Role of NOD1/NOD2 receptors in Fusobacterium nucleatum mediated NETosis

Polymorphonuclear neutrophils (PMNs) are indispensable in fighting infectious microbes by adopting various antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs). Although the role and importance of PMNs in periodontal disease are well established, the specific molecular mechanisms involved in NET formation are yet to be characterized. In the present study, we sought to determine the role of periodontal pathogen on NET formation by utilizing Fusobacterium nucleatum. Our data demonstrates that F. nucleatum activates neutrophils and induces robust NETosis in a time-dependent manner via the upregulation of the Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. Furthermore, CRISPR/Cas9 knockout of HL-60 cells and the use of ligands/inhibitors confirmed the involvement of NOD1 and NOD2 receptors in F. nucleatum-mediated NET formation. When treated with NOD1 and NOD2 inhibitors, we observed a significant downregulation of peptidylarginine deiminase 4 (PAD4) activity. In addition, neutrophils showed a significant increase and decrease of myeloperoxidase (MPO) and neutrophil elastase (NE) when treated with NOD1/NOD2 ligands and inhibitors, respectively. Taken together, CRISPR/Cas9 knockout of NOD1/NOD2 HL-60 cells and inhibitors of NOD signaling confirmed the role of NLRs in F. nucleatum-mediated NETosis. Our data demonstrates an important pathway linking NOD1 and NOD2 to NETosis by F. nucleatum, a prominent microbe in periodontal biofilms. This is the first study to elucidate the role of NOD-like receptors in NETosis and their downstream signaling network.