Vesicular Messages from Dental Biofilms for Neutrophils.

The encounter between dental biofilm and neutrophils in periodontitis remains elusive, although it apparently plays a crucial role in the periodontal pathology and constitutes a key topic of periodontology. Dental biofilm and neutrophils were isolated from orally healthy persons and patients with periodontitis. We investigated biofilm and its particle-shedding phenomenon with electron microscopy and nanoparticle tracking analysis (NTA); biofilm shedding-neutrophil interactions were examined ex vivo with epi-fluorescence microscopy. For this purpose, we used acellular dental biofilm shedding, purified lipopolysaccharide (LPS), and phorbol 12-myristate 13-acetate (PMA) as activators, and the interleukin 8 receptor beta (CXCR2) inhibitor and the anti-interleukin 8 receptor alpha (CXCR1) antibody as modulators. The shedding of acellular dental biofilms overwhelmingly consists of bacterial extracellular vesicles (BEVs). The latter induced the moderate formation of neutrophil extracellular traps (NETs) in orally healthy subjects and a strong formation in patients with periodontitis. A CXCR2 inhibitor and an anti-CXCR1 antibody had a minor effect on NET formation. Neutrophils from patients with periodontitis exhibited NET hyper-responsiveness. BEVs were stronger inducers of NET formation than purified LPS and PMA. A plateau of neutrophil responsiveness is reached above the age of 40 years, indicating the abrupt switch of maladaptive trained immunity (TI) into the activated modus. Our results suggest that dental biofilms consist of and disseminate immense amounts of outer membrane vesicles (OMVs), which initiate NET formation via a non-canonical cytosolic LPS/caspase-4/11/Gasdermin D pathway. This modus of NET formation is independent of neutrophil elastase (NE), myeloperoxidase (MPO), peptidylarginine deiminase 4 (PAD4), and toll-like receptors (TLR). In periodontitis, the hyper-responsiveness of neutrophils to BEVs and the increased NET formation appear to be a consequence of TI.

The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans.

Extracellular traps (ETs) are reticulate structures of extracellular DNA associated with antimicrobial molecules. Their formation by phagocytes (mainly by neutrophils: NETs) has been identified as an essential element of vertebrate innate immune defense. However, as ETs are also toxic to host cells and potent triggers of autoimmunity, their role between pathogen defense and human pathogenesis is ambiguous, and they contribute to a variety of acute and chronic inflammatory diseases. Since the discovery of ET formation (ETosis) a decade ago, evidence has accumulated that most reaction cascades leading to ET release involve ROS. An important new facet was added when it became apparent that ETosis might be directly linked to, or be a variant of, the autophagy cell death pathway. The present review analyzes the evidence to date on the interplay between ROS, autophagy and ETosis, and highlights and discusses several further aspects of the ROS-ET relationship that are incompletely understood. These aspects include the role of NADPH oxidase-derived ROS, the molecular requirements of NADPH oxidase-dependent ETosis, the roles of NADPH oxidase subtypes, extracellular ROS and of ROS from sources other than NADPH oxidase, and the present evidence for ROS-independent ETosis. We conclude that ROS interact with ETosis in a multidimensional manner, with influence on whether ETosis shows beneficial or detrimental effects.

Neutrophil extracellular trap (NET) formation characterises stable and exacerbated COPD and correlates with airflow limitation.

BACKGROUND: COPD is a progressive disease of the airways that is characterized by neutrophilic inflammation, a condition known to promote the excessive formation of neutrophil extracellular traps (NETs). The presence of large amounts of NETs has recently been demonstrated for a variety of inflammatory lung diseases including cystic fibrosis, asthma and exacerbated COPD. OBJECTIVE: We test whether excessive NET generation is restricted to exacerbation of COPD or whether it also occurs during stable periods of the disease, and whether NET presence and amount correlates with the severity of airflow limitation. PATIENTS, MATERIALS AND METHODS: Sputum samples from four study groups were examined: COPD patients during acute exacerbation, patients with stable disease, and smoking and non-smoking controls without airflow limitation. Sputum induction followed the ECLIPSE protocol. Confocal laser microscopy (CLSM) and electron microscopy were used to analyse samples. Immunolabelling and fluorescent DNA staining were applied to trace NETs and related marker proteins. CLSM specimens served for quantitative evaluation. RESULTS: Sputum of COPD patients is clearly characterised by NETs and NET-forming neutrophils. The presence of large amounts of NET is associated with disease severity (p < 0.001): over 90 % in exacerbated COPD, 45 % in stable COPD, and 25 % in smoking controls, but less than 5% in non-smokers. Quantification of NET-covered areas in sputum preparations confirms these results. CONCLUSIONS: NET formation is not confined to exacerbation but also present in stable COPD and correlates with the severity of airflow limitation. We infer that NETs are a major contributor to chronic inflammatory and lung tissue damage in COPD.

