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

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Notch is a critical regulator of angiogenesis, vascular differentiation, and vascular integrity. We investigated whether Notch signaling affects macrophage function during retinal angiogenesis in mice. Retinal macrophage recruitment and localization in mice with myeloid-specific loss of Notch1 was altered, as these macrophages failed to localize at the leading edge of the vascular plexus and at vascular branchpoints. Furthermore, these retinas were characterized by elongated endothelial cell sprouts that failed to anastomose with neighboring sprouts. The role of Notch signaling in macrophages in the developing retina has not previously been assessed.In this study, we show that Notch signaling is important for macrophage recruitment and localization in the developing retina. We also found an increased frequency of elongated sprouts that did not anastomose with neighboring sprouts in retinas in mice with myeloid-specific loss of Notch1. Furthermore, we show Notch signal activation in macrophages that interact with Dll4-positive tip cells, and that macrophages with Notch signaling are found predominately at the vascular front and in association with vascular branchpoints. These data suggest a novel way that Notch signaling regulates retinal angiogenesis. MethodsNotch1 mutant mice have been described. 5 Mice with a conditional allele of Notch1 (Notch1 flox ) 6 and the myeloid-specific Cre recombinase driver line (LysMCre) 7 were obtained from The Jackson Laboratory. Transgenic Notch reporter mice (TNR), harboring an enhanced GFP sequence under the control of 4 tandem copies of the CBF1 binding site consensus sequence 8 were also obtained from The Jackson Laboratory. All procedures were carried out according to approved protocols and guidelines established by the Columbia University Institutional Animal Care and Use Committee. Eyes from postnatal day 5 mice were fixed for 2 hours in 4% paraformaldehyde. Retinas were permeabilized in 1% BSA and 0.5% Triton X-100 overnight at 4°C and washed in PBLEC buffer (1% Triton X-100, 0.1mM MgCl 2 , 0.1mM CaCl 2 , 0.1mM MnCl 2 in PBS pH 6.8), then incubated overnight in PBLEC plus isolectin-B4 (Sigma-Aldrich), anti-F4/80 (Abcam), anti-GFP (Invitrogen), or anti-Dll4 (R&D Systems). After washing, retinas were incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen), washed, and mounted on slides with Vectashield (Dako) mounting medium for visualization using a LSM Meta 510 or Nikon A1R MP Multiphoton confocal microscope. Results/discussionPrevious studies have shown that loss of Dll4 in mice leads to excessive sprouting during retinal angiogenesis. 3,4 We first investigated whether decreased expression of Notch1 would lead to a similar defect. We found increased vascular density in retinas from Notch1 ϩ/Ϫ mice compared with control littermates ( Figure 1A,C and data not shown), supporting the model where Notch1 and Dll4 in endothelial cells regulate sprouting. Because macrophages are present in the retina and are known to express Notch1, 9,10 we assessed macrophage r...

We investigated whether Notch signaling plays a role in regulating macrophage responses to inflammation. In a wound healing assay, macrophage recruitment was decreased in Notch1+/− mice, and the wounds were characterized by decreased TNF-α expression. As wound healing progressed, Notch1+/− wounds had increased vascularization and collagen deposition compared with wild-type wounds. In mice with myeloid-specific Notch1 deletion, wounds had decreased macrophage recruitment as well as decreased TNF-α expression, indicating the specific role of Notch1 in the inflammatory response in these cells. In vitro, we found that vascular endothelial growth factor receptor-1 (VEGFR-1) was upregulated in macrophages in response to LPS/IFN-γ and that this upregulation depended on Notch signaling. Furthermore, macrophages from Notch1+/− mice had decreased expression of VEGFR-1 compared with macrophages from wild-type mice, whereas VEGFR-1 expression in Notch4−/− macrophages was normal. Inhibition of Notch signaling decreased induction of the inflammatory cytokines IL-6, IL-12, CXCL10, MCP-1, monokine induced by IFN-γ, and TNF-α in macrophages in response to LPS/IFN-γ. Additionally, macrophages from Notch1+/− mice demonstrated decreased induction of IL-6, IL-12, and TNF-α in response to stimulation compared with wild-type mice. Thus, both pharmacological inhibition of Notch and genetic analysis demonstrate that Notch1 regulates VEGFR-1 and cytokine expression in macrophages. We have also established that Notch1 is important for the inflammatory response during wound healing in mice.