Sara Brilha

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.

Hypoxia and tissue destruction in pulmonary TB

Background It is unknown whether lesions in human TB are hypoxic or whether this influences disease pathology. Human TB is characterised by extensive lung destruction driven by host matrix metalloproteinases (MMPs), particularly collagenases such as matrix metalloproteinase-1 (MMP-1). Methods We investigated tissue hypoxia in five patients with PET imaging using the tracer [18F]-fluoromisonidazole ([18F]FMISO) and by immunohistochemistry. We studied the regulation of MMP secretion in primary human cell culture model systems in normoxia, hypoxia, chemical hypoxia and by small interfering RNA (siRNA) inhibition. Results [18F]FMISO accumulated in regions of TB consolidation and around pulmonary cavities, demonstrating for the first time severe tissue hypoxia in man. Patlak analysis of dynamic PET data showed heterogeneous levels of hypoxia within and between patients. In Mycobacterium tuberculosis (M.tb)-infected human macrophages, hypoxia (1% pO2) upregulated MMP-1 gene expression 170-fold, driving secretion and caseinolytic activity. Dimethyloxalyl glycine (DMOG), a small molecule inhibitor which stabilises the transcription factor hypoxia-inducible factor (HIF)-1α, similarly upregulated MMP-1. Hypoxia did not affect mycobacterial replication. Hypoxia increased MMP-1 expression in primary respiratory epithelial cells via intercellular networks regulated by TB. HIF-1α and NF-κB regulated increased MMP-1 activity in hypoxia. Furthermore, M.tb infection drove HIF-1α accumulation even in normoxia. In human TB lung biopsies, epithelioid macrophages and multinucleate giant cells express HIF-1α. HIF-1α blockade, including by targeted siRNA, inhibited TB-driven MMP-1 gene expression and secretion. Conclusions Human TB lesions are severely hypoxic and M.tb drives HIF-1α accumulation, synergistically increasing collagenase activity which will lead to lung destruction and cavitation.

Membrane Type 1 Matrix Metalloproteinase Regulates Monocyte Migration and Collagen Destruction in Tuberculosis

Tuberculosis (TB) remains a global pandemic and drug resistance is rising. Multicellular granuloma formation is the pathological hallmark of Mycobacterium tuberculosis infection. The membrane type 1 matrix metalloproteinase (MT1-MMP or MMP-14) is a collagenase that is key in leukocyte migration and collagen destruction. In patients with TB, induced sputum MT1-MMP mRNA levels were increased 5.1-fold compared with matched controls and correlated positively with extent of lung infiltration on chest radiographs (r = 0.483; p < 0.05). M. tuberculosis infection of primary human monocytes increased MT1-MMP surface expression 31.7-fold and gene expression 24.5-fold. M. tuberculosis–infected monocytes degraded collagen matrix in an MT1-MMP–dependent manner, and MT1-MMP neutralization decreased collagen degradation by 73%. In human TB granulomas, MT1-MMP immunoreactivity was observed in macrophages throughout the granuloma. Monocyte–monocyte networks caused a 17.5-fold increase in MT1-MMP surface expression dependent on p38 MAPK and G protein–coupled receptor-dependent signaling. Monocytes migrating toward agarose beads impregnated with conditioned media from M. tuberculosis–infected monocytes expressed MT1-MMP. Neutralization of MT1-MMP activity decreased this M. tuberculosis network–dependent monocyte migration by 44%. Taken together, we demonstrate that MT1-MMP is central to two key elements of TB pathogenesis, causing collagen degradation and regulating monocyte migration.

Complex regulation of neutrophil-derived MMP-9 secretion in central nervous system tuberculosis

BackgroundCentral nervous system tuberculosis (CNS-TB) may be fatal even with treatment. Neutrophils are the key mediators of TB immunopathology, and raised CSF matrix metalloproteinase-9 (MMP-9) which correlates to neutrophil count in CNS-TB is associated with neurological deficit and death. The mechanisms by which neutrophils drive TB-associated CNS matrix destruction are not clearly defined.MethodsHuman brain biopsies with histologically proven CNS-TB were stained for neutrophils, neutrophil elastase, and MMP-9. Neutrophil MMP-9 secretion and gene expression were analyzed using Luminex and real-time PCR. Type IV collagen degradation was evaluated using confocal microscopy and quantitative fluorescent assays. Intracellular signaling pathways were investigated by immunoblotting and chemical inhibitors.ResultsMMP-9-expressing neutrophils were present in tuberculous granulomas in CNS-TB and neutrophil-derived MMP-9 secretion was upregulated by Mycobacterium tuberculosis (M.tb). Concurrent direct stimulation by M.tb and activation via monocyte-dependent networks had an additive effect on neutrophil MMP-9 secretion. Destruction of type IV collagen, a key component of the blood-brain barrier, was inhibited by neutralizing neutrophil MMP-9. Monocyte-neutrophil networks driving MMP-9 secretion in TB were regulated by MAP-kinase and Akt-PI3 kinase pathways and the transcription factor NF-kB. TNFα neutralization suppressed MMP-9 secretion to baseline while dexamethasone did not.ConclusionsMultiple signaling paths regulate neutrophil-derived MMP-9 secretion, which is increased in CNS-TB. These paths may be better targets for host-directed therapies than steroids currently used in CNS-TB.

