Intracellular growth of Mycobacterium tuberculosis after macrophage cell death leads to serial killing of host cells

A hallmark of pulmonary tuberculosis is the formation of macrophage-rich granulomas. These may restrict Mycobacterium tuberculosis (Mtb) growth, or progress to central necrosis and cavitation, facilitating pathogen growth. To determine factors leading to Mtb proliferation and host cell death, we used live cell imaging to track Mtb infection outcomes in individual primary human macrophages. Internalization of Mtb aggregates caused macrophage death, and phagocytosis of large aggregates was more cytotoxic than multiple small aggregates containing similar numbers of bacilli. Macrophage death did not result in clearance of Mtb. Rather, it led to accelerated intracellular Mtb growth regardless of prior activation or macrophage type. In contrast, bacillary replication was controlled in live phagocytes. Mtb grew as a clump in dead cells, and macrophages which internalized dead infected cells were very likely to die themselves, leading to a cell death cascade. This demonstrates how pathogen virulence can be achieved through numbers and aggregation states. DOI: http://dx.doi.org/10.7554/eLife.22028.00

Immunological insights into cigarette smoking-induced cardiovascular disease risk.

Smoking is one of the most prominent addictions of the modern world, and one of the leading preventable causes of death worldwide. Although the number of tobacco smokers is believed to be at a historic low, electronic cigarette use has been on a dramatic rise over the past decades. Used as a replacement for cigarette smoking, electronic cigarettes were thought to reduce the negative effects of burning tobacco. Nonetheless, the delivery of nicotine by electronic cigarettes, the most prominent component of cigarette smoke (CS) is still delivering the same negative outcomes, albeit to a lesser extent than CS. Smoking has been shown to affect both the structural and functional aspects of major organs, including the lungs and vasculature. Although the deleterious effects of smoking on these organs individually is well-known, it is likely that the adverse effects of smoking on these organs will have long-lasting effects on the cardiovascular system. In addition, smoking has been shown to play an independent role in the homeostasis of the immune system, leading to major sequela. Both the adaptive and the innate immune system have been explored regarding CS and have been demonstrated to be altered in a way that promotes inflammatory signals, leading to an increase in autoimmune diseases, inflammatory diseases, and cancer. Although the mechanism of action of CS has not been fully understood, disease pathways have been explored in both branches of the immune system. The pathophysiologically altered immune system during smoking and its correlation with cardiovascular diseases is not fully understood. Here we highlight some of the important pathological mechanisms that involve cigarette smoking and its many components on cardiovascular disease and the immune systems in order to have a better understanding of the mechanisms at play

Circulating stem cells and cardiovascular outcomes: from basic science to the clinic

The cardiovascular and haematopoietic systems have fundamental inter-relationships during development, as well as in health and disease of the adult organism. Although haematopoietic stem cells (HSCs) emerge from a specialized haemogenic endothelium in the embryo, persistence of haemangioblasts in adulthood is debated. Rather, the vast majority of circulating stem cells (CSCs) is composed of bone marrow-derived HSCs and the downstream haematopoietic stem/progenitors (HSPCs). A fraction of these cells, known as endothelial progenitor cells (EPCs), has endothelial specification and vascular tropism. In general, the levels of HSCs, HSPCs, and EPCs are considered indicative of the endogenous regenerative capacity of the organism as a whole and, particularly, of the cardiovascular system. In the last two decades, the research on CSCs has focused on their physiologic role in tissue/organ homoeostasis, their potential application in cell therapies, and their use as clinical biomarkers. In this review, we provide background information on the biology of CSCs and discuss in detail the clinical implications of changing CSC levels in patients with cardiovascular risk factors or established cardiovascular disease. Of particular interest is the mounting evidence available in the literature on the close relationships between reduced levels of CSCs and adverse cardiovascular outcomes in different cohorts of patients. We also discuss potential mechanisms that explain this association. Beyond CSCs' ability to participate in cardiovascular repair, levels of CSCs need to be interpreted in the context of the broader connections between haematopoiesis and cardiovascular function, including the role of clonal haematopoiesis and inflammatory myelopoiesis

Oxidative Stress in Neutrophils: Implications for Diabetic Cardiovascular Complications

