40 research outputs found
Experimentelle Untersuchungen zu Pathophysiologie und TherapieansÀtzen bei Asthma bronchiale
Asthma bronchiale ist ein heterogenes Erkrankungsbild mit unterschiedlichen PhĂ€notypen. In dieser Arbeit wurden zentrale Pathomechanismen des allergischen Type-2-High Asthma bronchiale erforscht. Mithilfe neu etablierter prĂ€klinischer Modelle und unter Einsatz innovativer Methoden und Technologien erfolgte zudem die experimentelle Untersuchung möglicher neuer Therapiestrategien fĂŒr das Asthma bronchiale. Diese umfassen die pharmakologische Inhibition der Nicht-Rezeptor-Tyrosinkinase Milztyrosinkinase (Syk) sowie die Aktivierung des Pattern-Recognition-Rezeptors NOD1.
Syk-Inhibition in der Maus blockierte fĂŒr das allergische Asthma bronchiale typische pathologische Charakteristika inklusive pulmonaler TH2-Inflammation, AtemwegshyperreagibilitĂ€t, pulmonaler Kollagen-Deposition und glattmuskulĂ€rer Proliferation. ZusĂ€tzlich charakterisierten wir Syk als zentralen Signaltransduktor diverser klinisch relevanter bronchokonstriktorischer Stimuli unabhĂ€ngig von Inflammation. Syk-Inhibition hatte zudem in prĂ€konstringierten Atemwegen eine rasche Bronchodilatation zur Folge. Syk stellt somit sowohl fĂŒr die Akuttherapie als auch fĂŒr die Dauertherapie des Asthma bronchiale eine vielversprechende Zielstruktur dar.
NOD1-Aktivierung reduzierte im murinen Modell der allergischen Atemwegsinflammation die Anzahl der eosinophilen Granulozyten in der bronchoalveolÀren Lavage sowie die AtemwegshyperreagibilitÀt. Mechanistisch zeigte sich eine Reduktion der Allergen-spezifischen T-Zell-Proliferation infolge der NOD1-Ligation. Folglich eignet sich neben weiteren Pattern-Recognition-Rezeptoren, die aktuell als mögliche Angriffspunkte in der Behandlung des Asthma bronchiale diskutiert werden, auch der NOD1-Rezeptor zur weiteren prÀklinischen Evaluation.
Des Weiteren untersuchten wir 25-Hydroxyvitamin D3 (25(OH)D) als mögliches Adjuvans in der Allergen-spezifischen Immuntherapie (SIT). Unsere experimentellen Daten deuten auf eine verstĂ€rkte Toleranz-Entwicklung infolge der additiven 25(OH)D-Gabe in 25(OH)D-defizienten MĂ€use hin. Diese Resultate stehen im Einklang mit weiteren prĂ€klinischen Arbeiten. Hier sind weiterfĂŒhrende klinische Studien gefordert.
Grundlegende Arbeiten zur Signaltransduktion der hypoxisch pulmonalen Vasokonstriktion (HPV) erweiterten unser VerstĂ€ndnis dieses wichtigen physiologischen Mechanismus, der im Rahmen des Ventilations/Perfusions-Mismatch bei Asthma bronchiale eine Rolle spielt. Die HPV ist auch bei anderen Krankheitsbildern von Relevanz. Die von uns aufgezeigte Rolle von Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) im Matching von Ventilation und Perfusion lieferte eine neue ErklĂ€rung fĂŒr die Genese der HypoxĂ€mie bei Patienten mit zystischer Fibrose. Da anhaltende HPV bei fortwĂ€hrender Hypoxie zur Genese der pulmonalen Hypertonie beitragen kann, bilden die von uns etablierten Konzepte zudem die Basis fĂŒr mögliche innovative therapeutische Angriffspunkte fĂŒr die Behandlung von Patienten mit pulmonaler Hypertonie, wie beispielsweise die NADPH-Oxidase-Untereinheit p22phox
PKCα Deficiency in Mice Is Associated with Pulmonary Vascular Hyperresponsiveness to Thromboxane A2 and Increased Thromboxane Receptor Expression
Pulmonary vascular hyperresponsiveness is a main characteristic of pulmonary
arterial hypertension (PAH). In PAH patients, elevated levels of the
vasoconstrictors thromboxane A2 (TXA2), endothelin (ET)-1 and serotonin
further contribute to pulmonary hypertension. Protein kinase C (PKC) isozyme
alpha (PKCα) is a known modulator of smooth muscle cell contraction. However,
the effects of PKCα deficiency on pulmonary vasoconstriction have not yet been
investigated. Thus, the role of PKCα in pulmonary vascular responsiveness to
the TXA2 analog U46619, ET-1, serotonin and acute hypoxia was investigated in
isolated lungs of PKCα-/- mice and corresponding wild-type mice, with or
without prior administration of the PKC inhibitor bisindolylmaleimide I or
Gö6976. mRNA was quantified from microdissected intrapulmonary arteries. We
found that broad-spectrum PKC inhibition reduced pulmonary vascular
responsiveness to ET-1 and acute hypoxia and, by trend, to U46619.
