Panton-Valentine leukocidin-induced neutrophil extracellular traps lack antimicrobial activity and are readily induced in patients with recurrent PVL + -Staphylococcus aureus infections.

Staphylococcus aureus strains that produce the toxin Panton-Valentine leukocidin (PVL-SA) frequently cause recurrent skin and soft tissue infections. PVL binds to and kills human neutrophils, resulting in the formation of neutrophil extracellular traps (NETs), but the pathomechanism has not been extensively studied. Furthermore, it is unclear why some individuals colonized with PVL-SA experience recurring infections whereas others are asymptomatic. We thus aimed to (1) investigate how PVL exerts its pathogenicity on neutrophils and (2) identify factors that could help to explain the predisposition of patients with recurring infections. We provide genetic and pharmacological evidence that PVL-induced NET formation is independent of NADPH oxidase and reactive oxygen species production. Moreover, through NET proteome analysis we identified that the protein content of PVL-induced NETs is different from NETs induced by mitogen or the microbial toxin nigericin. The abundance of the proteins cathelicidin (CAMP), elastase (NE), and proteinase 3 (PRTN3) was lower on PVL-induced NETs, and as such they were unable to kill S. aureus. Furthermore, we found that neutrophils from affected patients express higher levels of CD45, one of the PVL receptors, and are more susceptible to be killed at a low PVL concentration than control neutrophils. Neutrophils from patients that experience recurring PVL-positive infections may thus be more sensitive to PVL-induced NET formation, which might impair their ability to combat the infection.

Histone H3 clipping is a novel signature of human neutrophil extracellular traps.

Neutrophils are critical to host defence, executing diverse strategies to perform their antimicrobial and regulatory functions. One tactic is the production of neutrophil extracellular traps (NETs). In response to certain stimuli, neutrophils decondense their lobulated nucleus and release chromatin into the extracellular space through a process called NETosis. However, NETosis, and the subsequent degradation of NETs, can become dysregulated. NETs are proposed to play a role in infectious as well as many non-infection related diseases including cancer, thrombosis, autoimmunity and neurological disease. Consequently, there is a need to develop specific tools for the study of these structures in disease contexts. In this study, we identified a NET-specific histone H3 cleavage event and harnessed this to develop a cleavage site-specific antibody for the detection of human NETs. By microscopy, this antibody distinguishes NETs from chromatin in purified and mixed cell samples. It also detects NETs in tissue sections. We propose this antibody as a new tool to detect and quantify NETs.

Neutrophil phenotypes and functions in cancer: A consensus statement.

Neutrophils are the first responders to infection and inflammation and are thus a critical component of innate immune defense. Understanding the behavior of neutrophils as they act within various inflammatory contexts has provided insights into their role in sterile and infectious diseases; however, the field of neutrophils in cancer is comparatively young. Here, we summarize key concepts and current knowledge gaps related to the diverse roles of neutrophils throughout cancer progression. We discuss sources of neutrophil heterogeneity in cancer and provide recommendations on nomenclature for neutrophil states that are distinct in maturation and activation. We address discrepancies in the literature that highlight a need for technical standards that ought to be considered between laboratories. Finally, we review emerging questions in neutrophil biology and innate immunity in cancer. Overall, we emphasize that neutrophils are a more diverse population than previously appreciated and that their role in cancer may present novel unexplored opportunities to treat cancer.

Basic science under threat: Lessons from the Skirball Institute.

Support for basic science has been eclipsed by initiatives aimed at specific medical problems. The latest example is the dismantling of the Skirball Institute at NYU School of Medicine. Here, we reflect on the achievements and mission underlying the Skirball to gain insight into the dividends of maintaining a basic science vision within the academic enterprises.

The Neutrophil.

