Die rechtliche Natur der Zivilehe

[Abstract fehlt

ADAM8 signaling drives neutrophil migration and ARDS severity

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8–/– mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain

Novel protein biomarkers for pneumonia and acute exacerbations in COPD: a pilot study

IntroductionCommunity-acquired pneumonia (CAP) and acute exacerbations of chronic obstructive pulmonary disease (AECOPD) result in high morbidity, mortality, and socio-economic burden. The usage of easily accessible biomarkers informing on disease entity, severity, prognosis, and pathophysiological endotypes is limited in clinical practice. Here, we have analyzed selected plasma markers for their value in differential diagnosis and severity grading in a clinical cohort.MethodsA pilot cohort of hospitalized patients suffering from CAP (n = 27), AECOPD (n = 10), and healthy subjects (n = 22) were characterized clinically. Clinical scores (PSI, CURB, CRB65, GOLD I-IV, and GOLD ABCD) were obtained, and interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-2-receptor (IL-2R), lipopolysaccharide-binding protein (LBP), resistin, thrombospondin-1 (TSP-1), lactotransferrin (LTF), neutrophil gelatinase-associated lipocalin (NGAL), neutrophil-elastase-2 (ELA2), hepatocyte growth factor (HGF), soluble Fas (sFas), as well as TNF-related apoptosis-inducing ligand (TRAIL) were measured in plasma.ResultsIn CAP patients and healthy volunteers, we found significantly different levels of ELA2, HGF, IL-2R, IL-6, IL-8, LBP, resistin, LTF, and TRAIL. The panel of LBP, sFas, and TRAIL could discriminate between uncomplicated and severe CAP. AECOPD patients showed significantly different levels of LTF and TRAIL compared to healthy subjects. Ensemble feature selection revealed that CAP and AECOPD can be discriminated by IL-6, resistin, together with IL-2R. These factors even allow the differentiation between COPD patients suffering from an exacerbation or pneumonia.DiscussionTaken together, we identified immune mediators in patient plasma that provide information on differential diagnosis and disease severity and can therefore serve as biomarkers. Further studies are required for validation in bigger cohorts

IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis

IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond

MicroRNAs in alternative and classic activation of macrophages

Die Polarisierung von Makrophagen resultiert in einer Vielzahl von Subtypen, z.B. M1 oder M2. In dieser Studie lege ich die Makrophagen-Polarisierung im allergischen Asthma mit Fokus auf microRNA (miRNA) dar. Nach Etablierung von Subtyp–charakteristischen mRNA und miRNA Expressionsprofilen in vitro polarisierter, humaner blutstämmiger Makrophagen wurden diese Profile genutzt, um den Polarisierungsstatus isolierter Lungenmakrophagen in einem Mausmodell des Asthmas zu ermitteln. Es wurden humane blutstämmige Makrophagen in vitro durch IFNg/LPS (M1) bzw. IL4/IL13 (M2) polarisiert. M1-assoziierte Gene waren TNFa, IL6 und IL1b, während in M2 Makrophagen eine Induktion von CD209 und PPARg beobachtet wurde. Herauf-regulierte miRNAs waren z.B. hsa-miR-187-3p, hsa-miR-155-5p und hsa-miR-146a-5p (M1) bzw. hsa-miR-193b-3p und hsa-miR-511-5p (M2). Eine in silico Vorhersage von mRNA/miRNA Interaktionspartnern wurde in einem Luciferase Reportermodell überprüft, das u.a. hsa-miR-187-3p als Regulator von SH2B2 und hsa-miR-187-3p sowie hsa-miR-155-5p als kooperative Regulatoren von LAMP2 bestätigte. Unter physiologischen Bedingungen regulierte hsa-miR-187-3p die SH2B2 mRNA herunter, aber es lag kein Einfluß von hsa-miR-187-3p oder hsa-miR-155-5p auf LAMP2 mRNA oder Protein vor. Weiterhin wurden die miRNA Profile von murinen Makrophagen erhoben, die aus der bronchoalveolären Lavage und aus Lungengewebe gewonnen wurden. Diese Profile wurden in einer vergleichenden Analyse von gesunden Mäusen und Mäusen mit akuter Ovalbumin-induzierter eosinophiler Atemwegsentzündung eingesetzt. In Reaktion auf Ovalbumin regulierte miRNAs waren z.B. mmu-miR-21a-5p und mmu-miR-155-5p (heraufreguliert), sowie mmu-miR-126-3p und mmu-miR-146a-5p (herunterreguliert). Sowohl M1 als auch M2 assoziierte Muster zeigten sich vor allem in der reziproken Regulation von mmu-miR-155-5p (heraufreguliert) und mmu-miR-146a-5p (herunterreguliert).Macrophage polarization gives rise to a plethora of macrophage subtypes, e.g. M1 or M2. In this thesis, I aim to point out some features of macrophage polarization in allergic asthma with a focus on microRNA (miRNA). The chosen approach started with the establishment of subtype-characteristic mRNA and miRNA profiles of in vitro polarized human macrophages. In a second step, the miRNA patterns were used to interpret the polarization status of isolated lung macrophages from a murine model of asthma. In vitro polarization of human blood-derived macrophages was performed by IFNgLPS (M1) and IL4/IL13 (M2), and global mRNA and miRNA profiling ensued. M1-induced genes included TNFa, IL6 and IL1b, whereas in M2 macrophages, CD209 and PPARg were induced. M1-associated miRNAs were hsa-miR-187-3p, hsa-miR-155-5p and hsa-miR-146a-5p, while hsa-miR-193b-3p and hsa-miR-511-5p were induced in M2 macrophages. In silico prediction of mRNA/miRNA interaction partners was experimentally validated in a luciferase-based reporter assay that confirmed hsa-miR-187-3p as a regulator of SH2B2 and the pair of hsa-miR-187-3p and hsa-miR-155-5p as cooperative regulators of LAMP2. Under physiologic conditions, hsa-miR-187-3p was able to down-regulate SH2B2 transcript, but there was no impact of either hsa-miR-187-3p or hsa-miR-155-5p on LAMP2 mRNA or protein. Furthermore, the miRNA profiles of murine macrophages from the bronchoalveolar lavage fluid and from lung tissue were established and compared between healthy mice and mice with acute Ovalbumin-induced eosinophilic airway inflammation. Individual miRNAs responding to Ovalbumin were e.g. mmu-miR-21a-5p and mmu-miR-155-5p (up-regulated), and mmu-miR-126-3p and mmu-miR-146a-5p (down-regulated). Characteristics of both M1- and M2-associated miRNA patterns were most evident in the concomitant reciprocal expression of mmu-miR-155-5p (up-regulated) and mmu-miR-146a-5p (down-regulated)

microRNA-125a-3p is regulated by MyD88 in Legionella pneumophila infection and targets NTAN1.

Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. It is highly adapted to intracellular replication and manipulates host cell functions like vesicle trafficking and mRNA translation to its own advantage. However, it is still unknown to what extent microRNAs (miRNAs) are involved in the Legionella-host cell interaction.WT and MyD88-/- murine bone marrow-derived macrophages (BMM) were infected with L. pneumophila, the transcriptome was analyzed by high throughput qPCR array (microRNAs) and conventional qPCR (mRNAs), and mRNA-miRNA interaction was validated by luciferase assays with 3´-UTR mutations and western blot.L. pneumophila infection caused a pro-inflammatory reaction and significant miRNA changes in murine macrophages. In MyD88-/- cells, induction of inflammatory markers, such as Ccxl1/Kc, Il6 and miR-146a-5p was reduced. Induction of miR-125a-3p was completely abrogated in MyD88-/- cells. Target prediction analyses revealed N-terminal asparagine amidase 1 (NTAN1), a factor from the n-end rule pathway, to be a putative target of miR-125a-3p. This interaction could be confirmed by luciferase assay and western blot.Taken together, we characterized the miRNA regulation in L. pneumophila infection with regard to MyD88 signaling and identified NTAN1 as a target of miR-125a-3p. This finding unravels a yet unknown feature of Legionella-host cell interaction, potentially relevant for new treatment options