Analogously, selective inhibition of conventional PKC isozymes or PKCα
deficiency reduced ET-1-evoked pulmonary vasoconstriction. The pulmonary
vasopressor response to serotonin was unaffected by either broad PKC
inhibition or PKCα deficiency. Surprisingly, PKCα-/- mice showed pulmonary
vascular hyperresponsiveness to U46619 and increased TXA2 receptor (TP
receptor) expression in the intrapulmonary arteries. To conclude, PKCα
regulates ET-1-induced pulmonary vasoconstriction. However, PKCα deficiency
leads to pulmonary vascular hyperresponsiveness to TXA2, possibly via
increased pulmonary arterial TP receptor expression
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 VILIassociated 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) 1â3 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 hypoxi
Dissection of a Type I Interferon Pathway in Controlling Bacterial Intracellular Infection in Mice
Defense mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defense pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNÎČ in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated up-regulation of IFN-stimulated genes and a cell-autonomous defense 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 defense against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria
Role of Ryanodine Type 2 Receptors in Elementary Ca 2+ Signaling in Arteries and Vascular Adaptive Responses
Background: Hypertension is the major risk factor for cardiovascular disease, the most common cause of death worldwide. Resistance arteries are capable of adapting their diameter independently in response to pressure and flow-associated shear stress. Ryanodine receptors (RyRs) are major Ca2+-release channels in the sarcoplasmic reticulum membrane of myocytes that contribute to the regulation of contractility. Vascular smooth muscle cells exhibit 3 different RyR isoforms (RyR1, RyR2, and RyR3), but the impact of individual RyR isoforms on adaptive vascular responses is largely unknown. Herein, we generated tamoxifen-inducible smooth muscle cell-specific RyR2-deficient mice and tested the hypothesis that vascular smooth muscle cell RyR2s play a specific role in elementary Ca2+ signaling and adaptive vascular responses to vascular pressure and/or flow.
Methods and Results: Targeted deletion of the Ryr2 gene resulted in a complete loss of sarcoplasmic reticulum-mediated Ca2+-release events and associated Ca2+-activated, large-conductance K+ channel currents in peripheral arteries, leading to increased myogenic tone and systemic blood pressure. In the absence of RyR2, the pulmonary artery pressure response to sustained hypoxia was enhanced, but flow-dependent effects, including blood flow recovery in ischemic hind limbs, were unaffected.
Conclusions: Our results establish that RyR2-mediated Ca2+-release events in VSCM s specifically regulate myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. They further suggest that vascular smooth muscle cell-expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases
CD169/SIGLEC1 is expressed on circulating monocytes in COVID-19 and expression levels are associated with disease severity
Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Type I interferons are important in the defense of viral infections. Recently, neutralizing IgG auto-antibodies against type I interferons were found in patients with severe COVID-19 infection. Here, we analyzed expression of CD169/SIGLEC1, a well described downstream molecule in interferon signaling, and found increased monocytic CD169/SIGLEC1 expression levels in patients with mild, acute COVID-19, compared to patients with severe disease. We recommend further clinical studies to evaluate the value of CD169/SIGLEC1 expression in patients with COVID-19 with or without auto-antibodies against type I interferons
IFNs Modify the Proteome of <i>Legionella</i>-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid
Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria
Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study
Purpose: Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course.
