Abstract :
[en] Neutrophils are important actors of the immune system, particularly through the release of cytokines in the inflammatory environment. This process must be highly orchestrated to avoid cell overactivation and unwanted tissue damage. However, this elegant regulation is still only partially known and poorly characterized. Increasing evidence over the years show that Ca2+ is actively involved in cytokine secretion but our knowledge on the relationship between these two phenomena needs to be filled. To this end, in this study, we investigated the Ca2+-dependent mechanisms underlying cytokine secretion in neutrophils.
For that, the differentiated myeloid cell line HL-60 (dHL-60) was used as a cell model since primary neutrophils are unable to be genetically modified. Our results showed that the mobilization of several cytokines, notably IL-8, was up-regulated in a time-dependent manner by a pro-inflammatory stimulus (fMLF). Experiments of intracellular flow cytometry staining provided evidence on the presence of preformed IL-8 as well as de novo synthesis of IL-8. Changes in intracellular Ca2+ levels, resulting from an extracellular Ca2+ entry, is shown to be indispensable for efficient secretion of CCL2, CCL3, CCL4 and IL-8, even if an additional signal appears to be required for the release of IL-8. Ca2+-dependent cytokine secretion was associated to the store-operated Ca2+ entry (SOCE) mechanism and probably relies on the Ca2+ sensor STIM1.
Since Ca2+-binding proteins S100A8/A9 have been previously reported to be key actors in the regulation of neutrophil NADPH oxidase activation, we hypothesized that Ca2+ signals could be converted into cytokine secretion through intracellular S100A8/A9. Knockdown studies performed in mouse (Hoxb8 cells) and human (dHL-60) neutrophil models confirmed the involvement of S100A8/A9 in the regulation of cytokine secretion. Moreover, our data support the fact that a part of cytokine secretion occurs through the degranulation process.
Finally, we investigated the post-transcriptional mechanism involved in the regulation of S100A8/A9 expression and thus, in the control of cytokine secretion. Based on prediction network analysis, miR-132-5p was identified as a potential regulator of S100A8/A9. Stable overexpression of miR-132-5p in dHL-60 cells caused a strong inhibition of S100A8/A9 expression and IL-8 secretion underlining the preponderant role of miR-132-5p-regulated S100A8/A9 expression in the pro-inflammatory response.
To summarize, for the first time, we prove that Ca2+-dependent cytokine secretion is associated with SOCE and is regulated by intracellular S100A8/A9, which is negatively modulated by miR-132-5p to prevent excessive neutrophil activation and host damage.
