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.

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.

NETs Are Double-Edged Swords with the Potential to Aggravate or Resolve Periodontal Inflammation.

Periodontitis is a general term for diseases characterised by inflammatory destruction of tooth-supporting tissues, gradual destruction of the marginal periodontal ligament and resorption of alveolar bone. Early-onset periodontitis is due to disturbed neutrophil extracellular trap (NET) formation and clearance. Indeed, mutations that inactivate the cysteine proteases cathepsin C result in the massive periodontal damage seen in patients with deficient NET formation. In contrast, exaggerated NET formation due to polymorphonuclear neutrophil (PMN) hyper-responsiveness drives the pathology of late-onset periodontitis by damaging and ulcerating the gingival epithelium and retarding epithelial healing. Despite the gingival regeneration, periodontitis progression ends with almost complete loss of the periodontal ligament and subsequent tooth loss. Thus, NETs help to maintain periodontal health, and their dysregulation, either insufficiency or surplus, causes heavy periodontal pathology and edentulism.

Janus-Faced Neutrophil Extracellular Traps in Periodontitis.

Periodontitis is characterized by PMN infiltration and formation of neutrophil extracellular traps (NETs). However, their functional role for periodontal health remains complex and partially understood. The main function of NETs appears to be evacuation of dental plaque pathogen-associated molecular patterns. The inability to produce NETs is concomitant with aggressive periodontitis. But in cases with exaggerated NET production, NETs are unable to maintain periodontal health and bystander damages occur. This pathology can be also demonstrated in animal models using lipopolysaccharide as PMN activator. The progress of periodontitis appears to be a consequence of the formation of gingival pockets obstructing the evacuation of both pathogen-associated and damage-associated molecular patterns, which are responsible for the self-perpetuation of inflammation. Thus, besides the pathogenic effects of the periodontal bacteria, the dysregulation of PMN activation appears to play a main role in the periodontal pathology. Consequently, modulation of PMN activation might be a useful approach to periodontal therapy.

Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response.

Free DNA in cystic fibrosis airway fluids correlates with airflow obstruction.

Chronic obstructive lung disease determines morbidity and mortality of patients with cystic fibrosis (CF). CF airways are characterized by a nonresolving neutrophilic inflammation. After pathogen contact or prolonged activation, neutrophils release DNA fibres decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). NETs have been described to act in a beneficial way for innate host defense by bactericidal, fungicidal, and virucidal actions. On the other hand, excessive NET formation has been linked to the pathogenesis of autoinflammatory and autoimmune disease conditions. We quantified free DNA structures characteristic of NETs in airway fluids of CF patients and a mouse model with CF-like lung disease. Free DNA levels correlated with airflow obstruction, fungal colonization, and CXC chemokine levels in CF patients and CF-like mice. When viewed in combination, our results demonstrate that neutrophilic inflammation in CF airways is associated with abundant free DNA characteristic for NETosis, and suggest that free DNA may be implicated in lung function decline in patients with CF.

Neutrophils express distinct RNA receptors in a non-canonical way.

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.

Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy.

BACKGROUND: Cystic fibrosis (CF) lung disease is characterized by perpetuated neutrophilic inflammation with progressive tissue destruction. Neutrophils represent the major cellular fraction in CF airway fluids and are known to form neutrophil extracellular traps (NETs) upon stimulation. Large amounts of extracellular DNA-NETs are present in CF airway fluids. However, the structural contribution of NETs to the matrix composition of CF airway fluid remains poorly understood. We hypothesized that CF airway fluids consist of distinct DNA-NETs that are associated to subcellular structures. METHODOLOGY/PRINCIPAL FINDINGS: We employed atomic force microcopy (AFM) and scanning electron microcopy to ultrastructurally characterize the nature of CF sputum and the role of NETs within the extracellular CF sputum matrix. These studies demonstrate that CF sputum is predominantly composed of a high-density meshwork of NETs and NETosis-derived material. Treatment of CF sputum with different DNases degraded CF NETs and efficiently liquefied the mucous-like structure of CF sputum. Quantitative analysis of AFM results showed the presence of three globular fractions within CF sputum and the larger two ones featured characteristics of neutrophil ectosomes. CONCLUSIONS/SIGNIFICANCE: These studies suggest that excessive NET formation represents the major factor underlying the gel-like structure of CF sputum and provide evidence that CF-NETs contain ectosome-like structures that could represent targets for future therapeutic approaches.

