Elucidating the mechanism of mitochondrial superoxide production in pro-inflammatory macrophages
type: CreativeWork
description: Pro-inflammatory macrophages produce reactive oxygen species (ROS) which act as bactericidal agents and redox signals. Recently, mitochondria were identified as an important source of ROS in pro-inflammatory macrophages and it has been proposed that lipopolysaccharide (LPS) stimulated macrophages produce mitochondria derived superoxide by reverse electron transport (RET) at complex I. However, this has not been demonstrated and the mechanism of mitochondrial superoxide production is unknown. Furthermore, the influence of LPS induced metabolic reprogramming on mitochondrial ROS (mtROS) production has not been elucidated. Therefore, the aim of this thesis was to determine the mechanism of mitochondrial superoxide production and how this is driven by LPS induced metabolic reprogramming. To address this question, I measured LPS induced mitochondrial superoxide production with a mitochondria targeted superoxide probe and performed a temporal analysis of LPS induced metabolic reprogramming. I also used bone marrow derived macrophages (BMDMs) expressing the alternative oxidase (AOX) to determine the influence of an oxidised CoQ pool on mitochondrial superoxide production. Using this approach, I demonstrated that LPS induced mitochondrial superoxide production is driven by an elevated proton motive force (∆p), measured as mitochondrial membrane potential (∆ψm), and a reduced CoQ pool. The key metabolic changes that give rise to these conditions are repurposing of ATP production from oxidative phosphorylation to glycolysis, which reduces the reliance on F1FO-ATP synthase activity resulting in an elevated ∆p, and succinate oxidation which maintains a reduced CoQ pool. Since a high ∆p and reduced CoQ pool could drive mitochondrial superoxide production at either complex I or complex III of the electron transport chain, I next set out to determine the site and mechanism of mtROS production. To do this, I used BMDMs from a mouse homoplasmic for the *ND6 G14600A* mtDNA mutation. …
abstract: Pro-inflammatory macrophages produce reactive oxygen species (ROS) which act as bactericidal agents and redox signals. Recently, mitochondria were identified as an important source of ROS in pro-inflammatory macrophages and it has been proposed that lipopolysaccharide (LPS) stimulated macrophages produce mitochondria derived superoxide by reverse electron transport (RET) at complex I. However, this has not been demonstrated and the mechanism of mitochondrial superoxide production is unknown. Furthermore, the influence of LPS induced metabolic reprogramming on mitochondrial ROS (mtROS) production has not been elucidated. Therefore, the aim of this thesis was to determine the mechanism of mitochondrial superoxide production and how this is driven by LPS induced metabolic reprogramming. To address this question, I measured LPS induced mitochondrial superoxide production with a mitochondria targeted superoxide probe and performed a temporal analysis of LPS induced metabolic reprogramming. I also used bone marrow derived macrophages (BMDMs) expressing the alternative oxidase (AOX) to determine the influence of an oxidised CoQ pool on mitochondrial superoxide production. Using this approach, I demonstrated that LPS induced mitochondrial superoxide production is driven by an elevated proton motive force (∆p), measured as mitochondrial membrane potential (∆ψm), and a reduced CoQ pool. The key metabolic changes that give rise to these conditions are repurposing of ATP production from oxidative phosphorylation to glycolysis, which reduces the reliance on F1FO-ATP synthase activity resulting in an elevated ∆p, and succinate oxidation which maintains a reduced CoQ pool. Since a high ∆p and reduced CoQ pool could drive mitochondrial superoxide production at either complex I or complex III of the electron transport chain, I next set out to determine the site and mechanism of mtROS production. To do this, I used BMDMs from a mouse homoplasmic for the *ND6 G14600A* mtDNA mutation. …
author: Casey, Alva
language: eng
keywords: complex I, macrophage, mitochondria, reactive oxygen species, RET, reverse electron transport, ROS
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/ac5ab43d-36ff-4ddb-8d85-e08295d2f43f/download
license: http://purl.org/NET/rdflicense/allrightsreserved
date published: 2023-12-31
date modified: 2026-07-22T22:24:14Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.109461