K Hellwig

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.

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.

Intermedin Stabilized Endothelial Barrier Function and Attenuated Ventilator-induced Lung Injury in Mice

Background Even protective ventilation may aggravate or induce lung failure, particularly in preinjured lungs. Thus, new adjuvant pharmacologic strategies are needed to minimize ventilator-induced lung injury (VILI). Intermedin/Adrenomedullin-2 (IMD) stabilized pulmonary endothelial barrier function in vitro. We hypothesized that IMD may attenuate VILI-associated lung permeability in vivo. Methodology/Principal Findings Human pulmonary microvascular endothelial cell (HPMVEC) monolayers were incubated with IMD, and transcellular electrical resistance was measured to quantify endothelial barrier function. Expression and localization of endogenous pulmonary IMD, and its receptor complexes composed of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) 1–3 were analyzed by qRT-PCR and immunofluorescence in non ventilated mouse lungs and in lungs ventilated for 6 h. In untreated and IMD treated mice, lung permeability, pulmonary leukocyte recruitment and cytokine levels were assessed after mechanical ventilation. Further, the impact of IMD on pulmonary vasoconstriction was investigated in precision cut lung slices (PCLS) and in isolated perfused and ventilated mouse lungs. IMD stabilized endothelial barrier function in HPMVECs. Mechanical ventilation reduced the expression of RAMP3, but not of IMD, CRLR, and RAMP1 and 2. Mechanical ventilation induced lung hyperpermeability, which was ameliorated by IMD treatment. Oxygenation was not improved by IMD, which may be attributed to impaired hypoxic vasoconstriction due to IMD treatment. IMD had minor impact on pulmonary leukocyte recruitment and did not reduce cytokine levels in VILI. Conclusions/Significance IMD may possibly provide a new approach to attenuate VILI.

Moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia

OBJECTIVES Anti-inflammatory functions of antibiotics may counteract deleterious hyperinflammation in pneumonia. Moxifloxacin reportedly exhibits immunomodulatory properties, but experimental evidence in pneumonia is lacking. Therefore, we investigated moxifloxacin in comparison with ampicillin regarding pneumonia-associated pulmonary and systemic inflammation and lung injury. METHODS Ex vivo infected human lung tissue and mice with pneumococcal pneumonia were examined regarding local inflammatory response and bacterial growth. In vivo, clinical course of the disease, leucocyte dynamics, pulmonary vascular permeability, lung pathology and systemic inflammation were investigated. In addition, transcellular electrical resistance of thrombin-stimulated endothelial cell monolayers was quantified. RESULTS Moxifloxacin reduced cytokine production in TNF-α-stimulated, but not in pneumococci-infected, human lung tissue. In vivo, moxifloxacin treatment resulted in reduced bacterial load as compared with ampicillin, whereas inflammatory parameters and lung pathology were not different. Moxifloxacin-treated mice developed less pulmonary vascular permeability during pneumonia, but neither combination therapy with moxifloxacin and ampicillin in vivo nor examination of endothelial monolayer integrity in vitro supported direct barrier-stabilizing effects of moxifloxacin. CONCLUSIONS The current experimental data do not support the hypothesis that moxifloxacin exhibits potent anti-inflammatory properties in pneumococcal pneumonia.

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.