Abstract
Neutrophils, in cooperation with serum, are vital gatekeepers of a host’s microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with “target” particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.
Highlights
Heat exposure of serum can inhibit some or all viral activity in the serum while leaving properties like the pH, antibody content, and ionic composition largely unchanged
Whereas the amount of complement fragments deposited by 56uC-treated serum onto the zymosan surface was diminished to the level of the negative control (Fig. 1A), the amount of detected IgG remained as high as the positive control at this temperature (Fig. 1B)
We confronted initially quiescent human neutrophils with zymosan particles that were incubated in buffers containing differentially heat-treated autologous serum
Summary
Heat exposure of serum can inhibit some or all viral activity in the serum while leaving properties like the pH, antibody content, and ionic composition largely unchanged Exploiting this effect, serum heat treatment is a common method to protect laboratory personnel against infectious agents like HIV, and has many other applications in clinical immunology or cell-culture biology [1,2]. Heat treatment of serum at 56uC for 30 minutes resulted in a 50% reduction of migration of human peripheral blood leukocyte in a Boyden chamber, which was attributed to the inhibition of casein- and C3a-dependent chemotactic pathways [10]. A systematic assessment of the effects of temperature-dependent serum heat treatment on both the chemotactic as well as the phagocytic activity of the same type of human immune cell is missing
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