Neutrophils are immune cells with unusual biological features that furnish potent antimicrobial properties. These cells phagocytose and subsequently kill prokaryotic and eukaryotic organisms very efficiently. Importantly, it is not only their ability to attack microbes within a constrained intracellular compartment that endows neutrophils with antimicrobial function. They can unleash their effectors into the extracellular space, where, even post-mortem, their killing machinery can endure and remain functional. The antimicrobial activity of neutrophils must not be misconstrued as being microbe specific and should be viewed more generally as biotoxic. Outside of fighting infections, neutrophils can harness their noxious machinery in other contexts, like cancer. Inappropriate or dysregulated neutrophil activation damages the host and contributes to autoimmune and inflammatory disease. Here we review a number of topics related to neutrophil biology based on contemporary findings.

The cytosolic DNA sensor cGAS recognizes neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) are structures consisting of chromatin and antimicrobial molecules that are released by neutrophils during a form of regulated cell death called NETosis. NETs trap invading pathogens, promote coagulation, and activate myeloid cells to produce type I interferons (IFNs), proinflammatory cytokines that regulate the immune system. Here, we showed that macrophages and other myeloid cells phagocytosed NETs. Once in phagosomes, NETs translocated to the cytosol, where the DNA backbones of these structures activated the innate immune sensor cyclic GMP-AMP synthase (cGAS) and induced type I IFN production. The NET-associated serine protease neutrophil elastase (NE) mediated the activation of this pathway. We showed that NET induction in mice treated with the lectin concanavalin A, a model of autoimmune hepatitis, resulted in cGAS-dependent stimulation of an IFN response, suggesting that NETs activated cGAS in vivo. Thus, our findings suggest that cGAS is a sensor of NETs, mediating immune cell activation during infection.

Linker histone H1.2 and H1.4 affect the neutrophil lineage determination.

Neutrophils are important innate immune cells that tackle invading pathogens with different effector mechanisms. They acquire this antimicrobial potential during their maturation in the bone marrow, where they differentiate from hematopoietic stem cells in a process called granulopoiesis. Mature neutrophils are terminally differentiated and short-lived with a high turnover rate. Here, we show a critical role for linker histone H1 on the differentiation and function of neutrophils using a genome-wide CRISPR/Cas9 screen in the human cell line PLB-985. We systematically disrupted expression of somatic H1 subtypes to show that individual H1 subtypes affect PLB-985 maturation in opposite ways. Loss of H1.2 and H1.4 induced an eosinophil-like transcriptional program, thereby negatively regulating the differentiation into the neutrophil lineage. Importantly, H1 subtypes also affect neutrophil differentiation and the eosinophil-directed bias of murine bone marrow stem cells, demonstrating an unexpected subtype-specific role for H1 in granulopoiesis.

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.

Rotation Invariant Clustering of 3D Cell Nuclei Shapes.

Cellular imaging with confocal fluorescence laser microscopy gave rise to many new insights into the cellular machinery. One interesting observation suggests that morphology of cell nucleus plays a key role for neutrophilic function, which is an essential part of the innate immune system of most mammals. Due to the increasing availability of high resolution 3D images coming from the microscope, machine learning becomes a promising tool for automatically discovering underlying hidden structures. Here, the major difficulty consists of selecting an appropriate representation for characterizing the morphology of cell nucleus. In this work we tackle this problem and propose a fully unsupervised mechanism for finding structure in high-throughput 3D image data. The key component of our approach is based on Generic Fourier Transform (GFT) for 2D images, which for 3D involves spherical coordinate transformation prior to fast Discrete Fourier Transformation. On top on GFT we apply dimensionality reduction with Principal Component Analysis, followed by generative cluster analysis with a Gaussian Mixture Model. We validate our new approach first on a synthetic 3D-MNIST dataset with random rotations, where quantitative and qualitative results confirm the applicability of the proposed pipeline for exploring shape space in a purely unsupervised manner. Then we apply our proposed technique to a new collected dataset of high resolution 3D images of neutrophile nuclei suggesting a clustering model with six significant clusters of morphological cell nuclei prototypes. We visualize differences in the cell shape clusters by providing prototypical examples of neutrophilic cell nuclei.

Neutrophils: New insights and open questions.

