Notch Signaling In Macrophages Research Articles

RND3 Potentiates Proinflammatory Activation through NOTCH Signaling in Activated Macrophages.

Macrophage activation is a complex process with multiple control elements that ensures an adequate response to the aggressor pathogens and, on the other hand, avoids an excess of inflammatory activity that could cause tissue damage. In this study, we have identified RND3, a small GTP-binding protein, as a new element in the complex signaling process that leads to macrophage activation. We show that RND3 expression is transiently induced in macrophages activated through Toll receptors and potentiated by IFN-γ. We also demonstrate that RND3 increases NOTCH signaling in macrophages by favoring NOTCH1 expression and its nuclear activity; however, Rnd3 expression seems to be inhibited by NOTCH signaling, setting up a negative regulatory feedback loop. Moreover, increased RND3 protein levels seem to potentiate NFκB and STAT1 transcriptional activity resulting in increased expression of proinflammatory genes, such as Tnf-α, Irf-1, or Cxcl-10. Altogether, our results indicate that RND3 seems to be a new regulatory element which could control the activation of macrophages, able to fine tune the inflammatory response through NOTCH.

NOTCH signaling inhibition after DAPT treatment exacerbates alveolar echinococcosis hepatic fibrosis by blocking M1 and enhancing M2 polarization.

Alveolar echinococcosis (AE) is a lethal helminthic liver disease caused by persistent infection with Echinococcus multilocularis (E. multilocularis). Although more and more attention has been paid to the macrophages in E. multilocularis infection, the mechanism of macrophage polarization, a critical player in liver immunity, is seldom studied. NOTCH signaling is involved in cell survival and macrophage-mediated inflammation, but the role of NOTCH signaling in AE has been equally elusive. In this study, liver tissue samples from AE patients were collected and an E. multilocularis infected mouse model with or without blocking NOTCH signaling was established to analyze the NOTCH signaling, fibrotic and inflammatory response of the liver after E. multilocularis infection. Changes in polarization and origin of hepatic macrophages were analyzed by flow cytometry. In vitro qRT-PCR and Western blot assays were performed to analyze key receptors and ligands in NOTCH signaling. Our data demonstrated that hepatic fibrosis develops after AE, and the overall blockade of NOTCH signaling caused by DAPT treatment exacerbates the levels of hepatic fibrosis and alters the polarization and origin of hepatic macrophages. Blocking NOTCH signaling in macrophages after E. multilocularis infection downregulates M1 and upregulates M2 expression. The downregulation of NTCH3 and DLL-3 in the NOTCH signaling pathway is significant. Therefore, NOTCH3/DLL3 may be the key pathway in NOTCH signaling regulating macrophage polarization affecting fibrosis caused by AE.

Myeloid-specific blockade of Notch signaling ameliorates nonalcoholic fatty liver disease in mice.

Rationale: Macrophages play a central role in the development and progression of nonalcoholic fatty liver disease (NAFLD). Studies have shown that Notch signaling mediated by transcription factor recombination signal binding protein for immunoglobulin kappa J region (RBP-J), is implicated in macrophage activation and plasticity. Naturally, we asked whether Notch signaling in macrophages plays a role in NAFLD, whether regulating Notch signaling in macrophages could serve as a therapeutic strategy to treat NAFLD. Methods: Immunofluorescence staining was used to detect the changes of macrophage Notch signaling in the livers of human patients with NAFLD and choline deficient amino acid-defined (CDAA) diet-fed mice. Lyz2-Cre RBP-Jflox or wild-type C57BL/6 male mice were fed with CDAA or high fat diet (HFD) to induce experimental steatohepatitis or steatosis, respectively. Liver histology examinations were performed using hematoxylin-eosin (H&E), Oil Red O staining, Sirius red staining and immunohistochemistry staining for F4/80, Col1α1 and αSMA. The expression of inflammatory factors, fibrosis or lipid metabolism associated genes were evaluated by quantitative reverse transcription (qRT)-PCR, Western blot or enzyme-linked immunosorbent assay (ELISA). The mRNA expression of liver samples was profiled by using RNA-seq. A hairpin-type decoy oligodeoxynucleotides (ODNs) for transcription factor RBP-J was loaded into bEnd.3-derived exosomes by electroporating. Mice with experimental NAFLD were treated with exosomes loading RBP-J decoy ODNs via tail vein injection. In vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. Results: The results showed that Notch signaling was activated in hepatic macrophages in human with NAFLD or in CDAA-fed mice. Myeloid-specific RBP-J deficiency decreased the expression of inflammatory factors interleukin-1 beta (IL1β) and tumor necrosis factor alpha (TNFα), attenuated experimental steatohepatitis in mice. Furthermore, we found that Notch blockade attenuated lipid accumulation in hepatocytes by inhibiting the expression of IL1β and TNFα in macrophages in vitro. Meanwhile, we observed that tail vein-injected exosomes were mainly taken up by hepatic macrophages in mice with steatohepatitis. RBP-J decoy ODNs delivered by exosomes could efficiently inhibit Notch signaling in hepatic macrophages in vivo and ameliorate steatohepatitis or steatosis in CDAA or HFD mice, respectively. Conclusions: Combined, macrophage RBP-J promotes the progression of NAFLD at least partially through regulating the expression of pro-inflammatory cytokines IL1β and TNFα. Infusion of exosomes loaded with RBP-J decoy ODNs might be a promising therapy to treat NAFLD.

