Novel Insight into the Role of the S100A8/A9 Protein Complex in the Regulation of Neutrophil Functions

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 ▲]