TRAUMA-INDUCED REPROGRAMMING: CHANGES IN LIPID RAFT PROTEIN CONTENT
EMSL Project ID
16491
Abstract
Background: Following severe trauma mononuclear cells are reprogrammed leading to alterations in innate immunity. These phenotypes are responsible for increased susceptibility to invading organisms leading to the development of organ failure. This state has been recreated in vitro by subjecting mononuclear cells to factors induced by trauma, including platelet activating factor (PAF), oxidant stress and complement 5a (C5a). Although the mechanism(s) responsible for this reprogramming remain unknown, previous work has demonstrated that this process may be associated with alterations in the protein content within specific plasma membrane microdomains that are rich in cholesterol and sphingolipids termed lipid rafts. Following injury, we hypothesize that factors induced by trauma result in the production of the lipid mediator ceramide from lipid rafts. Ceramide once produced fuses within rafts leading to the formation of macrodomains resulting in changes in membrane fluidity. Due to these changes, various proteins are recruited to the lipid raft resulting in the formation of focal adhesion-like complexes that contain some but not all of the Toll-like receptor (TLR) components. Methods: Differentiated THP-1 cells will be subjected to lipopolysaccharide (LPS) stimulation for various periods of time up to 60 minutes. Selected cells will be pre-treated with PAF, hydrogen peroxide or C5a for periods of time up to 30-60 minutes. Lipid raft protein extraction will be performed using sucrose gradient centrifugation. Harvested proteins will then be used for analysis using the LC-ESI-MS system. These subsequent analysis will be performed as a NCRR Collaborative research project with Dr. David G. Camp of PNNL. Results/Discussion: It is our hypothesis that assembly of these complexes and changes in lipid raft content are responsible for subsequent reprogramming that induces enhanced activation in response to subsequent infection. Based on these in vitro observations, it is our hope to then explore potential changes that occur in severely injured trauma patients in order to determine potential prognostic and therapeutic targets.
Project Details
Start Date
2006-01-01
End Date
2009-01-04
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members