Early Secretory Antigenic Target-6 Drives Matrix Metalloproteinase-10 Gene Expression and Secretion in Tuberculosis

&NA; Tuberculosis (TB) causes disease worldwide, and multidrug resistance is an increasing problem. Matrix metalloproteinases (MMPs), particularly the collagenase MMP‐1, cause lung extracellular matrix destruction, which drives disease transmission and morbidity. The role in such tissue damage of the stromelysin MMP‐10, a key activator of the collagenase MMP‐1, was investigated in direct Mycobacterium tuberculosis (Mtb)‐infected macrophages and in conditioned medium from Mtb‐infected monocyte‐stimulated cells. Mtb infection increased MMP‐10 secretion from primary human macrophages 29‐fold, whereas Mtb‐infected monocytes increased secretion by 4.5‐fold from pulmonary epithelial cells and 10.5‐fold from fibroblasts. Inhibition of MMP‐10 activity decreased collagen breakdown. In two independent cohorts of patients with TB from different continents, MMP‐10 was increased in both induced sputum and bronchoalveolar lavage fluid compared with control subjects and patients with other respiratory diseases (both P < 0.05). Mtb drove 3.5‐fold greater MMP‐10 secretion from human macrophages than the vaccine strain bacillus Calmette‐Guerin (P < 0.001), whereas both mycobacteria up‐regulated TNF‐&agr; secretion equally. Using overlapping, short, linear peptides covering the sequence of early secretory antigenic target‐6, a virulence factor secreted by Mtb, but not bacillus Calmette‐Guerin, we found that stimulation of human macrophages with a single specific 15‐amino acid peptide sequence drove threefold greater MMP‐10 secretion than any other peptide (P < 0.001). Mtb‐driven MMP‐10 secretion was inhibited in a dose‐dependent manner by p38 and extracellular signal‐related kinase mitogen‐activated protein kinase blockade (P < 0.001 and P < 0.01 respectively), but it was not affected by inhibition of NF‐&kgr;B. In summary, Mtb activates inflammatory and stromal cells to secrete MMP‐10, and this is partly driven by the virulence factor early secretory antigenic target‐6, implicating it in TB‐associated tissue destruction.

Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis

Central nervous system tuberculosis (CNS TB) has a high mortality and morbidity associated with severe inflammation. The blood-brain barrier (BBB) protects the brain from inflammation but the mechanisms causing BBB damage in CNS TB are uncharacterized. We demonstrate that Mycobacterium tuberculosis (Mtb) causes breakdown of type IV collagen and decreases tight junction protein (TJP) expression in a co-culture model of the BBB. This increases permeability, surface expression of endothelial adhesion molecules and leukocyte transmigration. TJP breakdown was driven by Mtb-dependent secretion of matrix metalloproteinase (MMP)-9. TJP expression is regulated by Sonic hedgehog (Shh) through transcription factor Gli-1. In our model, the hedgehog pathway was downregulated by Mtb-stimulation, but Shh levels in astrocytes were unchanged. However, Scube2, a glycoprotein regulating astrocyte Shh release was decreased, inhibiting Shh delivery to brain endothelial cells. Activation of the hedgehog pathway by addition of a Smoothened agonist or by addition of exogenous Shh, or neutralizing MMP-9 activity, decreased permeability and increased TJP expression in the Mtb-stimulated BBB co-cultures. In summary, the BBB is disrupted by downregulation of the Shh pathway and breakdown of TJPs, secondary to increased MMP-9 activity which suggests that these pathways are potential novel targets for host directed therapy in CNS TB.

Epigenetic Regulation of Matrix Metalloproteinase-1 and -3 Expression in Mycobacterium tuberculosis Infection

In pulmonary tuberculosis (TB), the inflammatory immune response against Mycobacterium tuberculosis (Mtb) is associated with tissue destruction and cavitation, which drives disease transmission, chronic lung disease, and mortality. Matrix metalloproteinase (MMP)-1 is a host enzyme critical for the development of cavitation. MMP expression has been shown to be epigenetically regulated in other inflammatory diseases, but the importance of such mechanisms in Mtb-associated induction of MMP-1 is unknown. We investigated the role of changes in histone acetylation in Mtb-induced MMP expression using inhibitors of histone deacetylases (HDACs) and histone acetyltransferases (HAT), HDAC siRNA, promoter-reporter constructs, and chromatin immunoprecipitation assays. Mtb infection decreased Class I HDAC gene expression by over 50% in primary human monocyte-derived macrophages but not in normal human bronchial epithelial cells (NHBEs). Non-selective inhibition of HDAC activity decreased MMP-1/-3 expression by Mtb-stimulated macrophages and NHBEs, while class I HDAC inhibition increased MMP-1 secretion by Mtb-stimulated NHBEs. MMP-3 expression, but not MMP-1, was downregulated by siRNA silencing of HDAC1. Inhibition of HAT activity also significantly decreased MMP-1/-3 secretion by Mtb-infected macrophages. The MMP-1 promoter region between −2,001 and −2,942 base pairs from the transcriptional start site was key in control of Mtb-driven MMP-1 gene expression. Histone H3 and H4 acetylation and RNA Pol II binding in the MMP-1 promoter region were increased in stimulated NHBEs. In summary, epigenetic modification of histone acetylation via HDAC and HAT activity has a key regulatory role in Mtb-dependent gene expression and secretion of MMP-1 and -3, enzymes which drive human immunopathology. Manipulation of epigenetic regulatory mechanisms may have potential as a host-directed therapy to improve outcomes in the era of rising TB drug resistance.

