NLRP3 protects alveolar barrier integrity by an inflammasome-independent increase of epithelial cell adherence

Bacterial pneumonia is a major cause of acute lung injury and acute respiratory distress syndrome, characterized by alveolar barrier disruption. NLRP3 is best known for its ability to form inflammasomes and to regulate IL- 1β and IL-18 production in myeloid cells. Here we show that NLRP3 protects the integrity of the alveolar barrier in a mouse model of Streptococcus pneumoniae-induced pneumonia, and ex vivo upon treatment of isolated perfused and ventilated lungs with the purified bacterial toxin, pneumolysin. We reveal that the preserving effect of NLRP3 on the lung barrier is independent of inflammasomes, IL-1β and IL-18. NLRP3 improves the integrity of alveolar epithelial cell monolayers by enhancing cellular adherence. Collectively, our study uncovers a novel function of NLRP3 by demonstrating that it protects epithelial barrier function independently of inflammasomes

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early proinflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia

Acute Moraxella catarrhalis Airway Infection of Chronically Smoke-Exposed Mice Increases Mechanisms of Emphysema Development : A Pilot Study

In chronic obstructive pulmonary disease (COPD), acute exacerbations and emphysema development are characteristics for disease pathology. COPD is complicated by infectious exacerbations with acute worsening of respiratory symptoms with Moraxella catarrhalis as one of the most frequent pathogens. Although cigarette smoke (CS) is the primary risk factor, additional molecular mechanisms for emphysema development induced by bacterial infections are incompletely understood. We investigated the impact of M. catarrhalis on emphysema development in CS exposed mice and asked whether an additional infection would induce a solubilization of pro-apoptotic and proinflammatory endothelial monocyte-activating-protein-2 (EMAPII) to exert its activities in the pulmonary microvasculature and other parts of the lungs not exposed directly to CS. Mice were exposed to smoke (6 or 9 months) and/or infected with M. catarrhalis. Lungs, bronchoalveolar lavage fluid (BALF), and plasma were analyzed. CS exposure reduced ciliated area, caused rarefaction of the lungs, and induced apoptosis. EMAPII was increased independent of prior smoke exposure in BALF of infected mice. Importantly, acute M. catarrhalis infection increased release of matrixmetalloproteases-9 and -12, which are involved in emphysema development and comprise a mechanism of EMAPII release. Our data suggest that acute M. catarrhalis infection represents an independent risk factor for emphysema development in smoke-exposed mice

The role of pneumolysin for the development of acute lung injury in pneumococcal pneumonia

