Induction of NFκB responses by the S100A9 protein is TLR4-dependent.

Interactions between danger and pathogen-associated molecular patterns (DAMP and PAMP) and pattern recognition receptors such as Toll-like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of NFκB and cytokine secretion. In this report, we investigated the capacity of LPS-free S100A9 (DAMP) protein to activate human and mouse cells compared to lipoprotein-free LPS (PAMP). Firstly, we showed that LPS and S100A9 were able to increase NFκB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP-1 cells and mouse bone marrow-derived dendritic cells (BM-DC). Surprisingly, although S100A9 triggered a weaker cytokine response compared to LPS, we found that S100A9 more potently induced IκBα degradation and hence NFkB activation. Both the S100A9- and LPS-induced response was completely absent in TLR4 knock-out mice, while it was only slightly affected in RAGE knock-out mice. Also, we showed that LPS and S100A9 NFkB induction were strongly reduced in the presence of specific inhibitors of TLR-signaling. Chloroquine reduced S100A9 but not LPS signaling, indicating that S100A9 may need to be internalized in order to be fully active as a TLR4 inducer. This was confirmed using A488-labeled S100A9 that was internalized in THP-1 cells, showing a raise in fluorescence after 30 minutes at 37°C. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NFκB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NFκB in a qualitative distinct manner. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd, Immunology