Sources and significance of neutrophil heterogeneity

Abstract

The neutrophil is a white blood cell indispensable for the human defense against pathogenic bacteria and fungi. However, if this cell aims its antimicrobial weapons at healthy tissue, it will afflict severe damage to the human body. Hence, where, when, and how neutrophils are activated is finely balanced to keep the body healthy. It has long been thought that every neutrophil is similar and that every neutrophil responds similarly to an activating signal. However, recent research has discovered various subtypes of the neutrophil. The research presented in this thesis investigated the sources and significance of neutrophil heterogeneity. In Chapter 2 we described a novel assay to assess heterogeneity in the antibacterial capacity of neutrophils. In this in vitro assay, neutrophils were co-cultured with Staphylococcus aureus bacteria in a three-dimensional fibrin network to better mimic the in vivo conditions of antibacterial defense. A defect in antibacterial defense was easily detected as a decreased capacity to contain bacterial growth. In Chapter 3 I quantified heterogeneity in the pH reached inside the neutrophil phagolysosome after phagocytosis. The intraphagosomal pH level is known to directly influence microbial survival and to shape the activity of various antimicrobial molecules in the phagolysosome. Our results suggested that the neutrophil seems able to tune the intraphagosomal defense mechanisms to match the heterogeneity amongst pathogens. In Chapter 4 we investigated whether heterogeneous neutrophil subsets exhibited heterogeneous migration capacity. The three neutrophil subsets found in the circulation upon acute systemic inflammation display functional and phenotypical heterogeneity. The CD16low, banded subset is characterized by superior antibacterial capacity but also by an immature nucleus with incomplete segmentation. We hypothesized that this relatively bulky nucleus would hamper neutrophil migration. However, all three subsets migrated with similar efficiency in in vitro models of transendothelial or interstitial migration. Notably, the CD62Llow, hypersegmented subset displayed reduced rear release and deposited more membrane vesicles. In Chapter 5 we summarized several methods to prepare laboratory mice for intravital microscopy. Intravital microscopy is an important tool to study dynamic cellular processes in their natural environment, for example the neutrophil response at a site of infection. In Chapter 6 we considered whether the source of circulatory neutrophil heterogeneity during acute inflammation may be found in neutrophil recruitment from heterogeneous tissue environments. A murine endotoxemia model was used to study neutrophil heterogeneity throughout different tissue sites and neutrophil redistribution upon acute inflammation. We confirmed that acute inflammation induced a banded neutrophil subset in the circulation, but we did not distinguish a CD62Llow neutrophil subset comparable to the CD62Llow, hypersegmented neutrophil subset described in the human endotoxemia model. In Chapter 7 we studied the kinetics of neutrophil progenitor proliferation and maturation in human bone marrow. The new knowledge obtained in this research is expected to contribute to future therapies that activate or inhibit a specific neutrophil subtype at a specific location or time point, to treat a range of infections and immune diseases.