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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

Neurophil MMP-8 associates with NETs.(A and B) Neutrophils were infected with M.tb MOI of 10 for 4 hours and NETs stained with DAPI (blue), anti-histone 2B (H2B, green) or anti-MMP-8 (green), while M.tb was stained with anti-M.tb Ab (red). Mtb induces NET formation which do not adhere to the shape of the neutrophil nuclei (White arrows). Scale bars represent 25 μm. (C) Induced sputum NETs were greater in patients with TB than healthy controls (n = 10 both groups analyzed by Student’s t-test). (D) NETs marker citrulline H3 is present in induced sputum of TB patients but not in healthy controls. Blot representative of n = 2 both groups.
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ppat.1004917.g003: Neurophil MMP-8 associates with NETs.(A and B) Neutrophils were infected with M.tb MOI of 10 for 4 hours and NETs stained with DAPI (blue), anti-histone 2B (H2B, green) or anti-MMP-8 (green), while M.tb was stained with anti-M.tb Ab (red). Mtb induces NET formation which do not adhere to the shape of the neutrophil nuclei (White arrows). Scale bars represent 25 μm. (C) Induced sputum NETs were greater in patients with TB than healthy controls (n = 10 both groups analyzed by Student’s t-test). (D) NETs marker citrulline H3 is present in induced sputum of TB patients but not in healthy controls. Blot representative of n = 2 both groups.

Mentions: Next we showed that neutrophils generate NETs when stimulated with M.tb in vitro (S4A Fig), and NETs were digested by DNAse (S4B and S4C Fig). Neutrophil extracellular traps (NETs) are scaffolds containing DNA, histones and antimicrobial granule proteins. We demonstrated for the first time that MMP-8 co-localizes with NETs (Fig 3A and 3B). Next, we evaluated NETs in induced sputum from TB patients and healthy controls from a clinical study [33] (S1 Table). Sputum from TB patients had increased NET concentrations of 1548 mg/ml (± standard error 256 mg/ml) compared to controls at 372 mg/ml (± S.E. 150mg/ml) (Fig 3C, P<0.001). Citrulline H3, an established marker of NETs [34, 35] was detected in induced sputum of TB patients and not in healthy controls (Fig 3D). This was not due to dead or dying cells since these neutrophils do not contain citrulline H3 (S4D 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)

Neurophil MMP-8 associates with NETs.(A and B) Neutrophils were infected with M.tb MOI of 10 for 4 hours and NETs stained with DAPI (blue), anti-histone 2B (H2B, green) or anti-MMP-8 (green), while M.tb was stained with anti-M.tb Ab (red). Mtb induces NET formation which do not adhere to the shape of the neutrophil nuclei (White arrows). Scale bars represent 25 μm. (C) Induced sputum NETs were greater in patients with TB than healthy controls (n = 10 both groups analyzed by Student’s t-test). (D) NETs marker citrulline H3 is present in induced sputum of TB patients but not in healthy controls. Blot representative of n = 2 both groups.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4440706&req=5

ppat.1004917.g003: Neurophil MMP-8 associates with NETs.(A and B) Neutrophils were infected with M.tb MOI of 10 for 4 hours and NETs stained with DAPI (blue), anti-histone 2B (H2B, green) or anti-MMP-8 (green), while M.tb was stained with anti-M.tb Ab (red). Mtb induces NET formation which do not adhere to the shape of the neutrophil nuclei (White arrows). Scale bars represent 25 μm. (C) Induced sputum NETs were greater in patients with TB than healthy controls (n = 10 both groups analyzed by Student’s t-test). (D) NETs marker citrulline H3 is present in induced sputum of TB patients but not in healthy controls. Blot representative of n = 2 both groups.
Mentions: Next we showed that neutrophils generate NETs when stimulated with M.tb in vitro (S4A Fig), and NETs were digested by DNAse (S4B and S4C Fig). Neutrophil extracellular traps (NETs) are scaffolds containing DNA, histones and antimicrobial granule proteins. We demonstrated for the first time that MMP-8 co-localizes with NETs (Fig 3A and 3B). Next, we evaluated NETs in induced sputum from TB patients and healthy controls from a clinical study [33] (S1 Table). Sputum from TB patients had increased NET concentrations of 1548 mg/ml (± standard error 256 mg/ml) compared to controls at 372 mg/ml (± S.E. 150mg/ml) (Fig 3C, P<0.001). Citrulline H3, an established marker of NETs [34, 35] was detected in induced sputum of TB patients and not in healthy controls (Fig 3D). This was not due to dead or dying cells since these neutrophils do not contain citrulline H3 (S4D 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