ESAT-6 Drives MMP-10 Gene Expression and Secretion in Tuberculosis.

&NA; Tuberculosis (TB) causes disease worldwide, and multidrug resistance is an increasing problem. Matrix metalloproteinases (MMPs), particularly the collagenase MMP‐1, cause lung extracellular matrix destruction, which drives disease transmission and morbidity. The role in such tissue damage of the stromelysin MMP‐10, a key activator of the collagenase MMP‐1, was investigated in direct Mycobacterium tuberculosis (Mtb)‐infected macrophages and in conditioned medium from Mtb‐infected monocyte‐stimulated cells. Mtb infection increased MMP‐10 secretion from primary human macrophages 29‐fold, whereas Mtb‐infected monocytes increased secretion by 4.5‐fold from pulmonary epithelial cells and 10.5‐fold from fibroblasts. Inhibition of MMP‐10 activity decreased collagen breakdown. In two independent cohorts of patients with TB from different continents, MMP‐10 was increased in both induced sputum and bronchoalveolar lavage fluid compared with control subjects and patients with other respiratory diseases (both P < 0.05). Mtb drove 3.5‐fold greater MMP‐10 secretion from human macrophages than the vaccine strain bacillus Calmette‐Guerin (P < 0.001), whereas both mycobacteria up‐regulated TNF‐&agr; secretion equally. Using overlapping, short, linear peptides covering the sequence of early secretory antigenic target‐6, a virulence factor secreted by Mtb, but not bacillus Calmette‐Guerin, we found that stimulation of human macrophages with a single specific 15‐amino acid peptide sequence drove threefold greater MMP‐10 secretion than any other peptide (P < 0.001). Mtb‐driven MMP‐10 secretion was inhibited in a dose‐dependent manner by p38 and extracellular signal‐related kinase mitogen‐activated protein kinase blockade (P < 0.001 and P < 0.01 respectively), but it was not affected by inhibition of NF‐&kgr;B. In summary, Mtb activates inflammatory and stromal cells to secrete MMP‐10, and this is partly driven by the virulence factor early secretory antigenic target‐6, implicating it in TB‐associated tissue destruction.

Monocyte Adhesion, Migration, and Extracellular Matrix Breakdown Is Regulated by Integrin αVβ3 in Mycobacterium tuberculosis Infection

In tuberculosis (TB), the innate inflammatory immune response drives tissue destruction, morbidity, and mortality. Monocytes secrete matrix metalloproteinases (MMPs), which have key roles in local tissue destruction and cavitation. We hypothesized that integrin signaling might regulate monocyte MMP secretion in pulmonary TB during cell adhesion to the extracellular matrix (ECM). Adhesion to type I collagen and fibronectin by Mycobacterium tuberculosis–stimulated monocytes increased MMP-1 gene expression by 2.6-fold and 4.3-fold respectively, and secretion by 60% (from 1208.1 ± 186 to 1934.4 ± 135 pg/ml; p < 0.0001) and 63% (1970.3 ± 95 pg/ml; p < 0.001). MMP-10 secretion increased by 90% with binding to type I collagen and 55% with fibronectin, whereas MMP-7 increased 57% with collagen. The ECM did not affect the secretion of tissue inhibitors of metalloproteinases-1 or -2. Integrin αVβ3 surface expression was specifically upregulated in stimulated monocytes and was further increased after adhesion to type I collagen. Binding of either β3 or αV integrin subunits increased MMP-1/10 secretion in M. tuberculosis–stimulated monocytes. In a cohort of TB patients, significantly increased integrin β3 mRNA accumulation in induced sputum was detected, to our knowledge, for the first time, compared with control subjects (p < 0.05). Integrin αVβ3 colocalized with areas of increased and functionally active MMP-1 on infected monocytes, and αVβ3 blockade markedly decreased type I collagen breakdown, and impaired both monocyte adhesion and leukocyte migration in a transwell system (p < 0.0001). In summary, our data demonstrate that M. tuberculosis stimulation upregulates integrin αVβ3 expression on monocytes, which upregulates secretion of MMP-1 and -10 on adhesion to the ECM. This leads to increased monocyte recruitment and collagenase activity, which will drive inflammatory tissue damage.

Author Correction: Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an in vitro model of CNS tuberculosis

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