A Role for Syntaxin 3 in the Secretion of IL-6 from Dendritic Cells Following Activation of Toll-Like Receptors

Abstract

The role of dendritic cells (DCs) in directing the immune response is due in part to their capacity to produce a range of cytokines. Importantly, DCs are a source of cytokines, which can promote T cell survival and T helper cell differentiation. While it has become evident that soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors (SNAREs) are involved in membrane fusion and ultimately cytokine release, little is known about which members of this family facilitate the secretion of specific cytokines from DCs. We profiled mRNA of 18 SNARE proteins in DCs in response to activation with a panel of three Toll-like receptors (TLR) ligands and show differential expression of SNAREs in response to their stimulus and subsequent secretion patterns. Of interest, STX3 mRNA was up-regulated in response to TLR4 and TLR7 activation but not TLR2 activation. This correlated with secretion of IL-6 and MIP-1α. Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels. Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators. Given the role of IL-6 in Th17 differentiation, these findings suggest the potential of STX3 as therapeutic target in inflammatory disease.

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules

“…C). This rate increased on average about twofold to ~ 0.0027 bursts·s −1 ·μm −2 upon overnight stimulation with LPS, likely due to the increased expression of trafficking proteins and the rerouting of intracellular trafficking . These are coarse estimates, with a 10‐fold variation among cells (Fig.…”

Endosomal and Phagosomal SNAREs

“…The Qa-SNARE Stx3 is essential (170), and its abolishment leads to defects in secretion (61,94) and endosomal trafficking (52). Stx3 is present at the plasma membrane (94,104,109,122,312) and at various intracellular compartments (88,108,109,313,341).…”

Membrane trafficking as an active regulator of constitutively secreted cytokines

“…As a consequence, activated dendritic cells increase the delivery rate of IL-6-containing vesicles at the plasma membrane about two-fold, as shown by quantitative imaging (Verboogen et al, 2018b). At the posttranslational level, the SNARE protein syntaxin-3, which is involved in IL-6 release, moves from intracellular compartments to the plasma membrane in LPS-activated macrophages (Collins et al, 2014). Moreover, Förster resonance energy transfer (FRET) coupled to fluorescence lifetime imaging microscopy (FLIM) revealed that LPS activation of dendritic cells results in increased formation of the complex between the SNARE protein VAMP3which is also involved in IL-6 releaseand syntaxin-4 at the plasma membrane (Verboogen et al, 2017).…”

“…Moreover, Förster resonance energy transfer (FRET) coupled to fluorescence lifetime imaging microscopy (FLIM) revealed that LPS activation of dendritic cells results in increased formation of the complex between the SNARE protein VAMP3which is also involved in IL-6 releaseand syntaxin-4 at the plasma membrane (Verboogen et al, 2017). At transcriptional and translational levels, the expression of SNAREs and many other proteins involved in trafficking of cytokines is upregulated in activated macrophages and dendritic cells (Chiaruttini et al, 2016;Collins et al, 2014;Manderson et al, 2007;Mori et al, 2011;Murray et al, 2005a;Pagan et al, 2003). Although not directly proven, this is likely to result in an increased rate of exocytic trafficking that is required to secrete all the newly synthesized cytokines.…”

“…Interestingly, not only does their downregulation by RNAi or the overexpression of dominant-negative mutants lead to reduced secretion of TNF-αbut their overexpression has the opposite effect (Mori et al, 2011;Murray et al, 2005a,b;Pagan et al, 2003), indicating that the levels of these trafficking proteins can still be rate-limiting for secretion. Second, post-Golgi trafficking of IL-6 is mediated by the SNARE proteins VAMP3, syntaxin-3, syntaxin-6 and Vti1b, and their overexpression increases IL-6 production, whereas its knockdown following RNAi results in a reduction of IL-6 release (Collins et al, 2014;Manderson et al, 2007;Verboogen et al, 2017). Finally, for IL-12, its secretion from late endosomal and/or lysosomal compartments is sensitive to the levels of VAMP7, because knockout or knockdown of VAMP7 leads to reduced IL-12 secretion (Chiaruttini et al, 2016).…”