presentation_1_Trauma-Induced Damage-Associated Molecular Patterns-Mediated Remote Organ Injury and Immunosuppression in the Acutely Ill Patient.PDF

<p>Trauma is one of the leading causes of death and disability in the world. Multiple trauma or isolated traumatic brain injury are both indicative of human tissue damage. In the early phase after trauma, damage-associated molecular patterns (DAMPs) are released and give rise to sterile systemic inflammatory response syndrome (SIRS) and organ failure. Later, protracted inflammation following sepsis will favor hospital-acquired infection and will worsen patient’s outcome through immunosuppression. Throughout medical care or surgical procedures, severe trauma patients will be subjected to endogenous or exogenous DAMPs. In this review, we summarize the current knowledge regarding DAMP-mediated SIRS or immunosuppression and the clinical consequences in terms of organ failure and infections.</p

Gating strategy used to identify blood DC subsets and intracellular cytokines production in DCs in whole blood stimulated with TLR ligands.

<p>Whole blood samples were incubated with TLR3, 4, 7/8 or 9 ligands for 3.5-hour and then stained for identification of myeloid DC (HLA-DR+, Lin-, CD11c+, CD123-) and plasmacytoid DC (HLA-DR+, lin−, CD11c−, CD123+) together with intracellular cytokine production (TNFα, IL-12, IFNα).</p

Time course of circulating mDCs and pDCs numbers in patients with aneurysmal subarachnoid hemorrhage.

<p>Comparison of circulating (A) myeloid DC and (B) plasmacytoid DC counts in SAH patients (N = 21) on days 2, 5 and 10 compared with HC (N = 11). Plots represent median (Interquartile ranges). HC: healthy controls. SAH: aneurysmal subarachnoid hemorrhage. DCs: dendritic cells. * <i>P</i><0.05, ** <i>P</i><0.01.</p

Hypoxia Modulates Infection of Epithelial Cells by <em>Pseudomonas aeruginosa</em>

<div><p><em>Pseudomonas aeruginosa (P. aeruginosa)</em> is an opportunistic pathogen commonly associated with lung and wound infections. Hypoxia is a frequent feature of the microenvironment of infected tissues which induces the expression of genes associated with innate immunity and inflammation in host cells primarily through the activation of the hypoxia-inducible factor (HIF) and Nuclear factor kappaB (NF-κB) pathways which are regulated by oxygen-dependent prolyl-hydroxylases. Hypoxia also affects virulence and antibiotic resistance in bacterial pathogens. However, less is known about the impact of hypoxia on host-pathogen interactions such as bacterial adhesion and infection. In the current study, we demonstrate that hypoxia decreases the internalization of <em>P. aeruginosa</em> into cultured epithelial cells resulting in decreased host cell death. This response can also be elicited by the hydroxylase inhibitor Dimethyloxallyl Glycine (DMOG). Reducing HIF-2α expression or Rho kinase activity diminished the effects of hypoxia on <em>P. aeruginosa</em> infection. Furthermore, in an in vivo pneumonia infection model, application of DMOG 48 h before infection with <em>P. aeruginosa</em> significantly reduced mortality. Thus, hypoxia reduces <em>P. aeruginosa</em> internalization into epithelial cells and pharmacologic manipulation of the host pathways involved may represent new therapeutic targets in the treatment of <em>P. aeruginosa</em> infection.</p> </div

Time course of TLR-induced productions of TNF-α and IFN-α in circulating pDCs from patients with aneurysmal subarachnoid hemorrhage.

<p>Intracellular cytokine measurement was performed in circulating pDCs from SAH patients (N = 21) on days 2, 5 and 10 after brain injury and from HC (N = 11). The percentages of pDCs expressing TNF-α or IFN-α were assessed after a 3.5-hour <i>ex vivo</i> stimulation with (<b>A</b>) CL097 (TLR-7/8 agonist) or (<b>B</b>) CpG (TLR-9 agonist). The percentage of positive pDCs without TLR-stimulation was below 1% (data not shown). The results are presented as percentages of pDCs expressing TNF-α (%TNF-α⁺) or IFN-α (%IFN-α⁺). Plots represent median (Interquartile ranges). HC: healthy controls. IFN-α: interferon. pDCs: plasmacytoid dendritic cells. SAH: aneurysmal subarachnoid hemorrhage. TNF-α: tumour necrosis factor -α. *<i>P</i><0.05.</p

Time course of TLR-induced production of TNF-α and IL-12 in circulating mDCs from patients with aneurysmal subarachnoid hemorrhage.

