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Title: Tissue specificity of MMP gene expression and secretion in tuberculosis
Author: Dos Santos Brilha, Sara Sofia
ISNI:       0000 0004 6423 1393
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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In tuberculosis (TB), matrix metalloproteinases (MMPs) have a major role in tissue destruction and cavitation. The composition of each extracellular matrix (ECM) gives specificity to tissues and is important in control of local immune responses by acting as a “molecular postcode”. Hence, it is likely to have important implications in TB. During central nervous system (CNS) infection, the effect of TB on blood-brain barrier (BBB) function is unknown. I hypothesised that the ECM environment may determine the MMP response during innate immune activation in pulmonary and CNS TB. I aimed to investigate the effect of adhesion to the lung’s ECM in regulation of MMP expression and activity. I also aimed to develop a BBB cellular model and investigate the role of Mycobacterium tuberculosis (Mtb)-driven MMP secretion on BBB function. In a model of pulmonary TB, human bronchial epithelial cells (NHBEs) and monocytes were exposed to ECM components and stimulated with conditioned medium from Mtb-infected monocytes (CoMtb) or infected with Mtb. Type I collagen (Coll-I) matrix was shown to decrease MMP-1 mRNA accumulation by 48% and collagenolytic activity compared to NHBEs in the absence of matrix. MMP-1 co-localised with integrin α2β1 resulting in enhanced cell migration in wound healing assays. In contrast, soluble Coll-I led to integrin α2β1 occupancy without clustering and caused a 7-fold increase in collagenolytic activity but decreased migration. In Mtb-infected monocytes, adhesion to ECM components increased MMP-1 secretion by over 60%. Fibronectin and Coll-I also increased MMP-10 by 55% and 90% respectively. Surface expression of integrin αVβ3 was upregulated by Mtb-infection and adhesion to Coll-I. Activation of integrin αVβ3 mimicked the effect of Coll-I MMP secretion, while its inhibition impaired monocyte migration. CoMtb-stimulation of the BBB model decreased trans-endothelial electrical resistance from 154±1.2ohm.cm2 to 111.6±4.7ohm.cm2, while Papp increased 3-fold. Coll-IV and tight junction proteins (TJPs) degradation was also detected. MMP concentrations increased 125-fold for MMP-1 (0.35±2 to 43.8±5.1ng/mL) and 619-fold for MMP-9 (0.072±0.014 to 44.6±8.9ng/ml). Treatment with the MMP inhibitor Ro32-3555 prevented BBB disruption. Neutrophil and monocyte transmigration increased 60% and 80% respectively and returned to control levels with MMP blockade. MMP-9 was shown to be responsible for BBB disruption and its inhibition by antibodies prevented BBB disruption. The Hedgehog pathway was downregulated during infection, resulting in decrease TJPs gene expression, which contributes to BBB dysfunction. In summary, the ECM regulates both epithelial and monocyte expression of MMP-1 via integrins signalling. In the CNS, MMP-9 drives tissue destruction and BBB disruption which may be a potential reversible event in CNS TB immunopathology.
Supervisor: Friedland, Jon S. Sponsor: Fundação para a Ciência e a Tecnologia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral