Neutrophil Extracellular Traps Initiate Gallstone Formation.

The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.

Neutrophil Depletion Changes the N-Glycosylation Pattern of IgG in Experimental Murine Sepsis.

Sepsis is a life-threatening condition with a rising disease burden worldwide. It is a multifactorial disease and is defined as a dysregulated host response to infection. Neutrophils have been shown to be involved in the pathogenesis of sepsis by exacerbating inflammation. However, the exact effector mechanism of action still remains a mystery. Changes in the glycosylation pattern of the immunoglobulin G (IgG) Fc region are described for several diseases including meningococcal sepsis. In this study, we investigated the possible contribution of neutrophils and neutrophil implication, potentially related to degranulation or neutrophil extracellular trap (NET) formation in changing the IgG Fc N-glycosylation pattern in a murine sepsis model. We have measured the serum level of cytokines/chemokines and immunoglobulins, the serum activity of neutrophil elastase (NE), and analyzed the IgG Fc glycosylation pattern by Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (LC-ESI-MS) and Lectin enzyme-linked immunosorbent assay (ELISA). We observed an increased activity of NE- and neutrophil-associated cytokines such as keratinocyte chemoattractant (KC) with the development of sepsis. Regarding the IgG Fc N-glycosylation, we observed an increase in fucosylation and α1,3-galactosylation and a decrease for sialyation. Interestingly, these changes were not uniform for all IgG subclasses. After depletion of neutrophils, we saw a change in the exposure of fucose and α2,6-linked sialic acid during the time course of our experimental sepsis model. In conclusion, neutrophils can influence changes in the IgG glycosylation pattern in experimental sepsis.

The Dual Role of Neutrophil Extracellular Traps (NETs) in Sepsis and Ischemia-Reperfusion Injury: Comparative Analysis across Murine Models.

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus).

Breaking the Gingival Barrier in Periodontitis.

The break of the epithelial barrier of gingiva has been a subject of minor interest, albeit playing a key role in periodontal pathology, transitory bacteraemia, and subsequent systemic low-grade inflammation (LGI). The significance of mechanically induced bacterial translocation in gingiva (e.g., via mastication and teeth brushing) has been disregarded despite the accumulated knowledge of mechanical force effects on tight junctions (TJs) and subsequent pathology in other epithelial tissues. Transitory bacteraemia is observed as a rule in gingival inflammation, but is rarely observed in clinically healthy gingiva. This implies that TJs of inflamed gingiva deteriorate, e.g., via a surplus of lipopolysaccharide (LPS), bacterial proteases, toxins, Oncostatin M (OSM), and neutrophil proteases. The inflammation-deteriorated gingival TJs rupture when exposed to physiological mechanical forces. This rupture is characterised by bacteraemia during and briefly after mastication and teeth brushing, i.e., it appears to be a dynamic process of short duration, endowed with quick repair mechanisms. In this review, we consider the bacterial, immune, and mechanical factors responsible for the increased permeability and break of the epithelial barrier of inflamed gingiva and the subsequent translocation of both viable bacteria and bacterial LPS during physiological mechanical forces, such as mastication and teeth brushing.

Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Neutrophils' Extracellular Trap Mechanisms: From Physiology to Pathology.

Neutrophils are an essential part of the innate immune system and the first line of defense against invading pathogens. They phagocytose, release granular contents, produce reactive oxygen species, and form neutrophil extracellular traps (NETs) to fight pathogens. With the characterization of NETs and their components, neutrophils were identified as players of the innate adaptive crosstalk. This has placed NETs at the center not only of physiological but also pathological processes. Aside from their role in pathogen uptake and clearance, NETs have been demonstrated to contribute to the resolution of inflammation by forming aggregated NETs able to degrade inflammatory mediators. On the other hand, NETs have the potential to foster severe pathological conditions. When homeostasis is disrupted, they occlude vessels and ducts, serve as sources of autoantigens and danger or damage associated molecular patterns, directly damage tissues, and exaggerate complement activity and inflammation. This review focusses on the understanding of NETs from their formation to their functions in both physiological and pathological processes.

