In an in vitro model of human tuberculosis, monocyte-microglial networks regulate matrix metalloproteinase-1 and -3 gene expression and secretion via a p38 mitogen activated protein kinase-dependent pathway.

BACKGROUND: Tuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. However, relatively little is known about the regulation of these MMPs in the CNS. METHODS: Using a cellular model of CNS TB, we stimulated a human microglial cell line (CHME3) with conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb). MMP-1 and MMP-3 secretion was detected using ELISAs confirmed with casein zymography or western blotting. Key results of a phospho-array profile that detects a wide range of kinase activity were confirmed with phospho-Western blotting. Chemical inhibition (SB203580) of microglial cells allowed investigation of expression and secretion of MMP-1 and MMP-3. Finally we used promoter reporter assays employing full length and MMP-3 promoter deletion constructs. Student's t-test was used for comparison of continuous variables and multiple intervention experiments were compared by one-way ANOVA with Tukey's correction for multiple pairwise comparisons. RESULTS: CoMTb up-regulated microglial MMP-1 and MMP-3 secretion in a dose- and time-dependent manner. The phospho-array profiling showed that the major increase in kinase activity due to CoMTb stimulation was in p38 mitogen activated protein kinase (MAPK), principally the α and γ subunits. p38 phosphorylation was detected at 15 minutes, with a second peak of activity at 120 minutes. High basal extracellular signal-regulated kinase activity was further increased by CoMTb. Secretion and expression of MMP-1 and MMP-3 were both p38 dependent. CoMTb stimulation of full length and MMP-3 promoter deletion constructs demonstrated up-regulation of activity in the wild type but a suppression site between -2183 and -1612 bp. CONCLUSIONS: Monocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 MAPK in tuberculosis. p38 is therefore a potential target for adjuvant therapy in CNS TB

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

BACKGROUND: Central 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. METHODS: Human 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. RESULTS: MMP-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. CONCLUSIONS: Multiple 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

Mycobacterium tuberculosis subverts negative regulatory pathways in human macrophages to drive immunopathology.

Tuberculosis remains a global pandemic and drives lung matrix destruction to transmit. Whilst pathways driving inflammatory responses in macrophages have been relatively well described, negative regulatory pathways are less well defined. We hypothesised that Mycobacterium tuberculosis (Mtb) specifically targets negative regulatory pathways to augment immunopathology. Inhibition of signalling through the PI3K/AKT/mTORC1 pathway increased matrix metalloproteinase-1 (MMP-1) gene expression and secretion, a collagenase central to TB pathogenesis, and multiple pro-inflammatory cytokines. In patients with confirmed pulmonary TB, PI3Kδ expression was absent within granulomas. Furthermore, Mtb infection suppressed PI3Kδ gene expression in macrophages. Interestingly, inhibition of the MNK pathway, downstream of pro-inflammatory p38 and ERK MAPKs, also increased MMP-1 secretion, whilst suppressing secretion of TH1 cytokines. Cross-talk between the PI3K and MNK pathways was demonstrated at the level of eIF4E phosphorylation. Mtb globally suppressed the MMP-inhibitory pathways in macrophages, reducing levels of mRNAs encoding PI3Kδ, mTORC-1 and MNK-1 via upregulation of miRNAs. Therefore, Mtb disrupts negative regulatory pathways at multiple levels in macrophages to drive a tissue-destructive phenotype that facilitates transmission

Distinct emphysema subtypes defined by quantitative CT analysis are associated with specific pulmonary matrix metalloproteinases.

