Clustering analyses of murine bone marrow-derived neutrophils reveal a phenotypic heterogeneity that can respond differentially to stimulation.

Neutrophils are granulocytic cells produced in the bone marrow from a granulocytic progenitor cell. During infection, the production of chemokines and cytokines induces the recruitment of neutrophils to the infected tissue to promote the clearance of microbial pathogens. Several studies have shown that different subpopulations of neutrophils can be identified during infection. However, no previous studies evaluated subpopulations of neutrophils purified from the bone marrow (BM), which are typically used to study the biology of these cells based on the assumption that the neutrophil population is homogeneous. In the present study, responses of purified BM-derived neutrophils to various stimuli such as PMA, LPS, and were evaluated using flow cytometry and bh-SNE analyses. Further, neutrophil population heterogeneity was assessed by clustering analyses. Our data suggest that purified BM-derived neutrophils were not a homogeneous cell population and were clustered into 12 subsets, each displaying a unique marker profile, where CD11b and CD62L emerged as pivotal markers for neutrophil function. Importantly, the subsets responded differentially to each stimulus, suggesting a nuanced activation pattern. Changes in biomarker expression were analyzed via Ingenuity Pathway Analysis (IPA) to unravel functional implications of the identified clusters, revealing subsets associated with different neutrophil functions, such as "Migration of neutrophils" or "Phagocytosis in neutrophils". This study contributes to understanding the diversity of purified BM-derived neutrophils and the implications of using these cellular preparations to raise conclusions about the functionality of these cells in various infection models.