Oxidized Phospholipids and Neutrophil Elastase Coordinately Play Critical Roles in NET Formation

Neutrophil extracellular traps (NETs) are web-like structures consisting of decondensed chromatin DNA and contents of granules, such as myeloperoxidase (MPO) and neutrophil elastase (NE). NETs are usually released from neutrophils undergoing NETosis, a neutrophil-specific cell death mode characterized by the collapse and disappearance of cell membranes and nuclear envelopes. It is well known that production of reactive oxygen species (ROS) triggers NETosis and NET formation. However, details of intracellular signaling downstream of ROS production during NETosis and NET formation remains uncertain. Here, we demonstrated that the peroxidation of phospholipids plays a critical role in NETosis and NET formation induced by phorbol 12-myristate13-acetate (PMA) or immune complex in vitro and by lipopolysaccharide (LPS) in vivo. This phospholipid peroxidation is mediated by the enzymatic activity of MPO. On the other hand, NE, which was previously reported to be released from granules to cytosol by MPO during NET formation, is not required for either the peroxidation of phospholipids or the execution of NETosis, but contributes to chromatin decondensation and nuclear swelling independently of MPO-mediated oxidized phospholipids. Analysis of isolated nuclei clearly demonstrated that oxidized phospholipids and NE differently yet synergistically execute chromatin decondensation and nuclear swelling, and the subsequent release of nuclear contents. These findings indicate the dual roles of MPO in NETosis and NET formation, and provide new insight into the molecular mechanism of these phenomena.

Abstract MP226: Netosis Is Required For S100a8/a9-induced Granulopoiesis After Myocardial Infarction

Myocardial infarction (MI) provokes a massive systemic inflammatory response characterized by enhanced infiltration of neutrophils to the ischemic heart via granulopoiesis in the bone marrow. We recently discovered that S100A8/A9 is released by infiltrating neutrophils in the infarct to induce granulopoiesis by interacting with TLR4 on naïve neutrophils, priming the NLPR3-inflammasome for release of IL-1β which then leads to increased granulopoiesis. However, the mechanism of S100A8/A9 release from the infiltrating-neutrophils remain unclear. We hypothesized that neutrophils release S100A8/A9 via NETosis, a form of cell-death that involves extrusion of decondensed chromatin along with its entire granular content including S100A8/A9. To validate this hypothesis we found that neutrophils sorted from mouse heart 24 hours post-MI were enriched for genetic signature for an Nox-independent NETosis, dominated by Padi4. Furthermore, we found a robust increase in citrullination of histone moieties, colocalization of S100A8/A9 with H3Cit, and increased S100A8/A9 levels in the heart as early as 6 hours after MI. Depletion of Padi4 in the mouse myeloid cells by bone marrow transplantation studies was also associated with suppressed granulopoiesis, fewer neutrophils in the blood and heart, and reduced cardiac and serum S100A8/A9 after MI. To validate these findings in humans, we measured various markers of NETosis and found a marked increase in serum double-stranded DNA, MPO (myeloperoxidase) elastase, which paralleled S100A8/A9 levels in STEMI patients from the time of admission through post-revascularization by percutaneous coronary intervention. Taken together we propose that interventions to reduce NETosis and/or S100A8/A9 release particularly during the acute inflammatory phase could represent a departure from the status quo in managing post-MI inflammation and the subsequent heart failure.

A Fragile Balance: Does Neutrophil Extracellular Trap Formation Drive Pulmonary Disease Progression?

Neutrophils act as the first line of defense during infection and inflammation. Once activated, they are able to fulfil numerous tasks to fight inflammatory insults while keeping a balanced immune response. Besides well-known functions, such as phagocytosis and degranulation, neutrophils are also able to release “neutrophil extracellular traps” (NETs). In response to most stimuli, the neutrophils release decondensed chromatin in a NADPH oxidase-dependent manner decorated with histones and granule proteins, such as neutrophil elastase, myeloperoxidase, and cathelicidins. Although primarily supposed to prevent microbial dissemination and fight infections, there is increasing evidence that an overwhelming NET response correlates with poor outcome in many diseases. Lung-related diseases especially, such as bacterial pneumonia, cystic fibrosis, chronic obstructive pulmonary disease, aspergillosis, influenza, and COVID-19, are often affected by massive NET formation. Highly vascularized areas as in the lung are susceptible to immunothrombotic events promoted by chromatin fibers. Keeping this fragile equilibrium seems to be the key for an appropriate immune response. Therapies targeting dysregulated NET formation might positively influence many disease progressions. This review highlights recent findings on the pathophysiological influence of NET formation in different bacterial, viral, and non-infectious lung diseases and summarizes medical treatment strategies.

