that Satb1 is required for normal differentiation of conventional dendritic cells (DCs). Furthermore, Satb1 governs the differentiation of inflammatory DCs by regulating major histocompatibility complex class II (MHC II) expression through Notch1 signaling. Mechanistically, Satb1 binds to the Notch1 promoter, activating Notch expression and driving RBPJ occupancy of the H2-Ab1 promoter, which activates MHC II transcription. However, tumor-driven, unremitting expression of Satb1 in activated Zbtb46(+) inflammatory DCs that infiltrate ovarian tumors results in an immunosuppressive phenotype characterized by increased secretion of tumor-promoting Galectin-1 and IL-6. In vivo silencing of Satb1 in tumor-associated DCs reverses their tumorigenic activity and boosts protective immunity. Therefore, dynamic fluctuations in Satb1 expression govern the generation and immunostimulatory activity of steady-state and inflammatory DCs, but continuous Satb1 overexpression in differentiated DCs converts them into tolerogenic/pro-inflammatory cells that contribute to malignant progression.

RBP-J is required for M2 macrophage polarization in response to chitin and mediates expression of a subset of M2 genes.

Development of alternatively activated (M2) macrophage phenotypes is a complex process that is coordinately regulated by a plethora of pathways and factors. Here, we report that RBP-J, a DNA-binding protein that integrates signals from multiple pathways including the Notch pathway, is critically involved in polarization of M2 macrophages. Mice deficient in RBP-J in the myeloid compartment exhibited impaired M2 phenotypes in vivo in a chitin-induced model of M2 polarization. Consistent with the in vivo findings, M2 polarization was partially compromised in vitro in Rbpj-deficient macrophages as demonstrated by reduced expression of a subset of M2 effector molecules including arginase 1. Functionally, myeloid Rbpj deficiency impaired M2 effector functions including recruitment of eosinophils and suppression of T cell proliferation. Collectively, we have identified RBP-J as an essential regulator of differentiation and function of alternatively activated macrophages.

Rheumatoid arthritis-associated RBPJ polymorphism alters memory CD4+ T cells.

Notch signaling has recently emerged as an important regulator of immune responses in autoimmune diseases. The recombination signal-binding protein for immunoglobulin kappa J region (RBPJ) is a transcriptional repressor, but converts into a transcriptional activator upon activation of the canonical Notch pathway. Genome-wide association studies of rheumatoid arthritis (RA) identified a susceptibility locus, rs874040(CC), which implicated the RBPJ gene. Here, chromatin state mapping generated using the chromHMM algorithm reveals strong enhancer regions containing DNase I hypersensitive sites overlapping the rs874040 linkage disequilibrium block in human memory, but not in naïve CD4(+) T cells. The rs874040 overlapping this chromatin state was associated with increased RBPJ expression in stimulated memory CD4(+) T cells from healthy subjects homozygous for the risk allele (CC) compared with memory CD4(+) T cells bearing the protective allele (GG). Transcriptomic analysis of rs874040(CC) memory T cells showed a repression of canonical Notch target genes IL (interleukin)-9, IL-17 and interferon (IFN)γ in the basal state. Interestingly, activation of the Notch pathway using soluble Notch ligand, Jagged2-Fc, induced IL-9 and IL-17A while delta-like 4Fc, another Notch ligand, induced higher IFNγ expression in the rs874040(CC) memory CD4(+) T cells compared with their rs874040(GG) counterparts. In RA, RBPJ expression is elevated in memory T cells from RA patients compared with control subjects, and this was associated with induced inflammatory cytokines IL-9, IL-17A and IFNγ in response to Notch ligation in vitro. These findings demonstrate that the rs874040(CC) allele skews memory T cells toward a pro-inflammatory phenotype involving Notch signaling, thus increasing the susceptibility to develop RA.

A genome-wide analysis identifies a notch-RBP-Jκ-IL-7Rα axis that controls IL-17-producing γδ T cell homeostasis in mice.