A Pleomorphic Puzzle: Heterogeneous Pulmonary Vascular Occlusions in Patients with COVID-19.

Vascular occlusions in patients with coronavirus diseases 2019 (COVID-19) have been frequently reported in severe outcomes mainly due to a dysregulation of neutrophils mediating neutrophil extracellular trap (NET) formation. Lung specimens from patients with COVID-19 have previously shown a dynamic morphology, categorized into three types of pleomorphic occurrence based on histological findings in this study. These vascular occlusions in lung specimens were also detected using native endogenous fluorescence or NEF in a label-free method. The three types of vascular occlusions exhibit morphology of DNA rich neutrophil elastase (NE) poor (type I), NE rich DNA poor (type II), and DNA and NE rich (type III) cohort of eleven patients with six males and five females. Age and gender have been presented in this study as influencing variables linking the occurrence of several occlusions with pleomorphic contents within a patient specimen and amongst them. This study reports the categorization of pleomorphic occlusions in patients with COVID-19 and the detection of these occlusions in a label-free method utilizing NEF.

Connection between Periodontitis-Induced Low-Grade Endotoxemia and Systemic Diseases: Neutrophils as Protagonists and Targets.

Periodontitis is considered a promoter of many systemic diseases, but the signaling pathways of this interconnection remain elusive. Recently, it became evident that certain microbial challenges promote a heightened response of myeloid cell populations to subsequent infections either with the same or other pathogens. This phenomenon involves changes in the cell epigenetic and transcription, and is referred to as ''trained immunity''. It acts via modulation of hematopoietic stem and progenitor cells (HSPCs). A main modulation driver is the sustained, persistent low-level transmission of lipopolysaccharide from the periodontal pocket into the peripheral blood. Subsequently, the neutrophil phenotype changes and neutrophils become hyper-responsive and prone to boosted formation of neutrophil extracellular traps (NET). Cytotoxic neutrophil proteases and histones are responsible for ulcer formations on the pocket epithelium, which foster bacteremia and endoxemia. The latter promote systemic low-grade inflammation (SLGI), a precondition for many systemic diseases and some of them, e.g., atherosclerosis, diabetes etc., can be triggered by SLGI alone. Either reverting the polarized neutrophils back to the homeostatic state or attenuation of neutrophil hyper-responsiveness in periodontitis might be an approach to diminish or even to prevent systemic diseases.

Long noncoding RNA HOTAIR is upregulated in an aggressive subgroup of gastrointestinal stromal tumors (GIST) and mediates the establishment of gene-specific DNA methylation patterns.

Aberrant alterations of DNA methylation are common events in oncogenesis. The origin of cancer-associated epigenetic defects is of interest for mechanistic understanding of malignant transformation and-in the long run-therapeutic modulation of DNA methylation in a locus-specific manner. Given the ability of certain long noncoding RNAs to operate as an interface between DNA and the epigenetic modification machinery which can interact with DNA methyltransferases, we hypothesized-considering HOTAIR as an example-that this transcript may contribute to gene specificity of DNA methylation. Using gastrointestinal stromal tumors (GISTs, n = 67) as a model, we confirmed upregulation of HOTAIR in tumors with high risk of recurrence and showed high abundance of the transcript in GIST cell lines. HOTAIR knockdown in GIST-T1 cells triggered transcriptional response of genes involved in the organization and disassembly of the extracellular matrix and, notably, induced global locus-specific alterations of DNA methylation patterns. Hypomethylation was induced at a total of 507 CpG sites, whereas 382 CpG dinucleotides underwent gain of methylation upon HOTAIR depletion. Importantly, orchestrated gain or loss of methylation at multiple individual CpG sites was shown for cancer-related DPP4, RASSF1, ALDH1A3, and other targets. Collectively, our data indicate that HOTAIR enables target specificity of DNA methylation in GIST and is capable of dual (hypo- and hypermethylation) regulation by a yet to be defined mechanism. The results further suggest the feasibility of manipulating DNA methylation in a targeted manner and are of interest in the context of epigenetic cancer therapy.

