Regulation of monocyte NADPH oxidase: Role of pattern recognition receptors

Activation of the NADPH oxidase enzyme complex results in the production of the oxygen free radical, superoxide anion (O2 .-). Superoxide anion is critical for host defense against fungal and bacterial pathogens and efficient immune responses; however, uncontrolled monocyte-derived O2.- may contribute to chronic inflammation and tissue injury. We have previously identified several pathways that regulate the activity of NADPH oxidase in human monocytes; however, the receptor(s) responsible for the activation of NADPH oxidase in primary human monocytes have not yet been determined. This study shows that pattern recognition receptors, namely Dectin-1 and Complement Receptor 3, are essential for regulating NADPH oxidase activity in Zymosan-activated human monocytes. We show that TLR2 and TLR4 are not required for NADPH oxidase activation by Zymosan. In addition this study focuses on Dectin-1 downstream signaling and complex formation with intracellular signaling proteins. Our findings in human monocytes are supportive of the prior recognized role of Src and Syk tyrosine kinases in regulating Dectin-1-mediated ROS production in murine macrophages. Our data also shows that Src and Syk are tyrosine phosphorylated in Zymosan-treated cells and that they both regulate each others activity. Furthermore, we focused on Dectin-1 complex formation with intracellular signaling proteins including Syk, Src and PKCdelta, protein kinases that regulates NADPH oxidase activity in human monocytes. This is the first study to show the involvement of PKCdelta in Dectin-1 signaling. We found that the activity of PKCdelta is required for its own complex formation with Dectin-1 as well as Syk-Dectin-1 interaction. In contrast, Src and Syk inhibitors had no effect on PKCdelta association with Dectin-1. Blocking the activity of Src inhibited phospho-Syk/Dectin-1 complex formation which supports the role of Src in regulating Syk tyrosine phosphorylation/activation. Our data confirms that Dectin-1, a pattern recognition receptor, is a key player in the regulation of NADPH oxidase in Zymosan-activated human monocytes and we introduce PKCdelta as a novel player in Dectin-1 signaling.;To expand on this project, the study also includes data reporting an endogenous pathophysiological protein, osteopontin, as a novel ligand for Dectin-1. We show here that osteopontin induces the oxidative burst in monocytes through Dectin-1. We also show data supporting binding of osteopontin to Dectin-1.;Taken together, our study provides new insights into Dectin-1 ligands and downstream signaling in primary human monocytes and highlights novel signaling pathways utilized in these important cells. Our findings are relevant for understanding the regulation of NADPH oxidase in the innate immune response and in chronic inflammatory diseases.