Significance: Neutrophil behavior and function are altered by hyperglycemia associated with diabetes. Aberrant activation by hyperglycemia causes neutrophils to respond with increased production of reactive oxidative species (ROS). Excess ROS, a signature of primed neutrophils, can intracellularly induce neutrophils to undergo NETosis, flooding surrounding tissues with ROS and damage-associated molecular patterns such as S100 calcium binding proteins (S100A8/A9). The cargo associated with NETosis also attracts more immune cells to the site and signals for increased immune cell production. This inflammatory response to diabetes can accelerate other associated conditions such as atherosclerosis and thrombosis, increasing the risk of cardiovascular disease. Recent Advances: As the prevalence of diabetes continues to grow, more attention has been focused on developing effective treatment options. Currently, glucose-lowering medications and insulin injections are the most widely utilized treatments. As the disease progresses, medications are usually stacked to maintain glucose at desired target levels, but this approach often fails and does not effectively reduce cardiovascular risk, even with the latest drugs. Critical Issues: Despite advances in treatment options, diabetes remains a progressive disease as glucose lowering alone has failed to abolish the associated cardiovascular complications. Future Directions: Significant interest is being generated in developing treatments that do not solely focus on glucose control but rather mitigate glucotoxicity. Several therapies have been proposed that target cellular dysfunction downstream of hyperglycemia, such as using antioxidants to scavenge ROS, inhibiting ROS production from NOX, and suppressing neutrophil release of S100A8/A9 proteins

Neutrophils in cardiovascular disease: warmongers, peacemakers, or both?

Neutrophils, the most abundant of all leucocytes and the first cells to arrive at the sites of sterile inflammation/injury act as a double-edged sword. On one hand, they inflict a significant collateral damage to the tissues and on the other hand, they help facilitate wound healing by a number of mechanisms. Recent studies have drastically changed the perception of neutrophils from being simple one-dimensional cells with an unrestrained mode of action to a cell type that display maturity and complex behaviour. It is now recognized that neutrophils are transcriptionally active and respond to plethora of signals by deploying a wide variety of cargo to influence the activity of other cells in the vicinity. Neutrophils can regulate macrophage behaviour, display innate immune memory, and play a major role in the resolution of inflammation in a context-dependent manner. In this review, we provide an update on the factors that regulate neutrophil production and the emerging dichotomous role of neutrophils in the context of cardiovascular diseases, particularly in atherosclerosis and the ensuing complications, myocardial infarction, and heart failure. Deciphering the complex behaviour of neutrophils during inflammation and resolution may provide novel insights and in turn facilitate the development of potential therapeutic strategies to manage cardiovascular disease

Syndecan-4 as a genetic determinant of the metabolic syndrome

Abstract Background Syndecan-4 (SDC4) is a member of the heparan sulfate proteoglycan family of cell-surface receptors. We and others previously reported that variation in the SDC4 gene was associated with several components of the metabolic syndrome, including intra-abdominal fat, fasting glucose and triglyceride levels, and hypertension, in human cohorts. Additionally, we demonstrated that high fat diet (HFD)-induced obese female mice with a Sdc4 genetic deletion had higher visceral adiposity and a worse metabolic profile than control mice. Here, we aimed to first investigate whether the mouse Sdc4 null mutation impacts metabolic phenotypes in a sex- and diet-dependent manner. We then tested whether SDC4 polymorphisms are related to the metabolic syndrome (MetS) in humans. Methods For the mouse experiment, Sdc4-deficient (Sdc4 −/− ) and wild-type (WT) mice were treated with 14-weeks of low-fat diet (LFD). Body composition, energy balance, and selected metabolic phenotypes were assessed. For the human genetic study, we used logistic regression models to test 11 SDC4 SNPs for association with the MetS and its components in a cohort of 274 (113 with MetS) elderly subjects from southern Italy. Results Following the dietary intervention in mice, we observed that the effects of the Sdc4 null mutation on several phenotypes were different from those previously reported in the mice kept on an HFD. Nonetheless, LFD-fed female Sdc4 −/− mice, but not males, displayed higher levels of triglycerides and lower insulin sensitivity at fasting than WT mice, as seen earlier in the HFD conditions. In the parallel human study, we found that carriers of SDC4 rs2228384 allele C and rs2072785 allele T had reduced risk of MetS. The opposite was true for carriers of the SDC4 rs1981429 allele G. Additionally, the SNPs were found related to fasting triglyceride levels and triglyceride glucose (TyG) index, a reliable indicator of insulin resistance, with sex-stratified analysis detecting the association of rs1981429 with these phenotypes only in females. Conclusions Altogether, our results suggest that SDC4 is an evolutionary conserved genetic determinant of MetS and that its genetic variation is associated with fasting triglyceride levels in a female-specific manner

The ESAT-6 Protein of <i>Mycobacterium tuberculosis</i> Interacts with Beta-2-Microglobulin (β2M) Affecting Antigen Presentation Function of Macrophage