Paediatric and adult soft tissue sarcomas with NTRK1 gene fusions: a subset of spindle cell sarcomas unified by a prominent myopericytic/haemangiopericytic pattern.

Neoplasms with a myopericytomatous pattern represent a morphological spectrum of lesions encompassing myopericytoma of the skin and soft tissue, angioleiomyoma, myofibromatosis/infantile haemangiopericytoma and putative neoplasms reported as malignant myopericytoma. Lack of reproducible phenotypic and genetic features of malignant myopericytic neoplasms have prevented the establishment of myopericytic sarcoma as an acceptable diagnostic category. Following detection of a LMNA-NTRK1 gene fusion in an index case of paediatric haemangiopericytoma-like sarcoma by combined whole-genome and RNA sequencing, we identified three additional sarcomas harbouring NTRK1 gene fusions, termed 'spindle cell sarcoma, NOS with myo/haemangiopericytic growth pattern'. The patients were two children aged 11 months and 2 years and two adults aged 51 and 80 years. While the tumours of the adults were strikingly myopericytoma-like, but with clear-cut atypical features, the paediatric cases were more akin to infantile myofibromatosis/haemangiopericytoma. All cases contained numerous thick-walled dysplastic-like vessels with segmental or diffuse nodular myxohyaline myo-intimal proliferations of smooth muscle actin-positive cells, occasionally associated with thrombosis. Immunohistochemistry showed variable expression of smooth muscle actin and CD34, but other mesenchymal markers, including STAT6, were negative. This study showed a novel variant of myo/haemangiopericytic sarcoma with recurrent NTRK1 gene fusions. Given the recent introduction of a novel therapeutic approach targeting NTRK fusion-positive neoplasms, recognition of this rare but likely under-reported sarcoma variant is strongly encouraged.

Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation.

Neutrophils, the most abundant leukocytes in the human body, are considered to be the first line of defense in the fight against microorganisms. In this fight neutrophils employ weaponry such as reactive oxygen species produced via the NADPH oxidase complex 2 together with the release of intracellular granules containing antimicrobial agents. The discovery that activated neutrophils release decondensed chromatin as DNase-sensitive neutrophil extracellular traps (NETs) lead to a renewed interest in these leukocytes and the function of NETs in vivo. In this review, we will focus on desirable as well as detrimental features of NETs by the example of gout and pancreatitis. In our models we observed that neutrophils drive the initiation of inflammation and are required for the resolution of inflammation.

Neutrophil Extracellular Traps: A Crucial Factor in Post-Surgical Abdominal Adhesion Formation.

Post-surgical abdominal adhesions, although poorly understood, are highly prevalent. The molecular processes underlying their formation remain elusive. This review aims to assess the relationship between neutrophil extracellular traps (NETs) and the generation of postoperative peritoneal adhesions and to discuss methods for mitigating peritoneal adhesions. A keyword or medical subject heading (MeSH) search for all original articles and reviews was performed in PubMed and Google Scholar. It included studies assessing peritoneal adhesion reformation after abdominal surgery from 2003 to 2023. After assessing for eligibility, the selected articles were evaluated using the Critical Appraisal Skills Programme checklist for qualitative research. The search yielded 127 full-text articles for assessment of eligibility, of which 7 studies met our criteria and were subjected to a detailed quality review using the Critical Appraisal Skills Programme (CASP) checklist. The selected studies offer a comprehensive analysis of adhesion pathogenesis with a special focus on the role of neutrophil extracellular traps (NETs) in the development of peritoneal adhesions. Current interventional strategies are examined, including the use of mechanical barriers, advances in regenerative medicine, and targeted molecular therapies. In particular, this review emphasizes the potential of NET-targeted interventions as promising strategies to mitigate postoperative adhesion development. Evidence suggests that in addition to their role in innate defense against infections and autoimmune diseases, NETs also play a crucial role in the formation of peritoneal adhesions after surgery. Therefore, therapeutic strategies that target NETs are emerging as significant considerations for researchers. Continued research is vital to fully elucidate the relationship between NETs and post-surgical adhesion formation to develop effective treatments.

