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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.


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AMPK regulates neutrophil MMP-8 secretion in TB in vitro.(A) Human phosphokinase array. Neutrophils were stimulated with CoMCont or CoMTB. Representative blot from n = 4 healthy donors. Red circle highlights increased AMPKα2 phosphorylation in CoMTB-stimulated cells, with densitometric analysis of components of AMPK pathway below. (B) CoMTB stimulation phosphorylates AMPKα1/2 analyzed by western blotting and gel densitometry. Neutrophils were stimulated for 30 minutes. Bars represent mean ± s.e.m from n = 3 donors. (C) Neutrophils were infected with M.tb MOI of 10 and cell lysates immunoblotted for phospho-AMPKα (T172) at defined time points. M.tb caused maximal phosphorylation at 120 mins. (D) Compound C (Comp C) pre-incubation for 30 minutes before CoMTB stimulation suppresses neutrophil MMP-8 secretion at 4 hours. (E) Compound C (Comp C) was pre-incubated for 30 minutes before stimulation with CoMCont or CoMTB for 24 hours with MMP-8 gene expression analyzed by real-time PCR normalized to GAPDH. Bars represent mean ± s.d. of an experiment performed in biological triplicates on at least 2 occasions. *P<0.05, ***P<0.001. Analysis was performed using one-way ANOVA with Tukey’s post-test.
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ppat.1004917.g005: AMPK regulates neutrophil MMP-8 secretion in TB in vitro.(A) Human phosphokinase array. Neutrophils were stimulated with CoMCont or CoMTB. Representative blot from n = 4 healthy donors. Red circle highlights increased AMPKα2 phosphorylation in CoMTB-stimulated cells, with densitometric analysis of components of AMPK pathway below. (B) CoMTB stimulation phosphorylates AMPKα1/2 analyzed by western blotting and gel densitometry. Neutrophils were stimulated for 30 minutes. Bars represent mean ± s.e.m from n = 3 donors. (C) Neutrophils were infected with M.tb MOI of 10 and cell lysates immunoblotted for phospho-AMPKα (T172) at defined time points. M.tb caused maximal phosphorylation at 120 mins. (D) Compound C (Comp C) pre-incubation for 30 minutes before CoMTB stimulation suppresses neutrophil MMP-8 secretion at 4 hours. (E) Compound C (Comp C) was pre-incubated for 30 minutes before stimulation with CoMCont or CoMTB for 24 hours with MMP-8 gene expression analyzed by real-time PCR normalized to GAPDH. Bars represent mean ± s.d. of an experiment performed in biological triplicates on at least 2 occasions. *P<0.05, ***P<0.001. Analysis was performed using one-way ANOVA with Tukey’s post-test.

Mentions: To investigate the key regulatory pathways of neutrophil MMP-8 secretion, we performed a screening human phosphokinase array and observed that the AMP-activated protein kinase (AMPK) pathway was consistently activated in M.tb-infected neutrophils, especially AMPKα2 (T172) (Fig 5A). This activation was confirmed by immunoblotting (Fig 5B). Components of the MAP-kinase, STAT pathways, p53 and Src family of kinases were also activated consistent with previous data [39–41] (S6A, S6B and S6C Fig). AMPK is considered a master regulator of cellular energy homeostasis, existing as a heterotrimeric complex comprising catalytic α-subunits and regulatory β- and γ-subunits [13]. Its activation sets off a cascade of catabolic pathways including glycolysis and ketogenesis which can lead to the wasting which is characteristic in TB patients. We demonstrated that AMPKα was activated by phosphorylation in neutrophils directly infected with M.tb (Fig 5C).


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)

AMPK regulates neutrophil MMP-8 secretion in TB in vitro.(A) Human phosphokinase array. Neutrophils were stimulated with CoMCont or CoMTB. Representative blot from n = 4 healthy donors. Red circle highlights increased AMPKα2 phosphorylation in CoMTB-stimulated cells, with densitometric analysis of components of AMPK pathway below. (B) CoMTB stimulation phosphorylates AMPKα1/2 analyzed by western blotting and gel densitometry. Neutrophils were stimulated for 30 minutes. Bars represent mean ± s.e.m from n = 3 donors. (C) Neutrophils were infected with M.tb MOI of 10 and cell lysates immunoblotted for phospho-AMPKα (T172) at defined time points. M.tb caused maximal phosphorylation at 120 mins. (D) Compound C (Comp C) pre-incubation for 30 minutes before CoMTB stimulation suppresses neutrophil MMP-8 secretion at 4 hours. (E) Compound C (Comp C) was pre-incubated for 30 minutes before stimulation with CoMCont or CoMTB for 24 hours with MMP-8 gene expression analyzed by real-time PCR normalized to GAPDH. Bars represent mean ± s.d. of an experiment performed in biological triplicates on at least 2 occasions. *P<0.05, ***P<0.001. Analysis was performed using one-way ANOVA with Tukey’s post-test.
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Related In: Results  -  Collection

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ppat.1004917.g005: AMPK regulates neutrophil MMP-8 secretion in TB in vitro.(A) Human phosphokinase array. Neutrophils were stimulated with CoMCont or CoMTB. Representative blot from n = 4 healthy donors. Red circle highlights increased AMPKα2 phosphorylation in CoMTB-stimulated cells, with densitometric analysis of components of AMPK pathway below. (B) CoMTB stimulation phosphorylates AMPKα1/2 analyzed by western blotting and gel densitometry. Neutrophils were stimulated for 30 minutes. Bars represent mean ± s.e.m from n = 3 donors. (C) Neutrophils were infected with M.tb MOI of 10 and cell lysates immunoblotted for phospho-AMPKα (T172) at defined time points. M.tb caused maximal phosphorylation at 120 mins. (D) Compound C (Comp C) pre-incubation for 30 minutes before CoMTB stimulation suppresses neutrophil MMP-8 secretion at 4 hours. (E) Compound C (Comp C) was pre-incubated for 30 minutes before stimulation with CoMCont or CoMTB for 24 hours with MMP-8 gene expression analyzed by real-time PCR normalized to GAPDH. Bars represent mean ± s.d. of an experiment performed in biological triplicates on at least 2 occasions. *P<0.05, ***P<0.001. Analysis was performed using one-way ANOVA with Tukey’s post-test.
Mentions: To investigate the key regulatory pathways of neutrophil MMP-8 secretion, we performed a screening human phosphokinase array and observed that the AMP-activated protein kinase (AMPK) pathway was consistently activated in M.tb-infected neutrophils, especially AMPKα2 (T172) (Fig 5A). This activation was confirmed by immunoblotting (Fig 5B). Components of the MAP-kinase, STAT pathways, p53 and Src family of kinases were also activated consistent with previous data [39–41] (S6A, S6B and S6C Fig). AMPK is considered a master regulator of cellular energy homeostasis, existing as a heterotrimeric complex comprising catalytic α-subunits and regulatory β- and γ-subunits [13]. Its activation sets off a cascade of catabolic pathways including glycolysis and ketogenesis which can lead to the wasting which is characteristic in TB patients. We demonstrated that AMPKα was activated by phosphorylation in neutrophils directly infected with M.tb (Fig 5C).

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