Einleitung und Aufgabenstellung In Deutschland erfolgen ca. 4% aller Klinikeinweisungen aufgrund einer ambulant erworbenen Pneumonie. Die Letalität dieser Erkrankung ist bei schweren Verläufen mit 5%-29% hoch (188). Weltweit ist die Pneumonie beim Menschen die häufigste Infektionskrankheit. Besonders Menschen mit geschwächtem Immunsystem und mit einer chronisch-obstruktiven Lungenerkrankung sind gefährdet, an einer Pneumonie zu erkranken. Ein komplizierter Pneumonieverlauf kann zum akuten Lungenversagen führen, dessen Charakteristika die Bildung eines Lungenödems durch Schädigung der alveolokapillären Barriere und die Entstehung einer pulmonalarteriellen Hypertonie sind. Die hierfür relevanten Pathomechanismen sind derzeit noch nicht hinreichend bekannt Streptococcus pneumoniae ist weltweit mit 16-24% und in Deutschland mit fast 50% der häufigste Erreger der ambulant erworbenen Pneumonie (1;191). Das zytolytische und proinflammatorische Toxin Pneumolysin ist ein wichtiger Pathogenitätsfaktor der Pneumokokken. Die Rolle des Pneumolysin bei Pneumokokkenpneumonie und Entstehung des akuten Lungenversagens ist noch unzureichend bekannt. Ziel der vorliegenden Dissertationsarbeit war es, die Rolle des Pneumolysin für die Entstehung des akuten Lungenversagens bei Pneumokokkenpneumonie zu untersuchen und die an der Pneumolysin-induzierten Lungenschädigung beteiligten Effektorsysteme zu identifizieren. Im Fokus der Studie standen insbesondere Untersuchungen zum Einfluss von Pneumolysin auf die alveolokapilläre Barriere und auf die pulmonalvaskuläre Hämodynamik. Material und Methoden Bei Mäusen wurde eine Pneumokokkenpneumonie induziert um, in vivo Störungen der alveolokapillären Barrierefunktion und die zelluläre Immunantwort im Krankheitsverlauf untersuchen zu können. Im ex vivo- Modell der isoliert ventilierten und blutfrei perfundierten Mauslunge fand zudem eine detaillierte Charakterisierung der Wirkung von rekombinantem Pneumolysin statt. Mit dieser Methode konnte das native, intakte Organ unabhängig von humoralen, metabolischen oder zentralnervösen Faktoren des Gesamtorganismus untersucht werden. Dabei bestand die Möglichkeit, nach Pneumolysin-Provokation hämodynamische Veränderungen der pulmonalen Strombahn, Änderungen der endothelialen Permeabilität, Anreicherung von Thromboxan B2 im venösen Perfusionsmedium und Mediatoren im Lungengewebe zu erfassen. Ergänzend wurden in vitro-Untersuchungen an humanen Endothel- und Epithelzellmonolayern zur Charakterisierung Toxin-induzierter Veränderungen auf zellulärer Ebene eingesetzt. Ergebnisse In der murinen Pneumokokkenpneumonie wurde eine pulmonale Hyperpermeabilität bereits 12 Stunden post inoculationem beobachtet und damit deutlich früher registriert, als der Einstrom neutrophiler Granulozyten in die Lunge. Die frühe Schädigung der alveolokapillären Barriere erfolgte somit offenbar unabhängig von einem pulmonalen Neutrophileneinstrom. Am Modell der isoliert perfundierten Mauslunge wurde der Beitrag von Pneumolysin an der Lungenschädigung detailliert analysiert. Aerosoliertes Pneumolysin verursachte eine mikrovaskuläre Permeabilitätserhöhung und intravaskuläres Pneumolysin sowohl eine mikrovaskuläre Permeabilitätserhöhung, als auch eine pulmonale Hypertonie. Des Weiteren erhöhte Pneumolysin dosisabhängig die endo- und epitheliale Permeabilität in vitro. Ferner konnte demonstriert werden, dass Plättchen-aktivierender Faktor (PAF) bei der Entstehung der Pneumolysin-induzierten pulmonalen Schädigungen eine Schlüsselrolle einnimmt. Exogenes PAF erhöhte ebenso wie Pneumolysin den pulmonalarteriellen Druck in isolierten Mauslungen. Pneumolysin erhöhte den PAF-Gehalt im Lungengewebe und den Thromboxan-Gehalt im pulmonalvenösen Perfusionsmedium. In Lungen PAF-Rezeptor-defizienter Mäuse führte die Perfusion mit Pneumolysin zu deutlich verringerter pulmonalvaskulärer Druckreaktion und Thromboxan-Freisetzung verglichen mit Wildtyp-Lungen. Durch den Einsatz spezifischer Inhibitoren wurde gezeigt, dass am weiteren Signalweg hauptsächlich der Thromboxan-Rezeptor und intrazellulär die phosphatidylcholin-spezifische Phospholipase C, sowie der Rho-Kinase Weg beteiligt sind. Weitere Experimente mit Lungen PAF-Rezeptor-defizienter Mäuse demonstrierten darüber hinaus auch eine wesentliche Beteiligung des PAF an der Pneumolysin-induzierten pulmonalen Hyperpermeabilität. Schlussfolgerung Zusammenfassend sprechen die Ergebnisse für eine wichtige Bedeutung des Pneumolysin für die Enstehung des akuten Lungenversagens