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.

The initial inflammatory response to bioactive implants is characterized by NETosis.

Implants trigger an inflammatory response, which is important for osseointegration. Here we studied neutrophil extracellular trap (NET) release of human neutrophils in response to sandblasted large-grit acid etched (SLA) implants using fluorescent, confocal laser scanning and scanning electron microscopy. Our studies demonstrate that human neutrophils rapidly adhered to SLA surfaces, which triggered histone citrullination and NET release. Further studies showed that albumin or acetylsalicylic acid had no significant effects on the inflammatory response to SLA surfaces. In contrast to bioinert materials, which do not osseointegrate, the bioactivity of SLA surfaces is coupled with the ability to release NETs. Further investigations are necessary for clarifying the role of NETosis for osseointegration.

New aspects on the structure of neutrophil extracellular traps from chronic obstructive pulmonary disease and in vitro generation.

Polymorphonuclear neutrophils have in recent years attracted new attention due to their ability to release neutrophil extracellular traps (NETs). These web-like extracellular structures deriving from nuclear chromatin have been depicted in ambiguous roles between antimicrobial defence and host tissue damage. NETs consist of DNA strands of varying thickness and are decorated with microbicidal and cytotoxic proteins. Their principal structure has in recent years been characterised at molecular and ultrastructural levels but many features that are of direct relevance to cytotoxicity are still incompletely understood. These include the extent of chromatin decondensation during NET formation and the relative amounts and spatial distribution of the microbicidal components within the NET. In the present work, we analyse the structure of NETs found in induced sputum of patients with acutely exacerbated chronic obstructive pulmonary disease (COPD) using confocal laser microscopy and electron microscopy. In vitro induced NETs from human neutrophils serve for purposes of comparison and extended analysis of NET structure. Results demonstrate that COPD sputa are characterised by the pronounced presence of NETs and NETotic neutrophils. We provide new evidence that chromatin decondensation during NETosis is most extensive and generates substantial amounts of double-helix DNA in 'beads-on-a-string' conformation. New information is also presented on the abundance and location of neutrophil elastase (NE) and citrullinated histone H3 (citH3). NE occurs in high densities in nearly all non-fibrous constituents of the NETs while citH3 is much less abundant. We conclude from the results that (i) NETosis is an integral part of COPD pathology; this is relevant to all future research on the etiology and therapy of the disease; and that (ii) release of 'beads-on-a-string' DNA studded with non-citrullinated histones is a common feature of in vivo NETosis; this is of relevance to both the antimicrobial and the cytotoxic effects of NETs.

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.

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.

Fibrin mimics neutrophil extracellular traps in SEM.

Neutrophil extracellular traps (NETs) are extracellular web-like structures produced by activated polymorphonuclear neutrophils. NETs kill bacteria extracellularly, but their role in human pathology remains largely unclear. One possible way of studying NETs is through the SEM approach. However, web-like structures observed with SEM in sites of inflammation have been interpreted either as NETs or as fibrin. Thus, the question arises whether a reliable SEM discrimination between NETs and fibrin is at all possible. NET samples were collected as purulent crevicular exudate from periodontal pockets. DNase-digested controls for SEM were employed to demonstrate the DNA backbone and immuno-staining for confocal laser scanning microscopy was used to show the citrullinated histones of NETs. Blood clot samples were treated in the same way as the exudate samples to demonstrate that fibrin and fibrinolysis can mimic NETs and DNA digestion, respectively. No discrimination between fibrin and NETs based on morphological criteria in SEM was possible. Furthermore, only a vague distinction between DNA digestion and fibrinolysis could be made. These findings unambiguously indicate that the discrimination between NETs and fibrin by means of SEM is untrustworthy for samples of inflammatory exudate.

Neutrophil fate in gingival crevicular fluid.