Notch signaling is an important regulator for the development and function of both αß and γδ T cells, whereas roles of Notch signaling in T cell maintenance remain unclear. We reported previously that the Notch-Hes1 pathway was involved in the intrathymic development of naturally occurring IL-17-producing (IL-17(+)) γδ T cells. To gain insight into additional roles for the Notch axis in the homeostasis of γδ T cells, we performed a genome-wide analysis of Notch target genes and identified the novel promoter site of IL-7Rα driven by the Notch-RBP-Jκ pathway. Constitutive Notch signaling had the potential to induce IL-7Rα expression on γδ T cells in vivo, as well as in vitro, whereas conditional deletion of RBP-Jκ abrogated IL-7Rα expression, but not Hes1 expression, by γδ T cells and selectively reduced the pool size of IL-7Rα(high) IL-17(+) γδ T cells in the periphery. In the absence of IL-7Rα-mediated signaling, IL-17(+) γδ T cells were barely maintained in adult mice. Addition of exogenous IL-7 in vitro selectively expanded IL-17(+) γδ T cells. Thus, our results revealed a novel role for the Notch-RBP-Jκ-IL-7Rα axis that is independent of Hes1 for homeostasis of IL-17(+) γδ T cells.

Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages.

Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for the characterisation of macrophage polarisation in situ. Furthermore, CD163 cannot be considered a reliable M2 marker when used on its own.

Notch signaling regulates expression of Mcl-1 and apoptosis in PPD-treated macrophages.

Macrophages are cellular targets for infection by bacteria and viruses. The fate of infected macrophages plays a key role in determining the outcome of the host immune response. Apoptotic cell death of macrophages is considered to be a protective host defense that eliminates pathogens and infected cells. In this study, we investigated the involvement of Notch signaling in regulating apoptosis in macrophages treated with tuberculin purified protein derivative (PPD). Murine bone marrow-derived macrophages (BMMs) treated with PPD or infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) induced upregulation of Notch1. This upregulation correlated well with the upregulation of the anti-apoptotic gene mcl-1 both at the transcriptional and translational levels. Decreased levels of Notch1 and Mcl-1 were observed in BMM treated with PPD when a gamma secretase inhibitor (GSI), which inhibits the processing of Notch receptors, was used. Moreover, silencing Notch1 in the macrophage-like cell line RAW264.7 decreased Mcl-1 protein expression, suggesting that Notch1 is critical for Mcl-1 expression in macrophages. A significant increase in apoptotic cells was observed upon treatment of BMM with PPD in the presence of GSI compared to the vehicle-control treated cells. Finally, analysis of the mcl-1 promoter in humans and mice revealed a conserved potential CSL/RBP-Jκ binding site. The association of Notch1 with the mcl-1 promoter was confirmed by chromatin immunoprecipitation. Taken together, these results indicate that Notch1 inhibits apoptosis of macrophages stimulated with PPD by directly controlling the mcl-1 promoter.

Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization.

Emerging concepts suggest that the functional phenotype of macrophages is regulated by transcription factors that define alternative activation states. We found that RBP-J, the main nuclear transducer of signaling via Notch receptors, augmented Toll-like receptor 4 (TLR4)-induced expression of key mediators of classically activated M1 macrophages and thus of innate immune responses to Listeria monocytogenes. Notch-RBP-J signaling controlled expression of the transcription factor IRF8 that induced downstream M1 macrophage-associated genes. RBP-J promoted the synthesis of IRF8 protein by selectively augmenting kinase IRAK2-dependent signaling via TLR4 to the kinase MNK1 and downstream translation-initiation control through eIF4E. Our results define a signaling network in which signaling via Notch-RBP-J and TLRs is integrated at the level of synthesis of IRF8 protein and identify a mechanism by which heterologous signaling pathways can regulate the TLR-induced inflammatory polarization of macrophages.

Serum autoantibody signature of ductal carcinoma in situ progression to invasive breast cancer.

