Neutrophils and the regulation of matrix metalloproteinases in Tuberculosis
File(s)
Author(s)
Ong, Catherine Wei Min
Type
Thesis
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.
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.
Version
Open Access
Date Issued
2013-05
Date Awarded
2013-09
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Friedland, Jon
Sponsor
Singapore. Ministry of Health
Publisher Department
Medicine
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)