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