PROJECT SUMMARY Pneumonia is a common and deadly disease, claiming four million lives per year worldwide. Immune defense in pneumonia primarily depends on neutrophils, which kill pathogens via phagocytosis or release of neutrophil extracellular traps (NETs). NETs are weblike structures of chromatin complexed with microbicidal proteins that help eliminate bacteria during pneumonia, but also damage host lung tissue. In the clinical setting, where antibiotic use ensures bacterial clearance, NETs are largely pathologic. However, there are no therapies that target NET formation. Chi3L1 is an evolutionarily ancient, but poorly understood lectin that binds to the chitin cell walls of fungi, nematodes, and insects. Previous studies have shown that Chi3L1 promotes defense against bacteria during lung infection through binding to the cytokine receptor IL13Rα2. Further work has shown that Chi3L1-IL13Rα2 signaling activates AKT and ERK pathways in numerous cell types during lung injury to prevent cell death. In neutrophils, AKT and ERK signaling similarly modulate cell death pathways, inhibiting apoptosis and stimulating NETosis. Therefore, we speculated that Chi3L1 could contribute to bacterial clearance in pneumonia by activating IL13Rα2, leading to downstream activation of AKT and ERK, inhibition of apoptosis, and formation of bactericidal NETs. To test this prediction, we induced pneumonia in mice with Pseudomonas aeruginosa, a nosocomial bacterial pathogen. In preliminary experiments, we showed that Chi3L1-deficient mice have higher bacterial burdens, and that this was not explained by impaired neutrophil recruitment. Instead, Chi3L1-deficient mice showed decreased NET formation. Furthermore, IL13Rα2-deficient mice demonstrated the same decrease in NETs. Thus, as hypothesized, Chi3L1-IL13Rα2 signaling does regulate NETosis. However, it remains unclear how P. aeruginosa, a bacterium surrounded by peptidoglycan (PG) – not a chitin cell wall – could promote NETosis via the chitin-binding lectin Chi3L1. Structural similarities between PG and chitin, along with the existence of protein families capable of recognizing both PG and chitin suggest a possible answer: that Chi3L1 binds to PG as well. Thus, we hypothesize that Chi3L1 binds to bacterial cell walls during pneumonia and promotes NETosis via IL13Rα2. To test this hypothesis, we propose the following two aims. 1) Using well-characterized in vitro assays for NET formation, we will evaluate whether Chi3L1 directly activates IL13Rα2 in isolated neutrophils. We will also test whether Chi3L1 stimulates NETosis via AKT and ERK, and whether it inhibits apoptosis. 2) Next, we will use flow cytometry and microscopy to test if Chi3L1 is able to bind peptidoglycan. Finally, using our in vitro assay for NET formation, we will test if Chi3L1 binding to its substrate modulates its NETogenic effects. We believe these studies have potential to produce important insight into how Chi3L1-IL13Rα2 signaling governs NET form...