Wolf Dietrich Krautgartner

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.

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.

Transcutaneous vaccination via laser microporation

Driven by constantly increasing knowledge about skin immunology, vaccine delivery via the cutaneous route has recently gained renewed interest. Considering its richness in immunocompetent cells, targeting antigens to the skin is considered to be more effective than intramuscular or subcutaneous injections. However, circumvention of the superficial layer of the skin, the stratum corneum, represents the major challenge for cutaneous immunization. An optimal delivery method has to be effective and reliable, but also highly adaptable to specific demands, should avoid the use of hypodermic needles and the requirement of specially trained healthcare workers. The P.L.E.A.S.E.® (Precise Laser Epidermal System) device employed in this study for creation of aqueous micropores in the skin fulfills these prerequisites by combining the precision of its laser scanning technology with the flexibility to vary the number, density and the depth of the micropores in a user-friendly manner. We investigated the potential of transcutaneous immunization via laser-generated micropores for induction of specific immune responses and compared the outcomes to conventional subcutaneous injection. By targeting different layers of the skin we were able to bias polarization of T cells, which could be modulated by addition of adjuvants. The P.L.E.A.S.E.® device represents a highly effective and versatile platform for transcutaneous vaccination.

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.

Delayed but functional neutrophil extracellular trap formation in neonates.

To the editor: Sepsis is one of the leading morbidity and mortality factors in newborns, occurring in more than 700 of every 100 000 live births.[1][1] Newborns seem to have a unique susceptibility to early bacterial infections[2][2] compared with adults, but the underlying pathomechanisms are

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.

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.

Neutrophils express distinct RNA receptors in a non-canonical way

Background: RNAs modulate immune responses. Neutrophils represent the major fraction of immune cells, but receptors by which neutrophils sense RNA are poorly characterized. Results: Neutrophils and differentiated HL-60 cells express the RNA receptors RIG-I, MDA-5, and TLR8. RIG-I and MDA-5 are localized in secretory vesicles. Conclusion: Neutrophils express a distinct pattern of RNA receptors. Significance: RNA receptors on neutrophils could have implications for RNA-based therapeutics. 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.

Transcutaneous immunotherapy via laser-generated micropores efficiently alleviates allergic asthma in Phl p 5–sensitized mice

Specific immunotherapy via the subcutaneous or oral route is associated with local and, in some cases, systemic side effects and suffers from low patient compliance. Due to its unique immunological features, the skin represents a promising target tissue for effective and painless treatment of type I allergy. The current study was performed to compare the efficacy of transcutaneous immunotherapy via laser‐generated micropores to subcutaneous injection.

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.