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Title: The effect of macrophage activation and apoptosis in the immune response to respiratory pathogens
Author: Morris, Lucy Victoria
ISNI:       0000 0004 6424 0222
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Macrophage activation is essential for macrophage function; however, the phenotypic profile of macrophages can become dysregulated during pulmonary disease. Two major causes of lower respiratory tract infections are Streptococcus pneumoniae (S. pneumoniae) and Non-Typeable Haemophilus influenzae (NTHi). These are problematic as current preventative treatments against these opportunistic pathogens are ineffective for those most vulnerable to infection. Macrophages can undergo apoptosis when infected with S. pneumoniae as part of the innate immune response. This apoptosis-associated bacterial clearance is reduced in alveolar macrophages (AMs) from COPD and HIV patient associated with increased expression of the anti-apoptotic protein Mcl-1. I hypothesized that macrophages from Mcl-1 over-expressing transgenic mice would display an altered macrophage activation profile which would affect macrophage effector functions after bacterial challenge. Given the prominence of NTHi infection in diseases with defective macrophage function I also predicted NTHi challenge would cause macrophage apoptosis associated killing, which is inhibited by over expression of Mcl-1, similar to previous observations for S. pneumoniae. Induction of classical and alternative macrophage activation was analysed between wild-type and transgenic Bone Marrow Derived Macrophages (BMDM) and determined in human Monocyte Derived Macrophages (MDM). The effect of macrophage activation on killing of S. pneumoniae, microbicidal production and apoptotic activity was then assessed in both BMDM and MDM models. A transcriptomics study was conducted to understand changes in gene expression between wild-type and transgenic AMs at 16 hours, a critical time point of S. pneumoniae challenge, at the onset of macrophage apoptosis associated killing. Macrophage killing and microbicidal production in MDMs after NTHi challenge were observed and apoptosis and Mcl-1 protein levels were assessed over a 72-hour time course. The effects of macrophage activation on NTHi challenge was also measured. I found that Mcl-1 did not alter macrophage activation but activation of BMDM and MDM with IFN-γ (M(IFN-γ)) caused enhanced clearance of intracellular bacteria at early and late time points of S. pneumoniae challenge. Furthermore, levels of apoptosis were increased in M(IFN-γ) at 16 hours of S. pneumoniae challenge. Transcriptomic analysis of wild-type and transgenic AMs after 16 hours of challenge revealed induction of a T-cell signature in transgenic samples. Challenge of MDMs with NTHi caused low levels of macrophage apoptosis at 20 hours of challenge which increased at 48 and 72 hours. Levels of Mcl-1 remained high at 16-20 hours then decreased from peak at 20 hours onwards with increasing levels of macrophage apoptosis. These results demonstrate the importance of IFN-γ production during S. pneumoniae challenge. They also highlight a potential role for T-cell involvement in transgenic macrophages when the apoptotic response is compromised. Finally, they give insights into an unexplored aspect of macrophage immunology after NTHi challenge, showing increased apoptosis at later timepoints of infection. These findings offer useful insights into host innate immunology and may provide groundwork for future therapeutic development.
Supervisor: Marriott, Helen ; Dockrell, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available