Midgut Volvulus Adds a Murine, Neutrophil-Driven Model of Septic Condition to the Experimental Toolbox.

BACKGROUND: Severe infections that culminate in sepsis are associated with high morbidity and mortality. Despite continuous efforts in basis science and clinical research, evidence based-therapy is mostly limited to basic causal and supportive measures. Adjuvant therapies often remain without clear evidence. The objective of this study was to evaluate the septic volvulus ischemia-reperfusion model in comparison to two already established models and the role of neutrophil extacellular traps (NETs) in this model. METHODS: The technique of the murine model of midgut volvulus was optimized and was compared to two established models of murine sepsis, namely cecal ligation and puncture (CLP) and intra-peritoneal (i.p.) injection of lipopolysaccharide (LPS). RESULTS: Midgut volvulus for 15 min caused a comparable mortality (38%) as CLP (55%) and peritoneal LPS injection (25%) at 48 h. While oxidative stress was comparable, levels of circulating free DNA (cfDNA), and splenic/hepatic and pulmonary translocation of bacteria were decreased and increased, respectively at 48 h. DNases were increased compared to the established models. Proteomic analysis revealed an upregulation of systemic Epo, IL-1b, Prdx5, Parp1, Ccl2 and IL-6 at 48 h in comparison to the healthy controls. DISCUSSION AND CONCLUSION: Midgut volvulus is a stable and physiological model for sepsis. Depending on the duration and subsequent tissue damage, it represents a combination of ischemia-reperfusion injury and hyperinflammation.

Neutrophil Extracellular Traps Drive Dacryolithiasis.

Mucopeptide concretions, previously called dacryoliths, are macroscopic stones that commonly obstruct the lacrimal sac. The mechanism behind dacryolithiasis remains unclear; however, the involvement of various immune cells, including neutrophils, has been confirmed. These findings remain limited, and no information on neutrophil extracellular traps (NETs), essentially involved in the pathogenesis of other lithiases, is available yet. Here, we employ microcomputed tomography, magnetic resonance tomography, histochemistry, mass spectrometry, and enzyme activity analyses to investigate the role of neutrophils and NETs in dacryolithiasis. We classify mucopeptide concretions into three types, with respect to the quantity of cellular and acellular material, polysaccharides, and mucosubstances. We propose the role of neutrophils and NETs within the existing model of gradual formation and growth of mucopeptide concretions, with neutrophils contributing to the initial stages of dacryolithiasis, as they localized on the inner (older) parts of the tissue. As NETs localized on the outer (newer) parts of the tissue, we link their role to the late stages of dacryolithiasis, presumably maintaining the proinflammatory environment and preventing efficient clearance. An abundance of IgG on the surface indicates the involvement of the adaptive immune system later as well. These findings bring new perspectives on dacryolithiasis, in which the innate and adaptive immune system are essentially involved.

Suppression of neutrophils by sodium exacerbates oxidative stress and arthritis.

Introduction: Typical Western diet, rich in salt, contributes to autoimmune disease development. However, conflicting reports exist about the effect of salt on neutrophil effector functions, also in the context of arthritis. Methods: We investigated the effect of sodium chloride (NaCl) on neutrophil viability and functions in vitro, and in vivo employing the murine K/BxN-serum transfer arthritis (STA) model. Results and discussion: The effects of NaCl and external reactive oxygen species (H2O2) were further examined on osteoclasts in vitro. Hypertonic sodium-rich media caused primary/secondary cell necrosis, altered the nuclear morphology, inhibited phagocytosis, degranulation, myeloperoxidase (MPO) peroxidation activity and neutrophil extracellular trap (NET) formation, while increasing total ROS production, mitochondrial ROS production, and neutrophil elastase (NE) activity. High salt diet (HSD) aggravated arthritis by increasing inflammation, bone erosion, and osteoclast differentiation, accompanied by increased NE expression and activity. Osteoclast differentiation was decreased with 25 mM NaCl or 100 nM H2O2 addition to isotonic media. In contrast to NaCl, external H2O2 had pro-resorptive effects in vitro. We postulate that in arthritis under HSD, increased bone erosion can be attributed to an enhanced oxidative milieu maintained by infiltrating neutrophils, rather than a direct effect of NaCl.

Moonlighting chromatin: when DNA escapes nuclear control.

Extracellular chromatin, for example in the form of neutrophil extracellular traps (NETs), is an important element that propels the pathological progression of a plethora of diseases. DNA drives the interferon system, serves as autoantigen, and forms the extracellular scaffold for proteins of the innate immune system. An insufficient clearance of extruded chromatin after the release of DNA from the nucleus into the extracellular milieu can perform a secret task of moonlighting in immune-inflammatory and occlusive disorders. Here, we discuss (I) the cellular events involved in the extracellular release of chromatin and NET formation, (II) the devastating consequence of a dysregulated NET formation, and (III) the imbalance between NET formation and clearance. We include the role of NET formation in the occlusion of vessels and ducts, in lung disease, in autoimmune diseases, in chronic oral disorders, in cancer, in the formation of adhesions, and in traumatic spinal cord injury. To develop effective therapies, it is of utmost importance to target pathways that cause decondensation of chromatin during exaggerated NET formation and aggregation. Alternatively, therapies that support the clearance of extracellular chromatin are conceivable.

Neutrophil extracellular traps and DNases orchestrate formation of peritoneal adhesions.

Peritoneal adhesions are poorly understood but highly prevalent conditions that can cause intestinal obstruction and pelvic pain requiring surgery. While there is consensus that stress-induced inflammation triggers peritoneal adhesions, the molecular processes of their formation still remain elusive. We performed murine models and analyzed human samples to monitor the formation of adhesions and the treatment with DNases. Various molecular analyses were used to evaluate the adhesions. The experimental peritoneal adhesions of the murine models and biopsy material from humans are largely based on neutrophil extracellular traps (NETs). Treatment with DNASE1 (Dornase alfa) and the human DNASE1L3 analog (NTR-10), significantly reduced peritoneal adhesions in experimental models. We conclude that NETs serve as essential scaffold for the formation of adhesions; DNases interfere with this process. Herein, we show that therapeutic application of DNases can be employed to prevent the formation of murine peritoneal adhesions. If this can be translated into the human situation requires clinical studies.

Formation and Clearance of NETs in Health and Disease.

Neutrophils are the most abundant innate immune cells in humans and the first line of defense against invading pathogens [...].

Periodontitis-Derived Dark-NETs in Severe Covid-19.

The frequent severe COVID-19 course in patients with periodontitis suggests a link of the aetiopathogenesis of both diseases. The formation of intravascular neutrophil extracellular traps (NETs) is crucial to the pathogenesis of severe COVID-19. Periodontitis is characterised by an increased level of circulating NETs, a propensity for increased NET formation, delayed NET clearance and low-grade endotoxemia (LGE). The latter has an enormous impact on innate immunity and susceptibility to infection with SARS-CoV-2. LPS binds the SARS-CoV-2 spike protein and this complex, which is more active than unbound LPS, precipitates massive NET formation. Thus, circulating NET formation is the common denominator in both COVID-19 and periodontitis and other diseases with low-grade endotoxemia like diabetes, obesity and cardiovascular diseases (CVD) also increase the risk to develop severe COVID-19. Here we discuss the role of propensity for increased NET formation, DNase I deficiency and low-grade endotoxaemia in periodontitis as aggravating factors for the severe course of COVID-19 and possible strategies for the diminution of increased levels of circulating periodontitis-derived NETs in COVID-19 with periodontitis comorbidity.

Induction of Ocular Surface Inflammation and Collection of Involved Tissues.

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.