Background S100A8/A9 heterodimers are well-known alarmins that, upon release from activated or necrotic phagocytes, promote inflammation by binding to Toll-like receptor 4 (TLR4). These proteins are highly expressed in synovial phagocytes during arthritis and proved to be reliable biomarkers for monitoring disease activity in RA. Interestingly, we now identify a novel immune-regulatory mechanism of S100A8 in human monocyte-derived dendritic cells (moDCs).
Objectives This study aims to analyze immune-regulatory functions of S100 proteins in human DCs.
Methods MoDCs are differentiated with or without exposure to S100A8 prior to maturation with LPS. After characterization of the activation status using flow cytometry, the ability of these cells to induce autologous CD4+, CD8+, and γδ T-cell proliferation is investigated. Cytokines, secreted during development are analyzed by Luminex cytokine arrays. The metabolic state of DCs is examined by using Seahorse XFp Analyzer assays. Finally, to identify molecular mechanisms leading to an immune-regulatory phenotype, the mRNA expression of moDCs is analyzed by genome-wide gene expression arrays.
Results Our results demonstrate that early exposure to S100A8 interferes with in-vitro differentiation of moDCs. Compared to controls S100A8-exposed moDCs show dramatically reduced surface expression of co-stimulatory molecules upon LPS-induced maturation. In addition, early treatment of moDCs with S100A8 alters the secretion of immune-regulatory cytokines and chemokines depending on the developmental state of moDCs. S100A8-induced effects on moDC maturation are not limited to TLR4 stimulation but rather trigger a common state of unresponsiveness. Furthermore, mitochondrial respiration and glycolytic function is diminished in S100A8-treated moDCs.
As a consequence, S100A8-exposed moDCs have a reduced potential to induce autologous T-cell proliferation. We can show that these differences are mainly caused by reduced surface expression of co-stimulatory molecules on S100A8-treated moDCs.
Mechanistically, genome-wide gene expression analysis reveals dramatic differences in gene expression between S100A8-exposed and conventionally differentiated moDCs. We demonstrate that S100A8 pre-treatment of moDCs significantly blocks LPS-induced gene expression during moDC activation. Interestingly, in-silico analysis of transcription factor networks predicts NFκB and C/EBPδ as master regulators of S100A8-induced effects in developing moDCs. C/EBPδ on protein level, indeed, shows reduced expression in S100A8-differentiated moDCs prior and after LPS-induced maturation when compared to conventionally differentiated moDCs.
Conclusions Taken together, our results demonstrate a novel regulatory mechanism of innate immunity to prevent overwhelming immune responses. Dysregulated repression of detrimental adaptive immune responses might very well contribute to the disease phenotype in auto-immune disorders with high systemic S100A8/A9 levels. Therefore, S100A8-differentiated immune-suppressive DCs potentially represent a promising therapeutic tool to treat auto-immune diseases in the future.
Disclosure of Interest None declared
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