Super-Low Dose Lipopolysaccharide Dysregulates Neutrophil Migratory Decision-Making

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.

Bistable Mathematical Model of Neutrophil Migratory Patterns After LPS-Induced Epigenetic Reprogramming

“…We are interested in determining the relationship between the receptor dynamics and migration pattern, and in quantifying the LPS dose resulting in neutrophils migration toward a pro-inflammatory chemoattractant, toward a pro-resolution chemoattractant, or in neutrophils dysregulation and oscillation (Fan and Malik, 2003 ; Liu et al, 2012 ; Byrne et al, 2014 ). The model will qualitatively match the experimental results of our previous work, where stimulation with a super-low concentration of LPS will result in greater BLT1 over FPR1, and stimulation with a high concentration of LPS will result in greater FPR1 over BLT1 (Boribong et al, 2019 ). We construct a model with bistable behavior, with the motif for bistability coming from the non-linear mutual inhibition of GRK2 and GRK5 (see Figure 1 ).…”

“…In previous research, we used a microfluidic competitive chemotaxis-chip device to measure the migratory decision-making process of dHL-60 cells, a model neutrophil cell line, 5 h after they were pre-challenged with super-low-dose (1 ng/mL) and high-dose (100 ng/mL) of LPS in the presence of two competing chemoattractants, LTB4 and fMLP (Boribong et al, 2019 ). Challenging the cells with a super-low dose of LPS resulted in fMLP/LTB4 ratio of 0.8672.…”

“…In previous work, we showed that training with high dose LPS (100 ng/mL) enhances neutrophil migration toward the end-target, bacterial derived, source signal fMLP. By contrast, training with super-low dose LPS (1 ng/mL) alters neutrophil migratory phenotypes, which either migrate toward the intermediary signal LTB 4 or become dysregulated and exhibit oscillatory migratory patterns (Jones et al, 2016 ; Boribong et al, 2019 ). While the empirical data shows that neutrophils trained with LPS change migratory phenotype, it does not give information on the molecular mechanisms responsible for the difference in behavior.…”

TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

“…To further unravel the mechanism and signaling pathways by which TRPM7 channel and/or kinase blockade alters neutrophil function, we analyzed the effect of the different TRPM7 moieties on cellular signaling. Typically, chemotaxis and migration of neutrophils is regulated by LPS-triggered pathways followed by NFκB activation ( 32 , 33 ). For cell motility and chemotaxis also Erk1-dependent signaling cascades are essential ( 34 ).…”

Microfluidic devices for neutrophil chemotaxis studies

“…Neutrophil migration process is complex and guided by multi-chemoattractants released from injured tissues. Boribong et al [71] designed a microfluidic competitive chemotaxis-chip (µC 3 ) to measure neutrophil migration in a competitive chemoattractant environment completing chemoattractant gradients in the central channel where cells were exposed (Fig. 3C).…”