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Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

Ong CW, Elkington PT, Brilha S, Ugarte-Gil C, Tome-Esteban MT, Tezera LB, Pabisiak PJ, Moores RC, Sathyamoorthy T, Patel V, Gilman RH, Porter JC, Friedland JS - PLoS Pathog. (2015)

Bottom Line: The mechanism of matrix destruction resulting in cavitation is not well defined.Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes.These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Immunity, Hammersmith Campus, Imperial College London, London, United Kingdom; Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

ABSTRACT
Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

No MeSH data available.


Related in: MedlinePlus

Neutrophil MMP-8 and -9 are upregulated in human TB.(A) Neutrophils were infected with M.tb MOI of 1. MMP-8 secretion was upregulated at 4h. (B) Increasing M.tb MOI caused greater neutrophil MMP-8, analyzed at 4h. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. (C and D) Human TB lung biopsy specimens stained with H&E and anti-neutrophil elastase shows neutrophil infiltration around the cavity wall. Both scale bars represent 5 mm. n = 5. (E and F) Magnified H&E and MMP-8 stains from Fig 1C inset shows neutrophils immunoreactive for MMP-8 around the cavity wall. Both scale bars represent 50 μm. (G) MMP-9 concentrations increase in a dose-dependent manner after M.tb infection at 4 hours. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. *** P<0.001 (H) Biopsy proven M.tb infected human lung specimens were stained for MMP-9 (inset from Fig 1C). Neutrophils were immunoreactive for MMP-9. Scale bar represents 50 μm. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test or One-way ANOVA with Tukey’s post-test. **P<0.01, ***P<0.001.
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ppat.1004917.g001: Neutrophil MMP-8 and -9 are upregulated in human TB.(A) Neutrophils were infected with M.tb MOI of 1. MMP-8 secretion was upregulated at 4h. (B) Increasing M.tb MOI caused greater neutrophil MMP-8, analyzed at 4h. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. (C and D) Human TB lung biopsy specimens stained with H&E and anti-neutrophil elastase shows neutrophil infiltration around the cavity wall. Both scale bars represent 5 mm. n = 5. (E and F) Magnified H&E and MMP-8 stains from Fig 1C inset shows neutrophils immunoreactive for MMP-8 around the cavity wall. Both scale bars represent 50 μm. (G) MMP-9 concentrations increase in a dose-dependent manner after M.tb infection at 4 hours. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. *** P<0.001 (H) Biopsy proven M.tb infected human lung specimens were stained for MMP-9 (inset from Fig 1C). Neutrophils were immunoreactive for MMP-9. Scale bar represents 50 μm. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test or One-way ANOVA with Tukey’s post-test. **P<0.01, ***P<0.001.

Mentions: First, we investigated MMP-8 secretion from primary human neutrophils stimulated by live, virulent M.tb. Neutrophil MMP-8 secretion increased over time and in a dose-dependent manner in response to higher M.tb multiplicity of infection (MOI) (Fig 1A and 1B). TIMP-1 and -2 are the MMP inhibitors secreted by neutrophils [25, 26]. TIMP-1 was not secreted in response to stimulation by M.tb (S1A Fig). TIMP-2 concentrations increased significantly but to a 20-fold lower concentration than MMP-8 (S1B Fig). We demonstrated that neutrophil MMP-8 secretion was blocked by the NF-kB p65 subunit inhibitor Helenalin (IC50 10–50 μM), in a dose-dependent manner and the effect was maximal at 100 μM (S1C Fig, P<0.001). The dose-dependent suppression of neutrophil MMP-8 was replicated with additional specific NF-kB inhibitors caffeic acid phenethyl ester (CAPE) [27] (S1D Fig) and SN50 [28] (S1E Fig). Neutrophil viability was greater than 95% for all conditions by FACS staining with Annexin V and live/dead dye (S2 Fig).


Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

Ong CW, Elkington PT, Brilha S, Ugarte-Gil C, Tome-Esteban MT, Tezera LB, Pabisiak PJ, Moores RC, Sathyamoorthy T, Patel V, Gilman RH, Porter JC, Friedland JS - PLoS Pathog. (2015)

Neutrophil MMP-8 and -9 are upregulated in human TB.(A) Neutrophils were infected with M.tb MOI of 1. MMP-8 secretion was upregulated at 4h. (B) Increasing M.tb MOI caused greater neutrophil MMP-8, analyzed at 4h. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. (C and D) Human TB lung biopsy specimens stained with H&E and anti-neutrophil elastase shows neutrophil infiltration around the cavity wall. Both scale bars represent 5 mm. n = 5. (E and F) Magnified H&E and MMP-8 stains from Fig 1C inset shows neutrophils immunoreactive for MMP-8 around the cavity wall. Both scale bars represent 50 μm. (G) MMP-9 concentrations increase in a dose-dependent manner after M.tb infection at 4 hours. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. *** P<0.001 (H) Biopsy proven M.tb infected human lung specimens were stained for MMP-9 (inset from Fig 1C). Neutrophils were immunoreactive for MMP-9. Scale bar represents 50 μm. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test or One-way ANOVA with Tukey’s post-test. **P<0.01, ***P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440706&req=5

ppat.1004917.g001: Neutrophil MMP-8 and -9 are upregulated in human TB.(A) Neutrophils were infected with M.tb MOI of 1. MMP-8 secretion was upregulated at 4h. (B) Increasing M.tb MOI caused greater neutrophil MMP-8, analyzed at 4h. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. (C and D) Human TB lung biopsy specimens stained with H&E and anti-neutrophil elastase shows neutrophil infiltration around the cavity wall. Both scale bars represent 5 mm. n = 5. (E and F) Magnified H&E and MMP-8 stains from Fig 1C inset shows neutrophils immunoreactive for MMP-8 around the cavity wall. Both scale bars represent 50 μm. (G) MMP-9 concentrations increase in a dose-dependent manner after M.tb infection at 4 hours. Bars represent mean ± s.d. of experiments performed in triplicate and data are representative of a minimum of 2 independent experiments. *** P<0.001 (H) Biopsy proven M.tb infected human lung specimens were stained for MMP-9 (inset from Fig 1C). Neutrophils were immunoreactive for MMP-9. Scale bar represents 50 μm. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test or One-way ANOVA with Tukey’s post-test. **P<0.01, ***P<0.001.
Mentions: First, we investigated MMP-8 secretion from primary human neutrophils stimulated by live, virulent M.tb. Neutrophil MMP-8 secretion increased over time and in a dose-dependent manner in response to higher M.tb multiplicity of infection (MOI) (Fig 1A and 1B). TIMP-1 and -2 are the MMP inhibitors secreted by neutrophils [25, 26]. TIMP-1 was not secreted in response to stimulation by M.tb (S1A Fig). TIMP-2 concentrations increased significantly but to a 20-fold lower concentration than MMP-8 (S1B Fig). We demonstrated that neutrophil MMP-8 secretion was blocked by the NF-kB p65 subunit inhibitor Helenalin (IC50 10–50 μM), in a dose-dependent manner and the effect was maximal at 100 μM (S1C Fig, P<0.001). The dose-dependent suppression of neutrophil MMP-8 was replicated with additional specific NF-kB inhibitors caffeic acid phenethyl ester (CAPE) [27] (S1D Fig) and SN50 [28] (S1E Fig). Neutrophil viability was greater than 95% for all conditions by FACS staining with Annexin V and live/dead dye (S2 Fig).

Bottom Line: The mechanism of matrix destruction resulting in cavitation is not well defined.Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes.These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

View Article: PubMed Central - PubMed

Affiliation: Infectious Diseases and Immunity, Hammersmith Campus, Imperial College London, London, United Kingdom; Division of Infectious Diseases, Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

ABSTRACT
Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

No MeSH data available.


Related in: MedlinePlus