Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.693929
Title: Neutrophils and the regulation of matrix metalloproteinases in tuberculosis
Author: Ong, Catherine Wei Min
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
Tuberculosis (TB) continues to kill globally and this is compounded by increasing drug-resistance. Neutrophil influx and tissue destruction are hallmarks of TB and this is detrimental to the host. A matrix degrading phenotype causes tissue damage in TB whereby the activity of matrix metalloproteinases (MMPs) is unopposed by their specific tissue inhibitors (TIMPs). The hypothesis is that neutrophils have a key role in causing MMP-dependent tissue destruction in human TB. The aims are to investigate MMP and TIMP secretion from M.tuberculosis (M.tb ) infected neutrophils, neutrophil networks regulating MMP secretion and MMP and TIMP activity regulation in neutrophils in vivo. Using an in-vitro model, healthy primary human neutrophils were infected in vitro with M.tb or stimulated with conditioned media from M.tb- infected monocytes (CoMTB). MMP-8/-9 and TIMP-1/-2 secretion were analysed by Luminex array and zymography. Intracellular signalling pathways were investigated by phosphoarray, western blot and inhibitors. Gene expression was studied by real-time PCR. Neutrophil extracellular traps (NETS) were examined by immunofluorescence. DQ collagen degradation was examined by confocal microscopy and quantitative fluorescence assay. These studies were extended to investigate MMP-8 and -9 secretion from patients with AMPK mutation to evaluate if AMPK regulates neutrophil MMP secretion. Induced sputum samples from 108 patients and controls were analyzed. Immunohistochemistry of human TB lung specimens was performed. Neutrophil MMP-8 and -9 secretion are up-regulated 3 and 5 fold by M.tb respectively while CoMTB stimulation caused a 2 and 3 fold increase in MMP-8 and -9 (all p<0.001). The MAPK, PI3-kinase and AMPK pathway regulate neutrophil MMP secretion in CoMTB-stimulated neutrophils but not M.tb-infected cells. M.tb-infected neutrophils degrade collagen, which is abolished by the MMP inhibitor doxycycline. M.tb-driven NET formation is associated with MMP-8 and -9. MMP-8 and -9 concentrations are elevated in induced sputum of TB patients compared to controls (both p<0.001) and correlate with neutrophil markers myeloperoxidase and neutrophil gelatinase associated lipocalin. MMP-8 concentration correlated with clinical TB severity score (r=0.55, p<0.0001) and CXR score (r=0.52, p<0.0001). Induced sputum from TB patients has increased collagenase activity which is suppressed by MMP-8 neutralisation. MMP-8 secretion is decreased in AMPK patients with CoMTB stimulation. Immunohistochemistry of human TB lung specimens confirmed cellular findings. M.tb drives neutrophil MMP-8/-9 gene expression and secretion following direct infection and in monocyte-dependent networks. Pathways regulating neutrophil MMP secretion are stimulus specific. In vivo findings underscore the crucial role that neutrophils have in tissue destruction in TB.
Supervisor: Friedland, Jon Sponsor: Ministry of Health ; Singapore
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.693929  DOI: Not available
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