Characterization of lung interstitial neutrophils

Project Summary Neutrophils are major players in the innate immune system, known for their role in fighting infections by engulfing and destroying invading pathogens, including those in the lungs. Neutrophil heterogeneity is a key component in innate immune regulation. Distinct mature neutrophil subpopulations, defined by their signature gene expression profiles, phenotypes, behaviors, and functional states, exist even prior to encountering pathogens. Understanding the unique features and origins of these functional subpopulations will enable the effective and safe manipulation of their specific functions for personalized medicine. The objective of this proposed project is to investigate the origin and characterize the functional state of the lung interstitial neutrophil (LIN) subset. Preliminary scRNA-Seq analysis revealed a tightly regulated neutrophil reprogramming trajectory during lung infection and inflammation, based on gene expression patterns. The preliminary studies identified the functional heterogeneity of neutrophils across three different lung compartments: the vasculature, the interstitium, and the alveoli, and discovered that neutrophils lodged in the interstitium for an extended period before migrating to the alveoli. Based on these findings, it was hypothesized that transcriptional reprogramming leads to the formation of a specialized LIN subset defined by its unique gene expression profile. These neutrophils are highly specialized, with significant bactericidal activity, making the lung interstitium a primary site for bacterial elimination. Accordingly, expanding the LIN population could be a viable therapeutic strategy for enhancing neutrophil-mediated host defense, while potentially detrimental in sterile inflammation-induced acute lung injury. To advance the understanding of the function, fate, pathophysiological role, and origin of LINs and explore their therapeutic potential, three specific aims will be investigated: Aim 1 will characterize the functions and fate of LINs. In addition, neutrophil- mediated bacterial killing in the interstitium will be visualized and confirmed by fluorescent imaging. Aim 2 will elucidate the pathophysiological role of LINs in lung infection and inflammation. The LIN population will be expanded by inhibiting CCR2-mediated neutrophil transepithelial migration, and its effect on host bactericidal activity and lung injury will be tested in a neutropenia-related bacterial pneumonia model and an acid aspiration-induced acute lung injury (AA-ALI) model. Aim 3 will further investigate the microenvironment- induced transcriptional reprogramming that confers specialized functions to LINs. Additional scRNA-Seq analyses will be performed to gain a deeper understanding of neutrophil reprogramming in the interstitium and to reveal potential temporal and stimulus-specific reprogramming schemes. Together, the results from this study will establish LINs as a specialized neutrophil subpopulation with unique phenotype and function, and as a novel therapeutic target for treating lung infections and inflammation.