A Bioenergetics Study of Mitochondrial Function in Bone Marrow Derived Macrophages
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Al Khodair, Sahar Abdulrahman, A Bioenergetics Study of Mitochondrial Function in Bone Marrow Derived Macrophages, Trinity College Dublin.School of Biochemistry & Immunology, 2022Download Item:
Abstract:
Mitochondria are organelles involved in the generation of ATP, storage of intracellular calcium ions, regulation of the apoptotic pathway as well as numerous cell signalling pathways. Moreover, mitochondria are an important source of reactive oxygen species (ROS), which are molecules containing oxygen that can readily oxidize other molecules. Immune activation of macrophages with LPS is known to initiate a metabolic switching from oxidative phosphorylation to glycolysis; however, the bioenergetic mechanisms that underlie such events are not fully known. In this study, the effect of LPS treatment of mouse iBMDMs and primary BMDMs on electron transport chain activities, oxygen consumption rates, ROS production and the production of inflammatory cytokines, including IL-1ß and TNF-α were investigated.
The data from electron transfer complexes assays and oxygen respiration assays indicated that the stimulation of iBMDMs and BMDMs by LPS significantly decreased the activities of complexes I and II and citrate synthase up to 80% in a time-dependent manner, which may underlie time-dependent decreases in observed oxygen respiration. Importantly, this study showed that the LPS-related inhibition of complex II was reversible and that of complex I was time-dependent but irreversible. In activated macrophages, itaconate is one of the most highly induced metabolites. In this study, itaconate was found to be a time-dependent and reversible inhibitor of complex I and II activities in iBMDMs and BMDMs. The type of itaconate-induced inhibition of complex II was competitive and the Ki values calculated to be 0.014 mM and 0.039 mM in iBMDMs and BMDMs, respectively. These studies suggest that the itaconate concentrations that are known to exist in LPS-activatied macrophages are sufficient to reversibly inhibit complex I and II activities, but that the mechanism for LPS-induced irreversible inhibition of complex I activity remains unknown.
Metabolic control analysis (MCA) was used to investigate the spread of control among complex I and complex II/III over oxygen consumption in iBMDMs and BMDMs. The results provide information on the control possessed by the complexes and the level by which the complexes must be inhibited before deleterious effect are imposed on mitochondrial respiration. The results show that complex I possessed the highest level of control of the complexes examined over OCRs in iBMDMs and BMDMs. In iBMDMs the complex I and II/III energy thresholds were high at ~60 and ~80%, respectively. However, in BMDMs a lower inhibition thresholds of 40% and 60% were found for compelxes I and II/III, respectively. These results suggest that the 40% and 60% decrease in complex I specific activities, observed at 4h and 24h following LPS-activation in BMDMs, are sufficient to cause the corresponding decreases in mitochondrial respiration. Simarily the 50% and 80% decreases in complex II/III activities at 4h and 24h following LPS-activation will also overcome inhibition thresholds and reduce respiration rates in BMDMs.
As previous studies hypothesised that mitochondrial reactive oxygen species (ROS) are a driving force for cytokine secretion in LPS-activated macrophages, experiments were performed to assay both parameters during ETC complex titrations. While ROS levels were found to be 2-fold higher in iBMDMs compared to BMDMs, LPS-activation increased ROS only in BMDMs. Both rotenone and antimycin were found to increase ROS by ~1.4-fold only at concentrations >20nM, which also exceed the inhibition thresholds. However, at lower concentrations of 5 nM which do not exceed inhibition thresholds, IL-1ß secretion was found to be significantly reduced, especially in inflammasome-activated (LPS + ATP) conditions in BMDMs. Overall, these results suggest that slight perturbations of complex I or II/III activities are sufficient to regulate respiratory flux through the ETC and subsequently decrease cytokine secretion from LPS-activated macrophages. This may be the mechanism in which LPS-activated macrophages decrease complex I and II/III activities and feedback regulate the secretion of pro-inflammatory cytokines.
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Saudi Education Ministry
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https://tcdlocalportal.tcd.ie/pls/EnterApex/f?p=800:71:0::::P71_USERNAME:ALKHODASDescription:
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Author: Al Khodair, Sahar Abdulrahman
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Davey, GavinPublisher:
Trinity College Dublin. School of Biochemistry & Immunology. Discipline of BiochemistryType of material:
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