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Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways.

Green JA, Dholakia S, Janczar K, Ong CW, Moores R, Fry J, Elkington PT, Roncaroli F, Friedland JS - J Neuroinflammation (2011)

Bottom Line: Inhibition of the p38 MAP kinase pathway resulted in a 228% increase in MMP-2 secretion (P < 0.01).In contrast ERK MAP kinase inhibition further decreased MMP-2 secretion by 76.6% (P < 0.05).Inhibition of the NFκB pathway resulted in 301% higher MMP-2 secretion than CoMTb alone (P < 0.01).

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Infectious Diseases and Immunity and the Imperial College Wellcome Trust Centre for Clinical Tropical Medicine, Hammersmith Campus, Imperial College London, London, W12 0NN, UK. justin.green@imperial.ac.uk

ABSTRACT
Tuberculosis (TB) of the central nervous system (CNS) is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2) which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb), but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P < 0.01), dependent upon TNF-α. In human CNS TB brain biopsies but not controls the p38 pathway was activated in microglia/macrophages. Inhibition of the p38 MAP kinase pathway resulted in a 228% increase in MMP-2 secretion (P < 0.01). In contrast ERK MAP kinase inhibition further decreased MMP-2 secretion by 76.6% (P < 0.05). Inhibition of the NFκB pathway resulted in 301% higher MMP-2 secretion than CoMTb alone (P < 0.01). Caspase 8 restored MMP-2 secretion to basal levels. However, this caspase-dependent regulation of MMP-2 was independent of p38 and NFκB pathways; p38 phosphorylation was increased and p50/p65 NFκB nuclear trafficking unaffected by caspase 8 inhibition. In summary, suppression of microglial MMP-2 secretion by M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

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Suppression of MMP-2 by CoMTb is mediated by TNF-α. (A), CoMTb was preincubated with increasing doses of anti-TNF-α Ab for 2 h. (B), TNF-α suppresses MMP-2 secretion. Cells were stimulated with rhTNF-α 1-100 ng/ml. Note representative gelatin zymogram has been cut to correspond to the adjacent graph (thick black line). (C), IL-1β does not contribute to CoMTb induced MMP-2 secretion. Cells were preincubated with IL-1Ra for 2 h. Inhibition of IL-1β activity did not affect MMP-2 secretion. (D), IL-1β alone does not suppress MMP-2 secretion. Microglial cells were stimulated with rhIL-1β at concentrations of 1-100 ng/ml. Densitometric analysis of gelatin zymography is shown with a representative gelatin zymogram for A-D. For all experiments bars represent mean values ± SD of three samples, representative of at least duplicate experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison. *p < 0.05, **p < 0.01.
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Figure 2: Suppression of MMP-2 by CoMTb is mediated by TNF-α. (A), CoMTb was preincubated with increasing doses of anti-TNF-α Ab for 2 h. (B), TNF-α suppresses MMP-2 secretion. Cells were stimulated with rhTNF-α 1-100 ng/ml. Note representative gelatin zymogram has been cut to correspond to the adjacent graph (thick black line). (C), IL-1β does not contribute to CoMTb induced MMP-2 secretion. Cells were preincubated with IL-1Ra for 2 h. Inhibition of IL-1β activity did not affect MMP-2 secretion. (D), IL-1β alone does not suppress MMP-2 secretion. Microglial cells were stimulated with rhIL-1β at concentrations of 1-100 ng/ml. Densitometric analysis of gelatin zymography is shown with a representative gelatin zymogram for A-D. For all experiments bars represent mean values ± SD of three samples, representative of at least duplicate experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison. *p < 0.05, **p < 0.01.

Mentions: The effect of pre-incubation of CoMTb for two hours with increasing concentrations of TNF-α neutralizing antibody before stimulating microglia was investigated (Figure 2A). Previously we have demonstrated that a matched isotype control antibody has no inhibitory activity [41]. MMP-2 secretion was restored in a dose-dependent manner. 100 ng/ml recombinant TNF-α suppressed MMP-2 secretion by 61.0% similar to the level observed with CoMTb (Figure 2B). As we have shown that the TNF-α concentration in 1:5 CoMTb is approximately 2 ng/ml [33,37] the data show that TNF-α is necessary, but not sufficient, to cause CoMTb-induced MMP-2 suppression. Next, we investigated IL-1β which is present in CoMTb and important in CNS TB pathogenesis [20]. Pre-incubating microglial cells with the inhibitor IL-1Ra or stimulation with recombinant IL-1β did not alter MMP-2 secretion (Figure 2C &2D). There was neither an additive nor synergistic effect of adding these two recombinant cytokines concurrently (Additional file 1A). Soluble factors derived from M.tb culture (Tb medium) did not synergize with TNF-α to further suppress MMP-2 secretion since we demonstrated that there was no additional effect of adding filtered supernatant from cultured M. tuberculosis to TNF-α (Additional file 1B). Dexamethasone also did not regulate MMP-2 secretion (Additional file 2) nor did addition of IL-6, Oncostatin M or inhibition of G-protein coupled signaling via pertussis blockade experiments (data not shown).


Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways.

