Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis

Sylvain Pagie,Béatrice Charreau,Nathalie Gérard

doi:10.1186/s12964-017-0214-x

Sylvain Pagie, Béatrice Charreau + Show 1 more

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https://doi.org/10.1186/s12964-017-0214-x

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Abstract

BackgroundNotch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis. In mammals, four Notch receptors (Notch 1–4) can interact with five distinct ligands [Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4]. We previously reported that Notch activation is modulated in endothelial cells and monocytes during inflammation and showed that inflammation upregulates DLL4 on endothelial cells. DLL4 promotes differentiation of blood monocytes into proinflammatory M1 macrophages. Here, we further investigated the ability of DLL4 to interfere with the polarization of blood monocytes into immunosuppressive M2 macrophages.MethodsHuman blood monocytes were differentiated in vitro into M0 macrophages and then polarized into M1 or M2 macrophages with LPS/IFNγ and IL-4, respectively. Polarization steps were performed in the presence of immobilized recombinant DLL4. Immune phenotype and apoptosis of macrophage subsets were analyzed and quantified by flow cytometry. Regulatory effects of DLL4 on gene expression, cell signaling and apoptotic pathways were investigated by QPCR and western blots.ResultsThe phenotype of M2 macrophages was subject to specific alterations in the presence of recombinant DLL4. DLL4 inhibits the upregulation of IL-4 induced M2 markers such as CD11b, CD206, and CD200R. Survival of macrophages upon M2 polarization was also strongly reduced in the presence of DLL4. DLL4 induces a caspase3/7-dependent apoptosis during M2 but not M1 macrophage polarization. The Notch ligand DLL1 has no apoptotic effect. Both DLL4 signaling via Notch1 as well as DLL4-mediated apoptosis are Notch-dependent. Fully differentiated M2 macrophages became resistant to DLL4 action. Mechanistically, DLL4 selectively upregulates gene expression in macrophages upon M2 polarization, thereby affecting the Notch pattern (Notch1, 3, Jag1), activity (HES1), and transcription (IRF5, STAT1). The pro-apoptotic effectors Bax and Bak and the BH3-only proteins Bid and Bim seem to convey DLL4 apoptotic signal.ConclusionInterplay between the DLL4/Notch and IL-4/IL-4R signaling pathways impairs M2 differentiation. Thus, DLL4 may drive a Notch-dependent selection process not only by promoting M1 macrophage differentiation but also by preventing M2 macrophage differentiation through inhibition of M2-specific gene expression and apoptotic cell death.

Highlights

Notch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis
DLL4 impairs IL-4-mediated differentiation into M2 macrophage To investigate the interplay between Notch and macrophage phenotype, monocytes were purified from blood samples issued from healthy volunteers (EFS des Pays de la Loire, Nantes, France)
Monocytes were sequentially differentiated into M0 macrophages using M-CSF for 7 days and polarized into M1 macrophages with IFNγ and LPS or into M2 macrophages (M2a subtype) using IL-4 for 48 h, as previously described [23]

Summary

Introduction

Notch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis. DLL4 promotes differentiation of blood monocytes into proinflammatory M1 macrophages. We further investigated the ability of DLL4 to interfere with the polarization of blood monocytes into immunosuppressive M2 macrophages. An important feature of the monocyte/macrophage lineage is their diversity and plasticity. These varying functional phenotypes may be acquired through specific environmental cues [1,2,3]. The molecular basis of macrophage polarization is complex and includes transcription factors, signaling pathways, and epigenetic as well as posttranscriptional regulation. A balance between the Signal Transducer and Activator of Transcription (STAT) proteins STAT1 and STAT3/6 regulates M1 versus M2 polarization defining their inflammatory and immunosuppressive functions, respectively. Macrophage differentiation is a dynamic process and macrophages can switch from one phenotype to another according to requirements [5, 6]

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