<p>Intracellular cytokine measurement was performed in circulating mDCs from SAH patients (N = 21) on days 2, 5 and 10 and from HC (N = 11). The percentages of mDCs expressing TNF-α or IL-12 were assessed after a 3.5-hour <i>ex vivo</i> stimulation with (<b>A</b>) polyIC (TLR-3 agonist), (<b>B</b>) lipopolysaccharide (TLR-4 agonist) or (<b>C</b>) CL097 (TLR-7/8 agonist). The percentage of positive DCs without TLR-stimulation was below 1% (data not shown). The results are presented as percentages of mDCs expressing TNF-α (%TNF-α⁺) or IL-12 (% IL-12⁺). Plots represent median (Interquartile ranges). HC: healthy controls. mDCs: myeloid dendritic cells. SAH: aneurysmal subarachnoid hemorrhage. TNF-α: tumour necrosis factor -α. IL-12: intreleukin-12. * <i>P</i><0.05.</p

Exploratory comparison of mDC and pDC status on day 2 in survivors and non-survivors.

<p>(<b>A</b>) The number of circulating myeloid DCs and plasmacytoid DCs were compared on day 2 between 13 survivors and 8 non-survivors. On day 2, mDCs and pDCs were stimulated <i>ex vivo</i> with polyIC (TLR-3 agonist), lipopolysaccharide (TLR-4 agonist), CL097 (TLR-7/8 agonist) and CpG (TLR-9 agonist) for 3.5 hours. (B) The percentages of mDCs expressing TNF-α or IL-12 and (C) the percentages of pDCs expressing TNF-α or IFN-α were compared between survivors and non-survivors. The percentage of positive DCs without TLR stimulation was below 1% (data not shown). The results are presented as percentages of DCs expressing TNF-α (%TNF-α⁺), IL-12 (%IL-12⁺) or IFN-α (%IFN-α⁺). Plots represent median (Interquartile ranges). * <i>P</i><0.05.</p

Characteristics of the study population.

<p>ARDS: acute respiratory distress syndrome, ICU: intensive care unit, SAH: subarachnoid hemorrhage, SAPS: simplified acute physiological score, WFNS: World Federation of Neurological Surgeons.</p

HIF-2α is partly responsible for decreased internalization of <i>P. aeruginosa</i> in hypoxia.

<p>A: Knock down of HIF-1α and HIF-2α protein in HepG2 cells stably expressing shRNA directed against HIF-1α and HIF-2α shown by immunoblot. B: Antibiotic protection assay in normoxia with <i>P. aeruginosa</i> in HepG2 cells (see A). Internalized <i>P. aeruginosa</i> are shown as % of normoxic control. Data represent mean ± SEM of 3 independent experiments. C: Antibiotic protection assay in hypoxia with <i>P. aeruginosa</i> in HepG2 cells (see A). Internalized <i>P. aeruginosa</i> are shown as % of normoxic control. Data represent mean ± SEM of 3 independent experiments (** p<0.01, *** p<0.0001).</p

Bacterial internalization is decreased in DMOG treated cells.

<p>To mimic hypoxia, A549 cells were pre-treated with 1 mM of the pan-hydroxylase inhibitor DMOG or its' solvent DMSO for 24 h and throughout following infection with the <i>P. aeruginosa</i> reference strain ATCC 27853 (A), a CF pathogen <i>B. cenocepacia</i>, <i>J2315</i> (B) and two clinical <i>P. aeruginosa</i> isolates from CF patients, S7587, mucoid and non-mucoid <i>P. aeruginosa</i> (C and D). Data represent mean ± SEM of 3 independent experiments throughout (* p<0.05; *** p<0.0001).</p