Green JA, Dholakia S, Janczar K, Ong CW, Moores R, Fry J, Elkington PT, Roncaroli F, Friedland JS - J Neuroinflammation (2011)

Suppression of MMP-2 by CoMTb is mediated by TNF-α. (A), CoMTb was preincubated with increasing doses of anti-TNF-α Ab for 2 h. (B), TNF-α suppresses MMP-2 secretion. Cells were stimulated with rhTNF-α 1-100 ng/ml. Note representative gelatin zymogram has been cut to correspond to the adjacent graph (thick black line). (C), IL-1β does not contribute to CoMTb induced MMP-2 secretion. Cells were preincubated with IL-1Ra for 2 h. Inhibition of IL-1β activity did not affect MMP-2 secretion. (D), IL-1β alone does not suppress MMP-2 secretion. Microglial cells were stimulated with rhIL-1β at concentrations of 1-100 ng/ml. Densitometric analysis of gelatin zymography is shown with a representative gelatin zymogram for A-D. For all experiments bars represent mean values ± SD of three samples, representative of at least duplicate experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison. *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Suppression of MMP-2 by CoMTb is mediated by TNF-α. (A), CoMTb was preincubated with increasing doses of anti-TNF-α Ab for 2 h. (B), TNF-α suppresses MMP-2 secretion. Cells were stimulated with rhTNF-α 1-100 ng/ml. Note representative gelatin zymogram has been cut to correspond to the adjacent graph (thick black line). (C), IL-1β does not contribute to CoMTb induced MMP-2 secretion. Cells were preincubated with IL-1Ra for 2 h. Inhibition of IL-1β activity did not affect MMP-2 secretion. (D), IL-1β alone does not suppress MMP-2 secretion. Microglial cells were stimulated with rhIL-1β at concentrations of 1-100 ng/ml. Densitometric analysis of gelatin zymography is shown with a representative gelatin zymogram for A-D. For all experiments bars represent mean values ± SD of three samples, representative of at least duplicate experiments performed in triplicate. Data were analyzed by one-way analysis of variance followed by Tukey's multiple comparison. *p < 0.05, **p < 0.01.
Mentions: The effect of pre-incubation of CoMTb for two hours with increasing concentrations of TNF-α neutralizing antibody before stimulating microglia was investigated (Figure 2A). Previously we have demonstrated that a matched isotype control antibody has no inhibitory activity [41]. MMP-2 secretion was restored in a dose-dependent manner. 100 ng/ml recombinant TNF-α suppressed MMP-2 secretion by 61.0% similar to the level observed with CoMTb (Figure 2B). As we have shown that the TNF-α concentration in 1:5 CoMTb is approximately 2 ng/ml [33,37] the data show that TNF-α is necessary, but not sufficient, to cause CoMTb-induced MMP-2 suppression. Next, we investigated IL-1β which is present in CoMTb and important in CNS TB pathogenesis [20]. Pre-incubating microglial cells with the inhibitor IL-1Ra or stimulation with recombinant IL-1β did not alter MMP-2 secretion (Figure 2C &2D). There was neither an additive nor synergistic effect of adding these two recombinant cytokines concurrently (Additional file 1A). Soluble factors derived from M.tb culture (Tb medium) did not synergize with TNF-α to further suppress MMP-2 secretion since we demonstrated that there was no additional effect of adding filtered supernatant from cultured M. tuberculosis to TNF-α (Additional file 1B). Dexamethasone also did not regulate MMP-2 secretion (Additional file 2) nor did addition of IL-6, Oncostatin M or inhibition of G-protein coupled signaling via pertussis blockade experiments (data not shown).

Bottom Line: Inhibition of the p38 MAP kinase pathway resulted in a 228% increase in MMP-2 secretion (P < 0.01).In contrast ERK MAP kinase inhibition further decreased MMP-2 secretion by 76.6% (P < 0.05).Inhibition of the NFκB pathway resulted in 301% higher MMP-2 secretion than CoMTb alone (P < 0.01).

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Infectious Diseases and Immunity and the Imperial College Wellcome Trust Centre for Clinical Tropical Medicine, Hammersmith Campus, Imperial College London, London, W12 0NN, UK. justin.green@imperial.ac.uk

ABSTRACT
Tuberculosis (TB) of the central nervous system (CNS) is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2) which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb), but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P < 0.01), dependent upon TNF-α. In human CNS TB brain biopsies but not controls the p38 pathway was activated in microglia/macrophages. Inhibition of the p38 MAP kinase pathway resulted in a 228% increase in MMP-2 secretion (P < 0.01). In contrast ERK MAP kinase inhibition further decreased MMP-2 secretion by 76.6% (P < 0.05). Inhibition of the NFκB pathway resulted in 301% higher MMP-2 secretion than CoMTb alone (P < 0.01). Caspase 8 restored MMP-2 secretion to basal levels. However, this caspase-dependent regulation of MMP-2 was independent of p38 and NFκB pathways; p38 phosphorylation was increased and p50/p65 NFκB nuclear trafficking unaffected by caspase 8 inhibition. In summary, suppression of microglial MMP-2 secretion by M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

Show MeSH
Related in: MedlinePlus