Activin A levels are raised during human tuberculosis and blockade of the activin signaling axis influences murine responses to M. tuberculosis infection.

Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis-infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

Weaker protection against tuberculosis in BCG-vaccinated male 129 S2 mice compared to females.

BCG - the only available vaccine against tuberculosis (TB) - was first given to babies 100 years ago in 1921. While it is effective against TB meningitis and disseminated TB, its efficacy against pulmonary TB is variable, notably in adults and adolescents. TB remains one of the world's leading health problems, with a higher prevalence among men. Male sex is associated with increased susceptibility to Mycobacterium tuberculosis in mice, but sex-specific responses to BCG vaccination have not been examined. In this study we vaccinated TB-susceptible 129 S2 mice with BCG and challenged with low-dose M. tuberculosis H37Rv by aerosol infection. BCG was protective against TB in both sexes, as unvaccinated mice lost weight more rapidly than vaccinated ones and suffered from worse lung pathology. However, female mice were better protected than males, showing lower lung bacterial burdens and less weight loss. Overall, vaccinated female mice had increased numbers of T cells and less myeloid cells in the lungs compared to vaccinated males. Principal component analysis of measured features revealed that mice grouped according to timepoint, sex and vaccination status. The features that had the biggest impact on grouping overall included numbers of CD8 T cells, CD8 central memory T cells and CD4 T effector cells, with neutrophil and CD11b+GR-1- cell numbers having a big impact at day 29. Hierarchical clustering confirmed that the main difference in global immune response was due to mouse sex, with only a few misgrouped mice. In conclusion, we found sex-specific differences in response to M. tuberculosis H37Rv -challenge in BCG-vaccinated 129 S2 mice. This highlights the need to include both male and female mice in preclinical testing of vaccine candidates.

Role of Premycofactocin Synthase in Growth, Microaerophilic Adaptation, and Metabolism of Mycobacterium tuberculosis.

Mycofactocin is a new class of peptide-derived redox cofactors present in a selected group of bacteria including Mycobacterium tuberculosis. Mycofactocin biosynthesis requires at least six genes, including mftD, encoding putative lactate dehydrogenase, which catalyzes the penultimate biosynthetic step. Cellular functions remained unknown until recent reports on the significance of mycofactocin in primary alcohol metabolism. Here, we show that mftD transcript levels were increased in hypoxia-adapted M. tuberculosis; however, mftD functionality was found likely dispensable for l-lactate metabolism. Targeted deletion of mftD reduced the survival of M. tuberculosis in in vitro and in vivo hypoxia models but increased the bacterial growth in glucose-containing broth as well as in the lungs and spleens, albeit modestly, of aerosol-infected C57BL/6J mice. The cause of this growth advantage remains unestablished; however, the mftD-deficient M. tuberculosis strain had reduced NAD(H)/NADP(H) levels and glucose-6-phosphate dehydrogenase activity with no impairment in phthiocerol dimycocerosate lipid synthesis. An ultrastructural examination of parental and mycofactocin biosynthesis gene mutants in M. tuberculosis, M. marinum, and M. smegmatis showed no altered cell morphology and size except the presence of outer membrane-bound fibril-like features only in a mutant subpopulation. A cell surface-protein analysis of M. smegmatis mycofactocin biosynthesis mutants with trypsin revealed differential abundances of a subset of proteins that are known to interact with mycofactocin and their homologs that can enhance protein aggregation or amyloid-like fibrils in riboflavin-starved eukaryotic cells. In sum, phenotypic analyses of the mutant strain implicate the significance of MftD/mycofactocin in M. tuberculosis growth and persistence in its host. IMPORTANCE Characterization of proteins with unknown functions is a critical research priority as the intracellular growth and metabolic state of Mycobacterium tuberculosis, the causative agent of tuberculosis, remain poorly understood. Mycofactocin is a peptide-derived redox cofactor present in almost all mycobacterial species; however, its functional relevance in M. tuberculosis pathogenesis and host survival has never been studied experimentally. In this study, we examine the phenotypes of an M. tuberculosis mutant strain lacking a key mycofactocin biosynthesis gene in in vitro and disease-relevant mouse models. Our results pinpoint the multifaceted role of mycofactocin in M. tuberculosis growth, hypoxia adaptation, glucose metabolism, and redox homeostasis. This evidence strongly implies that mycofactocin could fulfill specialized biochemical functions that increase the survival fitness of mycobacteria within their specific niche.