Excessive Activation of Notch Signaling in Macrophages Promote Kidney Inflammation, Fibrosis, and Necroptosis.

Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease (ESRD). Existing treatments cannot control the progression of diabetic nephropathy very well. In diabetic nephropathy, Many monocytes and macrophages infiltrate kidney tissue. However, the role of these cells in the pathogenesis of diabetic nephropathy has not been fully elucidated. In this study, we analyzed patient kidney biopsy specimens, diabetic nephropathy model animals. Meanwhile, we cocultured cells and found that in diabetic nephropathy, damaged intrinsic renal cells (glomerular mesangial cells and renal tubular epithelial cells) recruited monocytes/macrophages to the area of tissue damage to defend against and clear cell damage. This process often involved the activation of different types of macrophages. Interestingly, the infiltrating macrophages were mainly M1 (CD68+iNOS+) macrophages. In diabetic nephropathy, crosstalk between the Notch pathway and NF-κB signaling in macrophages contributed to the polarization of macrophages. Hyperpolarized macrophages secreted large amounts of inflammatory cytokines and exacerbated the inflammatory response, extracellular matrix secretion, fibrosis, and necroptosis of intrinsic kidney cells. Additionally, macrophage depletion therapy with clodronate liposomes and inhibition of the Notch pathway in macrophages alleviated the pathological changes in kidney cells. This study provides new information regarding diabetic nephropathy-related renal inflammation, the causes of macrophage polarization, and therapeutic targets for diabetic nephropathy.

Sepsis downregulates aortic Notch signaling to produce vascular hyporeactivity in mice

Inhibition of Notch signaling in macrophages is known to reduce inflammation, however, its role in regulating vascular hyporeactivity in sepsis is unknown. Thus we aimed to evaluate the effect of sepsis on vascular Notch signaling. Polymicrobial sepsis was induced by caecal ligation and puncture (CLP) in mice. mRNA expressions of Notch receptors (Notch1,3) and ligands (Jag1, Dll4), and downstream effector genes (Hey1, MLCK, MYPT1) were assessed by RT-qPCR. Protein level of activated Notch (NICD) was assessed by Western blot and immuno-histochemistry. Isometric tension in isolated aortic rings was measured by wire myography.CLP down-regulated aortic expression of Notch3, Jag1 and Dll4 as compared to control mice. Additionally, the protein level of NICD was found to be lesser in aortic tissue sections from CLP mice. Expression of Hey1 and MLCK were attenuated whereas MYPT1 expression was increased in septic mouse aorta. DAPT pretreatment did not improve CLP-induced vascular hyporeactivity to NA, CaCl2 and high K+ (80 mM), rather significantly attenuated the aortic response to these vasoconstrictors in control mice. Treatment with 1400 W reversed attenuated Notch3 (but not Jag1 and MLCK) expression in septic mouse aorta. In conclusion, sepsis significantly attenuated the Notch (especially Notch3) signaling in mouse aorta along with reduction in contractile gene expression and vasoconstriction response. Further, iNOS/NO pathway was involved in sepsis-induced down-regulation of Notch3 receptor. Thus systemic inhibition of Notch signaling during sepsis may have serious impact on sepsis-induced vascular hyporeactivity.

