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

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-α 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-κB. 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.

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.

A review of the literature regarding nutritional supplements and their effect on vaginal flora and preterm birth.

PURPOSE OF REVIEW: The aim of this review was to evaluate recently published review articles which examine the use of nutritional supplements to prevent preterm birth (PTB) by modifying vaginal bacteria. RECENT FINDINGS: Probiotics, vitamin D and vitamin C were all identified as nutritional supplements that have the potential to alter bacterial flora and consequently reduce PTB and treat or prevent genital infections. Evidence shows that probiotics may reduce the incidence of PTB as well as being effective at treating bacterial vaginosis, a known cause for PTB. Low vitamin D levels may be associated with bacterial vaginosis, although no evidence was identified which demonstrated that vitamin D supplementation reduced the risk of having bacterial vaginosis or PTB.There is little evidence regarding vitamin C supplementation, although it does suggest a possible benefit with regard to preterm rupture of membranes; however, this did not appear to reduce rates of PTB. SUMMARY: Although there is evidence that taking probiotics in pregnancy may reduce the incidence of PTB, it is mainly derived from small, poor quality studies. Vitamin D and vitamin C may have potential benefits, but these remain to be proven. Large randomized controlled trials are needed to more accurately evaluate the potential benefits of these low-cost interventions for reducing PTB and its consequences.

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.

Mycobacterium tuberculosis upregulates microglial matrix metalloproteinase-1 and -3 expression and secretion via NF-kappaB- and Activator Protein-1-dependent monocyte networks.

Inflammatory tissue destruction is central to pathology in CNS tuberculosis (TB). We hypothesized that microglial-derived matrix metalloproteinases (MMPs) have a key role in driving such damage. Analysis of all of the MMPs demonstrated that conditioned medium from Mycobacterium tuberculosis-infected human monocytes (CoMTb) stimulated greater MMP-1, -3, and -9 gene expression in human microglial cells than direct infection. In patients with CNS TB, MMP-1/-3 immunoreactivity was demonstrated in the center of brain granulomas. Concurrently, CoMTb decreased expression of the inhibitors, tissue inhibitor of metalloproteinase-2, -3, and -4. MMP-1/-3 secretion was significantly inhibited by dexamethasone, which reduces mortality in CNS TB. Surface-enhanced laser desorption ionization time-of-flight analysis of CoMTb showed that TNF-alpha and IL-1beta are necessary but not sufficient for upregulating MMP-1 secretion and act synergistically to drive MMP-3 secretion. Chemical inhibition and promoter-reporter analyses showed that NF-kappaB and AP-1 c-Jun/FosB heterodimers regulate CoMTb-induced MMP-1/-3 secretion. Furthermore, NF-kappaB p65 and AP-1 c-Jun subunits were upregulated in biopsy granulomas from patients with cerebral TB. In summary, functionally unopposed, network-dependent microglial MMP-1/-3 gene expression and secretion regulated by NF-kappaB and AP-1 subunits were demonstrated in vitro and, for the first time, in CNS TB patients. Dexamethasone suppression of MMP-1/-3 gene expression provides a novel mechanism explaining the benefit of steroid therapy in these patients.

Mycobacterium tuberculosis-infected human monocytes down-regulate microglial MMP-2 secretion in CNS tuberculosis via TNFα, NFκB, p38 and caspase 8 dependent pathways.

