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 IL13R?2. Further work has shown that Chi3L1-IL13R?2 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 IL13R?2, 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, IL13R?2-deficient mice demonstrated the same decrease in NETs. Thus, as hypothesized, Chi3L1-IL13R?2 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 IL13R?2. 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 IL13R?2 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-IL13R?2 signaling governs NET formation, and to reveal targets for therapeutic inhibition of NETosis during pneumonia.

Public Health Relevance

The aim of this grant is to determine if the immunomodulatory protein Chi3L1 affects the function of neutrophils, which are immune cells that help to fight bacteria during pneumonia, but also cause collateral damage to the lung. We will answer this question using mouse models of infection and assays on isolated neutrophils, with the ultimate goal of developing Chi3L1-based therapies that limit lung injury during pneumonia.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32HL154641-01
Application #
10068943
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Kalantari, Roya
Project Start
2021-01-15
Project End
Budget Start
2021-01-15
Budget End
Support Year
1
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520