Exosome-mediated delivery of RBP-J decoy oligodeoxynucleotides ameliorates hepatic fibrosis in mice.

Rationale: Macrophages play multi-dimensional roles in hepatic fibrosis. Studies have implicated Notch signaling mediated by the transcription factor RBP-J in macrophage activation and plasticity. Additionally, we have previously shown that myeloid-specific disruption of RBP-J can ameliorate hepatic fibrosis in mice. Accordingly, we next asked whether blocking Notch signaling in macrophages could serve as a therapeutic strategy to treat hepatic fibrosis. In this study, we used a combination of transcription factor decoy oligodeoxynucleotides (ODNs) and exosomes to test this possibility.Methods: Hairpin-type decoy oligodeoxynucleotides (ODNs) were designed for the transcription factor RBP-J. The effects of RBP-J decoy ODNs on Notch signaling were evaluated by western blot, quantitative RT-PCR, luciferase reporter assays, and electrophoretic mobility shift assays. ODNs were loaded into HEK293T-derived exosomes by electroporation. A hepatic fibrosis mouse model was established by the intraperitoneal injection of carbon tetrachloride or bile duct ligation. Mice with hepatic fibrosis were administered exosomes loaded with RBP-J decoy ODNs via tail vein injection. The in vivo distribution of exosomes was analyzed by fluorescence labeling and imaging. Liver histology was examined using hematoxylin and eosin, Sirius red, and Masson staining, as well as immunohistochemical staining for Col1α1 and αSMA.Results: We found that RBP-J decoy ODNs could be efficiently loaded into exosomes and inhibit the activation of Notch signaling. Furthermore, exosomes administered via the tail vein were found to be primarily taken up by hepatic macrophages in mice with liver fibrosis. Importantly, RBP-J decoy ODNs delivered by exosomes could efficiently inhibit Notch signaling in macrophages and ameliorate hepatic fibrosis in mice.Conclusions: Combined, our data showed that the infusion of exosomes loaded with RBP-J decoy ODNs represents a promising therapeutic strategy for the treatment of hepatic fibrosis.

Notch Signaling Ligand Jagged1 Enhances Macrophage-Mediated Response to Helicobacter pylori.

Helicobacter pylori (H. pylori) is one of the gram-negative bacteria that mainly colonize the stomach mucosa and cause many gastrointestinal diseases, such as gastritis, peptic ulcer, and gastric cancer. Macrophages play a key role in eradicating H. pylori. Recent data have shown that Notch signaling could modulate the activation and bactericidal activities of macrophages. However, the role of Notch signaling in macrophages against H. pylori remains unclear. In the present study, in the co-culture model of macrophages with H. pylori, the inhibition of Notch signaling using γ-secretase decreased the expression of inducible nitric oxide synthase (iNOS) and its product, nitric oxide (NO), and downregulated the secretion of pro-inflammatory cytokine and attenuated phagocytosis and bactericidal activities of macrophages to H. pylori. Furthermore, we identified that Jagged1, one of Notch signaling ligands, was both upregulated in mRNA and protein level in activated macrophages induced by H. pylori. Clinical specimens showed that the number of Jagged1+ macrophages in the stomach mucosa from H. pylori-infected patients was significantly higher than that in healthy control. The overexpression of Jagged1 promoted bactericidal activities of macrophages against H. pylori and siRNA-Jagged1 presented the opposite effect. Besides, the addition of exogenous rJagged1 facilitated the pro-inflammatory mediators of macrophages against H. pylori, but the treatment of anti-Jagged1 neutralizing antibody attenuated it. Taken together, these results suggest that Jagged1 is a promoting molecule for macrophages against H. pylori, which will provide insight for exploring Jagged1 as a novel therapeutic target for the control of H. pylori infection.

