Identification of Potential Urinary Metabolite Biomarkers of Pseudomonas aeruginosa Ventilator-Associated Pneumonia.

Introduction: Ventilator-associated pneumonia (VAP) caused by Pseudomonas aeruginosa is a major cause of morbidity and mortality in hospital intensive care units (ICU). Rapid identification of P. aeruginosa-derived markers in easily accessible patients' samples can enable an early detection of P. aeruginosa VAP (VAP-PA), thereby stewarding antibiotic use and improving clinical outcomes. Methods: Metabolites were analysed using liquid chromatography-mass spectrometry (LC-MS) in prospectively collected urine samples from mechanically ventilated patients admitted to the Antwerp University Hospital ICU. Patients were followed from the start of mechanical ventilation (n = 100 patients) till the time of clinical diagnosis of VAP (n = 13). Patients (n = 8) in whom diagnosis of VAP was further confirmed by culturing respiratory samples and urine samples were studied for semi-quantitative metabolomics. Results: We first show that multivariate analyses highly discriminated VAP-PA from VAP-non-PA as well as from the pre-infection groups (R 2 = .97 and .98, respectively). A further univariate analysis identified 58 metabolites that were significantly elevated or uniquely present in VAP-PA compared to the VAP-non-PA and pre-infection groups (P < .05). These comprised both a known metabolite of histidine as well as a novel nicotine metabolite. Most interestingly, we identified 3 metabolites that were not only highly upregulated for, but were also highly specific to, VAP-PA, as these metabolites were completely absent in all pre-infection timepoints and in VAP-non-PA group. Conclusions: Considerable differences exist between urine metabolites in VAP-PA compared to VAP due to other bacterial aetiologies as well to non-VAP (pre-infection) timepoints. The unique urinary metabolic biomarkers we describe here, if further validated, could serve as highly specific diagnostic biomarkers of VAP-PA.

Mechanical Ventilation Impairs IL-17 Cytokine Family Expression in Ventilator-Associated Pneumonia.

Mechanical ventilation (MV) is the primary risk factor for the development of ventilator-associated pneumonia (VAP). Besides inducing a pro-inflammatory T-helper (Th)-1 cytokine response, MV also induces an anti-inflammatory Th2 cytokine response, marked by increased IL-4 secretion and reduced bacterial phagocytic capacity of rodent lung macrophages. Since IL-4 is known to downregulate both Th1 and Th17 cytokines, the latter is important in mediating mucosal immunity and combating bacterial and fungal growth, we studied and showed here in a rat model of MV that Th17 cytokines (IL-17A, IL-17F, and IL-22) were significantly upregulated in the lung as a response to different MV strategies currently utilized in clinic. To study whether the increased IL-4 levels are associated with downregulation of the anti-bacterial Th17 cytokines, we subsequently challenged mechanically ventilated rats with an intratracheal inoculation of Pseudomonas aeruginosa (VAP model) and showed a dramatic downregulation of IL-17A, IL-17F, and IL-22, compared to animals receiving the same bacterial burden without MV. For the studied Th1 cytokines (IFN, TNF, IL-6, and IL-1), only IFN showed a significant decrease as a consequence of bacterial infection in mechanically ventilated rats. We further studied IL-17A, the most studied IL-17 family member, in intensive care unit (ICU) pneumonia patients and showed that VAP patients had significantly lower levels of IL-17A in the endotracheal aspirate compared to patients entering ICU with pre-existing pneumonia. These translational data, obtained both in animal models and in humans, suggest that a deficient anti-bacterial Th17 response in the lung during MV is associated with VAP development.

Hypersynchronicity in the default mode-like network in a neurodevelopmental animal model with relevance for schizophrenia.

BACKGROUND: Immune activation during pregnancy is an important risk factor for schizophrenia. Brain dysconnectivity and NMDA receptor (NMDAR) hypofunction have been postulated to be central to schizophrenia pathophysiology. The aim of this study was to investigate resting-state functional connectivity (resting-state functional MRI-rsfMRI), microstructure (diffusion tension imaging-DTI) and response to NMDAR antagonist (pharmacological fMRI-phMRI) using multimodal MRI in offspring of pregnant dams exposed to immune challenge (maternal immune activation-MIA model), and determine whether these neuroimaging readouts correlate with schizophrenia-related behaviour. METHODS: Pregnant rats were injected with Poly I:C or saline on gestational day 15. The maternal weight response was assessed. Since previous research has shown behavioural deficits can differ between MIA offspring dependent on the maternal response to immune stimulus, offspring were divided into three groups: controls (saline, n = 11), offspring of dams that gained weight (Poly I:C WG, n = 12) and offspring of dams that lost weight post-MIA (Poly I:C WL, n = 16). Male adult offspring were subjected to rsfMRI, DTI, phMRI with NMDAR antagonist, behavioural testing and histological assessment. RESULTS: Poly I:C WL offspring exhibited increased functional connectivity in default mode-like network (DMN). Poly I:C WG offspring showed the most pronounced attenuation in NMDAR antagonist response versus controls. DTI revealed no differences in Poly I:C offspring versus controls. Poly I:C offspring exhibited anxiety. CONCLUSIONS: MIA offspring displayed a differential pathophysiology depending on the maternal response to immune challenge. While Poly I:C WL offspring displayed hypersynchronicity in the DMN, altered NMDAR antagonist response was most pronounced in Poly I:C WG offspring.

