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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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MMP-2 secretion and gene expression is suppressed by CoMTb. (A), Microglial cells were stimulated with control medium (open bars), CoMCont (diagonal hatched bars), CoMTb (solid bars) or infected with M.tb (grey bars) at an MOI of 1-10. 72 h supernatants were analyzed by Luminex with a representative gelatin zymogram shown. (B), Kinetics of CoM stimulated MMP-2 secretion analyzed by Luminex (representative zymogram shown). (C), MMP-2 gene expression is suppressed by CoMTb. mRNA from microglia stimulated for 24 h was analyzed by RT-PCR, normalized to 18S RNA and expressed as fold change relative to 24 h CoMCont mRNA. The mean ± SD from 3 experiments are shown analyzed by Student's t-test. (D), Astrocyte-microglial signaling networks do not suppress MMP-2 secretion. Conditioned medium from primary human astrocytes was prepared from control (CoACont) and M.tb infected cells (CoATb) and used to stimulate microglia. 72 h cell culture medium MMP-2 secretion was analyzed by Luminex (representative zymogram shown). A, B & D, Bars represent mean values ± SD of 3 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 1: MMP-2 secretion and gene expression is suppressed by CoMTb. (A), Microglial cells were stimulated with control medium (open bars), CoMCont (diagonal hatched bars), CoMTb (solid bars) or infected with M.tb (grey bars) at an MOI of 1-10. 72 h supernatants were analyzed by Luminex with a representative gelatin zymogram shown. (B), Kinetics of CoM stimulated MMP-2 secretion analyzed by Luminex (representative zymogram shown). (C), MMP-2 gene expression is suppressed by CoMTb. mRNA from microglia stimulated for 24 h was analyzed by RT-PCR, normalized to 18S RNA and expressed as fold change relative to 24 h CoMCont mRNA. The mean ± SD from 3 experiments are shown analyzed by Student's t-test. (D), Astrocyte-microglial signaling networks do not suppress MMP-2 secretion. Conditioned medium from primary human astrocytes was prepared from control (CoACont) and M.tb infected cells (CoATb) and used to stimulate microglia. 72 h cell culture medium MMP-2 secretion was analyzed by Luminex (representative zymogram shown). A, B & D, Bars represent mean values ± SD of 3 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: Microglia were stimulated with 1:5 diluted CoMCont, CoMTb or infected with M.tb at MOIs of 0.1, 1 and 10. CoMTb caused a 72.8% suppression of MMP-2 secretion (Figure 1A, P < 0.01). There was a 31% decrease in MMP-2 secretion due to direct infection of microglia at an MOI of 10 compared to control (P < 0.01), an infectious load-dependent effect but no effect on cell viability was demonstrated. CoMTb suppression of MMP-2 was evident by 24 hours, significant by 72 hours and sustained over 96 hours (Figure 1B). CoMTb decreased MMP-2 mRNA accumulation by 50% at 24 hours (Figure 1C, P < 0.05). This effect of CoMTb on microglia was specific since conditioned media from M.tb infected primary human astrocytes (CoATb, Figure 1D) had little effect and microglia (data not shown) had no effect on MMP-2 secretion.


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)

MMP-2 secretion and gene expression is suppressed by CoMTb. (A), Microglial cells were stimulated with control medium (open bars), CoMCont (diagonal hatched bars), CoMTb (solid bars) or infected with M.tb (grey bars) at an MOI of 1-10. 72 h supernatants were analyzed by Luminex with a representative gelatin zymogram shown. (B), Kinetics of CoM stimulated MMP-2 secretion analyzed by Luminex (representative zymogram shown). (C), MMP-2 gene expression is suppressed by CoMTb. mRNA from microglia stimulated for 24 h was analyzed by RT-PCR, normalized to 18S RNA and expressed as fold change relative to 24 h CoMCont mRNA. The mean ± SD from 3 experiments are shown analyzed by Student's t-test. (D), Astrocyte-microglial signaling networks do not suppress MMP-2 secretion. Conditioned medium from primary human astrocytes was prepared from control (CoACont) and M.tb infected cells (CoATb) and used to stimulate microglia. 72 h cell culture medium MMP-2 secretion was analyzed by Luminex (representative zymogram shown). A, B & D, Bars represent mean values ± SD of 3 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 1: MMP-2 secretion and gene expression is suppressed by CoMTb. (A), Microglial cells were stimulated with control medium (open bars), CoMCont (diagonal hatched bars), CoMTb (solid bars) or infected with M.tb (grey bars) at an MOI of 1-10. 72 h supernatants were analyzed by Luminex with a representative gelatin zymogram shown. (B), Kinetics of CoM stimulated MMP-2 secretion analyzed by Luminex (representative zymogram shown). (C), MMP-2 gene expression is suppressed by CoMTb. mRNA from microglia stimulated for 24 h was analyzed by RT-PCR, normalized to 18S RNA and expressed as fold change relative to 24 h CoMCont mRNA. The mean ± SD from 3 experiments are shown analyzed by Student's t-test. (D), Astrocyte-microglial signaling networks do not suppress MMP-2 secretion. Conditioned medium from primary human astrocytes was prepared from control (CoACont) and M.tb infected cells (CoATb) and used to stimulate microglia. 72 h cell culture medium MMP-2 secretion was analyzed by Luminex (representative zymogram shown). A, B & D, Bars represent mean values ± SD of 3 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: Microglia were stimulated with 1:5 diluted CoMCont, CoMTb or infected with M.tb at MOIs of 0.1, 1 and 10. CoMTb caused a 72.8% suppression of MMP-2 secretion (Figure 1A, P < 0.01). There was a 31% decrease in MMP-2 secretion due to direct infection of microglia at an MOI of 10 compared to control (P < 0.01), an infectious load-dependent effect but no effect on cell viability was demonstrated. CoMTb suppression of MMP-2 was evident by 24 hours, significant by 72 hours and sustained over 96 hours (Figure 1B). CoMTb decreased MMP-2 mRNA accumulation by 50% at 24 hours (Figure 1C, P < 0.05). This effect of CoMTb on microglia was specific since conditioned media from M.tb infected primary human astrocytes (CoATb, Figure 1D) had little effect and microglia (data not shown) had no effect on MMP-2 secretion.

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