BACKGROUND: Emphysema is characterised by distinct pathological sub-types, but little is known about the divergent underlying aetiology. Matrix-metalloproteinases (MMPs) are proteolytic enzymes that can degrade the extracellular matrix and have been identified as potentially important in the development of emphysema. However, the relationship between MMPs and emphysema sub-type is unknown. We investigated the role of MMPs and their inhibitors in the development of emphysema sub-types by quantifying levels and determining relationships with these sub-types in mild-moderate COPD patients and ex/current smokers with preserved lung function. METHODS: Twenty-four mild-moderate COPD and 8 ex/current smokers with preserved lung function underwent high resolution CT and distinct emphysema sub-types were quantified using novel local histogram-based assessment of lung density. We analysed levels of MMPs and tissue inhibitors of MMPs (TIMPs) in bronchoalveolar lavage (BAL) and assessed their relationship with these emphysema sub-types. RESULTS: The most prevalent emphysema subtypes in COPD subjects were mild and moderate centrilobular (CLE) emphysema, while only small amounts of severe centrilobular emphysema, paraseptal emphysema (PSE) and panlobular emphysema (PLE) were present. MMP-3, and -10 associated with all emphysema sub-types other than mild CLE, while MMP-7 and -8 had associations with moderate and severe CLE and PSE. MMP-9 also had associations with moderate CLE and paraseptal emphysema. Mild CLE occurred in substantial quantities irrespective of whether airflow obstruction was present and did not show any associations with MMPs. CONCLUSION: Multiple MMPs are directly associated with emphysema sub-types identified by CT imaging, apart from mild CLE. This suggests that MMPs play a significant role in the tissue destruction seen in the more severe sub-types of emphysema, whereas early emphysematous change may be driven by a different mechanism. TRIAL REGISTRATION: Trial registration number NCT01701869

Matrix Degradation in Human Immunodeficiency Virus Type 1-Associated Tuberculosis and Tuberculosis Immune Reconstitution Inflammatory Syndrome: A Prospective Observational Study.

Background: Extensive immunopathology occurs in human immunodeficiency virus (HIV)/tuberculosis (TB) coinfection, but the underlying molecular mechanisms are not well-defined. Excessive matrix metalloproteinase (MMP) activity is emerging as a key process but has not been systematically studied in HIV-associated TB. Methods: We performed a cross-sectional study of matrix turnover in HIV type 1 (HIV-1)-infected and -uninfected TB patients and controls, and a prospective cohort study of HIV-1-infected TB patients at risk of TB immune reconstitution inflammatory syndrome (TB-IRIS), in Cape Town, South Africa. Sputum and plasma MMP concentrations were quantified by Luminex, plasma procollagen III N-terminal propeptide (PIIINP) by enzyme-linked immunosorbent assay, and urinary lipoarabinomannan (LAM) by Alere Determine TB LAM assay. Peripheral blood mononuclear cells from healthy donors were cultured with Mycobacterium tuberculosis and extracellular matrix in a 3D model of TB granuloma formation. Results: MMP activity differed between HIV-1-infected and -uninfected TB patients and corresponded with specific TB clinical phenotypes. HIV-1-infected TB patients had reduced pulmonary MMP concentrations, associated with reduced cavitation, but increased plasma PIIINP, compared to HIV-1-uninfected TB patients. Elevated extrapulmonary extracellular matrix turnover was associated with TB-IRIS, both before and during TB-IRIS onset. The predominant collagenase was MMP-8, which was likely neutrophil derived and M. tuberculosis-antigen driven. Mycobacterium tuberculosis-induced matrix degradation was suppressed by the MMP inhibitor doxycycline in vitro. Conclusions: MMP activity in TB differs by HIV-1 status and compartment, and releases matrix degradation products. Matrix turnover in HIV-1-infected patients is increased before and during TB-IRIS, informing novel diagnostic strategies. MMP inhibition is a potential host-directed therapy strategy for prevention and treatment of TB-IRIS

Hypoxia increases neutrophil-driven matrix destruction after exposure to Mycobacterium tuberculosis.

The importance of neutrophils in the pathology of tuberculosis (TB) has been recently established. We demonstrated that TB lesions in man are hypoxic, but how neutrophils in hypoxia influence lung tissue damage is unknown. We investigated the effect of hypoxia on neutrophil-derived enzymes and tissue destruction in TB. Human neutrophils were stimulated with M. tuberculosis (M.tb) or conditioned media from M.tb-infected monocytes (CoMTB). Neutrophil matrix metalloproteinase-8/-9 and elastase secretion were analysed by luminex array and gelatin zymography, gene expression by qPCR and cell viability by flow cytometry. Matrix destruction was investigated by confocal microscopy and functional assays and neutrophil extracellular traps (NETs) by fluorescence assay. In hypoxia, neutrophil MMP-8 secretion and gene expression were up-regulated by CoMTB. MMP-9 activity and neutrophil elastase (NE) secretion were also increased in hypoxia. Hypoxia inhibited NET formation and both neutrophil apoptosis and necrosis after direct stimulation by M.tb. Hypoxia increased TB-dependent neutrophil-mediated matrix destruction of Type I collagen, gelatin and elastin, the main structural proteins of the human lung. Dimethyloxalylglycin (DMOG), which stabilizes hypoxia-inducible factor-1α, increased neutrophil MMP-8 and -9 secretion. Hypoxia in our cellular model of TB up-regulated pathways that increase neutrophil secretion of MMPs that are implicated in matrix destruction

