Traction Force Microscopy of Human Neutrophils During Critical Illness

Abstract

Mapping of tractional forces produced by human neutrophils enables a precise understanding of mechanisms responsible for well-regulated cell motility. In collaboration with the Henann Lab at Brown University's Department of Solid Mechanics and the Franck Lab at University of Wisconsin's College of Engineering, we have developed a novel method of quantifying cellular force generation. Previously, Traction Force Microscopy (TFM) methods have required a 3D imaging modality to capture 3D forces. Here, we demonstrate a new method that can capture 3D forces using only a 2D imaging modality. Therefore, both forces in-plane with the substrate as well as forces out-of-plane to the substrate can now be seen with a standard epifluorescent microscope. Our technique involves embedding a single layer of fiducial markers under the surface of a soft fibronectin-coated polyacrylamide gel, followed by our previously published single particle tracking algorithm (T-PT) and a novel finite element method to quantify forces from substrate displacement. To validate this system, PMNs were obtained from healthy donors with ex vivo activation and from septic donors. Sepsis is a potentially fatal systemic inflammatory response to infection that can progress to multiorgan system failure. We hypothesize that ex vivo activation of PMNs from healthy donors and different substrate stiffnesses will lead to differential force production. On soft substrates (1kPa), when comparing PMN force generation from healthy and septic donors, we found that neutrophils from septic donors produce greater forces. However, ex vivo activation of PMNs from healthy donors with Lipopolysaccharide (LPS) had no effect, while a stimulant cocktail of LPS, Interferon Gamma (IFN-g), and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) decreased force generation. Further, ex vivo activation with Phorbol Myristate Acetate (PMA) and N-Formylmethionine-leucyl-phenylalanine (fMLP) increased force generation. However, on stiff substrates (10kPa) LPS, PMA, and fMLP all had no effect on force generation, while the stimulant cocktail decreased total forces. Taken together with our novel TFM technique, these findings indicate that PMNs from septic patients are abnormal in their spatiotemporal generation of 3D forces. Further, secondary stimulation leads to differential dysregulation of force generation that is also mechanosensitive to substrate stiffness. Because there is still the need to further elucidate the deleterious molecular mechanisms during PMN-mediated sepsis, our methods will help bridge the divide between understanding dysregulation both in its biochemical and its mechanical interactions with the microenvironment.