3D Displacement Fields of Lipopolysaccharide Activated Neutrophil Chemotactic Behavior

Undergraduate #373
Discipline: Technology and Engineering
Subcategory: Biomedical Engineering

Rodolfo Amezcua - California State University, Long Beach

Sepsis is a systematic inflammatory immune response to infection that strikes more than twenty million people worldwide and claims over 250,000 American lives per year. Under severe conditions this syndrome is commonly associated with organ dysfunction, and is postulated that excessive neutrophil recruitment and their altered chemotactic response during sepsis plays a critical role. Thus modification of neutrophil function is of interest because of its therapeutic potential for septic patients; however, the cellular mechanisms responsible for this altered chemotactic behavior are not well understood. Under septic conditions, neutrophils are exposed to both N-formyl-methionine-leucine-phenylalanine (fMLP) and inflammatory response mediator chemoattractants, and have shown to exhibit intracellular signaling hierarchy of these competing gradients [1]. I hypothesize lipopolysaccharide (LPS) activated neutrophils subjected to a competing fMLP and interlekin-8 gradient will generate smaller 3D displacement fields towards fMLP than naive neutrophils due to activation of both G protein-coupled receptor (GPCR) and Toll-like receptor 4 (TRL4) pathways. By using a previously developed dual gradient chemotaxis chamber system capable of producing reliable linear gradients across 3D collagen gels [2], we were able to induce neutrophil chemotaxis. To determine 3D displacement fields, fluorescent microspheres are embedded in the collage gel. The displacement of these beads is tracked via confocal time-lapse microscopy and a digital volume correlation algorithm [3]. Our results show LPS activated neutrophils maintain a signaling hierarchy between fMLP and IL-8, but experience a reduction in directionality towards fMLP. These results will serve for further quantitative measures that consider kinematic movement such as contractility, mean volume change, and rotation [4].
References: [1] B. Heit, S. Tavener, E. Raharjo, and P. Kubes. An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. The Journal of cell biology, 159(1):91-102, Oct 14 2002.
[2] D.A Stout, J. Toyjanova, and Christian Franck. Planar gradient diffusion system to investigate chemotaxis in a 3d collagen matrix. JoVE, (100):e52948-e52948, 2015.
[3] E. Bar-Kochba, J. Toyjanova, E. Andrews, K.S. Kim, and C. Franck. A fast iterative digital volume correlation algorithm for large deformations. Experimental Mechanics, 55(1):261{274, 2015.
[4] D.A. Stout, E. Bar-Kochba, J.B Estrada, J. Toyjanova, H. Kesari, J.S. Reichner, and C. Franck. Mean deformation metrics for quantifying 3d cell matrix interactions without requiring information about matrix material properties. Proceedings of the National Academy of Sciences, 113(11):2898-2903, 2016.

Funder Acknowledgement(s): This study was supported, in part, by the Louis Stokes Alliance for Minority Participation program.

Faculty Advisor: David Stout, david.stout@csulb.edu

Role: I conducted experiments of neutrophils in our chemotaxis system and acquired confocal microscopy volumetric image data. I took the image data and used the digital volume correlation algorithm described to determine 3D displacement fields.