The Relationship Between Extracellular Mitochondria and Inflammatory Response

Abstract

Mitochondria are known to have both pro- and anti-inflammatory functions, especially when they are found outside of the cell. There is little known about the relationship between pro-inflammatory molecules and processes involved in the packaging and release of mitochondria from a cell. The purpose of this thesis is to establish inflammatory agents that activate movement of mitochondria out of the cell, and to determine if reactive oxygen species function as a messenger within this mechanism. A pilot study involving the induction of acute respiratory distress syndrome (ARDS) using lipopolysaccharide (LPS) in pigs was used to establish LPS and interleukin-6 as inflammatory agents. Literature was referenced to also establish protamine sulfate, an antagonist of the anticoagulant drug heparin, as an inflammatory agent. Cultured human cardiomyocytes were then treated using LPS, interleukin-6, protamine, and heparin-protamine complex to determine which substance instigated exocytosis of mitochondria. These cells had green fluorescent plasmids (GFP) transfected onto their mitochondria prior to treatment, allowing the mitochondrial concentration from the growth media of each sample to be detected using the CLARIOstar Plus microplate reader. Using this method, we determined our positive control (hydrogen peroxide), protamine, and heparin-protamine complex treatments resulted in a significant increase in extracellular mitochondria when compared to our negative control. The next experiment involved comparing these significant treatment groups with and without pretreatment using n-Acetyl L-cysteine (NAC), a strong antioxidant, to determine if the removal of reactive oxygen species would influence extracellular mitochondrial concentration, but the results were not conclusive. Increasing sample size may produce more conclusive results using this model.