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

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

AMPK regulates neutrophil MMP-8 secretion in patients.

<p>(<b>A</b>) Neutrophils are present in human TB lung cavity wall and stain positive for neutrophil elastase. Scale bar 250μm. (<b>B</b>) Neutrophils express phospho-AMPKα (T172) in the nuclei (red arrows) (inset of A). Scale bar 50μm. (<b>C</b>) Neutrophils from healthy donors and patients with AMPK deficiency were stimulated with <i>M</i>.<i>tb</i> MOI of 10, CoMCont or CoMTB for 30 minutes. In patients, AMPK is constitutively phosphorylated, resulting in a functional deficiency. Blot representative of 6 healthy controls and AMPK patients. (<b>D</b>) Neutrophils from healthy donors and patients with AMPK deficiency were stimulated with CoMTB. <i>n</i> = 3 both groups. Neutrophils derived from AMPK patients secreted less MMP-8 when stimulated than cells from healthy donors. Experiments were performed in biological triplicates and each point represents a sample. Analysis was performed using one-way ANOVA or two-tailed t-test. ** <i>P</i><0.01, ***<i>P</i><0.001.</p

Neutrophil MMP-8 and -9 are upregulated in human TB.

<p>(<b>A</b>) Neutrophils were infected with <i>M</i>.<i>tb</i> MOI of 1. MMP-8 secretion was upregulated at 4h. (<b>B</b>) Increasing <i>M</i>.<i>tb</i> 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. (<b>C and D</b>) 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. (<b>E and F</b>) Magnified H&E and MMP-8 stains from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004917#ppat.1004917.g001" target="_blank">Fig 1C</a> inset shows neutrophils immunoreactive for MMP-8 around the cavity wall. Both scale bars represent 50 μm. (<b>G</b>) MMP-9 concentrations increase in a dose-dependent manner after <i>M</i>.<i>tb</i> 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. *** <i>P</i><0.001 (<b>H</b>) Biopsy proven <i>M</i>.<i>tb</i> infected human lung specimens were stained for MMP-9 (inset from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004917#ppat.1004917.g001" target="_blank">Fig 1C</a>). 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. **<i>P</i><0.01, ***<i>P</i><0.001.</p

<i>M</i>.<i>tb</i> and CoMTB-stimulated neutrophils degrade collagen.

<p>(<b>A</b>) Neutrophils were stimulated with either <i>M</i>.<i>tb</i> MOI of 10, CoMCont or CoMTB for 4 hours. <i>M</i>.<i>tb</i> and CoMTB up-regulated MMP-8 secretion analyzed by ELISA. (<b>B</b>) Cell-free supernatants from (A) were incubated with Type I DQ collagen. Bars represent mean ± s.d. of experiments performed in triplicate and are representative of a minimum of 2 independent experiments. (<b>C</b>) CoMTB caused increased collagen breakdown by neutrophils, resulting in greater fluorescence of DQ collagen. (<b>D</b>) Neutrophils were infected with <i>M</i>.<i>tb</i> MOI 10, fixed and <i>M</i>.<i>tb</i> stained with <i>anti-M</i>.<i>tb</i> Ab. Infected cells degraded DQ collagen, increasing fluorescence. (<b>E</b>) Cell-free supernatants from neutrophils infected with <i>M</i>.<i>tb</i> at MOI 10 were added with doxycyline to Type I DQ collagen. Doxycycline suppressed collagenase activity in a dose-responsive manner. Bars represent mean ± s.d of an experiment performed in biological triplicates and represents 2–3 independent experiments. Analysis performed using One-way ANOVA with Tukey’s post-test. ** <i>P</i><0.01, ***<i>P</i><0.001.</p

Induced sputum samples of pulmonary TB patients have increased collagenase activity due to neutrophil-derived MMP-8.

<p>(<b>A and B</b>) Induced sputum MPO and NGAL was analyzed by ELISA from <i>n</i> = 51 TB patients and <i>n</i> = 57 healthy controls. MPO and NGAL were increased in patients with pulmonary TB. (<b>C and D</b>) Induced sputum MMP-8 closely correlated with both MPO and NGAL in TB patients, performed using Spearman’s correlation coefficient. (<b>E</b>) Induced sputum collagenase activity is increased in TB patients. (<i>n</i> = 11 each group). Subsets analyzed were representative of the whole cohort analyzed by Mann-Whitney test. (<b>F</b>) Confocal microscopy shows increased DQ collagen degradation in induced sputum of TB patient relative to control. Image is representative of <i>n</i> = 3 each group. Scale bars represent 50μm. (<b>G</b>) Induced sputum MMP-8 and collagenase activity correlate, analyzed by Spearman’s correlation coefficient (<i>n</i> = 22). (<b>H</b>) MMP-8 neutralization suppresses induced sputum collagenase activity from TB patients. MMP-8 neutralizing antibody was added to activated induced sputum with Type I DQ collagen (<i>n</i> = 11). Box and whiskers represent 10–90^th percentile with comparison using Wilcoxon-Sign rank test. (<b>I</b>) Induced sputum MMP-8 were higher in patients with pulmonary cavities than those without. * <i>P</i>< 0.05, **<i>P</i><0.01, ****<i>P</i><0.0001.</p

Neurophil MMP-8 associates with NETs.

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

AMPK regulates neutrophil MMP-8 secretion in TB <i>in vitro</i>.

<p>(<b>A</b>) Human phosphokinase array. Neutrophils were stimulated with CoMCont or CoMTB. Representative blot from <i>n</i> = 4 healthy donors. Red circle highlights increased AMPKα2 phosphorylation in CoMTB-stimulated cells, with densitometric analysis of components of AMPK pathway below. (<b>B</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 <i>n</i> = 3 donors. (<b>C</b>) Neutrophils were infected with <i>M</i>.<i>tb</i> MOI of 10 and cell lysates immunoblotted for phospho-AMPKα (T172) at defined time points. <i>M</i>.<i>tb</i> caused maximal phosphorylation at 120 mins. (<b>D</b>) Compound C (Comp C) pre-incubation for 30 minutes before CoMTB stimulation suppresses neutrophil MMP-8 secretion at 4 hours. (<b>E</b>) 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. *<i>P</i><0.05, ***<i>P</i><0.001. Analysis was performed using one-way ANOVA with Tukey’s post-test.</p