Methods: A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed.
Results: Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10-1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00-16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV patients.
Conclusions: Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19
Experimental investigations of the role of the sphingosine kinase/ sphingosine-1-phosphate system in pulmonary inflammation
Hintergrund: Sphingosin-1-Phosphat (S1P) fungiert in zahlreichen Signalwegen
als zentraler bioaktiver Mediator, sowohl intrazellulÀr als Second Messenger
als auch extrazellulĂ€r ĂŒber eine Familie G-Protein-gekoppelter Rezeptoren,
S1P1-S1P5. Die Synthese von S1P wird in der Lunge primÀr durch die
Sphingosinkinase-1 (SphK1) katalysiert. Das SphK/S1P-System spielt eine
zentrale Rolle in der Regulation des pulmonalen GefĂ€Ă- und Atemwegstonus sowie
in der Modulation proinflammatorischer Prozesse wie Migration und Aktivierung
verschiedener Leukozyten-Subpopulationen. Immunologische Prozesse scheinen in
der Pathogenese der pulmonalarteriellen Hypertonie (PAH) eine zentrale Rolle
zu spielen. Bei MĂ€usen fĂŒhrt die pulmonale TH2-Inflammation zu morphologischen
und funktionellen VerĂ€nderungen, die typisch fĂŒr die PAH sind, einschlieĂlich
perivaskulÀrer Leukozyteninfiltration, pulmonalarteriellem Remodeling und
pulmonalvaskulÀrer HyperreagibilitÀt (PVH) auf vasokonstriktorische Stimuli.
In dieser Arbeit wurde die Rolle des SphK/S1P-Systems in der akuten und
chronischen pulmonalen TH2-Inflammation mit Hilfe SphK1- (SphK1-/-) und
S1P4-defizienter MĂ€use (S1P4-/-) untersucht. Zudem erfolgte erstmals eine
detaillierte Charakterisierung des humanen SphK/S1P-Expressionsprofils der
Lunge, inklusive vergleichender Analysen zwischen Patienten mit und ohne
chronisch obstruktive Lungenerkrankung (COPD). Methoden: SphK1-/-- und
Wildtyp-MĂ€use (WT) wurden gegenĂŒber Ovalbumin (OVA) sensibilisiert und nach
akutem oder chronischem Regime atemwegsexponiert. Neben vergleichenden
Analysen funktioneller pulmonaler Parameter in isoliert perfundierten und
ventilierten Mauslungen (IPML) erfolgten Untersuchungen der bronchoalveolÀren
Lavage (BAL) sowie differenzierte histologische Analysen des
pulmonalarteriellen Remodelings. S1P4-/-- und WT-MĂ€use wurden unter akuter
pulmonaler TH2-Inflammation sowie in TH1- und TH2-assoziierten
HypersensitivitÀts-Modellen untersucht. Neben funktionellen Untersuchungen
(IPML) erfolgten Zytokinanalysen und diverse In-vitro-Studien. Unter Einsatz
humanen Lungengewebes wurden die mRNA-Expressionsprofile beider SphK-
Isoformen, der S1P-Rezeptor-Familie sowie der S1P-degradierenden Enzyme von 25
COPD- und 24 Nicht-COPD-Patienten vergleichend analysiert. Ergebnisse: Die
SphK1-Defizienz fĂŒhrte im Vergleich zu den WT-Tieren zu einer verringerten
akuten pulmonalen TH2-Inflammation und reduzierter AtemwegshyperreagibilitÀt
(AHR) bei unverÀnderter PVH. Bei chronischer Inflammation war hingegen eine
deutlich elevierte PVH in den SphK-/--MĂ€usen detektierbar sowie ein
ausgeprÀgtes pulmonalarterielles Remodeling, charakterisiert durch intimale
Neomuskularisation. Die S1P4-Defizienz hatte eine Aggravierung der akuten
pulmonalen TH2-Inflammation und AHR zur Folge, bei zugleich reduzierten
Interleukin (IL)-17-Spiegeln der BAL. Die PVH zeigte sich unverÀndert. In den
HypersensitivitÀts-Modellen zeigten sich eine erhöhte TH2-und eine verminderte
TH1-Antwort bei S1P4-Defizienz. In-vitro-Untersuchungen ergaben reduzierte
IL-17-Spiegel nach CD4+-Zell-Aktivierung durch S1P4-/- dendritische Zellen.