Neutrophils are the first line of defense against bacteria and fungi and help combat parasites and viruses. They are necessary for mammalian life, and their failure to recover after myeloablation is fatal. Neutrophils are short-lived, effective killing machines. Their life span is significantly extended under infectious and inflammatory conditions. Neutrophils take their cues directly from the infectious organism, from tissue macrophages and other elements of the immune system. Here, we review how neutrophils traffic to sites of infection or tissue injury, how they trap and kill bacteria, how they shape innate and adaptive immune responses, and the pathophysiology of monogenic neutrophil disorders.

Gasdermin D plays a vital role in the generation of neutrophil extracellular traps.

The death of a cell is an inevitable part of its biology. During homeostasis, most cells die through apoptosis. If homeostasis is disturbed, cell death can switch to proinflammatory forms of death, such as necroptosis, pyroptosis, or NETosis. We demonstrate that the formation of neutrophil extracellular traps (NETs), a special form of neutrophil cell death that releases chromatin structures to the extracellular space, is dependent on gasdermin D (GSDMD). GSDMD is a pore-forming protein and an executor of pyroptosis. We screened a chemical library and found a small molecule based on the pyrazolo-oxazepine scaffold that efficiently blocks NET formation and GSDMD-mediated pyroptotic cell death in human cells. During NETosis, GSDMD is proteolytically activated by neutrophil proteases and, in turn, affects protease activation and nuclear expansion in a feed-forward loop. In addition to the central role of GSDMD in pyroptosis, we propose that GSDMD also plays an essential function in NETosis.

The role of neutrophil extracellular traps in rheumatic diseases.

Rheumatic diseases are a collection of disorders defined by the presence of inflammation and destruction of joints and internal organs. A common feature of these diseases is the presence of autoantibodies targeting molecules commonly expressed in neutrophils. These preformed mediators are released by neutrophils but not by other immune cells such as macrophages. Neutrophils, major players in the host innate immune response, initiate a cell death mechanism termed neutrophil extracellular trap (NET) formation as a way to ensnare pathogens. NETs are also a source of released self-molecules found in rheumatic diseases. Subsequently, research on the role of NETs in the onset, progression and resolution of inflammation in rheumatic diseases has intensified. This Review has two aims. First, it aims to highlight the mechanisms required for the generation of NETs, the research landscape of which is rapidly changing. Second, it aims to discuss the role of neutrophils and NETs in systemic lupus erythematosus, vasculitis (specifically anti-neutrophil cytoplasmic autoantibody-associated vasculitis), rheumatoid arthritis and gout. Our goal is to clarify the field of NET research in rheumatic diseases in the hope of improving the therapeutic approaches utilized for these diseases.

Neutrophil Extracellular Traps: The Biology of Chromatin Externalization.

Neutrophils are essential to the homeostatic mission of safeguarding host tissues, responding rapidly and diversely to breaches of the host's barriers to infection, and returning tissues to a sterile state. In response to specific stimuli, neutrophils extrude modified chromatin structures decorated with specific cytoplasmic and granular proteins called neutrophil extracellular traps (NETs). Several pathways lead to this unique form of cell death (NETosis). Extracellular chromatin may have evolved to defend eukaryotic organisms against infection, and its release has at least three functions: trapping and killing of microbes, amplifying immune responses, and inducing coagulation. Here we review neutrophil development and heterogeneity with a focus on NETs, NET formation, and their relevance in host defense and disease.

The bacterial pigment pyocyanin inhibits the NLRP3 inflammasome through intracellular reactive oxygen and nitrogen species.