Tuberculosis (TB) of the central nervous system (CNS) is a deadly disease characterized by extensive tissue destruction, driven by molecules such as Matrix Metalloproteinase-2 (MMP-2) which targets CNS-specific substrates. In a simplified cellular model of CNS TB, we demonstrated that conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb), but not direct infection, unexpectedly down-regulates constitutive microglial MMP-2 gene expression and secretion by 72.8% at 24 hours, sustained up to 96 hours (P < 0.01), dependent upon TNF-α. In human CNS TB brain biopsies but not controls the p38 pathway was activated in microglia/macrophages. Inhibition of the p38 MAP kinase pathway resulted in a 228% increase in MMP-2 secretion (P < 0.01). In contrast ERK MAP kinase inhibition further decreased MMP-2 secretion by 76.6% (P < 0.05). Inhibition of the NFκB pathway resulted in 301% higher MMP-2 secretion than CoMTb alone (P < 0.01). Caspase 8 restored MMP-2 secretion to basal levels. However, this caspase-dependent regulation of MMP-2 was independent of p38 and NFκB pathways; p38 phosphorylation was increased and p50/p65 NFκB nuclear trafficking unaffected by caspase 8 inhibition. In summary, suppression of microglial MMP-2 secretion by M.tb-infected monocyte-dependent networks paradoxically involves the pro-inflammatory mediators TNF-α, p38 MAP kinase and NFκB in addition to a novel caspase 8-dependent pathway.

How to investigate and manage a patient with a Staphylococcus aureus bacteraemia.

Staphylococcus aureus bacteraemia is common, and associated with significant morbidity and mortality as a result of its high relapse rate and the risk of complicated infection. A positive blood culture for S. aureus should prompt a thorough patient assessment to identify a potential focus of infection, and the risk factors for the development or presence of complicated infection. Clinical management depends on the patient's characteristics and presenting features. This article gives a systematic approach to the patient with S. aureus bacteraemia, including points to look for on history and examination, the markers of complicated infection, and when to request transoesophageal echocardiography and further imaging. Treatment principles outlined include the rationale for choice of antibiotic treatment and need to involve infection specialists.

A severe case of methaemoglobinaemia in a Brazilian hairdresser.

Methaemoglobinaemia is an extremely rare condition with multiple causes, both genetic and acquired. We present a severe case of methaemoglobinaemia occurring in a Brazilian hairdresser working in the UK. She presented after several days of preparing popular 'Brazilian blowdry' treatments for customers at a hair salon. She had been exposed to multiple volatile chemicals, including formaldehyde, without any respiratory protection, and we postulate that this may have caused her illness. If so, this would be the first published case of methaemoglobinaemia caused by exposure to the volatile components of beauty products.

Snapshot PCR surveillance for SARS-CoV-2 in hospital staff in England.

BACKGROUND: Significant nosocomial transmission of SARS-CoV-2 has been demonstrated. Understanding the prevalence of SARS-CoV-2 carriage amongst HCWs at work is necessary to inform the development of HCW screening programmes to control nosocomial spread. METHODS: Cross-sectional 'snapshot' survey from April-May 2020; HCWs recruited from six UK hospitals. Participants self-completed a health questionnaire and underwent a combined viral nose and throat swab, tested by Polymerase Chain Reaction (PCR) for SARS-CoV-2 with viral culture on majority of positive samples. FINDINGS: Point prevalence of SARS-CoV-2 carriage across the sites was 2.0% (23/1152 participants), median cycle threshold value 35.70 (IQR:32.42-37.57). 17 were previously symptomatic, two currently symptomatic (isolated anosmia and sore throat); the remainder declared no prior or current symptoms. Symptoms in the past month were associated with threefold increased odds of testing positive (aOR 3.46, 95%CI 1.38-8.67; p = 0.008). SARS-CoV-2 virus was isolated from only one (5%) of nineteen cultured samples. A large proportion (39%) of participants reported symptoms in the past month. INTERPRETATION: The point-prevalence is similar to previous estimates for HCWs in April 2020, though a magnitude higher than in the general population. Based upon interpretation of symptom history and testing results including viral culture, the majority of those testing positive were unlikely to be infectious at time of sampling. Development of screening programmes must balance the potential to identify additional cases based upon likely prevalence, expanding the symptoms list to encourage HCW testing, with resource implications and risks of excluding those unlikely to be infectious with positive tests. FUNDING: Public Health England.

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.