Abstract
Neutrophil recruitment guided by chemotactic cues is a central event in host defense against infection and tissue injury. While the mechanisms underlying neutrophil chemotaxis have been extensively studied, these are just recently being addressed by using high-content approaches or surface-bound chemotactic gradients (haptotaxis) in vitro. Here, we report a haptotaxis assay, based on the classic under-agarose assay, which combines an optical patterning technique to generate surface-bound formyl peptide gradients as well as an automated imaging and analysis of a large number of migration trajectories. We show that human neutrophils migrate on covalently-bound formyl-peptide gradients, which influence the speed and frequency of neutrophil penetration under the agarose. Analysis revealed that neutrophils migrating on surface-bound patterns accumulate in the region of the highest peptide concentration, thereby mimicking in vivo events. We propose the use of a chemotactic precision index, gyration tensors and neutrophil penetration rate for characterizing haptotaxis. This high-content assay provides a simple approach that can be applied for studying molecular mechanisms underlying haptotaxis on user-defined gradient shape.
Highlights
Neutrophils are rapidly recruited into infected or injured tissues where they play a pivotal role in host defense against invading pathogens and in wound healing[1]
We have developed a framework for a high-content haptotaxis assay based on the well-established under-agarose assay (Fig. 1)
Alignment of gradients to the edge of the cell seeding well and time-lapse imaging allowed the analysis of a large number of migrating neutrophils under diverse conditions
Summary
Neutrophils are rapidly recruited into infected or injured tissues where they play a pivotal role in host defense against invading pathogens and in wound healing[1]. Since neutrophils encounter many chemoattractants, they prioritize and integrate chemotactic cues assuring preferential migration toward the inflamed site[4, 5] Neutrophils recognize chemokines, such as CXCL8 (IL-8), which are thought to be bound to the surface of endothelial cells, forming a stable gradient to guide neutrophils toward the site of inflammation[6,7,8,9]. Formyl peptides of bacterial or mitochondrial origin[12] through the formyl peptide receptors 1 (FPR1) and FPR2/lipoxin A4 receptor (FPR2/ALX) activate the p38 mitogen-activated protein kinase pathway, which dominates over the phosphoinositide 3-kinase pathway[10] and direct neutrophils to arrive at the inflamed site[3] Based on these characteristics, CXCL8 is classified as an intermediate chemotactic cue, whereas formyl peptides, such as N-formyl-Met-Leu-Phe (fMLF) a natural peptide or its synthetic analogs such as formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fNLFNTK) are end-target cues[4, 13, 14]. Combining the functional data with mathematical analysis evaluating chemotaxis indices currently in use, we propose penetration rate as a novel index for quantification of haptotaxis
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