Abstract
BackgroundTuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. However, relatively little is known about the regulation of these MMPs in the CNS.MethodsUsing a cellular model of CNS TB, we stimulated a human microglial cell line (CHME3) with conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb). MMP-1 and MMP-3 secretion was detected using ELISAs confirmed with casein zymography or western blotting. Key results of a phospho-array profile that detects a wide range of kinase activity were confirmed with phospho-Western blotting. Chemical inhibition (SB203580) of microglial cells allowed investigation of expression and secretion of MMP-1 and MMP-3. Finally we used promoter reporter assays employing full length and MMP-3 promoter deletion constructs. Student’s t-test was used for comparison of continuous variables and multiple intervention experiments were compared by one-way ANOVA with Tukey’s correction for multiple pairwise comparisons.ResultsCoMTb up-regulated microglial MMP-1 and MMP-3 secretion in a dose- and time-dependent manner. The phospho-array profiling showed that the major increase in kinase activity due to CoMTb stimulation was in p38 mitogen activated protein kinase (MAPK), principally the α and γ subunits. p38 phosphorylation was detected at 15 minutes, with a second peak of activity at 120 minutes. High basal extracellular signal-regulated kinase activity was further increased by CoMTb. Secretion and expression of MMP-1 and MMP-3 were both p38 dependent. CoMTb stimulation of full length and MMP-3 promoter deletion constructs demonstrated up-regulation of activity in the wild type but a suppression site between -2183 and -1612 bp.ConclusionsMonocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 MAPK in tuberculosis. p38 is therefore a potential target for adjuvant therapy in CNS TB.
Highlights
Tuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3
Monocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 mitogen activated protein kinase (MAPK) in tuberculosis. p38 is a potential target for adjuvant therapy in CNS TB
Monocyte-microglial networks drive MMP-1 and MMP-3 secretion from human microglia Increasing concentrations of CoMTb but not Conditioned media from control monocytes (CoMCont) resulted in greater secretion of MMP-1 and MMP-3 in a dose-dependent manner (Figure 1A and B)
Summary
Tuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. Monocytic cell-derived matrix metalloproteinases (M MPs) are central mediators of TB-driven pulmonary tissue destruction due to their ability to degrade fibrillar collagens [2] This family of 23 proteases with differing, but overlapping, substrates can degrade all elements of the extracellular matrix at neutral pH, differentiating them from other proteases that might be induced in inflammatory situations [3,4]. In the CNS, MMPs are known to have a number of substrates, such as dystroglycan, a transmembrane receptor that anchors astrocyte endfeet to the basement membrane via laminin binding [6]. Cleavage of these molecules contributes to blood-brain barrier breakdown. A specific and proven adjuvant to anti-tuberculous chemotherapy that improves patient outcomes, suppresses MMPs, but not TIMPs, in CNS TB patients [11,13]
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