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

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.