CD8 signaling in microglia/macrophage M1 polarization in a rat model of cerebral ischemia.

Classical or M1 activity of microglia/macrophages has been described in several neurodegenerative and brain inflammatory conditions and has also been linked to expansion of ischemic injury in post-stroke brain. While different pathways of M1 polarization have been suggested to occur in the post-stroke brain, the precise underlying mechanisms remain undefined. Using a transient middle cerebral artery occlusion (MCAO) rat model, we showed a progressive M2 to M1 polarization in the perilesional brain region with M1 cells becoming one of the dominant subsets by day 4 post-stroke. Comparing key receptors involved in M1 polarization (CD8, IFNγR, Clec4, FcγR, TLR3 and TLR4) and their signal transducers (Syk, Stat1, Irf3, and Traf6) at the day 4 time point, we showed a strong upregulation of CD8 along with SYK transducer in dissected perilesional brain tissue. We further showed that CD8 expression in the post-stroke brain was associated with activated (CD68+) macrophages and that progressive accumulation of CD8+CD68+ cells in the post-stroke brain coincided with increased iNOS (M1 marker) and reduced Arg1 (M2 marker) expression on these cells. In vitro ligand-based stimulation of the CD8 receptor caused increased iNOS expression and an enhanced capacity to phagocytose E. coli particles; and interestingly, CD8 stimulation was also able to repolarize IL4-treated M2 cells to an M1 phenotype. Our data suggest that increased CD8 signaling in the post-stroke brain is primarily associated with microglia/macrophages and can independently drive M1 polarization, and that modulation of CD8 signaling could be a potential target to limit secondary post-stroke brain damage.

Mechanical Ventilation Induces Interleukin 4 Secretion in Lungs and Reduces the Phagocytic Capacity of Lung Macrophages.

Patients receiving mechanical ventilation are at risk of developing ventilator-associated pneumonia. Here, we show that clinically utilized ventilation protocols in rats with 5 mL/kg or 8 mL/kg tidal volumes cause increased interleukin 4 (IL-4) expression, lowered ratio of TH1:TH2 transcriptional factors (Tbet:Gata3), and increased arginase 1-positive (Arg1+) macrophages and eosinophils in lungs. Macrophages from ventilated lungs had reduced ex vivo capacity toward phagocytosing bacteria. Ventilated animals, when further challenged with bacterial pneumonia, continued to show persistence of Arg1+ M2 macrophages as well as an increased bacterial burden compared with spontaneously breathing animals receiving the same bacterial dose. Increased IL-4 expression also occurred in a mouse ventilation model, and abrogation of IL-4 signaling restored lung bacterial burden in an IL-4Rα-/- ventilator-associated pneumonia model. Our data suggest that mechanical ventilation induces an immunosuppressive state in lungs, providing new insight in the development of ventilator-associated pneumonia.

Biofilm-Induced Type 2 Innate Immunity in a Cystic Fibrosis Model of Pseudomonas aeruginosa.

Biofilm-producing strains of Pseudomonas aeruginosa are a major cause of morbidity and mortality in cystic fibrosis (CF) patients. In these patients, increased levels of IL-17 as well as of IL-5 and IL-13 along with arginase (Arg)-positive macrophages have been observed in bronchoalveolar lavage fluid. While IL-17 is a strong proinflammatory cytokine associated with host defense against bacterial and fungal infections and is also elevated in several autoimmune diseases, IL-5/IL-13 and Arg1-positive M2 macrophages are part of the anti-inflammatory type 2 (Th2) immunity. To study whether increased IL-5 and IL-13 levels are related to biofilm formation, which is frequently observed in CF patients colonized by P. aeruginosa, we utilized an agarose bead-embedded P. aeruginosa rat model commonly employed in in vivo biofilm studies. We showed that "sterile" agarose bead instillation in rat notably increased lung transcript levels of IL-5 and IL-13 at two post-instillation study-points, day 1 and day 3. Concurrently, increased infiltration of type 2 innate cells such as eosinophils and Arg1 positive M2 activated macrophages (Arg1+CD68+) was also observed both at day 1 and day 3 while the proportion of M1 activated macrophages (iNOS+CD68+) at these time-points decreased. In contrast, P. aeruginosa-loaded beads caused a drastic elevation of proinflammatory Th1 (IFNγ, TNFα, IL-12a) and antibacterial Th17 (IL-17a, IL-17f, IL-22, IL-23a) cytokines along with a high influx of neutrophils and M1 macrophages, while Th2 cytokines (IL-5 and IL-13) drastically declined at day 1 post-infection. Interestingly, at day 3 post-infection, both Th1 and Th17 cytokines sharply declined and corroborated with decreased M1 and increased M2 macrophages. These data suggest that while IL-17 is linked to episodes of acute exacerbations of infection in CF patients, the increased Th2 cytokines and M2 macrophages observed in these patients are largely due to the biofilm matrix. The data presented here has important implications for clinical management of CF patients.