Visualization of Chromatin in the Yeast Nucleus and Nucleolus Using Hyperosmotic Shock

Unlike in most eukaryotic cells, the genetic information of budding yeast in the exponential growth phase is only present in the form of decondensed chromatin, a configuration that does not allow its visualization in cell nuclei conventionally prepared for transmission electron microscopy. In this work, we studied the distribution of chromatin and its relationships to the nucleolus using different cytochemical and immunocytological approaches applied to yeast cells subjected to hyperosmotic shock. Our results show that osmotic shock induces the formation of heterochromatin patches in the nucleoplasm and intranucleolar regions of the yeast nucleus. In the nucleolus, we further revealed the presence of osmotic shock-resistant DNA in the fibrillar cords which, in places, take on a pinnate appearance reminiscent of ribosomal genes in active transcription as observed after molecular spreading (“Christmas trees”). We also identified chromatin-associated granules whose size, composition and behaviour after osmotic shock are reminiscent of that of mammalian perichromatin granules. Altogether, these data reveal that it is possible to visualize heterochromatin in yeast and suggest that the yeast nucleus displays a less-effective compartmentalized organization than that of mammals.

Chromatin regulates expression of small RNAs to help maintain transposon methylome homeostasis in Arabidopsis

Background Eukaryotic genomes are partitioned into euchromatic and heterochromatic domains to regulate gene expression and other fundamental cellular processes. However, chromatin is dynamic during growth and development and must be properly re-established after its decondensation. Small interfering RNAs (siRNAs) promote heterochromatin formation, but little is known about how chromatin regulates siRNA expression. Results We demonstrate that thousands of transposable elements (TEs) produce exceptionally high levels of siRNAs in Arabidopsis thaliana embryos. TEs generate siRNAs throughout embryogenesis according to two distinct patterns depending on whether they are located in euchromatic or heterochromatic regions of the genome. siRNA precursors are transcribed in embryos, and siRNAs are required to direct the re-establishment of DNA methylation on TEs from which they are derived in the new generation. Decondensed chromatin also permits the production of 24-nt siRNAs from heterochromatic TEs during post-embryogenesis, and siRNA production from bipartite-classified TEs is controlled by their chromatin states. Conclusions Decondensation of heterochromatin in response to developmental, and perhaps environmental, cues promotes the transcription and function of siRNAs in plants. Our results indicate that chromatin-mediated siRNA transcription provides a cell-autonomous homeostatic control mechanism to help reconstitute pre-existing chromatin states during growth and development including those that ensure silencing of TEs in the future germ line.

The Role of Neutrophil NETosis in Organ Injury: Novel Inflammatory Cell Death Mechanisms

NETosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps (NET), where net-like structures of decondensed chromatin and proteases are produced by polymorphonuclear (PMN) granulocytes. These structures immobilise pathogens and restrict them with antimicrobial molecules, thus preventing their spread. Whilst NETs possess a fundamental anti-microbial function within the innate immune system under physiological circumstances, increasing evidence also indicates that NETosis occurs in the pathogenic process of other disease type, including but not limited to atherosclerosis, airway inflammation, Alzheimer’s and stroke. Here, we reviewed the role of NETosis in the development of organ injury, including injury to the brain, lung, heart, kidney, musculoskeletal system, gut and reproductive system, whilst therapeutic agents in blocking injuries induced by NETosis in its primitive stages were also discussed. This review provides novel insights into the involvement of NETosis in different organ injuries, and whilst potential therapeutic measures targeting NETosis remain a largely unexplored area, these warrant further investigation.

