Impaired cellular immunity because of dysregulation of Th1 cytokines is a major feature of AIDS. Interleukin-12 (IL-12) is an important Th1 cytokine, which plays a critical role in cellular immunity by producing IFNgamma from T and NK cells. Biologically active IL-12 is a 70 kD glycoprotein composed of two disulfide-linked subunits, p35 and p40. The p40 subunit is an appropriate indicator for IL-12 due to its expression in IL-12p70 producing cells including monocytes/macrophages in response to various stimuli, such as bacterial endotoxin, LPS. However, how LPS induces IL-12p40 in monocytes is not clear. A severely decreased IL-12p40 production has been found in PBMCs and macrophages from HIV positive individuals and is believed to be correlated to HIV-associated immunodeficiency. The mechanism underlying the impaired IL-12 production in HIV infected monocytes/macrophages remains unknown. HIV-1 Nef protein, an AIDS pathogenetic factor, has been shown to downregulate Th1 cytokines. In this study, I investigated the potential signaling pathways involved in the downregulation of IL-12p40 production by intracellular Nef in LPS-activated human monocytic cells. LPS activates monocytes by coupling to CD14/TLR4 receptor on the cell surface and activating MAPK pathways, calcium/PI3K pathways and several transcription factors including Ets2, NFkappaB, and AP-1. These signaling components have been shown to be implicated in downregulation of IL-12p40 production by several immunosuppressive drugs such as dexamethasone (Dxm), FK506 and cyclosporine A (CyA). By utilizing these immunosuppressive drugs and the specific inhibitors that are involved in MAPKs and calcium/PI3K signaling pathways. I demonstrated that Dxm and FK506/CyA mediated downregulation of IL-12p40 production is regulated by two distinct and independent signaling pathways, JNK and CaMKII activated PI3K pathways in LPS-stimulated human monocytic cells, in which, Dxm regulating via the JNK whereas FK506/CyA regulating via the CaMKII. By extensive mutative analyses of the IL-12p40 promoter, I further demonstrated that the cis-binding motifs of transcription factor AP-1 and NFkappaB were required for IL-12p40 transcription in both signaling pathways. I then focused on intracellular Nef as a potential repressive molecule in regulation of MAPKs, CaMKII activated PI3K and transcriptional factors involved in LPS-induced IL-12p40 expression. I demonstrated that intracellular Nef downregulates IL-12p40 in human monocytes by specifically inhibition the INK MAPK and transcription factor NFkappaB. These studies may provide new therapeutic tools for the treatment of inflammation and autoimmune diseases and a further understanding for significant functions of intracellular Nef in AIDS pathogenesis
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