The fate of the neutrophils within the inflammatory exudate in the periodontal crevice and their possible participation in the formation of neutrophil extracellular traps (NETs) are of clinical interest. However, the cytological analysis of clinical samples of inflammatory exudate is restricted by the obtainable quantities, which do not enable employing the routine approaches. Clinical examinations, ACLAR strip sampling, scanning electron microscopy, and confocal laser scanning microscopy were employed to analyze purulent crevicular exudate and gingival crevicular fluid in periodontitis. Bacteria, neutrophil activation, NETosis stages, and NETs were identified by molecular probe, expression of citrullinated histone H3, enzymatic digestion, and ultrastructurally. Crevicular neutrophils, all in diverse NETosis stages marked by the histone citrullination, and an abundance of NETs were found in both purulent crevicular exudate and gingival crevicular fluid. Largely varying quantities of dispersed crevicular bacteria were entrapped by NETs, but no phagocytized bacteria were evident in gingival crevicular fluid. The offered method enables for the first time the demonstration NETs in gingival crevicular fluid. The histone citrullination of all the floating crevicular neutrophils indicates that they all undergo NETosis.

A cover of glass: first report of biomineralized silicon in a ciliate, Maryna umbrellata (Ciliophora: Colpodea).

Using hydrofluoric acid, scanning electron microscope-assisted X-ray microanalysis, and energy-filtered transmission electron microscopy, we present the first definite proof of biomineralized silicon [(SiO(2))](n) in a ciliophoran protist, Maryna umbrellata, a common inhabitant of ephemeral pools. In the trophic specimen, the amorphic silicon (glass) granules are accumulated in the anterior half of the body. When entering the dormant stage, most glass granules are excreted to form the surface cover of the globular resting cyst. Most likely, the silicon granules are synthesized in vesicles of the Golgi apparatus. First, nanospheres with a size of 20-40 nm are formed in a fibrous matrix; they grow to be spongious complexes, eventually becoming amorphous glass granules with an average size of 819 nm x 630 nm. In the transmission electron microscope, the silicon granules show the characteristic fracture pattern of glass known from many other silicon-bearing organisms. A literature survey suggests that silicon is very rare in ciliates. The fine structure and genesis of silicon granules in M. umbrellata are very similar to those of other organisms, including vascular plants and animals, indicating a common mechanism. Light perception and protection against mechanical stress and predators might be functions of the silicon granules in M. umbrellata. The palaeontological significance of glass cysts in ciliates is also discussed.

Correlations between AFM and SEM imaging of acid-etched tooth enamel.

Enamel bond strength is an important factor in restorative dentistry and crucially depends on the enamel roughness. To increase roughness, different etching procedures are employed and profilometric estimations, with probe profilometers, including atomic force microscopy (AFM), have been made. However, no correlation between roughness and bond strength has been found. To search for a possible error source leading to the underestimation of enamel roughness when utilizing probe profilometers, the authors compared scanning electron microscopy and AFM images of acid-etched tooth enamel. The results showed that AFM imaging cannot correctly depict the acid-etched enamel surface, because of the high steepness of the enamel crystallites and the generation of convolute images. This leads to a large underestimation of the profilometric parameters measured with AFM, or other profilometers, on acid-etched tooth enamel surfaces.

Visualization of neutrophil extracellular traps in TEM.

Neutrophil extracellular traps (NETs) have recently been described as an important innate defence mechanism in inflammation. However, routine electron microscopic staining techniques faintly stain NETs and are therefore insufficient for enabling a distinction between these and the host cell debris as well as proteins regularly present at the site of inflammation. In order to test suitable electron microscopic staining techniques, NETs induced ex vivo via phorbol myristate were absorbed on formvar. Four types of drop-on-grid positive staining were used: osmium tetroxide (Os), osmium tetroxide-uranyl acetate-lead citrate (Os-U-Pb), ruthenium red-osmium tetroxide (RR-OsO4), and cuprolinic blue enhanced by sodium tungstate (CB-WO4). We observed no staining of NETs using Os, faint staining with Os-U-Pb, better but still weak staining with CB-WO4 and outstanding staining with RR-OsO4. Furthermore, RR-OsO4 staining also enables the observation of bacterial fimbriae-mediated adhesion, which is possibly responsible for the ability of NETs to bind bacteria. Thus, the offered RR-OsO4 staining technique may facilitate the study of the NETs-bacterial interactions.

Restorative therapy of primary teeth severely affected by amelogenesis imperfecta.