<div><p>ESAT-6, an abundantly secreted protein of <i>Mycobacterium tuberculosis</i> (<i>M. tuberculosis</i>) is an important virulence factor, inactivation of which leads to reduced virulence of <i>M. tuberculosis</i>. ESAT-6 alone, or in complex with its chaperone CFP-10 (ESAT-6:CFP-10), is known to modulate host immune responses; however, the detailed mechanisms are not well understood. The structure of ESAT-6 or ESAT-6:CFP-10 complex does not suggest presence of enzymatic or DNA-binding activities. Therefore, we hypothesized that the crucial role played by ESAT-6 in the virulence of mycobacteria could be due to its interaction with some host cellular factors. Using a yeast two-hybrid screening, we identified that ESAT-6 interacts with the host protein beta-2-microglobulin (β2M), which was further confirmed by other assays, like GST pull down, co-immunoprecipitation and surface plasmon resonance. The C-terminal six amino acid residues (90–95) of ESAT-6 were found to be essential for this interaction. ESAT-6, in complex with CFP-10, also interacts with β2M. We found that ESAT-6/ESAT-6:CFP-10 can enter into the endoplasmic reticulum where it sequesters β2M to inhibit cell surface expression of MHC-I-β2M complexes, resulting in downregulation of class I-mediated antigen presentation. Interestingly, the ESAT-6:β2M complex could be detected in pleural biopsies of individuals suffering from pleural tuberculosis. Our data highlight a novel mechanism by which <i>M. tuberculosis</i> may undermine the host adaptive immune responses to establish a successful infection. Identification of such novel interactions may help us in designing small molecule inhibitors as well as effective vaccine design against tuberculosis.</p></div

Soluble ESAT-6:CFP-10 increases the levels of free HLA class I heavy chain molecules.

<p>(A) PMA-differentiated THP-1 macrophages were treated with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each). After 2 hours, cells were washed and incubated with anti-HLA class I heavy chain mAb HC-10 followed by FITC conjugated anti-mouse secondary Ab. Surface expression of free HLA class I heavy chain molecules were studied by flow cytometry. Cells stained with appropriate isotype Ab were used as control. (B) Median fluorescence intensities of different experimental groups described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004446#ppat-1004446-g006" target="_blank">Figure 6A</a> were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages pre-treated with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each) were fixed, permeabilized and stained with HC-10 Ab followed by Alexa Fluor 594 conjugated anti-mouse secondary Ab (red). Matching isotype Ab was used as control. The nucleus was visualized by DAPI staining (blue). The stained cells were observed under a confocal microscope. (D) In another set of experiments, THP-1 macrophages were pre-treated for 30 minutes with 5 µM MG-132 followed by incubation with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each) for 2 hours. Free HLA class I molecules on the cell surface were stained with HC-10 Ab followed by staining with FITC conjugated anti-mouse secondary Ab and studied by flow cytometry. Isotype-matched Ab was used as control. (E) THP-1 macrophages treated with either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein (12.5 µM each) in the absence or presence MG-132 werefixed, permeabilized and stained with HC-10 Ab followed by Alexa Fluor 594 conjugated secondary anti-mouse Ab (red). Nucleus was stained with DAPI (blue) and cells were visualized under a confocal microscope. Data shown is representative of three independent experiments.</p

Exogenously added ESAT-6 or ESAT-6:CFP-10 complex can enter into ER.

<p>PMA-differentiated THP-1 macrophages were incubated with FITC-labelled ESAT-6:CFP-10 (green) either at 4°C (A) or 37°C (B) for about 120 minutes. Cells were then washed, fixed, permeabilised and stained for an ER marker calnexin using rabbit anti-calnexin Ab and Alexa Fluor 594 conjugated anti-rabbit secondary Ab (red). In another set of experiments KG-1 dendritic like cells (C) or PMA-differentiated THP-1 macrophages (D) or thioglycolate-elicited C57BL/6 mouse peritoneal macrophages (E) were treated with FITC-labelled ESAT-6 or ESAT-6:CFP-10 (green) for about 100 minutes at 37°C and then incubated with ER-Tracker dye (blue) for another 20 minutes and observed under the LSM 510 Meta confocal microscope. Results are representative of three different experiments.</p

Soluble ESAT-6:CFP-10 reduces surface levels of β2M-associated HLA class I molecules.

<p>(A) PMA-differentiated THP-1 macrophages were treated with 12.5 µM of either ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10 protein for 2 hours. Cells were stained with (W6/32) mAb followed by FITC conjugated anti-mouse secondary Ab. Expression of surface β2M conjugated HLA class I molecules was studied by flow cytometry. Isotype-matched Ab was used as control. (B) Median fluorescence intensities of different experimental groups of Figure 7A were calculated and the results are shown as mean ± SD of 3 different experiments. (C) THP-1 macrophages were either left untreated (control) or treated with 12.5 µM of ESAT-6:CFP-10 or ESAT-6ΔC:CFP-10. After 2 hours, cell were harvested and lysates were prepared. Equal amount of protein from each experimental group was incubated with W6/32 mAb bound to protein A/G agarose. Isotype matched Ab was used as control. Pulled-down complexes (Lanes 5–8) were resolved on a 15% glycine SDS-PAGE and transferred onto a nitrocellulose membrane which was probed with anti-β2M Ab. About 10% of the lysate was used as input controls (Lanes 1–4, upper panel). Equal loading in the input samples was also confirmed by probing the input controls with anti-β-actin Ab (Lanes 1–4, lower panel).</p