Trafficking Machinery is Rapidly Primed to Facilitate Polarised IL-6 Secretion in Dendritic Cells

Abstract

The mounting of an adaptive immune response is critical for removing pathogens from the body and generating immunological memory. Central to this process are myeloid cells, which sense pathogens through a variety of cell surface receptors, engulf and destroy pathogens and become activated. Activation is essential for the release of cytokines as well as the cell-surface presentation of pathogen-derived-antigens. Activation-induced cytokine release by myeloid cells requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is becoming increasingly clear that trafficking machinery has to be re-programmed through post-translational modifications to dynamically regulate cytokine secretory events. We demonstrate through quantitative total internal-resonance fluorescence (TIRF) microscopy that short-term stimulation with the pathogenic stimulus lipopolysaccharide (LPS) is sufficient to up-regulate IL-6 secretion rates in human blood monocyte-derived dendritic cells and that this secretion is asymmetric and thus polarised. Using bioinformatics analysis of our phosphoproteomic data, we demonstrate that LPS stimulation of monocyte-derived dendritic cells rapidly reprograms SNARE-associated membrane trafficking machinery, through phosphorylation/dephosphorylation events. Finally, we link this enhanced rate of secretion to the phosphorylation of the SNARE protein VAMP3 at serine 44 (48 in mice), by showing that this phosphorylation drives the release of VAMP3 by its chaperone WDFY2 and the complexing of VAMP3 with STX4 at the plasma membrane.