Walter Stoiber

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.

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

BackgroundCOPD 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.ObjectiveWe 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 methodsSputum 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.ResultsSputum 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.ConclusionsNET 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.

MyoD and Myogenin expression during myogenic phases in brown trout: A precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow‐growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast‐growing salmonid with exceptionally large embryos. Results confirm the three‐phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second‐ and third‐phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies. Developmental Dynamics 236:1106–1114, 2007.

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.

Phases of myogenic cell activation and possible role of dermomyotome cells in teleost muscle formation

Present knowledge indicates that fibre recruitment (hyperplasia) in developing teleost fish occurs in three distinct phases. However, the origin and relationship of the myogenic precursors activated during the different phases remains unclear. Here, we address this issue using molecular techniques on embryos and larvae of pearlfish, a large cyprinid species. Results provide comprehensive molecular characterisation of cell recruitment over the three phases of myogenesis, identifying muscle types as they arise. Specifically, we show that the myogenic cells arising during 2nd phase myogenesis are clearly different from the myogenic cells arising during the 3rd phase and that the dermomyotome is a major source of myogenic cells driving 2nd phase hyperplasia. These findings are discussed in relation to their implications for the generality of vertebrate developmental patterns. Developmental Dynamics 235:3132–3143, 2006.

Patterns of superficial fibre formation in the European pearlfish (Rutilus frisii meidingeri) provide a general template for slow muscle development in teleost fish

Abstract The debate about the pattern of muscle formation in teleost fish has recently been heightened in the literature. Here we examine superficial muscle development in the pearlfish, a cyprinid endemic to a small area of Central Europe, and uninfluenced by economic interest and breeding. Using light and electron microscopy, histochemistry and immunohistochemistry techniques, we report that: (1) Superficial fibre precursors originate close to the notochord, are part of the same cell population as the so-called muscle pioneer cells, and are transferred laterally to end up at the surface of the myotome. (2) Superficial fibre maturation is exceptionally rapid. Structural and enzymatic functionality is attained at a time when prospective deep fibres have not passed beyond the early myotube state. This strong contrast weakens as the embryo develops. (3) Apart from the muscle pioneers, the superficial fibres appear to be capable of functioning before they receive any direct innervation, implying that signals are transferred to these fibres via cell-to-cell junctions. We suggest that the capability of rapid superficial fibre maturation is a rather general feature among teleosts and may aid pre-hatch survival under a variable environment. Our results indicate that muscle formation in teleost fish may follow a common basic pattern that is open to considerable ontogenetic and phylogenetic modification in response to habitat conditions.

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.

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.

Postembryonic fast muscle growth of teleost fish depends upon a nonuniformly distributed population of mitotically active Pax7+ precursor cells.

Muscle development in teleost embryos has been shown to depend on myogenic cell recruitment from the dermomyotome (DM). However, little is known as to the cellular mechanisms that account for myotome growth after the dissociation of the DM. Here we combine immunolabeling for cell‐specific markers with quantitative analysis to determine the sources and patterns of activation of myogenic cells in pearlfish larvae. Results demonstrate that appearance of mitotically active myogenic precursors inside the myotome coincides with the dissociation of the DM. Such cells are preferentially aggregated within the posterior lateral fast muscle. We therefore propose a growth model in which a pool of proliferative DM‐derived precursors transferred to the posterior lateral fast muscle functions as an important source of myogenic cell spread to carry forward stratified fast muscle hyperplasia. This indicates that postembryonic teleost muscle growth includes a cellular mechanism that has no direct equivalent in the amniotes. Developmental Dynamics 238:2442–2448, 2009.