Notch signaling regulates apoptosis in Macrophages infected with mycobacteria

Abstract

Apoptosis of macrophages infected with pathogens is considered to be one of the important host immune responses. Previously, Mcl-1 expression was found to be up-regulated in monocytes/macrophages during infection with virulent Mycobacterium tuberculosis (Mtb), a causative agent of tuberculosis. Myeloid cell leukemia-1 (Mcl-1) is a pro-survival member of the Bcl-2 protein family. The regulatory mechanism of Mcl-1 expression in macrophages is not well characterized. In this study, upregulation of Mcl-1 in bone marrow derived macrophages (BMM ) and macrophage-like cell line RAW264.7 were observed when cells were treated with Mtb-derived purified protein derivatives (PPD) or infected with Mycobacterium bovis BCG (M. bovis BCG). The Notch signaling pathway is a highly conserved cell signaling pathway present in most multicellular organisms. Notch signaling modulates numerous cellular functions such as proliferation, adhesion, angiogenesis and apoptosis. Recent studies reported that Notch signaling is activated in macrophages during infection with M. bovis BCG. We found a well correlation between Mcl-1 and Notch1 expression in PPD-activated or M. bovis BCG-infected macrophages. In addition, cleaved Notch1, an indicator of activated Notch signaling pathway, was detected upon PPD treatment of macrophages, suggesting that Notch signaling is activated. Treatment with y- secretase inhibitor (GSI), a specific inhibitor of Notch signaling, or knockdown of Notch1 resulted in decreased expression of Mcl-1 and increased apoptosis of infected macrophages. Since a conserved potential binding site for CSL, a DNA binding protein and interacting partner with Notch, was identified in the promoter region of mcl-1, chromatin immunoprecipitation (ChIP) assay was used to determine whether Notch1 directly binds to mcl-1 promoter. ChIP assay clearly demonstrated that stimulation with PPD recruited intracellular Notch to the mcl-1 promoter. Taken together, these results suggested that Notch signaling directly regulates Mcl-1 expression in macrophages upon treatment with PPD or infection with M. bovis BCG. This new function of Notch1 in regulating Mcl-1 expression in antibacterial immunity may open a new strategy to manipulate host immune response against tuberculosis.