Using RNAi technique to investigate the roles of Notch1 gene in macrophages

Abstract

Notch gene encodes a protein family of conserved transmembrane receptors expressed on a cell surface. There are four Notch genes, i.e. Notch1, 2, 3 and 4 in veterbrates. Five ligands have been identified, i.e. Jagged-1 and-2 and Dll-1, 3 and 4. Notch signaling is initiated with the interaction of receptor and ligand. Notch signaling is involved in maintenance of stem cells, binary cell-fate decisions, induction of terminal differentiation and also in tumorigenesis. Macrophages are white blood cells performing important roles in both innate and acquired immunity. Previously, it was reported that Notch receptors are expressed in lipopolysaccharide (LPS)/interferon gamma (IFNγ) - activated macrophages. To investigate the role of Notch1 in macrophages in this study, RNAi technique was applied to specifically silence Notch1 expression in RAW264.7 macrophage-like cell line. Silencing of Notch1 resulted in cellular morphological alteration, and increased pro-inflammatory molecule production such as the nitric oxide and MHC class II and cytokines expression including IL-10, IL-12p40 and TNF. We unexpectedly found that one of the target genes of Notch signaling Deltex1 was significantly up-regulated in the absence of LPS / IFNγ stimulation. Knock down Notch1 in macrophages increased efficacy in bactericidal activities against both gram negative and positive bacteria. Therefore, Notch1 is involved in effector functions of macrophages during both innate and acquired immune responses. Hence, Notch1 might be the therapeutic target for manipulating macrophage functions.