FX11 limits Mycobacterium tuberculosis growth and potentiates bactericidal activity of isoniazid through host-directed activity.

Lactate dehydrogenase A (LDHA) mediates interconversion of pyruvate and lactate, and increased lactate turnover is exhibited by malignant and infected immune cells. Hypoxic lung granuloma in Mycobacterium tuberculosis-infected animals present elevated levels of Ldha and lactate. Such alterations in the metabolic milieu could influence the outcome of host-M. tuberculosis interactions. Given the central role of LDHA for tumorigenicity, targeting lactate metabolism is a promising approach for cancer therapy. Here, we sought to determine the importance of LDHA for tuberculosis (TB) disease progression and its potential as a target for host-directed therapy. To this end, we orally administered FX11, a known small-molecule NADH-competitive LDHA inhibitor, to M. tuberculosis-infected C57BL/6J mice and Nos2-/- mice with hypoxic necrotizing lung TB lesions. FX11 did not inhibit M. tuberculosis growth in aerobic/hypoxic liquid culture, but modestly reduced the pulmonary bacterial burden in C57BL/6J mice. Intriguingly, FX11 administration limited M. tuberculosis replication and onset of necrotic lung lesions in Nos2-/- mice. In this model, isoniazid (INH) monotherapy has been known to exhibit biphasic killing kinetics owing to the probable selection of an INH-tolerant bacterial subpopulation. However, adjunct FX11 treatment corrected this adverse effect and resulted in sustained bactericidal activity of INH against M. tuberculosis As a limitation, LDHA inhibition as an underlying cause of FX11-mediated effect could not be established as the on-target effect of FX11 in vivo was unconfirmed. Nevertheless, this proof-of-concept study encourages further investigation on the underlying mechanisms of LDHA inhibition and its significance in TB pathogenesis.

Immunofluorescent Detection of NET Components in Paraffin-Embedded Tissue.

Neutrophil extracellular traps (NETs) consist of decondensed chromatin fibers studded with granular and cytoplasmic proteins and peptides that are released by stimulated neutrophil granulocytes. If present in abundance (e.g., in large thrombi), NETs are depicted in H&E-stained tissue sections as pale bluish areas. Since no NET-specific antibodies exist, to unambiguously identify even small amounts of NETs in tissue, it is essential to demonstrate colocalization of nuclear and granular/cytoplasmic NET components which in unstimulated neutrophils are clearly separated. This requires good tissue preservation and a very defined immunolocalization, which can be achieved by using 2-3 µm thick sections of paraffin-embedded tissue. It provides sufficiently good tissue preservation for subcellular localization of two or more NET components, thereby allow to differentiate stimulated from unstimulated neutrophils and to clearly identify NETs. In this chapter, we will provide protocols for antigen retrieval and immunofluorescent labeling of NET components in paraffin-embedded tissue with commercially available antibodies.

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue.

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6-8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients. Neutrophils can combat invading microorganisms by phagocytosis, degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps drive inflammatory pathogenesis in malaria.

Neutrophils are essential innate immune cells that extrude chromatin in the form of neutrophil extracellular traps (NETs) when they die. This form of cell death has potent immunostimulatory activity. We show that heme-induced NETs are essential for malaria pathogenesis. Using patient samples and a mouse model, we define two mechanisms of NET-mediated inflammation of the vasculature: activation of emergency granulopoiesis via granulocyte colony-stimulating factor production and induction of the endothelial cytoadhesion receptor intercellular adhesion molecule-1. Soluble NET components facilitate parasite sequestration and mediate tissue destruction. We demonstrate that neutrophils have a key role in malaria immunopathology and propose inhibition of NETs as a treatment strategy in vascular infections.

Dnase1-deficient mice spontaneously develop a systemic lupus erythematosus-like disease.

Systemic lupus erythematosus (SLE) is an autoimmune disease that has high morbidity and can result in multi-organ damage. SLE is characterized by dysregulated activation of T- and B-lymphocytes and the production of autoantibodies directed against nuclear components. The endonuclease deoxyribonuclease 1 (DNase1) is abundant in blood and a subset of SLE patients have mutations in DNASE1. Furthermore, a report showed that Dnase1-deficient mice develop an SLE-like disease, but these mice also carry a deletion of the gene adjacent to Dnase1, which encodes the chaperone TRAP1/HSP75. We generated a murine strain deficient in Dnase1 with an intact Trap1 gene to examine if a lack of DNase1 is responsible for the development of a spontaneous SLE-like disease. We show that the Dnase1-deficient mice do indeed develop an SLE-like phenotype with elevated autoantibody production by 9 months and kidney damage by 12 months. Notably, this model recapitulates the female bias seen in human SLE patients since female Dnase1-deficient mice produced the highest concentrations of autoantibodies and had more severe kidney damage than males. Since there is currently no cure for SLE the protective role of DNase1 as demonstrated in our study remains of great therapeutic interest.

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.

Cell-Cycle Proteins Control Production of Neutrophil Extracellular Traps.

Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21Cip. CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.

Immunodetection of NETs in Paraffin-Embedded Tissue.

The pathogenic potential of neutrophil extracellular traps (NETs) was recently described, and their detection in tissue could serve as a prognostic marker. NETs are delicate and filigree structures; hence good tissue preservation is essential for their detection. Indeed, analysis of paraffin-embedded tissue has proven superior to the study of cryo sections. Though, under favorable conditions, the presence of NETs can be detected in tissue sections stained with histological dyes, definitive identification of NETs needs the colocalization of immunofluorescent signals for both nuclear and granular (or cytoplasmic) NET components. We tested diverse antigen retrieval methods and various combinations of commercially available antibodies and present here staining protocols to detect NETs in human and murine tissue sections.

Neutrophil extracellular traps: how to generate and visualize them.

Neutrophil granulocytes are the most abundant group of leukocytes in the peripheral blood. As professional phagocytes, they engulf bacteria and kill them intracellularly when their antimicrobial granules fuse with the phagosome. We found that neutrophils have an additional way of killing microorganisms: upon activation, they release granule proteins and chromatin that together form extracellular fibers that bind pathogens. These novel structures, or Neutrophil Extracellular Traps (NETs), degrade virulence factors and kill bacteria, fungi and parasites. The structural backbone of NETs is DNA, and they are quickly degraded in the presence of DNases. Thus, bacteria expressing DNases are more virulent. Using correlative microscopy combining TEM, SEM, immunofluorescence and live cell imaging techniques, we could show that upon stimulation, the nuclei of neutrophils lose their shape and the eu- and heterochromatin homogenize. Later, the nuclear envelope and the granule membranes disintegrate allowing the mixing of NET components. Finally, the NETs are released as the cell membrane breaks. This cell death program (NETosis) is distinct from apoptosis and necrosis and depends on the generation of Reactive Oxygen Species by NADPH oxidase. Neutrophil extracellular traps are abundant at sites of acute inflammation. NETs appear to be a form of innate immune response that bind microorganisms, prevent them from spreading, and ensure a high local concentration of antimicrobial agents to degrade virulence factors and kill pathogens thus allowing neutrophils to fulfill their antimicrobial function even beyond their life span. There is increasing evidence, however, that NETs are also involved in diseases that range from auto-immune syndromes to infertility. We describe methods to isolate Neutrophil Granulocytes from peripheral human blood and stimulate them to form NETs. Also we include protocols to visualize the NETs in light and electron microscopy.

Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans.

Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.