Project Summary Our work demonstrates that peptidoglycan (PGN), a major component of the cell wall of all Gram-positive bacteria, promotes inflammation and coagulation. Baboons responded to in vivo PGN challenge with features of systemic inflammation and disseminated intravascular coagulopathy (DIC) similar to those seen in patients with inhalation anthrax. We found evidence of activation of both the intrinsic and extrinsic coagulation pathways. In vitro, we found PGN stimulated robust cytokine production in human innate immune cells and prothrombinase activity in human platelets exposed to highly purified polymeric PGN derived from B. anthracis or Staphylococcus aureus. Innate immune cell responses required PGN recognition by surface Fcγ receptors, phagocytosis, digestion in lysosomes, and stimulation of cytoplasmic NOD sensors. In the last iteration of this grant, we showed that these processes were dependent on human serum opsonins, IgG and serum amyloid P. We also showed that PGN-stimulated human monocytes expressed tissue factor (TF), an initiator of the extrinsic coagulation pathway, in part by virtue of the proinflammatory cytokines. This project will provide mechanistic insight into PGN-stimulated pathologies. First, we will test whether and how the anthrax toxins affect the immune system activation events we have documented to date and listed above. Second, we will identify the pathways that regulate PGN-induced activation of the intrinsic coagulation pathway in vivo. Lastly, we will apply existing biological inhibitors to each pathway (intrinsic, extrinsic coagulation and complement) to ask which of these contributes most to the pathology, organ failure and death in PGN-challenged animals. The results will greatly inform treatment options for patients with Gram-positive bacteremia.