Inflammasomes are cytosolic complexes that mature and secrete the inflammatory cytokines interleukin 1ß (IL-1ß) and IL-18 and induce pyroptosis. The NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome detects many pathogen- and danger-associated molecular patterns, and reactive oxygen species (ROS)/reactive nitrogen species (RNS) have been implicated in its activation. The phenazine pyocyanin (PCN) is a virulence factor of Pseudomonas aeruginosa and generates superoxide in cells. Here we report that PCN inhibits IL-1ß and IL-18 release and pyroptosis upon NLRP3 inflammasome activation in macrophages by preventing speck formation and Caspase-1 maturation. Of note, PCN did not regulate the AIM2 (absent in melanoma 2) or NLRC4 inflammasomes or tumor necrosis factor (TNF) secretion. Imaging of the fluorescent glutathione redox potential sensor Grx1-roGFP2 indicated that PCN provokes cytosolic and nuclear but not mitochondrial redox changes. PCN-induced intracellular ROS/RNS inhibited the NLRP3 inflammasome posttranslationally, and hydrogen peroxide or peroxynitrite alone were sufficient to block its activation. We propose that cytosolic ROS/RNS inhibit the NLRP3 inflammasome and that PCN's anti-inflammatory activity may help P. aeruginosa evade immune recognition.

Copper Regulates the Canonical NLRP3 Inflammasome.

Inflammasomes are multimeric protein complexes that are activated through a NOD-like receptor and regulate the proteolytic activation of caspase-1 and cytokines, like IL-1ß. The NLRP3 inflammasome is implicated in many human pathologies including infections, autoinflammatory syndromes, chronic inflammation, and metabolic diseases; however, the molecular mechanisms of activation are not fully understood. In this study we show that NLRP3 inflammasome activation requires intracellular copper. A clinically approved copper chelator, tetrathiomolybdate, inhibited the canonical NLRP3 but not the AIM2, NLRC4, and NLRP1 inflammasomes or NF-κB-dependent priming. We demonstrate that NLRP3 inflammasome activation is blocked by removing copper from the active site of superoxide dismutase 1, recapitulating impaired inflammasome function in superoxide dismutase 1-deficient mice. This regulation is specific to macrophages, but not monocytes, both in mice and humans. In vivo, depletion of bioavailable copper resulted in attenuated caspase-1-dependent inflammation and reduced susceptibility to LPS-induced endotoxic shock. Our results indicate that targeting the intracellular copper homeostasis has potential for the treatment of NLRP3-dependent diseases.

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.

Diverse stimuli engage different neutrophil extracellular trap pathways.

Neutrophils release neutrophil extracellular traps (NETs) which ensnare pathogens and have pathogenic functions in diverse diseases. We examined the NETosis pathways induced by five stimuli; PMA, the calcium ionophore A23187, nigericin, Candida albicans and Group B Streptococcus. We studied NET production in neutrophils from healthy donors with inhibitors of molecules crucial to PMA-induced NETs including protein kinase C, calcium, reactive oxygen species, the enzymes myeloperoxidase (MPO) and neutrophil elastase. Additionally, neutrophils from chronic granulomatous disease patients, carrying mutations in the NADPH oxidase complex or a MPO-deficient patient were examined. We show that PMA, C. albicans and GBS use a related pathway for NET induction, whereas ionophores require an alternative pathway but that NETs produced by all stimuli are proteolytically active, kill bacteria and composed mainly of chromosomal DNA. Thus, we demonstrate that NETosis occurs through several signalling mechanisms, suggesting that extrusion of NETs is important in host defence.

Tetrahydroisoquinolines: New Inhibitors of Neutrophil Extracellular Trap (NET) Formation.

Neutrophils are short-lived leukocytes that migrate to sites of infection as part of the acute immune response, where they phagocytose, degranulate, and form neutrophil extracellular traps (NETs). During NET formation, the nuclear lobules of neutrophils disappear and the chromatin expands and, accessorized with neutrophilic granule proteins, is expelled. NETs can be pathogenic in, for example, sepsis, cancer, and autoimmune and cardiovascular diseases. Therefore, the identification of inhibitors of NET formation is of great interest. Screening of a focused library of natural-product-inspired compounds by using a previously validated phenotypic NET assay identified a group of tetrahydroisoquinolines as new NET formation inhibitors. This compound class opens up new avenues for the study of cellular death through NET formation (NETosis) at different stages, and might inspire new medicinal chemistry programs aimed at NET-dependent diseases.