bei Pneumokokkenpneumonie. Plättchen-aktivierender Faktor ist hierfür ein zentraler Mediator. Diese Erkenntnisse könnten zur Entwicklung neuer pharmakologischer Interventionsstrategien zur therapeutischen Beeinflussung des akuten Lungenversagens bei schwerer Pneumonie führen.Introduction and aim In Germany, community-acquired pneumonia (CAP) leads to approximately 4% of admissions to hospitals. In severe cases, lethality is still high (5%- 29%). Worldwide, pneumonia is the most common infectious disease. Especially people with immunodeficiency or with chronic obstructive pulmonary disease are at risk to acquire pneumonia. A major complication of severe pneumonia is acute respiratory failure, which is characterised by impairment of pulmonary microvascular barrier function (resulting in pulmonary edema) and pulmonary hypertension. The relevant pathways and mechanisms are not yet adequately described. Streptococcus pneumoniae is the most prevalent causal pathogen identified in community-acquired pneumonia worldwide. Pneumolysin (PLY), a cytolytic and proinflammatory toxin, is an important virulence factor of S. pneumoniae. The role of PLY in the development of acute lung injury in pneumococcal pneumonia has still to be defined. The primary objective of the current study was to investigate the role of pneumolysin in the development of acute lung injury in pneumococcal pneumonia and to analyse the pathways contributing to PLY-induced pulmonary impairment. The main focus of the study lay on the characterization of PLY-evoked alterations of alveolo- capillary barrier function and pulmonary hemodynamics. Material and methods Pneumococcal pneumonia was induced in mice in order to investigate the impairment of the alveolo-capillary barrier function and the cellular immune response in the course of the disease in vivo. To characterise the impact of recombinant PLY in detail, an ex vivo model of isolated ventilated and blood- free perfused mouse lungs was employed. The model allowed monitoring of lung parameters in intact organs, unaltered by systemic humoral, metabolic or central nervous influences. This experimental setup allowed for collection of data on changes in lung hemodynamics and fluid balance, on accumulation of thromboxane in the venous buffer effluate and on the concentration of mediators in the lung established after PLY-challenge. Furthermore, in vitro endothelial and epithelial cell monolayers were used to characterise morphologic changes induced by PLY. Results In murine pneumococcal pneumonia, lung hyperpermeability was already observed 12 hours after infection, which was considerably earlier than the migration of neutrophils into the lung. The early impairment of the alveolo-capillary barrier was therefore independent of pulmonary neutrophil influx. The model of isolated perfused mouse lung was used to analyse in detail the contribution of PLY to lung damage. Aerosolized PLY led to an increase of microvascular permeability and intravascular PLY caused an increase in microvascular permeability as well as pulmonary hypertension. Further PLY dose-dependently increased the permeability of endothelial and epithelial cells in vitro. In addition, platelet-activating factor (PAF) played a key role in PLY-induced pulmonary impairment. Both exogenous PAF and PLY increased the pulmonary arterial pressure in isolated mouse lungs. PLY increased the concentration of PAF in lung tissue and the concentration of thromboxane in venous buffer effluate. Perfusion with PLY evoked less pulmonary vascular pressure response and less release of thromboxan in isolated lungs of PAF receptor-deficient mice. Application of specific inhibitors demonstrated that in the downstream signaling pathway mainly the thromboxan receptor, the intracellular phosphatidylcholine-specific phospholipase C and the Rho/Rho-kinase were involved. Further experiments with lungs of PAF receptor-deficient mice also demonstrated a significant influence of PAF on PLY-induced pulmonary hyperpermeability independent from the pressor response. Conclusion In summary, the results demonstrate an important role for PLY in the development of acute lung injury in pneumococcal pneumonia, with PAF being a central mediator. The PAF-R and its downstream signalling pathways may thus provide perspectives for specific pharmacological interventions in severe pneumococcal pneumonia

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early proinflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia