Holger C. Müller

Systemic use of the endolysin Cpl-1 rescues mice with fatal pneumococcal pneumonia.

Objectives:Community-acquired pneumonia is a very common infectious disease associated with significant morbidity and mortality. Streptococcus pneumoniae is the predominant pathogen in this disease, and pneumococcal resistance to multiple antibiotics is increasing. The recently purified bacteriophage endolysin Cpl-1 rapidly and specifically kills pneumococci on contact. The aim of this study was to determine the therapeutic potential of Cpl-1 in a mouse model of severe pneumococcal pneumonia. Design:Controlled, in vivo laboratory study. Subjects:Female C57/Bl6 mice, 8–12 weeks old. Interventions:Mice were transnasally infected with pneumococci and therapeutically treated with Cpl-1 or amoxicillin by intraperitoneal injections starting 24 or 48 hours after infection. Measurements and Main Results:Judged from clinical appearance, decreased body weight, reduced dynamic lung compliance and Pao2/Fio2 ratio, and morphologic changes in the lungs, mice suffered from severe pneumonia at the onset of therapy. When treatment was commenced 24 hours after infection, 100% Cpl-1–treated and 86% amoxicillin-treated mice survived otherwise fatal pneumonia and showed rapid recovery. When treatment was started 48 hours after infection, mice had developed bacteremia, and three of seven (42%) Cpl-1–treated and five of seven (71%) amoxicillin-treated animals survived. Cpl-1 dramatically reduced pulmonary bacterial counts, and prevented bacteremia, systemic hypotension, and lactate increase when treatment commenced at 24 hours. In vivo, treatment with Cpl-1 or amoxicillin effectively reduced counts of penicillin-susceptible pneumococci. The inflammatory response in Cpl-1–and amoxicillin-treated mice was lower than in untreated mice, as determined by multiplex cytokine assay of lung and blood samples. In human epithelial cell cultures, lysed bacteria evoked less proinflammatory cytokine release and cell death, as compared with viable bacteria. Conclusions:Cpl-1 may provide a new therapeutic option in the treatment of pneumococcal pneumonia.

Streptococcus pneumoniae Stimulates a STING- and IFN Regulatory Factor 3-Dependent Type I IFN Production in Macrophages, which Regulates RANTES Production in Macrophages, Cocultured Alveolar Epithelial Cells, and Mouse Lungs

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. In this study, we examine an innate immune recognition pathway that senses pneumococcal infection, triggers type I IFN production, and regulates RANTES production. We found that human and murine alveolar macrophages as well as murine bone marrow macrophages, but not alveolar epithelial cells, produced type I IFNs upon infection with S. pneumoniae. This response was dependent on the pore-forming toxin pneumolysin and appeared to be mediated by a cytosolic DNA-sensing pathway involving the adapter molecule STING and the transcription factor IFN regulatory factor 3. Indeed, DNA was present in the cytosol during pneumococcal infection as indicated by the activation of the AIM2 inflammasome, which is known to sense microbial DNA. Type I IFNs produced by S. pneumoniae-infected macrophages positively regulated gene expression and RANTES production in macrophages and cocultured alveolar epithelial cells in vitro. Moreover, type I IFNs controlled RANTES production during pneumococcal pneumonia in vivo. In conclusion, we identified an immune sensing pathway detecting S. pneumoniae that triggers a type I IFN response and positively regulates RANTES production.

Dissection of a type I interferon pathway in controlling bacterial intracellular infection in mice

Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN‐dependent cell‐autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNβ in a STING‐ and IRF3‐ dependent manner. Paracrine type I IFNs stimulated upregulation of IFN‐stimulated genes and a cell‐autonomous defence pathway acting on replicating and non‐replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN‐stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria.

Simvastatin attenuates ventilator-induced lung injury in mice

IntroductionMechanical ventilation (MV) is a life saving intervention in acute respiratory failure without alternative. However, particularly in pre-injured lungs, even protective ventilation strategies may evoke ventilator-induced lung injury (VILI), which is characterized by pulmonary inflammation and vascular leakage. Adjuvant pharmacologic strategies in addition to lung protective ventilation to attenuate VILI are lacking. Simvastatin exhibited anti-inflammatory and endothelial barrier stabilizing properties in vitro and in vivo.MethodsMice were ventilated (12 ml/kg; six hours) and subjected to simvastatin (20 mg/kg) or sham treatment. Pulmonary microvascular leakage, oxygenation, pulmonary and systemic neutrophil and monocyte counts and cytokine release in lung and blood plasma were assessed. Further, lung tissue was analyzed by electron microscopy.ResultsMechanical ventilation induced VILI, displayed by increased pulmonary microvascular leakage and endothelial injury, pulmonary recruitment of neutrophils and Gr-1high monocytes, and by liberation of inflammatory cytokines in the lungs. Further, VILI associated systemic inflammation characterized by blood leukocytosis and elevated plasma cytokines was observed. Simvastatin treatment limited pulmonary endothelial injury, attenuated pulmonary hyperpermeability, prevented the recruitment of leukocytes to the lung, reduced pulmonary cytokine levels and improved oxygenation in mechanically ventilated mice.ConclusionsHigh-dose simvastatin attenuated VILI in mice by reducing MV-induced pulmonary inflammation and hyperpermeability.

Immunostimulation with Macrophage-Activating Lipopeptide-2 Increased Survival in Murine Pneumonia

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.