PURPOSE: The identification of markers associated with progression to invasive breast cancer (IBC) is a major factor that can guide physicians in the initial therapeutic decision and the management of ductal carcinoma in situ (DCIS). EXPERIMENTAL DESIGN: We examined autoantibody targets in 20 DCIS and 20 IBC patients using protein microarrays and identified humoral responses that can be used to distinguish the two groups. The five most differentially targeted antigens were selected to generate an autoantibody signature for the in situ to invasive breast cancer transition. This signature was next tested on 120 independent samples (61 DCIS and 59 IBC) using specific ELISA assays. The prognosis value of the autoantibody signature was finally evaluated in a cohort of DCIS patients followed for 5 years. RESULTS: A set of five autoantibody targets (RBP-Jκ, HMGN1, PSRC1, CIRBP, and ECHDC1) with the highest differential signal intensity found in the protein microarrays experiment was used to establish an autoantibody signature of the DCIS to IBC transition. Using ELISA, this signature significantly discriminated DCIS from IBC [area under the ROC curve (AUC) = 0.794, 95% confidence interval (CI): 0.674-0.877]. Interestingly, our panel could highly distinguish low-grade DCIS from high-grade DCIS exhibiting an AUC of 0.749 (95% CI: 0.581-0.866). Finally, using a Kaplan-Meier analysis, the autoantibody signature could significantly divide the DCIS patients into a poor prognosis group and a good prognosis group (P = 0.01). CONCLUSION: These results indicate the potential of autoantibody detection as a new prognostic test with possible clinical implications for the management of DCIS.

Accelerated acute allograft rejection accompanied by enhanced T-cell proliferation and attenuated Treg function in RBP-J deficient mice.

Acute allograft rejection (AAR) involves both the innate and the adaptive immune systems. As a critical pathway in peripheral T-cell differentiation and function, Notch signaling is potentially involved in the modulation of AAR, but its role in alloimmune responses has not been fully addressed. By using fully MHC-mismatched allograft transplantation model and T-cell specific RBP-J deficient mice, we examined the role of Notch/RBP-J pathway in alloimmune responses in vivo. AAR was significantly accelerated in RBP-J deficient mice compared with the wild-type controls, as demonstrated by the marked reduction in graft survival. The reduction in graft survival was associated with augmented alloantigen specific T-cell proliferation and increased number of Th1, Th2, and Th17 cells in the RBP-J deficient recipient mice. Furthermore, although the frequency of CD4(+)CD25(+)Foxp3(+) Tregs was intact in RBP-J knockout recipients, their ability to suppress Teff responses in vitro was significantly dampened. These findings suggest that Notch/RBP-J pathway may attenuate AAR by suppressing in vivo expansion of alloreactive T-cell proliferation and facilitating CD4(+)CD25(+) Treg suppression ability, indicating that Notch pathway could be exploited to limit T-cell-mediated AAR.

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

BACKGROUND: Dendritic 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. RESULTS: When 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. CONCLUSION: The 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.

Integrated regulation of Toll-like receptor responses by Notch and interferon-gamma pathways.

Toll-like receptor (TLR) responses are regulated to avoid toxicity and achieve coordinated responses appropriate for the cell environment. We found that Notch and TLR pathways cooperated to activate canonical Notch target genes, including transcriptional repressors Hes1 and Hey1, and to increase production of canonical TLR-induced cytokines TNF, IL-6, and IL-12. Cooperation by these pathways to increase target gene expression was mediated by the Notch-pathway component and transcription factor RBP-J, which also contributed to lethality after endotoxin injection. TLR- and Notch-induced Hes1 and Hey1 attenuated IL-6 and IL-12 production. This Hes1- and Hey1-mediated feedback inhibitory loop was abrogated by interferon-gamma (IFN-gamma), which blocked TLR-induced activation of canonical Notch target genes by inhibiting Notch2 signaling and downstream transcription. These findings identify new immune functions for RBP-J, Hes, and Hey proteins and provide insights into mechanisms by which Notch, TLR, and IFN-gamma signals are integrated to modulate specific effector functions in macrophages.