Abstract
Neutrophils are the first responders to infection and play a pivotal role in many inflammatory diseases, including sepsis. Recent studies have shown that lipopolysaccharide (LPS), a classical pattern recognition molecule, dynamically programs innate immune responses. In this study, we show that pre-treatment with super-low levels of LPS [1 ng/mL] significantly dysregulate neutrophil migratory phenotypes, including spontaneous migration and altering neutrophil decision-making. To quantify neutrophil migratory decision-making with single-cell resolution, we developed a novel microfluidic competitive chemotaxis-chip (μC3) that exposes cells in a central channel to competing chemoattractant gradients. In this reductionist approach, we use two chemoattractants: a pro-resolution (N-Formyl-Met-Leu-Phe, fMLP) and pro-inflammatory (Leukotriene B4, LTB4) chemoattractant to model how a neutrophil makes a decision to move toward an end target chemoattractant (e.g., bacterial infection) vs. an intermediary chemoattractant (e.g., inflammatory signal). We demonstrate that naïve neutrophils migrate toward the primary end target signal in higher percentages than toward the secondary intermediary signal. As expected, we found that training with high dose LPS [100 ng/mL] influences a higher percentage of neutrophils to migrate toward the end target signal, while reducing the percentage of neutrophils that migrate toward the intermediary signal. Surprisingly, super-low dose LPS [1 ng/mL] significantly changes the ratios of migrating cells and an increased percentage of cells migrate toward the intermediary signal. Significantly, there was also an increase in the numbers of spontaneously migrating neutrophils after treatment with super-low dose LPS. These results shed light onto the directional migratory decision-making of neutrophils exposed to inflammatory training signals. Understanding these mechanisms may lead to the development of pro-resolution therapies that correct the neutrophil compass and reduce off-target organ damage.
Highlights
Recent studies suggest that neutrophils are a key player in the development of sepsis, the current leading cause of death in hospitals [1,2,3,4]
We report a novel microfluidic competitive chemotaxis-chip that enables the measurement of neutrophil migration in the presence of dual gradients (Figures 1, 2A, Table 1)
We have designed a novel microfluidic competitive chemotaxischip that enables the formation of a dual, competitive chemoattractant gradient
Summary
Recent studies suggest that neutrophils are a key player in the development of sepsis, the current leading cause of death in hospitals [1,2,3,4]. Neutrophils are the most abundant white blood cells (∼60%) and are the first responders to infection and inflammation. Neutrophils can navigate effectively through complex tissue microenvironments toward pathogens and play a critical role in controlling infection under normal conditions [5]. Neutrophils migrate and accumulate in healthy organs instead of migrating toward the infection [2]. Lack of control of the tissue microenvironment and the complexity of tracking the trajectories of immune cells in vivo prohibits the study of cell migratory decision-making
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