Die Expressionsanalysen des humanen Lungengewebes wiesen eine bei COPD-
Patienten im Vergleich zu Nicht-COPD-Patienten verringerte Expression von S1P5
nach. Schlussfolgerung: Diese Daten zeigen erstmals eine deutliche
Dissoziation TH2-induzierter Atemwegs- und GefĂ€Ăpathologie: WĂ€hrend sich die
SphK1-Defizienz protektiv auf Inflammation und AHR auswirkt, kommt es zu einer
Aggravierung der PVH bei zugleich ausgeprÀgtem pulmonalvaskulÀrem Remodeling.
Neben der vielfach postulierten Relevanz in der Entstehung von Asthma
bronchiale, könnte das SphK/S1P-System eine zentrale Rolle in der Pathogenese
der PAH spielen. S1P4 scheint wesentlich an der Modulation der allergischen
Atemwegsinflammation und AHR des SphK/S1P-Systems beteiligt zu sein. Die stark
verringerten IL-17-Spiegel bei S1P4-Defizienz deuten auf eine Inhibition der
TH17-Zelldifferenzierung hin. Die Ursache und Bedeutung der verringerten
Expression von S1P5 bei COPD ist gegenwÀrtig unklar. Aufgrund des komplexen
Zusammenspiels der einzelnen Komponenten des SphK/S1P-Systems sind weitere
Untersuchungen erforderlich, um gezielte pharmakologische Interventionen des
SphK/S1P-Systems bei pulmonalen Erkrankungen wie PAH, Asthma bronchiale und
COPD etablieren zu können.Introduction: Pulmonary arterial hypertension (PAH) is a fatal condition
characterized by increased pulmonary vascular resistance, pulmonary arterial
remodeling and right heart failure. Pulmonary Th2 inflammation induces
morphological and functional changes which resemble those observed in PAH,
including perivascular inflammation, pulmonary vascular hyperresponsiveness
and pulmonary arterial remodeling. The bioactive sphingolipid
sphingosine-1-phosphate (S1P) acts through five G-protein coupled receptors,
S1P1-S1P5, as a vasoconstrictor and an important modulator of immune
signaling. In the lung, S1P is predominantly synthetized by sphingosine kinase
1 (SphK1). Methods: SphK1-deficient mice (SphK1-/-), S1P4-deficient mice
(S1P4-/-) and the corresponding wild-type mice (wt) were employed in acute and
chronic ovalbumin-evoked pulmonary inflammation. The effects of SphK1- and
S1P4-deficiency on inflammation, airway responsiveness, pulmonary vascular
responsiveness and pulmonary arterial remodeling were investigated. Results:
Acute inflammation was accompanied by airway and pulmonary vascular
hyperresponsiveness. Airway inflammation and hyperresponsiveness were reduced
in SphK1-/- mice, while being aggravated in S1P4-/- mice as compared to wt
mice. Pulmonary vascular hyperresponsiveness did not differ between strains.
After chronic Th2 inflammation, SphK1-/- mice showed an increased vascular
hyperresponsiveness compared with wt mice accompanied by remodeling of the
small and intra-acinar arteries. A linear regression between functional and
morphometric vascular data could be shown. Conclusion: The SphK/S1P system may
play a protective role in the pathogenesis of pulmonary arterial hypertension.
Moreover, this study provides further evidence for the relevance of the
SphK/S1P system in allergic airway disease and is the first demonstration of a
dissociation between airway and vascular pathology induced by Th2-type of
inflammation