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Title: Role of macrophages metabolic reprogramming and mitochondrial fission against Streptococcus pneumoniae
Author: Mohasin, M. D.
ISNI:       0000 0004 7226 8567
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2018
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Rationale & Hypothesis: Pneumonia is a leading cause of infection-related death and Streptococcus pneumoniae, the commonest cause, accounts for approximately one million deaths in children each year. Macrophages are key effectors of innate immune responses but the precise microbicidal mechanisms used to control S. pneumoniae are incompletely characterised. West et al. reported that TLR1/2/4 agonists augment intracellular bacterial killing through inducing mitochondrial reactive oxygen species (mROS). Recently, the Dockrell group has demonstrated that in addition macrophages use mROS as a component of the microbicidal response and mROS are important effectors of microbicidal responses in an apoptotic programme that contributes to intracellular bacteria killing when other canonical mechanisms are exhausted. I hypothesized that mitochondrial homeostasis would be altered in response to intracellular bacteria as an important element of the microbicidal response to S. pneumoniae. Methodology: Mouse bone marrow cells and human monocytes were differentiated into macrophages. Macrophages metabolic profiles, mROS generation, bacterial killing and mitochondrial fission/mitophagy were evaluated by XF24 flux analyser, flow-cytometry, gentamicin protection assay and Confocal/Electron microscopy/Immunoblotting, respectively. Findings: Measures of respiration during bacterial infection have been less studied than the use of microbial components such as LPS. Methods were optimised to study metabolic profiles in macrophages challenged with S. pneumoniae. The macrophage's bioenergetic profile demonstrated that S. pneumoniae significantly decreased mitochondrial respiration capacity and ATP-linked respiration but increased proton leakage, glycolytic respiration and mROS production. The HIV protein gp120 reduced ATP-linked oxygen consumption rate (OCR) and proton leakage while chronic obstructive pulmonary disease (COPD) reduced maximum respiration capacity, respiration reserve and ATP-linked OCR but increased proton leak. mROS were produced in close proximity to bacteria and to phagolysosomes as well as to nitric oxide production. Mitochondrial maximum respiration capacity was reduced following bacterial infection but this reduction was reversed by inhibiting mROS production. S. pneumoniae significantly increased mitochondrial fission resulting in reduced mitochondrial network complexity 12 hours after bacterial-challenge, before apoptosis induction. Fission was induced via a Drp1-independent non-canonical pathway. Fragmented mitochondria were co-localised or adjacent to an E3 ubiquitin ligase Parkin, phagolysosomes and intracellular bacteria but LC3B was not recruited and electron microscopy failed to identify evidence of mitophagy. mROS co-localized with mitochondria that had undergone fission. Fission was reversed by the PI-3 kinase inhibitor 3-methyladenine (3MA) but was not altered by the Drp1 inhibitor Mdivi-1. Inhibiting mitochondrial fission with 3MA decreased mROS production, apoptosis and intracellular bacterial killing. Conclusions: Mitochondrial fission occurs prior to apoptosis induction and enhances mROS production. Targeting mitochondrial homeostasis and fission represents potential cellular targets with which to modulate host innate immune responses against intracellular pathogens.
Supervisor: Dockrell, David ; Marriott, Helen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available