120 research outputs found
Innate Immune Recognition and Inflammasome Activation in Listeria Monocytogenes Infection
Listeria monocytogenes is an intracellular, Gram-positive bacterium that can cause life-threatening illness especially in immunocompromised individuals and newborns. The pathogen propagates within the cytosol of various host cells after escaping from the phagosomal compartment depending on the cytolysin listeriolysin O. While L. monocytogenes can manipulate the endocytic and many host-cell signaling cascades to its advantage, host cells are however capable of detecting Listeria infection at different cellular compartments by expressing innate immune receptors that trigger antibacterial defense pathways. These receptors include the Toll-like receptors, NOD-like receptors (NLRs), and cytosolic DNA sensors. Some NLRs as well as the DNA sensor AIM2 form multiprotein complexes called inflammasomes. Inflammasomes regulate caspase-1-dependent production of the key inflammatory cytokines IL-1Ī² and IL-18 as well as pyroptotic cell death in L. monocytogenes-infected cells. This review describes the current knowledge about innate immune sensing and inflammasome activation in Listeria infection
Pseudomonas aeruginosa Triggered Exosomal Release of ADAM10 Mediates Proteolytic Cleavage in Trans
Pneumonia is a life-threatening disease often caused by infection with Streptococcus pneumo niae and Pseudomonas aeruginosa. Many of the mediators (e.g., TNF, IL-6R) and junction molecules
(e.g., E-cadherin) orchestrating inflammatory cell recruitment and loss of barrier integrity are prote olytically cleaved through a disintegrin and metalloproteinases (ADAMs). We could show by Western
blot, surface expression analysis and measurement of proteolytic activity in cell-based assays, that
ADAM10 in epithelial cells is upregulated and activated upon infection with Pseudomonas aeruginosa
and Exotoxin A (ExoA), but not upon infection with Streptococcus pneumoniae. Targeting ADAM10
by pharmacological inhibition or gene silencing, we demonstrated that this activation was critical
for cleavage of E-cadherin and modulated permeability and epithelial integrity. Stimulation with
heat-inactivated bacteria revealed that the activation was based on the toxin repertoire rather than
the interaction with the bacterial particle itself. Furthermore, calcium imaging experiments showed
that the ExoA action was based on the induction of calcium influx. Investigating the extracellular
vesicles and their proteolytic activity, we could show that Pseudomonas aeruginosa triggered exosomal
release of ADAM10 and proteolytic cleavage in trans. This newly described mechanism could consti tute an essential mechanism causing systemic inflammation in patients suffering from Pseudomonas
aeruginosa-induced pneumonia stimulating future translational studies
Perioperative Anaesthesiological Management of Malignant Pleural Mesothelioma Patients Undergoing Extrapleural Pneumonectomy (EPP) and Extended Pleurectomy/Decortication ((E)PD)
Introduction: Macroscopic complete resection (MCR) within a multimodality treatment concept offers currently the best survival for malignant pleural mesothelioma patients. The current standardised therapy is within a multimodality approach including (neo-)adjuvant chemotherapy followed by macroscopic complete resection (MCR). However, MCR in form of extrapleural pneumonectomy (EPP) or extended pleurectomy/decortication ((E)PD) is correlated with significant morbidity and mortality if not performed in high volume centres as described previously according to the literature. In addition, there exist no standardised anaesthesiological protocol for this surgical approach according to the literature.
Methods: At our institution, diagnosed mesothelioma patients up to an International Mesothelioma Interest Group (IMIG) stage III receive induction chemotherapy followed by either EPP or (E)PD and in certain cases additional adjuvant therapy. In the period 1999-end 2019, 362 patients were intended to be treated and 303 underwent induction chemotherapy followed by MCR. MCR can be achieved either by EPP or (E)PD. Both procedures request a good teamwork between the surgeon and the anaesthesiologist.