NOTCH3 signaling is essential for NF-\u03baB activation in TLR-activated macrophages

Macrophage activation by Toll receptors is an essential event in the development of the response against pathogens. NOTCH signaling pathway is involved in the control of macrophage activation and the inflammatory processes. In this work, we have characterized NOTCH signaling in macrophages activated by Toll-like receptor (TLR) triggering and determined that DLL1 and DLL4 are the main ligands responsible for NOTCH signaling. We have identified ADAM10 as the main protease implicated in NOTCH processing and activation. We have also observed that furin, which processes NOTCH receptors, is induced by TLR signaling in a NOTCH-dependent manner. NOTCH3 is the only NOTCH receptor expressed in resting macrophages. Its expression increased rapidly in the first hours after TLR4 activation, followed by a gradual decrease, which was coincident with an elevation of the expression of the other NOTCH receptors. All NOTCH1, 2 and 3 contribute to the increased NOTCH signaling detected in activated macrophages. We also observed a crosstalk between NOTCH3 and NOTCH1 during macrophage activation. Finally, our results highlight the relevance of NOTCH3 in the activation of NF-κB, increasing p65 phosphorylation by p38 MAP kinase. Our data identify, for the first time, NOTCH3 as a relevant player in the control of inflammation.

Myeloid-specific blockade of Notch signaling alleviates murine pulmonary fibrosis through regulating monocyte-derived Ly6clo MHCIIhi alveolar macrophages recruitment and TGF-β secretion.

Macrophages in lung, including resident alveolar macrophages (AMs) and interstitial macrophages (IMs), and monocyte-derived macrophages, play important roles in pulmonary fibrosis (PF), but mechanisms underlying their differential regulation remain unclear. Recombination signal-binding protein Jκ (RBP-J)-mediated Notch signaling regulates macrophage development and phenotype. Here, using bleomycin-induced fibrosis model combined with myeloid-specific RBP-J disruption (RBP-JcKO ) mouse, we investigated the role of Notch signaling in macrophages during PF. Compared with the control, RBP-JcKO mice exhibited alleviated lung fibrosis as manifested by reduced collagen deposition and inflammation, and decreased TGF-β production. FACS analysis suggested that decreased Ly6clo MHCIIhi AMs might make the major contribution to attenuated fibrogenesis in RBP-JcKO mice, probably by reduced inflammatory factor release and enhanced matrix metalloproteinases expression. Using clodronate-mediated macrophage depletion in RBP-JckO mice, we demonstrated that embryonic-derived AMs play negligible role in lung fibrosis, which was further supported by adoptive transfer experiments. Moreover, on CCR2 knockout background, the effect of RBP-J deficiency on fibrogenesis was not elicited, suggesting that Notch regulated monocyte-derived AMs. Co-culture experiment showed that monocyte-derived AMs from RBP-JcKO mice exhibit reduced myofibroblast activation due to decreased TGF-β secretion. In conclusion, monocyte-derived Ly6clo MHCIIhi AMs, which are regulated by RBP-J-mediated Notch signaling, play an essential role in lung fibrosis.

Notch signal deficiency alleviates hypertrophic scar formation after wound healing through the inhibition of inflammation

Pathological scar is a common complication after wound healing. One of the most important factors that affects scar formation is inflammation. During this process, macrophages play a critical role in the wound healing process, as well as in scar formation. Notch signaling is reported to participate in inflammation and fibrosis; however, whether it affects scar formation is still unclear. In this study, RBP-J knockout mice, in which Notch signaling was down-regulated, and control mice were used, and a skin incision model was established. Sirius red staining and Masson staining suggested that RBP-J knockout could significantly reduce collagen sedimentation after wound healing. Western blot analysis and RT-PCR also confirmed the results. During wound healing, the expression of inflammatory cytokines and macrophage infiltration were decreased in RBP-J knockout mice. In vitro, it was also verified that RBP-J deficiency in macrophages effectively suppressed the expression of inflammatory cytokines and chemotaxis of macrophages after LPS stimulation. In conclusion, blocking Notch signaling in macrophages effectively alleviated scar formation by suppressing the inflammatory response and collagen sedimentation.

The Role of Notch Signaling in Macrophages during Inflammation and Infection: Implication in Rheumatoid Arthritis?

