The role of notch signaling pathway in polarization of macrophages in mice

Abstract

Notch signaling is a well conserved signaling pathway which is involved in regulation of cell fate determination, differentiation, proliferation and cell death of various cell types. Interaction between Notch receptors and ligands initiates the signaling cascade, resulting in transcription of its target genes. Previous studies demonstrated that Notch signaling regulates effector functions of innate immune cells, macrophages, such as production of TNFα and nitric oxide, partly through interaction with other signaling pathways. The phenotypes of macrophages are highly plastic, depending on the microenvironment they are surrounded. At least three types of different effector macrophages have been described, i.e. classically activated macrophages (CA-MФ), alternatively activated macrophages (AA-MФ) and regulatory macrophages (Reg-MФ). In these studies, we investigated the expression of Notch receptors and their ligands in different types of effector macrophages, and the relationship of Notch signaling pathway and other well characterized signaling pathways using murine bone marrow derived macrophages. Polarization of macrophages were confirmed by monitoring the expression of marker genes, arginase1 (Arg1), il12p40 and il10 for AA, CA and Reg-MФ, respectively. Western blot revealed that Notch1 is highly expressed in CA and regulatory macrophages. The level of Notch1 correlated with the appearance of cleaved Notch1 (Val1744) in these 2 types of macrophages. In addition, distinct patterns of ligand expression and Notch target gene, Hes1, in different types of macrophages were observed. Upon inhibition of Notch signaling by gamma secretase inhibitor (GSI), altering in gene expression of key markers for each type was observed. GSI treatment resulted in decreased il10, il12p40 and il23p19 mRNA expression in CA and Reg-MФ, but did not affect arg1 mRNA level in AA-MФ. Moreover, inhibiting Notch signaling affected the status of MAP kinase pathway. Reduced phosphorylation of ERK1/2 was seen in CA-MФ upon GSI treatment. Taken together, we proposed that Notch signaling plays an essential role in regulating differentiation of effector macrophages partly by interacting and regulating MAP kinase pathway.