115 research outputs found
Induction of KrĂĽppel-Like Factor 4 Mediates Polymorphonuclear Neutrophil Activation in Streptococcus pneumoniae Infection
The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae-dependent induction of the transcription factor Kruppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor alpha and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection
Bioprinted Multi-Cell Type Lung Model for the Study of Viral Inhibitors
Influenza A virus (IAV) continuously causes epidemics and claims numerous lives every year. The available treatment options are insufficient and the limited pertinence of animal models for human IAV infections is hampering the development of new therapeutics. Bioprinted tissue models support studying pathogenic mechanisms and pathogen-host interactions in a human micro tissue environment. Here, we describe a human lung model, which consisted of a bioprinted base of primary human lung fibroblasts together with monocytic THP-1 cells, on top of which alveolar epithelial A549 cells were printed. Cells were embedded in a hydrogel consisting of alginate, gelatin and collagen. These constructs were kept in long-term culture for 35 days and their viability, expression of specific cell markers and general rheological parameters were analyzed. When the models were challenged with a combination of the bacterial toxins LPS and ATP, a release of the proinflammatory cytokines IL-1β and IL-8 was observed, confirming that the model can generate an immune response. In virus inhibition assays with the bioprinted lung model, the replication of a seasonal IAV strain was restricted by treatment with an antiviral agent in a dose-dependent manner. The printed lung construct provides an alveolar model to investigate pulmonary pathogenic biology and to support development of new therapeutics not only for IAV, but also for other viruses
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) 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 hypoxi
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
Optimization of cell-laden bioinks for 3D bioprinting and efficient infection with influenza A virus
Bioprinting is a new technology, which arranges cells with high spatial resolution, but its potential to create models for viral infection studies has not yet been fully realized. The present study describes the optimization of a bioink composition for extrusion printing. The bioinks were biophysically characterized by rheological and electron micrographic measurements. Hydrogels consisting of alginate, gelatin and Matrigel were used to provide a scaffold for a 3D arrangement of human alveolar A549 cells. A blend containing 20% Matrigel provided the optimal conditions for spatial distribution and viability of the printed cells. Infection of the 3D model with a seasonal influenza A strain resulted in widespread distribution of the virus and a clustered infection pattern that is also observed in the natural lung but not in two-dimensional (2D) cell culture, which demonstrates the advantage of 3D printed constructs over conventional culture conditions. The bioink supported viral replication and proinflammatory interferon release of the infected cells. We consider our strategy to be paradigmatic for the generation of humanized 3D tissue models by bioprinting to study infections and develop new antiviral strategies.DFG, 325093850, Open Access Publizieren 2017 - 2018 / Technische Universität Berli
A novel European H5N8 influenza A virus has increased virulence in ducks but low zoonotic potential
We investigated in a unique setup of animal models and a human lung explant culture biological properties, including zoonotic potential, of a representative 2016 highly pathogenic avian influenza virus (HPAIV) H5N8, clade 2.3.4.4 group B (H5N8B), that spread rapidly in a huge and ongoing outbreak series in Europe and caused high mortality in waterfowl and domestic birds. HPAIV H5N8B showed increased virulence with rapid onset of severe disease and mortality in Pekin ducks due to pronounced neuro- and hepatotropism. Cross-species infection was evaluated in mice, ferrets, and in a human lung explant culture model. While the H5N8B isolate was highly virulent for Balb/c mice, virulence and transmissibility were grossly reduced in ferrets, which was mirrored by marginal replication in human lung cultures infected ex vivo. Our data indicate that the 2016 HPAIV H5N8B is avian-adapted with augmented virulence for waterfowl, but has low zoonotic potential. The here tested combination of animal studies with the inoculation of human explants provides a promising future workflow to evaluate zoonotic potential, mammalian replication competence and avian virulence of HPAIV.Peer Reviewe
protection by adrenomedullin
Ventilator-induced lung injury (VILI) contributes to morbidity and mortality
in acute respiratory distress syndrome (ARDS). Particularly pre-injured lungs
are susceptible to VILI despite protective ventilation. In a previous study,
the endogenous peptide adrenomedullin (AM) protected murine lungs from VILI.
We hypothesized that mechanical ventilation (MV) contributes to lung injury
and sepsis in pneumonia, and that AM may reduce lung injury and multiple organ
failure in ventilated mice with pneumococcal pneumonia. We analyzed in mice
the impact of MV in established pneumonia on lung injury, inflammation,
bacterial burden, hemodynamics and extrapulmonary organ injury, and assessed
the therapeutic potential of AM by starting treatment at intubation. In
pneumococcal pneumonia, MV increased lung permeability, and worsened lung
mechanics and oxygenation failure. MV dramatically increased lung and blood
cytokines but not lung leukocyte counts in pneumonia. MV induced systemic
leukocytopenia and liver, gut and kidney injury in mice with pneumonia. Lung
and blood bacterial burden was not affected by MV pneumonia and MV increased
lung AM expression, whereas receptor activity modifying protein (RAMP) 1-3
expression was increased in pneumonia and reduced by MV. Infusion of AM
protected against MV-induced lung injury (66% reduction of pulmonary
permeability p<0.01; prevention of pulmonary restriction) and against VILI-
induced liver and gut injury in pneumonia (91% reduction of AST levels p<0.05,
96% reduction of alanine aminotransaminase (ALT) levels p<0.05, abrogation of
histopathological changes and parenchymal apoptosis in liver and gut). MV
paved the way for the progression of pneumonia towards ARDS and sepsis by
aggravating lung injury and systemic hyperinflammation leading to liver,
kidney and gut injury. AM may be a promising therapeutic option to protect
against development of lung injury, sepsis and extrapulmonary organ injury in
mechanically ventilated individuals with severe pneumonia
The Novel Human Influenza A(H7N9) Virus Is Naturally Adapted to Efficient Growth in Human Lung Tissue
A novel influenza A virus (IAV) of the H7N9 subtype has been isolated from severely diseased patients with pneumonia and acute respiratory distress syndrome and, apparently, from healthy poultry in March 2013 in Eastern China. We evaluated replication, tropism, and cytokine induction of the A/Anhui/1/2013 (H7N9) virus isolated from a fatal human infection and two low-pathogenic avian H7 subtype viruses in a human lung organ culture system mimicking infection of the lower respiratory tract. The A(H7N9) patient isolate replicated similarly well as a seasonal IAV in explanted human lung tissue, whereas avian H7 subtype viruses propagated poorly. Interestingly, the avian H7 strains provoked a strong antiviral type I interferon (IFN-I) response, whereas the A(H7N9) virus induced only low IFN levels. Nevertheless, all viruses analyzed were detected predominantly in type II pneumocytes, indicating that the A(H7N9) virus does not differ in its cellular tropism from other avian or human influenza viruses. Tissue culture-based studies suggested that the low induction of the IFN-β promoter correlated with an efficient suppression by the viral NS1 protein. These findings demonstrate that the zoonotic A(H7N9) virus is unusually well adapted to efficient propagation in human alveolar tissue, which most likely contributes to the severity of lower respiratory tract disease seen in many patients
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