Notch signaling coordinates numerous cellular processes and has been implicated in many pathological conditions, including rheumatoid arthritis (RA). Although the role of Notch signaling in development, maturation, differentiation, and activation of lymphocytes has been comprehensively reported, less is known about its role in myeloid cells. Certainly, limited data are available about the role of Notch signaling in macrophages during inflammation and infection. In this review, we discuss the recent advances pertaining to the role of Notch signaling in differentiation, activation, and metabolism of macrophages during inflammation and infection. We also highlight the reciprocal interplay between Notch signaling and other signaling pathways in macrophages under different inflammatory and infectious conditions including pathogenesis of RA. Finally, we discuss approaches that could consider Notch signaling as a potential therapeutic target against infection- and inflammation-driven diseases.

Acetylation-Dependent Regulation of Notch Signaling in Macrophages by SIRT1 Affects Sepsis Development.

SIRT1 is reported to participate in macrophage differentiation and affect sepsis, and Notch signaling is widely reported to influence inflammation and macrophage activation. However, the specific mechanisms through which SIRT1 regulates sepsis and the relationship between SIRT1 and Notch signaling remain poorly elucidated. In this study, we found that SIRT1 levels were decreased in sepsis both in vitro and in vivo and that SIRT1 regulation of Notch signaling affected inflammation. In lipopolysaccharide (LPS)-induced sepsis, the levels of Notch signaling molecules, including Notch1, Notch2, Hes1, and intracellular domain of Notch (NICD), were increased. However, NICD could be deacetylated by SIRT1, and this led to the suppression of Notch signaling. Notably, in macrophages from myeloid-specific RBP-J−/− mice, in which Notch signaling is inhibited, pro-inflammatory cytokines were expressed at lower levels than in macrophages from wild-type littermates and in RBP-J−/− macrophages, and the NF-κB pathway was also inhibited. Accordingly, in the case of RBP-J−/− mice, LPS-induced inflammation and mortality were lower than in wild-type mice. Our results indicate that SIRT1 inhibits Notch signaling through NICD deacetylation and thus ultimately alleviates sepsis.

Notch Signaling in Macrophages in the Context of Cancer Immunity.

Macrophages play both tumor-suppressing and tumor-promoting roles depending on the microenvironment. Tumor-associated macrophages (TAMs) are often associated with poor prognosis in most, but not all cancer. Understanding how macrophages become TAMs and how TAMs interact with tumor cells and shape the outcome of cancer is one of the key areas of interest in cancer therapy research. Notch signaling is involved in macrophage activation and its effector functions. Notch signaling has been indicated to play roles in the regulation of macrophage activation in pro-inflammatory and wound-healing processes. Recent evidence points to the involvement of canonical Notch signaling in the differentiation of TAMs in a breast cancer model. On the other hand, hyperactivation of Notch signaling specifically in macrophages in tumors mass has been shown to suppress tumor growth in an animal model of cancer. Investigations into how Notch signaling is regulated in TAMs and translates into pro- or anti-tumor functions are still largely in their infancy. Therefore, in this review, we summarize the current understanding of the conflicting roles of Notch signaling in regulating the effector function of macrophages and the involvement of Notch signaling in TAM differentiation and function. Furthermore, how Notch signaling in TAMs affects the tumor microenvironment is reviewed. Finally, the direct or indirect cross-talk among TAMs, tumor cells and other cells in the tumor microenvironment via Notch signaling is discussed along with the possibility of its clinical application. Investigations into Notch signaling in macrophages may lead to a more effective way for immune intervention in the treatment of cancer in the future.

Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop.

