The role of neutrophil extracellular traps in lung epithelial cell death

Neutrophile erfüllen eine wichtige Rolle bei der angeborenen Immunabwehr, indem sie in den Körper eindringende Mikroorganismen bekämpfen. Ihre antimikrobielle Aktivitität wird durch Phagozytose, die Ausschüttung reaktiver Sauerstoffspezies ("reactive oxygen species", ROS) und lytische Enzyme vermittelt. Eine alternative Bekämpfung der Pathogene kann über die Ausbildung von sogenannten "Neutrophil Extracellular Traps" (NET) erfolgen, einer speziellen Art des Zelltods, bei dem die Neutrophilen DNA, Histone, verschiedene granuläre Proteine (wie z.B. Neutrophile Elastase), aber auch zytoplasmatische Komponenten netzartig herausschleudern. Obwohl die Ausbildung von NET in verschiedenen Organismen und pathophysiologischen Bedingungen und ihre bakterizide Wirkung dokumentiert wurde, ist wenig bekannt über die Wirkung von NET auf körpereigene Zellen und im speziellen bei der Pathogenese des akuten Lungenschadens (ALI). In dieser Arbeit wurde die Auswirkung der NET-Bildung auf Endothel- und Epithelzellen studiert, vor allem im Bezug auf funktionelle Veränderungen von alveolären Epithelzellen, die mit für den Gasaustausch in der Lunge verantwortlich sind. In einem Mausmodell des ALI konnte die Bildung von NET in Lungengewebe und Lavage der erkrankten Tiere gezeigt werden. Direkte Interaktion von NET mit endothelialen und epithelialen Zellen führte nicht nur zu Änderungen in der Permeabilität des Zellverbandes, sondern auch zu einer erhöhten Synthese pro-apoptotischer Proteine und wirkte sich in konzentrationsabhängiger Weise zytotoxisch auf die Zellen aus. Da der DNase-Verdau von NET keinen Einfluss auf deren zytotoxischen Effekt hatte, können NET-assoziierte Proteine dafür verantwortlich gemacht werden. Hier waren es vor allem Histone, die auch in isolierter Form zytotoxisch wirkten, was durch verschiedene Antikörper gegen Histone verhindert werden konnte. Da aktiviertes Protein C (APC) Histone degradiert, wurde die Histon-induzierte Zytotoxizität in vitro reduziert, nicht aber die NET-induzierte Zytotoxizität. Dies legt den Schluss nahe, dass Histone in NET gegen den Abbau durch APC geschützt sind. Die Inhibierung von Neutrophiler Elastase, einem weiteren Proteinbestandteil von NET, hatte keine Wirkung auf deren Zytotoxizität, allerdings konnte eine stärkere Elastase-Aktivität nach DNase-Behandlung von NET festgestellt werden. Die Inhibierung von Myeloperoxidase, einer weiteren Komponente der NET verningerte allerdings die NETinduzierte Zytotoxizität. Die Proteomanalyse von mit NET inkubierten Epithelzellen zeigte einen deutlichen Anstieg an Apoptose-relevanten Proteinen. Diese Arbeit zeigt, dass bestimmte Proteine in den NET und hier vor allem die Histone, aber auch Myeloperoxidase stark zytotoxisch auf Endothel- und Epithelzellen wirken. Damit können NET einer der Auslöser für die Zerstörung von Geweben und Organen bei entzündlichen Erkrankungen, wie beispielsweise des akuten Lungenversagens (ALI), sein.Neutrophils play an important role in innate immunity by defending the host against invaded microorganisms. Antimicrobial activity of neutrophils is mediated by phagocytosis, release of reactive oxygen species and lytic enzymes as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and certain granular proteins such as neutrophil elastase as well as some cytoplasmic proteins. NET formation in different organisms, its bactericidal activity, and its presence in several pathophysiological conditions have been documented; however, little is known about the direct effect of NET on the host cells and its potential role in the pathogenesis of acute lung injury (ALI). This study aims to decipher the influence of NET on host cells, particularly alveolar epithelial cells as major cells responsible for gas exchange in the lung. Herein, NET formation was documented in the lung tissue and the bronchoalveolar lavage fluid in a mouse model of ALI. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity, indicating that NET associated proteins are responsible for this effect. NET associated proteins were also identified by mass spectrometry. Following treatment of epithelial cells with NET, different proteins with roles in cell death processes were upregulated. Pre-incubation of NET with antibodies against histones significantly reduced NET-mediated cytotoxicity, suggesting that the cell-destructive activity of NET was at least in great part mediated by histones. Activated protein C (APC) did decrease the histones-induced cytotoxicity in a purified system, but did not change NETinduced cytotoxicity, indicating that histones in NET are protected against APC degradation. Inhibition of neutrophil elastase, another abundant component of NET, did not decrease NET-mediated cytotoxicity, although elastase activity increased after DNA digestion. Inhibition of myeloperoxidase, another granular component of NET, decreased NETmediated cytotoxicity. Moreover, in epithelial cells, NET induced a biphasic change in cellular permeability; at earlier stages decrease and at the later stages increase was observed. These data reveal the important role of protein components in NET, particularly histones and partly myeloperoxidase, which may lead to host cell cytotoxicity and may be one of the inducers of organ destruction associated with inflammatory diseases including ALI. Thus, NET in a spatio-temporal manner can influence the cell functions

