Abstract
Neutrophils are known to be the first responders to infection or injury. However, as inflammation progresses, other leukocytes become increasingly important in inflammation propagation, tissue reconstruction, and inflammation resolution. In recent years, there has been an increase in publications that analyze neutrophil behavior in vitro, but there remains a gap in the literature for in vitro technologies that enable quantitatively measuring interactions between different types of human leukocytes. Here, we used an in vitro platform that mimics inflammation by inducing neutrophil swarming to analyze the behavior of various leukocytes in a swarming setting. Using human peripheral blood leukocytes isolated directly from whole blood, we found that myeloid cells and lymphoid cells had different migratory behaviors. Myeloid cells, which are predominately neutrophils, exhibited swarming behavior. This behavior was not seen with lymphoid cells. We perturbed the peripheral blood leukocyte system by adding exogenous leukotriene B4 (LTB4) to the medium. Notably, only the myeloid cell compartment was significantly changed by the addition of LTB4. Additionally, LTB4 had no significant impact on myeloid cell migration during the recruitment phase of swarming. To further investigate the myeloid cell compartment, we isolated neutrophils and monocytes to analyze their interaction on the platform. We found that neutrophils increase monocyte migration toward the bioparticle clusters, as measured through speed, chemotactic index, track straightness, and swarm size. These results were confirmed with in vivo mouse experiments, where monocyte accumulation only occurred when neutrophils were present. Additionally, we found that both neutrophils and monocytes release the monocyte chemoattractant proteins CCL2 and CCL3 in the presence of Staphylococcus aureus bioparticles. Furthermore, extracellular vesicles from swarming neutrophils caused monocyte activation. These findings suggest that neutrophils play an essential role in the onset of inflammation not only by sealing off the site of infection or injury, but also by recruiting additional leukocytes to the site.
Highlights
Neutrophils are known to be the first responders to infection or injury in the body [1]
Upon adding the peripheral blood leukocytes (PBLs) to our in vitro microarray platform, we observed swarming-like behavior among the PBLs (Figure 1A, Video S1), similar to neutrophil swarming as previously reported [1, 3, 7]
We used the principle that lymphoid cells tend to have large nuclei and very little cytoplasm, while myeloid cells have comparatively higher cytoplasm to develop a method for distinguishing them
Summary
Neutrophils are known to be the first responders to infection or injury in the body [1]. Upon processing the chemotactic signal, neutrophils become activated This begins the recruitment ( called growth or exponential) phase of the swarm, where the activated neutrophils release a myriad of lipid mediators, cytokines, and extracellular vesicles (EVs) that activate and direct the migration of additional neutrophils, eliciting exponential growth of the swarm [3, 7]. Neutrophils migrate in and out of the swarm during this stage, but the overall swarm size remains approximately constant This phase marks pathogen clearance, extracellular matrix digestion, and tissue reconstruction [2, 4]. Cell debris is cleared away, inflammatory chemokines are degraded, and immune cells either undergo reverse migration or apoptosis [4], though this phase is often not explored in detail in swarming studies [1, 3, 5,6,7,8]
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