Adrenomedullin attenuates ventilator-induced lung injury in mice

Background Mechanical ventilation (MV) is a life-saving intervention in acute respiratory failure without any alternative. However, even protective ventilation strategies applying minimal mechanical stress may evoke ventilator-induced lung injury (VILI). Adjuvant pharmacological strategies in addition to lung-protective ventilation to attenuate VILI are lacking. Adrenomedullin exhibited endothelial barrier-stabilising properties in vitro and in vivo. Methods In untreated mice (female C57/Bl6 mice, 11–15 weeks old) and animals treated with adrenomedullin, lung permeability, local and systemic inflammation and markers of distal organ function were assessed following 2 or 6 h of mechanical ventilation with 100% oxygen and protective or moderately injurious ventilator settings, respectively. Results Adrenomedullin dramatically reduced lung permeability in VILI in mice, leading to improved oxygenation. Adrenomedullin treatment reduced myosin light chain phosphorylation, attenuated the accumulation of leucocytes in the lung and prevented the increase in lactate and creatinine levels in mice ventilated with high tidal volumes. Moreover, adrenomedullin protected against VILI even when treatment was initiated 2 h after the beginning of mechanical ventilation in a 6 h VILI mouse model. Conclusion Adjuvant treatment with adrenomedullin may be a promising new pharmacological approach to attenuate VILI.

The Sphingosine-1 Phosphate receptor agonist FTY720 dose dependently affected endothelial integrity in vitro and aggravated ventilator-induced lung injury in mice.

Lung barrier protection by Sphingosine-1 Phosphate (S1P) has been demonstrated experimentally, but recent evidence suggests barrier disruptive properties of high systemic S1P concentrations. The S1P analog FTY720 recently gained an FDA approval for treatment of multiple sclerosis. In case of FTY720 treated patients experiencing multiple organ dysfunction syndrome the drug may accumulate due to liver failure, and the patients may receive ventilator therapy. Whereas low doses of FTY720 enhanced endothelial barrier function, data on effects of increased FTY720 concentrations are lacking. We measured transcellular electrical resistance (TER) of human umbilical vein endothelial cell (HUVEC) monolayers, performed morphologic analysis and measured apoptosis by TUNEL staining and procaspase-3 degradation in HUVECs stimulated with FTY720 (0.01-100 μM). Healthy C57BL/6 mice and mice ventilated with 17 ml/kg tidal volume and 100% oxygen for 2 h were treated with 0.1 or 2 mg/kg FTY720 or solvent, and lung permeability, oxygenation and leukocyte counts in BAL and blood were quantified. Further, electron microscopic analysis of lung tissue was performed. We observed barrier protective effects of FTY720 on HUVEC cell layers at concentrations up to 1 μM while higher concentrations induced irreversible barrier breakdown accompanied by induction of apoptosis. Low FTY720 concentrations (0.1 mg/kg) reduced lung permeability in mechanically ventilated mice, but 2 mg/kg FTY720 increased pulmonary vascular permeability in ventilated mice accompanied by endothelial apoptosis, while not affecting permeability in non-ventilated mice. Moreover, hyperoxic mechanical ventilation sensitized the pulmonary vasculature to a barrier disrupting effect of FTY720, resulting in worsening of ventilator induced lung injury. In conclusion, the current data suggest FTY720 induced endothelial barrier dysfunction, which was probably caused by proapoptotic effects and enhanced by mechanical ventilation.

Ventilator-induced lung injury: Intermedin reduziert pulmonalvaskuläre Permeabilität im Mausmodell

Die maschinelle Beatmung birgt das Risiko, Lungenschaden (ventilator-induced lung injury; VILI) zu verursachen oder zu aggravieren. Insbesondere in vorgeschadigten Lungen konnen lungenprotektive Beatmungsstrategien VILI limitieren, jedoch nicht verhindern. Adjuvante pharmakologische Interventionen konnten VILI uber protektive Beatmung hinaus reduzieren. In vitro-Studien legen barrierestabilisierende Eigenschaften des endogenen Peptids Intermedin (IMD) nahe. Humane umbilikalvenose Endothelzellen (HUVECs) wurden mit IMD inkubiert und der transendotheliale elektrische Widerstand (TER) als Mas der Integritat der endothelialen Barriere gemessen. Der Einfluss von Beatmung auf die pulmonale Expression von endogenem IMD, CRLR und RAMP1–3 wurde mittels qPCR und Immunhistochemie analysiert. Ferner wurden Mause mit Tidalvolumina von 12ml/kg fur 6h beatmet und mit IMD (25µg/kg x h) oder Placebo behandelt. In HUVECs fuhrte IMD zur Stabilisierung der endothelialen Barrierefunktion. Beatmung induzierte pulmonalvaskulare Hyperpermeabilitat und eine pulmonale und systemische Inflammation. Endogenes IMD und seine Rezeptorkomplexe aus CRLR und RAMP1–3 waren im pulmonalvaskularen Endothel lokalisierbar. Durch Beatmung nahm die Expression von RAMP3 ab, wahrend IMD, CRLR und RAMP1–2 nicht messbar reguliert wurden. Exogenes IMD reduzierte die VILI-assoziierte Hyperpermeabilitat, ohne die Rekrutierung von Leukozyten in die Lunge zu beeinflussen. Im Rahmen dieser Untersuchung wurde unseres Wissens nach erstmals die Reduktion pulmonalvaskularer Permeabilitat durch IMD in vivo beobachtet.