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See detailNovel Insight into the Role of the S100A8/A9 Protein Complex in the Regulation of Neutrophil Functions
Jung, Nicolas UL

Doctoral thesis (2019)

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand calcium-binding proteins and are abundantly expressed in the cytosol of neutrophils. Mostly found under heterodimeric form, S100A8 ... [more ▼]

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand calcium-binding proteins and are abundantly expressed in the cytosol of neutrophils. Mostly found under heterodimeric form, S100A8/A9 have various intracellular and extracellular functions; they act as alarmins, amplifying the host inflammatory response. Our previous study showed that the intracellular activity of S100A8/A9 is carried by the phosphorylation of S100A9. Based on these results, we further investigated the importance of this post-translational modification on the extracellular activity of the protein complex and its impact on the inflammatory functions of neutrophils. First, we analyzed the phosphorylation state of secreted S100A8/A9 and the mechanism by which the protein complex is released into the extracellular space. Our results show that S100A9 is secreted under a phosphorylated form within the S100A8/A9 protein complex and this release is highly correlated to the process of NETosis. Next, we investigated the inflammatory response of neutrophil-like dHL-60 cells when stimulated with the phosphorylated and non-phosphorylated form of S100A8/A9. Our results indicate that only the phosphorylated form of S100A8/A9 increases the expression and secretion of various cytokines (e.g. TNFa, CCL4, CXCL8). Using receptor-neutralizing antibodies, we then determined the receptor and signaling pathways associated to S100A8/A9-P-induced cytokine secretion. The reduction of expression levels of the previously mentioned cytokines, after TLR4 blocking, point out that S100A8/A9-P-induced signaling is mediated in part by TLR4. Finally, we investigated the post-transcriptional response induced by S100A8/A9-P stimulation. Using miRNA-sequencing of S100A8/A9-P stimulated dHL-60 cells, we identified an upregulation of miR-146a-5p, miR-146b-5p and miR-155-5p expression. Since these three microRNAs have previously been described to regulate TLR4 signaling at various levels, we investigated their influence on the inflammatory response mediated by S100A8/A9-P. Stable overexpression of miR-146a-5p and miR-155-5p in dHL-60 cells resulted in the reduced S100A8/A9-P-mediated secretion of cytokines through the inhibition of key players in the TLR4 signaling pathways. To summarize, our results give new insight into the pro-inflammatory functions induced by S100A8/A9-P in neutrophils and reveal the potential of the phosphorylated protein complex as a major regulator of inflammation in chronic inflammatory diseases. [less ▲]

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See detailSecretion of the Phosphorylated Form of S100A9 from Neutrophils Is Essential for the Proinflammatory Functions of Extracellular S100A8/A9.
Schenten, Veronique; Plancon, Sebastien; Jung, Nicolas UL et al

in Frontiers in immunology (2018), 9

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca(2+)-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8 ... [more ▼]

S100A8 and S100A9 are members of the S100 family of cytoplasmic EF-hand Ca(2+)-binding proteins and are abundantly expressed in the cytosol of neutrophils. In addition to their intracellular roles, S100A8/A9 can be secreted in the extracellular environment and are considered as alarmins able to amplify the inflammatory response. The intracellular activity of S100A8/A9 was shown to be regulated by S100A9 phosphorylation, but the importance of this phosphorylation on the extracellular activity of S100A8/A9 has not yet been extensively studied. Our work focuses on the impact of the phosphorylation state of secreted S100A9 on the proinflammatory function of neutrophils. In a first step, we characterized the secretion of S100A8/A9 in different stimulatory conditions and investigated the phosphorylation state of secreted S100A9. Our results on neutrophil-like differentiated HL-60 (dHL-60) cells and purified human neutrophils showed a time-dependent secretion of S100A8/A9 when induced by phorbol 12-myristoyl 13-acetate and this secreted S100A9 was found in a phosphorylated form. Second, we evaluated the impact of this phosphorylation on proinflammatory cytokine expression and secretion in dHL-60 cells. Time course experiments with purified unphosphorylated or phosphorylated S100A8/A9 were performed and the expression and secretion levels of interleukin (IL)-1alpha, IL-1beta, IL-6, tumor necrosis factor alpha, CCL2, CCL3, CCL4, and CXCL8 were measured by real-time PCR and cytometry bead array, respectively. Our results demonstrate that only the phosphorylated form of the complex induces proinflammatory cytokine expression and secretion. For the first time, we provide evidence that S100A8/PhosphoS100A9 is inducing cytokine secretion through toll-like receptor 4 signaling. [less ▲]

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