Vascular miR‐181b controls tissue factor‐dependent thrombogenicity and inflammation in type 2 diabetes

BACKGROUND: Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood. METHODS: In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity. RESULTS: In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, D-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181-/- animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181-/- mice showed increased TF expression and FXa generation upon stimulation with LPS. CONCLUSIONS: miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis

Neutrophil Extracellular Traps Directly Induce Epithelial and Endothelial Cell Death: A Predominant Role of Histones

Neutrophils play an important role in innate immunity by defending the host organism against invading microorganisms. Antimicrobial activity of neutrophils is mediated by release of antimicrobial peptides, phagocytosis as well as formation of neutrophil extracellular traps (NET). These structures are composed of DNA, histones and granular proteins such as neutrophil elastase and myeloperoxidase. This study focused on the influence of NET on the host cell functions, particularly on human alveolar epithelial cells as the major cells responsible for gas exchange in the lung. Upon direct interaction with epithelial and endothelial cells, NET induced cytotoxic effects in a dose-dependent manner, and digestion of DNA in NET did not change NET-mediated cytotoxicity. Pre-incubation of NET with antibodies against histones, with polysialic acid or with myeloperoxidase inhibitor but not with elastase inhibitor reduced NET-mediated cytotoxicity, suggesting that histones and myeloperoxidase are responsible for NET-mediated cytotoxicity. Although activated protein C (APC) did decrease the histone-induced cytotoxicity in a purified system, it did not change NET-induced cytotoxicity, indicating that histone-dependent cytotoxicity of NET is protected against APC degradation. Moreover, in LPS-induced acute lung injury mouse model, NET formation was documented in the lung tissue as well as in the bronchoalveolar lavage fluid. These data reveal the important role of protein components in NET, particularly histones, which may lead to host cell cytotoxicity and may be involved in lung tissue destruction

Neutrophils: Between host defence, immune modulation, and tissue injury.

Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage

The Staphylococcus aureus Extracellular Adherence Protein Eap Is a DNA Binding Protein Capable of Blocking Neutrophil Extracellular Trap Formation

The extracellular adherence protein (Eap) of Staphylococcus aureus is a secreted protein known to exert a number of adhesive and immunomodulatory properties. Here we describe the intrinsic DNA binding activity of this multifunctional secretory factor. By using atomic force microscopy, we provide evidence that Eap can bind and aggregate DNA. While the origin of the DNA substrate (e.g., eukaryotic, bacterial, phage, and artificial DNA) seems to not be of major importance, the DNA structure (e.g., linear or circular) plays a critical role with respect to the ability of Eap to bind and condense DNA. Further functional assays corroborated the nature of Eap as a DNA binding protein, since Eap suppressed the formation of “neutrophil extracellular traps” (NETs), composed of DNA-histone scaffolds, which are thought to function as a neutrophil-mediated extracellular trapping mechanism. The DNA binding and aggregation activity of Eap may thereby protect S. aureus against a specific anti-microbial defense reaction from the host

Histones induce epithelial and endothelial cell death.

