OC 46.4 - Neutrophil extracellular traps induced by ex vivo trauma plasma mediate endotheliopathy

Monday, June 24, 2024

15:30 – 15:45 ICT

Room: 111 A-C

Presenting Author(s)

Benjamin W. Stocker, MD

General Surgery Resident
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Denver, Colorado, United States

Co-Author(s)

IL

Ian W. LaCroix

PhD Student
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Biochemistry and Molecular Genetics
Aurora, Colorado, United States
MK

Marguerite Kelher

Research Instructor
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States
LG

Lauren T. Gallagher

General Surgery Resident
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States
SM

Sanchayita Mitra

Research Instructor
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States
BR

Benjamin J. Ramser

General Surgery Resident
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States
WH

William Hallas

General Surgery Resident
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States
OT

Otto N. Thielen, MD

General Surgery Resident
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States
CE

Christopher Erickson

Postdoctoral Researcher
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Biochemistry and Molecular Genetics
Aurora, Colorado, United States
Ernest E. Moore, MD

Director of Research
Ernest E Moore Shock Trauma Center ay Denver Health
Denver, Colorado, United States
KH

Kirk C. Hansen

Professor
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Biochemistry and Molecular Genetics
Aurora, Colorado, United States
AD

Angelo D'Alessandro

Professor
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Biochemistry and Molecular Genetics
Aurora, Colorado, United States
CS

Christopher C. Silliman

Professor
Vitalant Research Institute
Aurora, Colorado, United States
MC

Mitchell J. Cohen

Professor
University of Colorado Anschutz Medical Campus, School of Medicine, Department of Surgery
Aurora, Colorado, United States

Background: Neutrophil extracellular traps (NETs), networks of decondensed chromatin and neutrophil enzymes, are mediators of infectious and sterile inflammation. Markers of NETs formation (NETosis) and endotheliopathy are increased following trauma, but actual NETosis and resultant endothelial dysfunction has not been directly demonstrated post-injury.

Aims: We investigated if NETs are induced after trauma and would cause endotheliopathy. We hypothesized that ex vivo trauma plasma would induce NETosis and subsequent endotheliopathy.

Methods: Plasma samples were collected on admission from minimally injured (MI:injury severity score(ISS) < 15, base deficit(BD) < 6) and severely injured patients (SI:ISS≥15, BD≥6). Isolated healthy neutrophils (PMNs) were exposed to either healthy or trauma plasma. Immunofluorescence microscopy was used to confirm NETosis. Soluble PMN releasates (SNT) and NETs fractions were isolated from PMN culture. Permeability of cultured endothelial cells exposed to isolated fractions was measured by Electric Cell-substrate Impedance Sensing (ECIS) and compared using area under the curve (AUC). Decreased AUC signifies increased permeability and models endotheliopathy. This study was approved by review board with waiver of consent. Funded by NIGMS, NIH, and Department of Defense.

Results: Immunofluorescence confirmed NETosis and greater stimulation of NETosis by SI plasma than other plasmas ex vivo (Figure 1). SNT from SI plasma-stimulated PMNs caused significantly greater endotheliopathy than healthy and MI SNT (Figure 2 A-B). NETs stimulated by SI plasma caused significantly greater endotheliopathy compared to healthy plasma and a trend towards greater endotheliopathy than MI plasma (Figure 2 C-D).

Conclusion(s): This study provides the first direct evidence that the trauma milieu found in ex vivo plasma stimulates NETosis which causes breakdown of the endothelial barrier. The plasma from severely injured patients caused the release of soluble factors and NETs from PMNs which induced significant endotheliopathy. Future work will include quantifying the level of NETosis and leveraging proteomics to characterize the unique composition of NETs created by differing stimuli.