Multi-Omic Analysis of Peripheral Circulating Neutrophils and the Effects of Transmigration.

Neutrophils, the most prevalent leukocytes in circulation, are integral to initiating and resolving inflammatory responses. While traditionally viewed as a homogeneous population, emerging studies have revealed significant phenotypic heterogeneity and functional versatility within the neutrophil population. However, developing a consensus on neutrophil markers for precise and comprehensive identification of neutrophil phenotypes at baseline is essential to deepen our understanding of their biology. In this study, we employed a combination of single-cell RNA sequencing (scRNA-seq) and advanced multiplex flow cytometry analyses to examine the transcriptional and proteomic landscapes of human neutrophils in healthy peripheral blood while employing functional assays to assess their migratory capabilities and phenotypic alterations post-transmigration. Through transcriptional and proteomic analyses, our findings identify three distinct neutrophil profiles, each characterized by specific effector functions and migratory patterns. These subsets demonstrated distinction in the expression of markers related to neutrophil maturation, aging, immune modulation, and inflammatory responses, suggesting a complex spectrum of neutrophil states in circulation and post-vascular transmigration. Our findings advance the field's understanding of neutrophil heterogeneity and offer a more nuanced view of their roles in homeostasis and the immune response.