The implications of neutrophil extracellular traps in the pathophysiology of atherosclerosis and atherothrombosis

Cardiovascular disease is the leading cause of mortality worldwide. Atherosclerosis constitutes most cardiovascular disease etiologies. Atherosclerosis is a chronic inflammatory and lipid-driven disease affecting the intima of blood vessels, resulting in an increase in its thickness and, therefore, narrowing of the arterial lumen. Many blood and immune cells have been shown to be implicated in atherosclerosis pathophysiology. Neutrophils are among those cells with their novel function of forming neutrophil extracellular traps. Neutrophil extracellular traps are mesh-like structures formed and released on activation of neutrophils. These structures consist of decondensed chromatin, histones, and other components, including nuclear and cellular proteins, cytoskeleton, proteases, and azurophilic granules. Neutrophil extracellular traps contain these elements and hold other circulating elements in the blood, such as tissue factor, fibrin, and other coagulation factors. Neutrophil extracellular traps are also implicated in the pathogenesis of atherothrombosis, which evolves as a consequence of atherosclerosis. In this review, we aim to demonstrate the process of neutrophil extracellular traps formation, release, and interaction with other blood cells, meaning it could be possible to use neutrophil extracellular traps as a therapeutic target in deceleration of atherosclerosis progression. Impact statement Fatal consequences of atherosclerosis and atherothrombosis give research in this field great importance. This review provides recent information about the implications of neutrophils in the pathophysiology of atherosclerosis and atherothrombosis via formation and release of neutrophil extracellular traps (NETs), thereby enhancing our understanding on how the process of atherosclerosis develops and how its consequences occur. Information provided in this review suggests NETs as a new therapeutic target and a rich point for research. This review gives answers to questions about the mechanisms of atherosclerosis and atherothrombosis progression through studying the implications of NETs in these processes.

Role of HMGB1 in the Interplay between NETosis and Thrombosis in Ischemic Stroke: A Review

Neutrophil extracellular traps (NETs) comprise decondensed chromatin, histones and neutrophil granular proteins and are involved in the response to infectious as well as non-infectious diseases. The prothrombotic activity of NETs has been reported in various thrombus-related diseases; this activity can be attributed to the fact that the NETs serve as a scaffold for cells and numerous coagulation factors and stimulate fibrin deposition. A crosstalk between NETs and thrombosis has been indicated to play a role in numerous thrombosis-related conditions including stroke. In cerebral ischemia, neutrophils are the first group of cells to infiltrate the damaged brain tissue, where they produce NETs in the brain parenchyma and within blood vessels, thereby aggravating inflammation. Increasing evidences suggest the connection between NETosis and thrombosis as a possible cause of “tPA resistance”, a problem encountered during the treatment of stroke patients. Several damage-associated molecular pattern molecules have been proven to induce NETosis and thrombosis, with high mobility group box 1 (HMGB1) playing a critical role. This review discusses NETosis and thrombosis and their crosstalk in various thrombosis-related diseases, focusing on the role of HMGB1 as a mediator in stroke. We also addresses the function of peptidylarginine deiminase 4 with respect to the interplay with HMGB1 in NET-induced thrombosis.

Chromatin Regulates Bipartite-Classified Small RNA Expression to Maintain Epigenome Homeostasis in Arabidopsis

Eukaryotic genomes are partitioned into euchromatic and heterochromatic domains to regulate gene expression and other fundamental cellular processes. However, chromatin is dynamic during growth and development, and must be properly re-established after its decondensation. Small interfering RNAs (siRNAs) promote heterochromatin formation in eukaryotes, but little is known about how chromatin regulates siRNA transcription. We demonstrated that thousands of transposable elements (TEs) produce exceptionally high levels of siRNAs in Arabidopsis thaliana embryos. Depending on whether they are located in euchromatic or heterochromatic regions of the genome, bipartite-classified TEs generate siRNAs throughout embryogenesis according to two distinct patterns. siRNAs are transcribed in embryos and required to direct the re-establishment of DNA methylation on TEs from which they are derived in the new generation. Decondensed chromatin also permits the production of 24-nt siRNAs from heterochromatic TEs during post-embryogenesis, and siRNA production from bipartite-classified TEs is controlled by their chromatin states. Decondensation of heterochromatin in response to developmental, and perhaps environmental, cues promotes the transcription and function of siRNAs in plants. Our results indicate that chromatin-mediated siRNA transcription provides a cell-autonomous homeostatic control mechanism to reconstitute pre-existing chromatin states during growth and development including those that ensure silencing of TEs in the future germ line.