Conclusion: Although, there has been a shift lately from EPP towards lung sparing procedure (E)PD, both surgical approaches are still performed to date and is a challenging procedure for both, the surgeon as well as the anaesthesiologist. Herewith, we present our institutional perioperative standard operating procedures for the surgical and anaesthesiological management of EPP or (E)PD according to international terms of reference
PKCĪ± 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 (PKCĪ±) is a known modulator of smooth muscle cell contraction. However,
the effects of PKCĪ± deficiency on pulmonary vasoconstriction have not yet been
investigated. Thus, the role of PKCĪ± in pulmonary vascular responsiveness to
the TXA2 analog U46619, ET-1, serotonin and acute hypoxia was investigated in
isolated lungs of PKCĪ±-/- mice and corresponding wild-type mice, with or
without prior administration of the PKC inhibitor bisindolylmaleimide I or
Gƶ6976. 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 PKCĪ±
deficiency reduced ET-1-evoked pulmonary vasoconstriction. The pulmonary
vasopressor response to serotonin was unaffected by either broad PKC
inhibition or PKCĪ± deficiency. Surprisingly, PKCĪ±-/- mice showed pulmonary
vascular hyperresponsiveness to U46619 and increased TXA2 receptor (TP
receptor) expression in the intrapulmonary arteries. To conclude, PKCĪ±
regulates ET-1-induced pulmonary vasoconstriction. However, PKCĪ± deficiency
leads to pulmonary vascular hyperresponsiveness to TXA2, possibly via
increased pulmonary arterial TP receptor expression
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-
1Ī² 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-1Ī² 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
Production of isotope-labeled proteins in insect cells for NMR
Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR applications for proteins, which have become accessible by this eukaryotic expression host
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 IFNĪ² 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
Population analysis of Legionella pneumophila reveals a basis for resistance to complement-mediated killing
Legionella pneumophila is the most common cause of the severe respiratory infection known as Legionnaires' disease. However, the microorganism is typically a symbiont of free-living amoeba, and our understanding of the bacterial factors that determine human pathogenicity is limited. Here we carried out a population genomic study of 902 L. pneumophila isolates from human clinical and environmental samples to examine their genetic diversity, global distribution and the basis for human pathogenicity. We find that the capacity for human disease is representative of the breadth of species diversity although some clones are more commonly associated with clinical infections. We identified a single gene (lag-1) to be most strongly associated with clinical isolates. lag-1, which encodes an O-acetyltransferase for lipopolysaccharide modification, has been distributed horizontally across all major phylogenetic clades of L. pneumophila by frequent recent recombination events. The gene confers resistance to complement-mediated killing in human serum by inhibiting deposition of classical pathway molecules on the bacterial surface. Furthermore, acquisition of lag-1 inhibits complement-dependent phagocytosis by human neutrophils, and promoted survival in a mouse model of pulmonary legionellosis. Thus, our results reveal L. pneumophila genetic traits linked to disease and provide a molecular basis for resistance to complement-mediated killing. The bacterium Legionella pneumophila can cause severe respiratory infection, but is typically a symbiont of free-living amoeba. Here, the authors analyse the genomes of 902 clinical and environmental isolates, and identify a bacterial gene that is strongly associated with human infection and confers resistance to complement-mediated killing.Peer reviewe
The common HAQ STING variant impairs cGAS-dependent antibacterial responses and is associated with susceptibility to Legionnairesā disease in humans
Abstract The cyclic GMP-AMP synthase (cGAS)-STING pathway is central for
innate immune sensing of various bacterial, viral and protozoal infections.
Recent studies identified the common HAQ and R232H alleles of TMEM173/STING,
but the functional consequences of these variants for primary infections are
unknown. Here we demonstrate that cGAS- and STING-deficient murine macrophages
as well as human cells of individuals carrying HAQ TMEM173/STING were severely
impaired in producing type I IFNs and pro-inflammatory cytokines in response
to Legionella pneumophila, bacterial DNA or cyclic dinucleotides (CDNs). In
contrast, R232H attenuated cytokine production only following stimulation with
bacterial CDN, but not in response to L. pneumophila or DNA. In a mouse model
of Legionnairesā disease, cGAS- and STING-deficient animals exhibited higher
bacterial loads as compared to wild-type mice. Moreover, the haplotype
frequency of HAQ TMEM173/STING, but not of R232H TMEM173/STING, was increased
in two independent cohorts of human Legionnairesā disease patients as compared
to healthy controls. Our study reveals that the cGAS-STING cascade contributes
to antibacterial defense against L. pneumophila in mice and men, and provides
important insight into how the common HAQ TMEM173/STING variant affects
antimicrobial immune responses and susceptibility to infection. Trial
registration ClinicalTrials.gov DRKS00005274, German Clinical Trials Register
Author summary Interferons (IFNs) and pro-inflammatory cytokines are key
regulators of gene expression and antibacterial defense during Legionella
pneumophila infection. Here we demonstrate that production of these mediators
was largely or partly dependent on the cyclic GMP-AMP synthase (cGAS)-STING
pathway in human and murine cells. Cells of individuals carrying the common
HAQ allele of TMEM173/STING were strongly impaired in their ability to respond
to L. pneumophila, bacterial DNA or cyclic dinucleotides (CDNs), whereas the
R232H allele was only attenuated in sensing of exogenous CDNs. Importantly,
cGAS and STING contributed to antibacterial defense in mice during L.
pneumophila lung infection, and the allele frequency of HAQ TMEM173/STING, but
not of R232H TMEM173/STING, was increased in two independent cohorts of human
Legionnairesā disease patients as compared to healthy controls. Hence, sensing
of bacterial DNA by the cGAS/STING pathway contributes to antibacterial
defense against L. pneumophila infection, and the hypomorphic variant HAQ
TMEM173/STING is associated with increased susceptibility to Legionnairesā
disease in humans
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