Neutrophils are the first line of defense in the cellular inflammatory response, and studying their behavior can lead to strategies for treating inflammatory disorders. Using quantitative tools and assays, we propose to investigate the fundamental mechano-chemical processes of adhesion and motility that control neutrophil extravasation from blood into tissue.
In aim 1, we will simulate integrin-mediated firm adhesion of a neutrophil by merging Adhesive Dynamics - a mechanically accurate method for modeling adhesion - with a stochastic simulation of inside-out signal transduction. These models will predict how the rate and extent of neutrophil firm adhesion are controlled by a neutrophil's internal molecular machinery. Furthermore, we will use stochastic signaling methods to analyze experiments performed in Project 3 in which a neutrophil held in a pipette is stimulated by impingement with a moleculariy-coated bead.
In aim 2, we will test predictions from the modeling in aim 1 by performing flow chamber adhesion experiments in which external and internal variables are systematically varied, to confirm that our quantitative understanding of the molecular control of adhesion is correct. Working closely with Projects 2 and 5, we will use pharmacological intervention, antibodies, and sIRNA technology to adjust neutrophil components and examine their effect on the transition to firm adhesion.
In aim 3, we will work with Core C and use traction force microscopy to examine the motility of neutrophils in well-defined gradients of chemoattractant. We have built a chamber that combines microfluidics, to impose well-defined chemoattractant gradients across cells, with traction force microscopy. The goal is to understand how speed and direction in neutrophil motility is related to force generation, and to understand how the molecular components in neutrophils control contractility. Previous work has shown that neutrophil directional motion is accompanied by strong loci of contractile traction stress in the uropod. Using pharmacological inhibition, antibodies and sIRNA technology, we will measure how key molecular players affect neutrophil polarity, the generation of traction stresses, and cell motion. In summary, our work will provide fundamental insights as to how molecular components control neutrophil function in inflammation.
An understanding of the fundamental principles governing the action of neutrophils would be helpful for designing therapies to treat inflammatory diseases. Further, inflammation is connected to other diseases such as cancer, understanding and manipulating neutrophil behavior would have wide impact across the health sciences.
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