Aberrant energy metabolism is critical for cancer progression. Tumor-associated macrophages (TAMs) can stimulate tumor angiogenesis and enhance cancer metastasis; however, the metabolic interaction between cancer cells and macrophages characterized by lactate shuttles remains unclear. Here, we showed that lactate activated human macrophages to a TAM-like phenotype and stimulated the secretion of CCL5 by activation of Notch signaling in macrophages. Reciprocally, CCL5 increased cell migration, induced cancer cell EMT, and promoted aerobic glycolysis in breast cancer cells, suggesting a positive metabolic feedback loop in the co-culture system. Inhibition of CCR5, the cognate receptor of CCL5, or neutralization of CCL5, broke the metabolic loop and decreased cancer cell migration and EMT. Inhibition of aerobic glycolysis significantly reduced breast cancer cell EMT, indicated that aerobic glycolysis was necessary for the invasive phenotype of cancer cells. We further showed that TGF-β signaling regulated the expression of CCR5 in the co-culture system, and CCL5 induced glycolysis by mediation of AMPK signaling. The expression of CCL5-CCR5 axis was highly associated with macrophage infiltration, TGF-β and p-AMPK in clinical samples. CCL5-CCR5 axis promoted breast cancer metastasis in vivo. Our findings suggested a pivotal role of CCL5-CCR5 axis in the metabolic communication between cancer cells and macrophages.

Blockade of Canonical Notch Signaling in Macrophages Alters the Inflammatory Response and Compounds Delayed Wound Healing in a Murine Model of Diabetes

Blockade of Canonical Notch Signaling in Macrophages Alters the Inflammatory Response and Compounds Delayed Wound Healing in a Murine Model of Diabetes

Blocking Notch signal in myeloid cells alleviates hepatic ischemia reperfusion injury by repressing the activation of NF-κB through CYLD.

Ischemia-reperfusion (I/R) is a major reason of hepatocyte injury during liver surgery and transplantation. Myeloid cells including macrophages and neutrophils play important roles in sustained tissue inflammation and damage, but the mechanisms regulating myeloid cells activity have been elusive. In this study, we investigate the role of Notch signaling in myeloid cells during hepatic I/R injury by using a mouse model of myeloid specific conditional knockout of RBP-J. Myeloid-specific RBP-J deletion alleviated hepatic I/R injury. RBP-J deletion in myeloid cells decreased hepatocytes apoptosis after hepatic I/R injury. Furthermore, myeloid-specific RBP-J deletion led to attenuated inflammation response in liver after I/R injury. Consistently, Notch blockade reduced the production of inflammatory cytokines by macrophages in vitro. We also found that blocking Notch signaling reduced NF-κB activation and increased cylindromatosis (CYLD) expression and knockdown of CYLD rescued reduction of inflammatory cytokines induced by Notch blockade in macrophages during I/R injury in vitro. On the other hand, activation of Notch signaling in macrophages led to increased inflammatory cytokine production and NF-κB activation and decreased CYLD expression in vitro. These data suggest that activation of Notch signaling in myeloid cells aggravates I/R injury, by enhancing the inflammation response by NF-κB through down regulation of CYLD.

Myeloid-Specific Blockade of Notch Signaling Attenuates Choroidal Neovascularization through Compromised Macrophage Infiltration and Polarization in Mice.

Macrophages have been recognized as an important inflammatory component in choroidal neovascularization (CNV). However, it is unclear how these cells are activated and polarized, how they affect angiogenesis and what the underlining mechanisms are during CNV. Notch signaling has been implicated in macrophage activation. Previously we have shown that inducible disruption of RBP-J, the critical transcription factor of Notch signaling, in adult mice results in enhanced CNV, but it is unclear what is the role of macrophage-specific Notch signaling in the development of CNV. In the current study, by using the myeloid specific RBP-J knockout mouse model combined with the laser-induced CNV model, we show that disruption of Notch signaling in macrophages displayed attenuated CNV growth, reduced macrophage infiltration and activation, and alleviated angiogenic response after laser induction. The inhibition of CNV occurred with reduced expression of VEGF and TNF-α in infiltrating inflammatory macrophages in myeloid specific RBP-J knockout mice. These changes might result in direct inhibition of EC lumen formation, as shown in an in vitro study. Therefore, clinical intervention of Notch signaling in CNV needs to pinpoint myeloid lineage to avoid the counteractive effects of global inhibition.

Impact of Notch1 Deletion in Macrophages on Proinflammatory Cytokine Production and the Outcome of Experimental Autoimmune Encephalomyelitis.