CXCR2 mediates NADPH oxidase-independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation.

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs). Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein-coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase-independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

Sinus floor elevation via hydraulic detachment and elevation of the Schneiderian membrane.

OBJECTIVES: Minor sinus floor elevation is a method with relatively high predictability but is technically demanding. Improvement of the technique and increase in the predictability are desirable. MATERIAL AND METHODS: A clinical protocol for minor sinus floor elevation with SLA-ITI (large grit acid-etched implants with diameter of 4.8 mm) is described. Using trephine instead of spiral burrs enables the harvesting of autogenous grafts from the implant socket and guarantees a perfect implant socket. The latter is necessary for optimal implant anchoring and for the hydraulic seal between socket and the osteotome. The whole allows a hydraulic detachment of the Schneiderian membrane, where the blood cushion gradually detaches and elevates the membrane, preventing its contact with the graft. RESULTS: Eight patients were successfully treated with the method described above. No membrane perforation occurred and an uneventful healing was observed in all patients. All implants were loaded prosthodontically 3 months after the implantation. CONCLUSIONS: The clinical protocol presented provides high predictability in clinical outcome, together with extremely low morbidity and shortened surgery.

Surface morphology of pocket epithelium.

The pocket epithelium in periodontitis differs from the clinically healthy epithelium in its increase in sulcular depth. However, closer surface morphological distinctions have not been described. To study the surface characteristics of pocket gingiva, the authors analyzed pocket and sulcular epithelium biopsies by scanning and transmission electron microscopy using cytochemical staining for visualization of bacterial adhesion. The clinically healthy and the marginal pocket epithelium were characterized by squamous epithelial cells joined by tight junctions and an inconspicuous surface lacking a distinctive papillary formation. The large quantity of bacteria that adhered to the clinically healthy and marginal pocket epithelium did not appear to elicit any significant defense response. The deeper part of the pocket epithelium revealed a wrinkled papillary relief, increased exfoliation of epithelial cells, leukocyte transmigration, and bacterial internalization, as well as internalization-induced epithelial apoptosis. The alteration of the deep pocket epithelium surface might be either genuine or due to environmental changes of the crevice, or both. Therefore, the periodontitis recovery after removing the deep pocket epithelium might now be related to the pathological alterations of the deep pocket epithelium.

Supramolecular pellicle precursors.

Saliva contacting with solid surfaces in the oral cavity forms a coat termed the pellicle. However, its formation is not fully understood. Although indications for the existence of supramolecular pellicle precursors have been reported, the possible relationship between them and pellicle formation is unclear. This study investigates the ability of supramolecular precursors to form the pellicle via interaction with a solid surface. Fixed and unfixed salivary globes were spread onto a microscopic grid and examined by transmission electron microscopy. Biochemical pretreatment of saliva revealed that neither disulphide links nor transglutaminase-mediated crosslinking are responsible for maintaining the salivary globes, i.e. supramolecular pellicle precursors. However, the detergent, sodium dodecyl sulphate, caused dissociation of the salivary globes, indicating their micellar nature. Saliva contacting a formvar film for 10 s did not form a complete surface coating, but single supramolecular pellicle precursors were observed attached to the surface. After extension of the contact time to 60 s, a surface layer was formed by clustering and fusion of the supramolecular pellicle precursors. The supramolecular pellicle precursors are unstable and attain a thermodynamically more favourable state by adhesion to a solid surface. As a result, a layer of fused precursors covering the solid surface is formed -- the salivary pellicle.