Pulmonary inflammatory response and immunomodulation to multiple trauma and hemorrhagic shock in pigs

Background Patients suffering from severe trauma experience substantial immunological stress. Lung injury is a known risk factor for the development of posttraumatic complications, but information on the long-term course of the pulmonary inflammatory response and treatment with mild hypothermia are scarce. Aim To investigate the pulmonary inflammatory response to multiple trauma and hemorrhagic shock in a porcine model of combined trauma and to assess the immunomodulatory properties of mild hypothermia. Methods Following induction of trauma (blunt chest trauma, liver laceration, tibia fracture), two degrees of hemorrhagic shock (45 and 50%) over 90 (n = 30) and 120 min. (n = 20) were induced. Animals were randomized to hypothermia (33°C) or normothermia (38°C). We evaluated bronchoalveolar lavage (BAL) fluid and tissue levels of cytokines and investigated changes in microRNA- and gene-expression as well as tissue apoptosis. Results We observed a significant induction of Interleukin (IL) 1β, IL-6, IL-8, and Cyclooxygenase-2 mRNA in lung tissue. Likewise, an increased IL-6 protein concentration could be detected in BAL-fluid, with a slight decrease of IL-6 protein in animals treated with hypothermia. Lower IL-10 protein levels in normothermia and higher IL-10 protein concentrations in hypothermia accompanied this trend. Tissue apoptosis increased after trauma. However, intervention with hypothermia did not result in a meaningful reduction of pro-inflammatory biomarkers or tissue apoptosis. Conclusion We observed signs of a time-dependent pulmonary inflammation and apoptosis at the site of severe trauma, and to a lower extent in the trauma-distant lung. Intervention with mild hypothermia had no considerable effect during 48 hours following trauma

Streptococcus pneumoniae disrupts the structure of the golgi apparatus and subsequent epithelial cytokine response in an H2O2-dependent manner

Abstract Background Lung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host–pathogen interactions during pneumococcal infections. Methods Following in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid. Results In vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment. Conclusion Streptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms. Video Abstrac

<i>Legionella pneumophila</i>-Derived Outer Membrane Vesicles Promote Bacterial Replication in Macrophages

<div><p>The formation and release of outer membrane vesicles (OMVs) is a phenomenon of Gram-negative bacteria. This includes <i>Legionella pneumophila</i> (<i>L</i>. <i>pneumophila</i>), a causative agent of severe pneumonia. Upon its transmission into the lung, <i>L</i>. <i>pneumophila</i> primarily infects and replicates within macrophages. Here, we analyzed the influence of <i>L</i>. <i>pneumophila</i> OMVs on macrophages. To this end, differentiated THP-1 cells were incubated with increasing doses of <i>Legionella</i> OMVs, leading to a TLR2-dependent classical activation of macrophages with the release of pro-inflammatory cytokines. Inhibition of TLR2 and NF-κB signaling reduced the induction of pro-inflammatory cytokines. Furthermore, treatment of THP-1 cells with OMVs prior to infection reduced replication of <i>L</i>. <i>pneumophila</i> in THP-1 cells. Blocking of TLR2 activation or heat denaturation of OMVs restored bacterial replication in the first 24 h of infection. With prolonged infection-time, OMV pre-treated macrophages became more permissive for bacterial replication than untreated cells and showed increased numbers of <i>Legionella</i>-containing vacuoles and reduced pro-inflammatory cytokine induction. Additionally, miRNA-146a was found to be transcriptionally induced by OMVs and to facilitate bacterial replication. Accordingly, IRAK-1, one of miRNA-146a’s targets, showed prolonged activation-dependent degradation, which rendered THP-1 cells more permissive for <i>Legionella</i> replication. In conclusion, <i>L</i>. <i>pneumophila</i> OMVs are initially potent pro-inflammatory stimulators of macrophages, acting via TLR2, IRAK-1, and NF-κB, while at later time points, OMVs facilitate <i>L</i>. <i>pneumophila</i> replication by miR-146a-dependent IRAK-1 suppression. OMVs might thereby promote spreading of <i>L</i>. <i>pneumophila</i> in the host.</p></div