Animal models of hospital-acquired pneumonia: current practices and future perspectives.

Lower respiratory tract infections are amongst the leading causes of mortality and morbidity worldwide. Especially in hospital settings and more particularly in critically ill ventilated patients, nosocomial pneumonia is one of the most serious infectious complications frequently caused by opportunistic pathogens. Pseudomonas aeruginosa is one of the most important causes of ventilator-associated pneumonia as well as the major cause of chronic pneumonia in cystic fibrosis patients. Animal models of pneumonia allow us to investigate distinct types of pneumonia at various disease stages, studies that are not possible in patients. Different animal models of pneumonia such as one-hit acute pneumonia models, ventilator-associated pneumonia models and biofilm pneumonia models associated with cystic fibrosis have been extensively studied and have considerably aided our understanding of disease pathogenesis and testing and developing new treatment strategies. The present review aims to guide investigators in choosing appropriate animal pneumonia models by describing and comparing the relevant characteristics of each model using P. aeruginosa as a model etiology for hospital-acquired pneumonia. Key to establishing and studying these animal models of infection are well-defined end-points that allow precise monitoring and characterization of disease development that could ultimately aid in translating these findings to patient populations in order to guide therapy. In this respect, and discussed here, is the development of humanized animal models of bacterial pneumonia that could offer unique advantages to study bacterial virulence factor expression and host cytokine production for translational purposes.

The endotracheal tube microbiome associated with Pseudomonas aeruginosa or Staphylococcus epidermidis.

Ventilator-associated pneumonia (VAP) is one of the commonest hospital-acquired infections associated with high mortality. VAP pathogenesis is closely linked to organisms colonizing the endotracheal tube (ETT) such as Staphylococcus epidermidis and Pseudomonas aeruginosa, the former a common commensal with pathogenic potential and the latter a known VAP pathogen. However, recent gut microbiome studies show that pathogens rarely function alone. Hence, we determined the ETT microbial consortium co-colonizing with S. epidermidis or P. aeruginosa to understand its importance in the development of VAP and for patient prognosis. Using bacterial 16S rRNA and fungal ITS-II sequencing on ETT biomass showing presence of P. aeruginosa and/or S. epidermidis on culture, we found that presence of P. aeruginosa correlated inversely with patient survival and with bacterial species diversity. A decision tree, using 16S rRNA and patient parameters, to predict patient survival was generated. Patients with a relative abundance of Pseudomonadaceae <4.6% and of Staphylococcaceae <70.8% had the highest chance of survival. When Pseudomonadaceae were >4.6%, age of patient <66.5 years was the most important predictor of patient survival. These data indicate that the composition of the ETT microbiome correlates with patient prognosis, and presence of P. aeruginosa is an important predictor of patient outcome.

P2X7 receptor antagonism reduces the severity of spontaneous seizures in a chronic model of temporal lobe epilepsy.

BACKGROUND: The available pharmacotherapy for patients with epilepsy primarily address the symptoms and are ineffective in about 40% of patients. Brain inflammation gained support as potential target for developing new therapies, especially the P2X7 receptor (P2X7R), involved in processing of IL-1ß, might be an interesting candidate. This study was designed to investigate the effect of a novel P2X7R antagonist on the severity and on the number of chronic spontaneous recurrent seizures (SRS), which was unexplored until now. METHODS: After one-week of vehicle treatment (20% HP-ß-cyclodextrin), JNJ-42253432 was administered subcutaneously for another week under continuous video-electroencephalography monitoring (n = 17) in Sprague Dawley rats 3 months after kainic acid-induced status epilepticus. The proportion of different seizure classes, as well as the number of SRS/day were calculated for the vehicle and treatment period. In addition, post-mortem microglial activation and astrogliosis were assessed. RESULTS: A significant decrease of the proportion of type 4-5 SRS (p < 0.05), while an increase of type 1-3 was demonstrated (p < 0.05) from the vehicle to the treatment period. There was no effect of the P2X7R antagonist on the number of SRS/day or the glial markers. CONCLUSIONS: The P2X7R antagonist gave rise to a less severe profile of the chronic seizure burden without suppressing the SRS frequency. More studies are needed to unravel the underlying mechanisms of the beneficial effect on seizure severity and whether the administration of the compound during early epileptogenesis could induce long-term disease-modifying effects.