<p>(A) A549 cells were treated for 16 h with different concentrations of histone type II-A, and the cell morphology was evaluated. (B) A549 cell numbers were counted after treatment with various concentrations of histones for 16 h. (C) HUVEC or A549 cells were treated with 200 µg/ml histones for 16 h or left untreated (control). (D) HUVEC were treated for 16 h with different concentrations of histones, and the extent of cytotoxity was measured. B and D are representative data of three independent experiments, and in A and C pictures are representative pictures from three independent experiments at 20× magnification.</p

NET formation in LPS-induced lung injury mouse model.

<p>(A) Immunofluorescence staining of lung sections from mice after 24 h intratracheal LPS administration was performed, as compared to the control section, for DNA/histone (red), neutrophil elastase (green) and DAPI (blue). The higher magnification views of the insets (1, 2 and 3), which were randomly chosen, showed co-localization of neutrophil elastase (green) and DNA/histone (red) in NET structures. (B) Immunofluorescence staining of sections from PBS- or LPS-treated mice was performed for DNA/histone (red), CD46 (green) as a cell membrane marker and DAPI (blue). The randomly chosen insets (4, 5 and 6) showed NET formation in LPS-treated lungs in higher magnification views as appeared by extracellular chromatin, disintegration of the cell membranes as well as weak signal for DAPI (indication of chromatin decondensation). DAPI alone and DNA/histone alone were also shown for the insets 4, 5 and 6. In A and B, yellow arrows indicate some of the tissue destruction areas adjacent to NET. Shown are representative pictures of >10 fields of tissue staining. (C) Immunofluorescence staining of lung sections from mice after 24 h intratracheal LPS administration was performed for myeloperoxidase (MPO, red), citrullinated histone H3 (Cit His3, green) and DAPI (blue). The higher magnification views (right column) of the selected areas showed co-localization of myeloperoxidase with citrullinated histone H3 which indicate NET formation.</p

Myeloperoxidase inhibition moderately decreases NET-induced cytotoxicity of epithelial cells.

<p>Nondigested or DNase-digested NET were pre-incubated without or with myeloperoxidase inhibitor (MPOI), followed by incubation of NET with epithelial cells, A549 or AT-II cells, for 16 h and quantification of cytotoxicty. MPOI alone (37 ng/ml) was not toxic for the epithelial cells. Shown are representative data of three (for AT-II cells, n = 2) independent experiments (mean SD), *<i>p</i><0.05 and ns = non-significant.</p

NET cause lung epithelial cell death in a concentration-dependent manner.

<p>(A) The morphology of A549 cells was evaluated after 4 or 16 h treatment with medium (control), NET or staurosporine. Shown are representative pictures of >8 independent experiments at 20× magnification. (B) Cell growth from (A) was quantified by measuring the difference between occupied cell area after 16 h and 4 h. (C) Multicaspase activity of A549 cells was measured after 16 h treatment with two concentrations of NET (3.4 and 10.1 µg/ml DNA-NET) or staurosporine. Shown are representative data of three independent experiments (mean SD), *<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001. (D) Immunofluorescence staining of A549 cells after 16 h treatment with two concentrations of NET (3.4 and 10.1 µg/ml DNA-NET) or staurosporine was performed for ethidium homodimer III (ethidium-HD, red), annexin V (green), and Hoechst 33342 (black and white). Shown are representative pictures of three independent experiments. (E) Percentage of ethidium-HD and annexin-V positive cells from (D) was evaluated by morphometry analysis.</p