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.

BackgroundIt 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).MethodsWe 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.ConclusionsHuman TB lesions are severely hypoxic and M.tb drives HIF-1α accumulation, synergistically increasing collagenase activity which will lead to lung destruction and cavitation.

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.

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.