Migration and resolution, lung microenvironment and mechanisms: examining the diverse responses of neutrophils during S. pneumoniae pneumonia and acute lung injury

Bacterial pneumonias remain important clinical problems with major morbidity and mortality. S. pneumoniae is the leading cause of community-acquired pneumonias and an important cause of ARDS and post-viral bacterial pneumonias. The lung's immune response is critical to clearance of S. pneumoniae, repair of the lung tissue, and return to homeostasis. Our studies have addressed many aspects of neutrophil kinetics and function and document the numerous ways in which neutrophils vary and can be categorized, based on cytokine production, surface markers, transcriptomes, age, or many other criteria, each of which describes the range of functions that neutrophils can perform. This proposal focuses on the mechanisms underlying the process through which neutrophils function to identify and repair the infection and injury and/or to further injure the lungs. Our overall working hypothesis is that during the immune response, neutrophils undergo changes in their transcriptomes that regulate their function and that the unique structure of the lungs creates microenvironments within the parenchyma and the alveoli/small airways that regulate the phenotype of neutrophils. We further postulate that spatial and temporal features of the innate immune response contribute to this phenotype. Aim 1 will determine the changes that neutrophils undergo as they are recruited and carry out their functions during S. pneumoniae pneumonia and the biological importance of these changes. We will determine the changes in the transcriptome of neutrophils in the systemic blood, pulmonary capillary blood, lung parenchyma and alveoli/airways at 6h, 18h, 2d and 4d after inoculation, identifying both spatial and temporal changes in neutrophil transcriptomes using scRNAseq/CITE-seq. We postulate that heterogeneity in endothelial and alveolar Type 1 and 2 cell transcriptomes will describe niches of migration. Protein expression of identified neutrophil proteins will be assessed using mass cytometry. The mechanisms underlying the phenotypes of neutrophils expressing IFNγ, IL-1α and/or TNF and of SiglecF+ neutrophils will be studied. Aim 2 will determine the changes in the lung microenvironment during the development and resolution of S. pneumoniae-induced inflammation and injury. The spatial transcriptome of alveoli and bronchioles/bronchi will be determined 6h, 18h, 2d and 4d after inoculation of S. pneumoniae. The effect of signaling induced by Type I and II IFNs, IL-1α/β and TNF on neutrophil transcriptomes and function will be identified. The hypothesis that the oxidative microenvironment experienced by myeloid cells contributes to transcriptomic heterogeneity and function will be tested. These studies will determine the changes in neutrophils as they function during pneumonia, will compare neutrophils defined by particular criteria, and will shed light on the changes and contributions of the lung microenvironment as inflammation develops and resolves. They will reveal novel context-dependent relationships and interactomes, providing insight into neutrophil function in the innate immune response and potential therapeutic opportunities for bacterial pneumonia. Project Number: 5R01HL175463-02 | Fiscal Year: 2026 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Claire Doerschuk | Institution: UNIV OF NORTH CAROLINA CHAPEL HILL, CHAPEL HILL, NC | Award Amount: $744,784 | Activity Code: R01 | Study Section: Lung Immunology and Infection Study Section[LII] View on NIH RePORTER: https://reporter.nih.gov/project-details/5R01HL17546302