Measuring proteome of 1,000 neutrophils directly isolated from inflammatory sites

Neutrophils are typically the earliest and most frequent inflammatory cells in sterile inflammation. However, to achieve a thorough comprehension of their involvement in pathophysiology, neutrophils must be characterized molecularly either as isolated cells or remaining on individual inflammatory site. While these cells have been extensively studied in different inflammatory situations using transcriptome analyses, this does not fully represent their molecular state due to their post-mitotic nature. High-throughput proteomic technologies can fill the gap by providing a comprehensive insight into their structural and functional architecture. The currently available approach for neutrophil proteomics analyzed millions of cells, which limits the analysis of scant population of these cells at individual inflammatory site, especially in case of chronic inflammation or steady-state infiltration. Although recent proteomics approaches are sensitive enough to analyze very low input samples, even at single-cell resolution, their application in understanding neutrophils (ten times lesser cell volume than HeLa cells) remains unexplored, presenting a necessity for further investigation.

We here present a pipeline for the proteome analysis of down to 1,000 human (~60ng) and mouse (~40ng) neutrophils isolated directly from inflammatory sites. This approach enabled identification of ~4800 and ~3400 proteins from 1,000 isolated circulating murine and human neutrophils with high reproducibility and 102-108 copies/cell. We also provide a searchable database for human and murine protein copy numbers, a few of which were also validated with quantitative flow cytometry approach. Our analysis demonstrated the activation of circulatory neutrophils upon stroke induction compared to sham controls and naive mice. These proteome changes were also observed in circulating neutrophils isolated from human patients compared to healthy controls. Further we found that post-stroke brain-infiltrated neutrophils exhibited a strongly distinct proteome compared to circulatory neutrophils in the same host. Brain-infiltrated neutrophils showed a metabolic switch towards increased mitochondrial activity and ROS-production to adapt glucose-deprived condition. Our approach was also efficient to demonstrate a functional shift of neutrophils transmigrated to oral cavity upon transient oral inflammation.

Overall, this method represents a significant leap in intricate neutrophil proteome characterization, even from minute samples, and holds great promise for expanding our understanding of neutrophil biology in health and disease. We further envisage to investigate neutrophil at single-cell resolution to reveal the heterogeneity and functional diversity of these intriguing immune cells.