PROJECT DESCRIPTION: Sepsis affects at least 1.7 million Americans annually, causing the death of 270,000 patients and including 30% of all hospital deaths. Unfortunately, there are no effective therapies for patients with sepsis and septic shock. Excessive neutrophil activation is a critical determinant of inflammation and tissue injury in sepsis. Therefore, targeting neutrophil activation, migration, and infiltration may be a rational strategy to reduce sepsis morbidity and mortality. During the last funding period of this MIRA project, we have made significant advances in the molecular mechanism of neutrophil and endothelial cell activation, their interaction, and neutrophil infiltration in the lungs in sepsis. We previously reported extracellular cold- inducible RNA-binding protein (eCIRP) as a new damage-associated molecular pattern molecule released in sepsis to increase inflammation and cause acute lung injury. To continue our MIRA program, we aim to explore the deep insights into neutrophils’ phenotypic and functional characteristics induced by eCIRP that may aggravate organ injury in sepsis. We have discovered a previously unknown neutrophil population with antigen-presenting, T-cell activating, and aged phenotypes, which we named antigen-presenting aged neutrophils (APANs). APANs produce IL-12, which polarizes Th1 cells to generate interferon-, thereby priming and inducing neutrophils to produce excessive neutrophil extracellular traps (NETs), causing further tissue injury. Adoptive transfer of APANs aggravated sepsis and increased the mortality of septic animals, suggesting that APANs play a critical role in sepsis pathobiology. However, APANs’ localization, induction, and effector functions in sepsis remain unknown, as does their contribution to the immune and cognitive dysfunction of sepsis survivors. We have recently predicted in silico and confirmed in vitro that our newly discovered stable RNA mimic A12 binds to eCIRP with high affinity, decreasing eCIRPs affinity for its receptor, inhibiting eCIRP’s ability to induce TNF release and to induce NETosis. Thus, A12 is a novel and potent eCIRP inhibitor with the potential to attenuate the detrimental effects of eCIRP-induced APANs in sepsis. As such, this renewal MIRA research program will address the following three key questions: 1) How does eCIRP induce APANs, and what are their effector functions? 2) How do APANs aggravate sepsis? 3) Does targeting eCIRP regulate APANs to mitigate sepsis? The proposed research will lead to a new direction for developing innovative therapeutics to treat patients suffering from sepsis and septic shock by preventing or modulating the novel hyperinflammatory neutrophil population of APANs.