Invariant Natural Killer T cell dynamics in HIV-associated tuberculosis.

RATIONALE: Tuberculosis (TB) is the leading cause of mortality and morbidity in people living with HIV infection. HIV-infected patients with TB disease are at risk of the paradoxical TB-associated immune reconstitution inflammatory syndrome (TB-IRIS) when they commence anti-retroviral therapy. However, the pathophysiology is incompletely understood and specific therapy is lacking. OBJECTIVES: We investigated the hypothesis that invariant Natural Killer T (iNKT) cells contribute to innate immune dysfunction associated with TB-IRIS. METHODS: In a cross-sectional study of 101 HIV-infected and -uninfected South African patients with active TB and controls, iNKT cells were enumerated using α-galactosylceramide-loaded CD1d tetramers and subsequently functionally characterised by flow cytometry. In a second study of 49 HIV-1-infected TB patients commencing anti-retroviral therapy, iNKT cells in TB-IRIS patients with non-IRIS controls were compared longitudinally. MEASUREMENTS AND MAIN RESULTS: Circulating iNKT cells were reduced in HIV-1 infection, most significantly the CD4+ subset, which was inversely associated with HIV-1 viral load. iNKT cells in HIV-associated TB had increased surface CD107a expression, indicating cytotoxic degranulation. Relatively increased iNKT cell frequency in HIV-infected patients with active TB was associated with development of TB-IRIS following anti-retroviral therapy initiation. iNKT cells in TB-IRIS were CD4+CD8- subset deplete and degranulated around the time of TB-IRIS onset. CONCLUSIONS: Reduced iNKT cell CD4+ subsets as a result of HIV-1 infection may skew iNKT cell functionality towards cytotoxicity. Increased CD4- cytotoxic iNKT cells may contribute to immunopathology in TB-IRIS

Optimisation of Bioluminescent Reporters for Use with Mycobacteria

BACKGROUND: Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI. METHODOLOGY/PRINCIPAL FINDINGS: One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging. CONCLUSIONS/SIGNIFICANCE: While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase

Gender-dependent differences in plasma matrix metalloproteinase-8 elevated in pulmonary tuberculosis.

Tuberculosis (TB) remains a global health pandemic and greater understanding of underlying pathogenesis is required to develop novel therapeutic and diagnostic approaches. Matrix metalloproteinases (MMPs) are emerging as key effectors of tissue destruction in TB but have not been comprehensively studied in plasma, nor have gender differences been investigated. We measured the plasma concentrations of MMPs in a carefully characterised, prospectively recruited clinical cohort of 380 individuals. The collagenases, MMP-1 and MMP-8, were elevated in plasma of patients with pulmonary TB relative to healthy controls, and MMP-7 (matrilysin) and MMP-9 (gelatinase B) were also increased. MMP-8 was TB-specific (p<0.001), not being elevated in symptomatic controls (symptoms suspicious of TB but active disease excluded). Plasma MMP-8 concentrations inversely correlated with body mass index. Plasma MMP-8 concentration was 1.51-fold higher in males than females with TB (p<0.05) and this difference was not due to greater disease severity in men. Gender-specific analysis of MMPs demonstrated consistent increase in MMP-1 and -8 in TB, but MMP-8 was a better discriminator for TB in men. Plasma collagenases are elevated in pulmonary TB and differ between men and women. Gender must be considered in investigation of TB immunopathology and development of novel diagnostic markers

Matrix metalloproteinase-9 activity and a downregulated Hedgehog pathway impair blood-brain barrier function in an <i>in vitro</i> 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