Phosphorylation of VAMP3 couples IL-6 exocytosis to dendritic cell activation.

Adaptive immunity is crucial for combating pathogens and generating immunological memory. Central to adaptive immunity are myeloid cells, which activate upon pathogen detection. Activation is essential for inflammatory cytokine release and requires a complex series of molecular events to facilitate cytokine expression. However, although the transcriptional machinery regulating cytokine expression is well defined, it is apparent that trafficking machinery also has to be reprogrammed to facilitate cytokine secretion. We demonstrate through quantitative total internal resonance fluorescence (TIRF) microscopy that short-term inflammatory stimulation with lipopolysaccharide (LPS) is sufficient to upregulate interleukin-6 (IL-6) secretion in dendritic cells. Through bioinformatic analysis of phosphoproteomic data, we demonstrate that the activation of dendritic cells rapidly reprograms SNARE-associated trafficking machinery. We link the enhanced rate of IL-6 secretion to phosphorylation of the SNARE protein VAMP3. This releases VAMP3 from the chaperone WDFY2, enabling the trafficking of IL-6-positive VAMP3-positive vesicles to the plasma membrane. VAMP3 then complexes with STX4, facilitating IL-6 secretion. Finally, we found that VAMP3-dependent IL-6 secretion is polarised, reconciling findings that, in dendritic cells, IL-6-positive vesicles are non-polarised, whereas VAMP3-positive vesicles are largely polarised.