Mechanisms of TRAIL expression by human plasmacytoid dendritic cells (168.29)

Abstract

Abstract One major mechanism of CD4+ T cell decline during HIV infection is due to the death ligand-mediated apoptosis of uninfected CD4+ T cells. TNF-Related Apoptosis Inducing Ligand (TRAIL) is one death ligand involved in this process and is expressed by a number of peripheral blood mononuclear cells upon HIV infection, including plasmacytoid dendritic cells (pDC). The mechanisms of TRAIL expression by pDC still need to be elucidated. To better characterize the expression of TRAIL, pDC were stimulated with TLR7 and TLR9 ligands with or without the addition of brefeldin A (BFA), a Golgi inhibitor. We directly stained pDC for surface TRAIL, and also stained for intracellular TRAIL by permeabilizing pDC with 0.1% Triton-X after overnight fixation with 2% PFA. Flow cytometry analysis revealed that with TLR7 and TLR9 agonists, there was an increase in TRAIL expression on the surface of pDC, while with BFA addition, there was a decrease in surface TRAIL expression accompanied with an increase in intracellular TRAIL expression. Using Amnis Image Stream, we were also able to visualize the location of intracellular and surface TRAIL expression on each pDC. This study showed that upon TLR7 and TLR9 stimulation, pDC upregulate TRAIL expression, and with the addition of BFA, TRAIL is prevented from reaching the surface, accumulating in the cytoplasm of pDC.