Project title: Matrix turnover in tuberculosis
Supervisor: Prof Jon S Friedland
Hypothesis: Tuberculosis (TB) upregulates extracellular matrix metalloprotease inducer (EMMPRIN)
to drive extracellular matrix remodelling, generating matrix degradation products (MDPs) that reflect
immunopathology and can be used in a biomarker set for TB diagnosis and treatment monitoring.
Background: TB kills 4600 people each day and is characterised by lung cavitation and fibrosis.
Matrix metalloproteinases (MMPs) are implicated in driving pathology, but the role of EMMPRIN and
regulation of the downstream matrix degradation products (MDPs) has not been studied in TB. MDPs
may be both diagnostic markers and markers, of response to standard treatment and treatment with
anti-MMP agents targeting the innate inflammatory response implicated in morbidity and mortality.
EMMPRIN is a cell-surface glycoprotein member of the immunoglobulin superfamily expressed on
multiple cell types (9), which can be cleaved to a soluble form. EMMPRIN appears to be a master
regulator of multiple MMPs and mediates invasion and metastasis in cancer but has not been
investigated in TB. Proteolysis of collagen and elastin fibrils by MMPs releases immunoreactive
peptides which have been studied successfully in other destructive pulmonary pathologies, but never
in TB. For example, N-terminal propeptides of types I and III collagen (PINP and PIIINP) are elevated
in sarcoidosis and desmosine, an amino acid unique to mature elastin, is elevated in patients with
cigarette-induced lung disease. Preliminary data shows PIIINP is elevated in plasma and sputum in
TB and reflects activation of the key collagenase MMP-1 which is central to TB immunopathology.
Specific Aims are to investigate:
1) The regulation of EMMPRIN and MDP gene expression and secretion in Mtb.
2) EMMPRIN and MDPs in a cross-sectional cohort of patients, including pulmonary TB, HIVassociated TB, MDR-TB and disseminated TB.
Research Plan
1) EMMPRIN and MDPs in a TB cell culture model: Up-regulation of EMMPRIN will be investigated
in Mtb-infected primary human monocytes/macrophages and in stromal cells (fibroblasts & epithelial
cells) stimulated with live, virulent Mtb or by conditioned medium from Mtb-infected monocytes
(CoMtb) to model intercellular networks. EMMPRIN release will be analyzed by ELISA and Luminex
array, cell surface expression by flow cytometry and gene expression by quantitative PCR. To
investigate MDP generation, cells will be cultured on collagen, fibronectin, or elastin coated plates to
provide MMP substrates. Cells will be directly infected or stimulated with CoMtb and MMP secretion
analyzed by ELISA and luminex and zymography. MDP accumulation will be determined by ELISA
and western blotting. Matrix degradation will be visualised and quantitated using DQ labelled
collagen, which gains fluorescence on cleavage, and fluorescent gelatin, which loses fluorescence.
2) EMMPRIN and MDPs in patients with TB:
6 cohorts of 40 patients, will be recruited to profile all MDPs elevated in TB in a collaborative study.
Sample size is calculated to show a significant difference on basis of preliminary data. For each
patient, clinical history, examination and baseline laboratory findings (HIV status, CD4 count) will be
recorded on an established proforma. A digital CXR will be performed and lung infiltration will be
scored by ImageJ analysis. The cohorts will be: i) Healthy controls. ii) Pulmonary TB, HIV -ve. iii)
Pulmonary TB, HIV +ve. iv) Extrapulmonary TB. v) MDR TB. vi) Respiratory symptomatics without
TB on clinical assessment, sputum Genexpert analysis and culture.
Sputum, peripheral blood mononuclear cells (PBMCs), plasma and urine will be stored. EMMPRIN
concentrations are measured by Luminex and cell surface expression by flow cytometry. MMPs and
MDPs are analyzed by Luminex and ELISA. Desmosine and other MDPs will be measured by mass
spectrometry to determine the entire profile of MDPs elevated in TB. The relationship between
EMMRPIN, MMPs and MDPs and clinical features will be determined. Modelling will be performed
using this data to establish the potential of MDPs and MMPs as biomarkers.
Summary: This project investigates EMMPRIN and MDPs in TB for the first time and provides a
broad training in translational research.
Selected References: Elkington et al., MMP-1 drives immunopathology in human tuberculosis and transgenic mice. J Clin
Invest 121, 1827; N. F. Walker et al., Doxycycline and HIV Infection Suppress Tuberculosis-induced Matrix
Metalloproteinases. Am J Respir Crit Care Med, 185, 9899; L. Lammi et al., Propeptide levels of type III and type I
procollagen in the serum and bronchoalveolar lavage fluid of patients with pulmonary sarcoidosis. Eur Respir J 10, 2725.