Regulation of neutrophil cytokine release and degranulation during inflammation: Role of SNARE fusion proteins

Neutrophil granulocytes are the first effector cells to be recruited to sites of infection. They rapidly release granule proteins and pro-inflammatory cytokines to efficiently kill intruding pathogens and recruit other immune cells. In exocytotic events, specific interactions of so-called soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins lead to the formation of complexes in order to mediate membrane fusion. Because of the excessive release of neutrophil-derived mediators leading to the development of chronic inflammatory disorders, we aimed in the present scientific work to investigate in more details the regulatory processes of mediator release in neutrophils during inflammation, with emphasis on SNARE proteins. The main objectives were i) to characterize the release of pro-inflammatory mediators, ii) to profile SNARE expression, iii) to determine the functional role of SNAREs and SNARE complexes in cytokine release and degranulation, and iv) to identify the intracellular localization of SNAREs. To characterize the pro-inflammatory response in neutrophils in regard to exocytosis, extensive kinetic studies were performed in a first step on LPS-stimulated primary neutrophils. A novel linear fitting approach was created to correlate the relationship between granule proteins and cytokines secreted to the inflammatory site. In a second step, SNARE expression levels were determined by whole-transcript analysis and the similar profiles in primary neutrophils as well as DMSO-differentiated HL-60 cells (dHL-60 cells), a neutrophil cell model, were underlined. Using an RNAi strategy, the SNARE syntaxin 3 (STX3) was identified as an essential actor in the release of the cytokines IL-1α, IL-1β, IL-12b, and CCL4. It was also involved in MMP-9 exocytosis from gelatinase granules where it could partly be localized. The SNARE SNAP29, which shares common localization with STX3, functionally affects the release of IL-12b, CCL2 and IL-8 as well as MMP-9, and represents a potential candidate to form cognate complexes with STX3. The knockdown of VAMP3, another SNARE candidate, showed deregulated secretion of IL-12b, CCL4, IL-8 as well as MMP-9. However, VAMP3 was located at the plasma membrane and was thus excluded as being part of the STX3-SNAP29 complex. Our findings provide first evidence that SNARE fusion proteins are involved in the release of IL- 12b, IL-1α, IL-1β, CCL4, IL-8, and CCL2 in a neutrophil-like cell model. The impact of SNAREs on gelatinase degranulation led us to hypothesize that cytokines might be packaged in these granules before subsequent exocytosis. !