Notch signaling is involved in regulating TLR-mediated responses in activated macrophages. In this study, we investigated the impact of Notch signaling in macrophages in an experimental autoimmune encephalomyelitis (EAE) model. To examine the impact of deficiency in Notch signaling in activated macrophages in EAE, an adoptive transfer of activated macrophages derived from Notch1(fl/fl) × Mx1cre(+/-) (Notch1 knockout [N1KO]) or CSL/Rbp-jκ(fl/fl) × Mx1cre(+/-) (CSL/RBP-Jκ KO) mice was performed prior to induction of EAE. Mice receiving activated N1KO macrophages showed decreased severity of EAE compared with mice receiving wild-type or CSL/RBP-Jκ KO macrophages. In vitro restimulation of splenocytes by myelin oligodendrocyte glycoprotein 35-55 peptide from these mice revealed that cells from mice receiving N1KO macrophages produced significantly less IL-17 compared with the control mice, whereas IFN-γ production was similar in both groups. We found that activated N1KO, but not CSL/RBP-Jκ KO, macrophages produced less IL-6 and had lower CD80 expression compared with wild-type and did not exhibit any defect in IL-12p40/70 production, whereas activated macrophages from CSL/RBP-Jκ KO mice phenocopied γ-secretase inhibitor treatment for reduced IL-12p40/70 production. Furthermore, the nuclear translocation of the NF-κB subunit c-Rel was compromised in γ-secretase inhibitor-treated and CSL/RBP-Jκ KO but not N1KO macrophages. These results suggest that Notch1 and CSL/RBP-Jκ in macrophages may affect the severity of EAE differently, possibly through modulating IL-6 and CD80 expression, which is involved in the Th17 but not Th1 response.

Modulation of Notch signaling by intracellular bacterial toxins (610.3)

The Notch signaling pathway has recently been recognized as a critical signaling pathway in macrophages. This study examines how Notch signaling functions in the context of infectious disease as well as how contrasting intracellular bacterial toxins modify this pathway. In this work, we investigate how the Notch signaling pathway is altered in macrophages by Bacillus anthracis edema toxin (ET) and Clostridium difficile toxin B (TcdB). In preliminary studies, ET was found to activate and augment Notch signaling in macrophages through a mechanism involving transducin‐like enhancer of split (TLE) and recombinant recognition sequence binding protein at the Jκ site (RBP‐J). Because ET, an immunosuppressive toxin, activates Notch signaling in macrophages, we hypothesized that TcdB, an inflammation stimulating toxin, may have an alternative effect on this signaling pathway. To address this possibility, the Notch signaling pathway was examined in primary macrophages exposed to TcdB, and results from these studies found that Notch signaling was suppressed by TcdB. Further analyses have suggested that the TcdB induced suppression of Notch signaling is through a mechanism involving the stimulation of interferon signaling. These results provide insight into mechanisms utilized by disparate bacterial toxins to modify Notch signaling in macrophages.

Direct regulation of Interleukin-6 expression by Notch signaling in macrophages (172.36)

Abstract Notch signaling is required for immune cell development. The intracellular domain of Notch (NIC) induces expression of target genes by binding to CSL, a DNA-binding protein. In macrophages, Notch signaling regulates inflammatory cytokines TNFα and IL-6. IL-6 is secreted by various cell types, including activated macrophages and functions in immune regulation and inflammation. The consensus CSL binding site in the promoter region of IL-6 was shown to negatively regulate IL-6 mRNA, but the role of Notch signaling in IL-6 expression in macrophages remained unclear. We investigated the role of Notch signaling in the regulation of the IL-6 transcript in macrophages. Our results showed that bone marrow derived macrophages (BMMΦ) and RAW264.7 cell lines activated with lipopolysaccharide (LPS) and interferon gamma (IFNγ) induced IL-6 mRNA expression and upregulation of Notch1. Inhibition of Notch signaling in BMMΦ using a gamma secretase inhibitor partially decreases the level of IL-6 mRNA and protein. In contrast, RAW264.7, transiently transfected with constitutively active Notch1, displayed a dramatic increase in IL-6 mRNA. Inhibition of NF-κB completely abrogated IL-6 mRNA expression induced by the over expression of NIC. Chromatin immunoprecipitation assay demonstrated that Notch1 binds to the promoter of IL-6 in activated RAW264.7 but not in unstimulated cells. These results strongly suggest that Notch1 positively regulates IL-6 expression via NF-κB in activated macrophages.