OBJECTIVE: Primary teeth severely affected by amelogenesis imperfecta (AI) often show an extensive loss of enamel. Such defects are difficult to restore with resin composites, since neither the correct anatomic form nor the marginal fit can be guaranteed. METHODS AND MATERIALS: After clinical and scanning electron microscopic examinations were performed on replica models of 5 patients with primary teeth affected by AI, impressions were made without previous preparation by rotary instruments. Composite crowns and veneers were manufactured and luted adhesively using the total bonding technique and low-viscosity resin composite. RESULTS: The pre-restorative scanning electron microscopic analysis showed that the dentinal tubules were exposed and that the border of the residual enamel was in the process of splitting. The preoperative oral examination had revealed tooth discoloration, masticatory disturbances, hypersensitivity, and speech problems. After placement of the restorations, patients reported improvements in tooth sensitivity, articulation, and mastication. CONCLUSIONS: A new protocol for restoration of primary teeth with an extensive loss of enamel is offered. It is quick and easy to perform, highly esthetic, and can be applied in children younger than 4 years old.

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.

Cytochemical differences in bacterial glycocalyx.

To examine new cytochemical aspects of the bacterial adhesion, a strain 41452/01 of the oral commensal Streptococcus sanguis and a wild strain of Staphylococcus aureus were grown with and without sucrose supplementation for 6 days. Osmiumtetraoxyde (OsO4), uranyl acetate (UA), ruthenium red (RR), cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs), and the tannic acid-metal salt technique (TAMST) were applied for electron microscopy. Cytochemically, only RR-positive fimbriae in S. sanguis were visualized. By contrast, some types of fimbriae staining were observed in S. aureus glycocalyx: RR-positive, OsO4-positive, tannophilic and CB-positive with ceasing point at 0.3 M MgCl2. The CB staining with CEC, used for the first time for visualization of glycoproteins of bacterial glycocalyx, also reveals intacellular CB-positive substances-probably the monomeric molecules, that is, subunits forming the fimbriae via extracellular assembly. Thus, glycosylated components of the biofilm matrix can be reliably related to single cells. The visualization of intracellular components by CB with CEC enables clear distinction between S. aureus and other bacteria, which do not produce CB-positive substances. The small quantities of tannophilic substances found in S. aureus makes the use of TAMST for the same purpose difficult. The present work protocol enables, for the first time, a partial cytochemical differentiation of the bacterial glycocalyx.

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.

Glycocalyx morphology of Candida albicans.

In this study, we examine new cytochemical aspects of the fimbria-mediated adhesion of the oral facultative pathogen Candida albicans. A wild-type strain of the yeast was grown with and without sucrose supplementation for 8 days. Osmium tetroxide, uranyl acetate (UA), ruthenium red (RR), and cupromeronic blue (CB) staining with critical electrolytic concentrations (CECs) and tannic acid-metal salt technique (TAMST) were applied to specimens separately or in combination for transmission electron microscopy (TEM) examination. Cytochemically, two types of fimbriae of C. albicans were distinguished: RR-positive fimbriae of polyanionic glycoconjugates and CB-positive fimbriae with a ceasing point of 0.3 M MgCl2 where no staining of sulfated carboxyl-rich and/or phospho-glycoconjugates occurred. Additionally, CB-positive intercellular fibers were observed, which seemed to be involved in intercellular adhesion. The present protocol enables, for the first time, a partial cytochemical differentiation between at least two kinds of yeast fimbriae.

Candida-induced stomatopyrosis and its relation to diabetes mellitus.

BACKGROUND: Glycaemic disorders and oral candidosis can be accompanied by burning mouth sensations. However, no clear relation between all three disorders is known. METHODS: Seventy-two native Upper-Austrians with burning mouth sensations were examined and smears for Candida estimation were taken from the spots where the sensations were felt. All patients with previously unknown diabetes mellitus (DM) were subjected to an oral glucose tolerance test (OGTT). Use of glucocorticoid-containing anti-asthmatic sprays and the body mass index (BMI) were determined. RESULTS: Of the examined non-inhalers of sprays, 52% had increased candidal density. A correlation between that increase and type 2 DM was found. The burning sensations in all patients with increased candidal density subsided completely after anti-mycotic therapy. CONCLUSION: The perception of burning sensations was hypothesised to occur via stimulation of the capsaicin (vanilloid) receptor by Candida metabolites. The Candida-induced stomatopyrosis should be regarded as a single symptom indicating (predisposition to or established) type 2 DM in non-inhalers of the concerned population.