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Additional file 2: Figure S1. Vascular inflammation reduces miR-181b expression and induces TF and VCAM1 expression in HMEC-1. HMEC-1 were cultured overnight and exposed to high glucose or stimulated with 10 ng/mL of TNFα. Treatment with 25mM glucose (A) and TNFα (B) caused a reduction of miR-181b expression, while mRNA expression of asTF (C), flTF (D), and VCAM1 (E) was induced after 4h and 6h post stimulation. Western blot analysis showed the protein induction of asTF (F), flTF (G), and VCAM1 (H) following 6 h or 24 h TNFα stimulation. n ≥ 3, p-values by ANOVA test with Dunn’s multiple comparison post hoc test.

Additional file 4: Figure S3. Original western blot showing protein abundance of NFκB, phospho-NFκB, histone 3, and GAPDH in nuclear and cytoplasmic extracts from THP-1 cell transfected with miR-181b or a control mimic under basal conditions or presence of LPS.

Additional file 5: Figure S4. No alteration of blood-borne TF activity in miR-181−/− animals. miR-181b expression in spleen tissue of wt and miR-181−/− animals (A). MV-derived FXa generation (B) and TAT complexes (C) in plasma of wt and miR-181−/− animals stimulated with LPS for 4 h. n = 4–5 animals per group, comparison by Mann Whitney or student’s t test.

Additional file 6: Figure S5. Healthy controls have higher miR-126 but not miR-181b expression than patients with type 2 diabetes. Expression of miR-181b (A) or miR-126 (B) in heathy controls and the cohort with poorly controlled type 2 diabetes. n = 20 for controls, n = 46 for poorly controlled diabetes, p-values by Mann Whitney test.

Additional file 3: Figure S2. miR-181b reduces KPNA4 expression in HMEC-1. KPNA4 mRNA (A) and protein (B) expression in HMEC-1 transfected with a control mimic or miR-181b. n ≥ 3, p-value by student’s t test.

Additional file 1: Table S1. Characteristics of healthy controls. BMI body mass index, HbA1c glycated hemoglobin. Values presented are means ± SD, median [IQR] or percentage.

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 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.

Neutrophile erfüllen eine wichtige Rolle bei der angeborenen Immunabwehr, indem sie inden Körper eindringende Mikroorganismen bekämpfen. Ihre antimikrobielle Aktivitität wirddurch Phagozytose, die Ausschüttung reaktiver Sauerstoffspezies ("reactive oxygen species",ROS) und lytische Enzyme vermittelt. Eine alternative Bekämpfung der Pathogene kann überdie Ausbildung von sogenannten "Neutrophil Extracellular Traps" (NET) erfolgen, einerspeziellen Art des Zelltods, bei dem die Neutrophilen DNA, Histone, verschiedene granuläreProteine (wie z.B. Neutrophile Elastase), aber auch zytoplasmatische Komponenten netzartigherausschleudern. Obwohl die Ausbildung von NET in verschiedenen Organismen undpathophysiologischen Bedingungen und ihre bakterizide Wirkung dokumentiert wurde, istwenig bekannt über die Wirkung von NET auf körpereigene Zellen und im speziellen bei derPathogenese des akuten Lungenschadens (ALI). In dieser Arbeit wurde die Auswirkung derNET-Bildung auf Endothel- und Epithelzellen studiert, vor allem im Bezug auf funktionelleVeränderungen von alveolären Epithelzellen, die mit für den Gasaustausch in der Lungeverantwortlich sind.In einem Mausmodell des ALI konnte die Bildung von NET in Lungengewebe und Lavageder erkrankten Tiere gezeigt werden. Direkte Interaktion von NET mit endothelialen undepithelialen Zellen führte nicht nur zu Änderungen in der Permeabilität des Zellverbandes,sondern auch zu einer erhöhten Synthese pro-apoptotischer Proteine und wirkte sich inkonzentrationsabhängiger Weise zytotoxisch auf die Zellen aus.Da der DNase-Verdau von NET keinen Einfluss auf deren zytotoxischen Effekt hatte, könnenNET-assoziierte Proteine dafür verantwortlich gemacht werden. Hier waren es vor allemHistone, die auch in isolierter Form zytotoxisch wirkten, was durch verschiedene Antikörpergegen Histone verhindert werden konnte. Da aktiviertes Protein C (APC) Histone degradiert,wurde die Histon-induzierte Zytotoxizität in vitro reduziert, nicht aber die NET-induzierteZytotoxizität. Dies legt den Schluss nahe, dass Histone in NET gegen den Abbau durch APCgeschützt sind. Die Inhibierung von Neutrophiler Elastase, einem weiteren Proteinbestandteilvon NET, hatte keine Wirkung auf deren Zytotoxizität, allerdings konnte eine stärkereElastase-Aktivität nach DNase-Behandlung von NET festgestellt werden. Die Inhibierungvon Myeloperoxidase, einer weiteren Komponente der NET verningerte allerdings die NETinduzierteZytotoxizität. Die Proteomanalyse von mit NET inkubierten Epithelzellen zeigteeinen deutlichen Anstieg an Apoptose-relevanten Proteinen.Diese Arbeit zeigt, dass bestimmte Proteine in den NET und hier vor allem die Histone, aberauch Myeloperoxidase stark zytotoxisch auf Endothel- und Epithelzellen wirken. Damitkönnen NET einer der Auslöser für die Zerstörung von Geweben und Organen beientzündlichen Erkrankungen, wie beispielsweise des akuten Lungenversagens (ALI), sein., Neutrophils play an important role in innate immunity by defending the host against invadedmicroorganisms. Antimicrobial activity of neutrophils is mediated by phagocytosis, release ofreactive oxygen species and lytic enzymes as well as formation of neutrophil extracellulartraps (NET). These structures are composed of DNA, histones and certain granular proteinssuch as neutrophil elastase as well as some cytoplasmic proteins. NET formation in differentorganisms, its bactericidal activity, and its presence in several pathophysiological conditionshave been documented; however, little is known about the direct effect of NET on the hostcells and its potential role in the pathogenesis of acute lung injury (ALI). This study aims todecipher the influence of NET on host cells, particularly alveolar epithelial cells as majorcells responsible for gas exchange in the lung.Herein, NET formation was documented in the lung tissue and the bronchoalveolar lavagefluid 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 NETdid not change NET-mediated cytotoxicity, indicating that NET associated proteins areresponsible for this effect. NET associated proteins were also identified by massspectrometry. Following treatment of epithelial cells with NET, different proteins with rolesin cell death processes were upregulated. Pre-incubation of NET with antibodies againsthistones significantly reduced NET-mediated cytotoxicity, suggesting that the cell-destructiveactivity 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 NETinducedcytotoxicity, indicating that histones in NET are protected against APC degradation.Inhibition of neutrophil elastase, another abundant component of NET, did not decreaseNET-mediated cytotoxicity, although elastase activity increased after DNA digestion.Inhibition of myeloperoxidase, another granular component of NET, decreased NETmediatedcytotoxicity. Moreover, in epithelial cells, NET induced a biphasic change incellular 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 andpartly myeloperoxidase, which may lead to host cell cytotoxicity and may be one of theinducers of organ destruction associated with inflammatory diseases including ALI. Thus,NET in a spatio-temporal manner can influence the cell functions.