Norbert Suttorp

Angiopoietin-2 sensitizes endothelial cells to TNF-α and has a crucial role in the induction of inflammation

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1–mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus–induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2−/− mice. Intravital microscopy showed normal TNF-α–induced leukocyte rolling in the vasculature of Angpt2−/−mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-α and modulating TNF-α–induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.

IFNβ induction by influenza A virus is mediated by RIG-I which is regulated by the viral NS1 protein

Influenza A virus causes epidemics of respiratory diseases in humans leading to thousands of death annually. One of its major virulence factors, the non‐structural protein 1 (NS1), exhibits interferon‐antagonistic properties. While epithelial cells of the respiratory tract are the primary targets of influenza virus, the virus‐sensing mechanisms in these cells eventually leading to IFNβ production are incompletely understood. Here we show that infection of epithelial cells with NS1‐deficient influenza A virus upregulated expression of two molecules that have been previously implicated in sensing of RNA viruses, the retinoic acid‐inducible gene I (RIG‐I) and the melanoma differentiation‐associated gene 5 (MDA5). Gene silencing and overexpression experiments demonstrated that RIG‐I, its adapter interferon‐beta promoter stimulator 1 (IPS‐1) and interferon‐regulated factor 3 (IRF3) were involved in influenza A virus‐mediated production of the antiviral IFNβ. In addition, we showed that the NS1 protein is capable to inhibit the RIG‐I‐induced signalling, a mechanism which corresponded to the observation that only NS1‐deficient but not the wild‐type virus induced high‐level production of IFNβ. In conclusion, we demonstrated a critical involvement of RIG‐I, IPS‐1 and IRF3 in influenza A virus infection of epithelial cells.

Procalcitonin predicts patients at low risk of death from community-acquired pneumonia across all CRB-65 classes

The aim of the present study was to investigate the prognostic value, in patients with community-acquired pneumonia (CAP), of procalcitonin (PCT) compared with the established inflammatory markers C-reactive protein (CRP) and leukocyte (WBC) count alone or in combination with the CRB-65 (confusion, respiratory rate ≥30 breaths·min−1, low blood pressure (systolic value <90 mmHg or diastolic value ≤60 mmHg) and age ≥65 yrs) score. In total, 1,671 patients with proven CAP were enrolled in the study. PCT, CRP, WBC and CRB-65 score were all determined on admission and patients were followed-up for 28 days for survival. In contrast to CRP and WBC, PCT levels markedly increased with the severity of CAP, as measured by the CRB-65 score. In 70 patients who died during follow-up, PCT levels on admission were significantly higher compared with levels in survivors. In receiver operating characteristic analysis for survival, the area under the curve (95% confidence interval) for PCT and CRB-65 was comparable (0.80 (0.75–0.84) versus 0.79 (0.74–0.84)), but each significantly higher compared with CRP (0.62 (0.54–0.68)) and WBC (0.61 (0.54–0.68)). PCT identified low-risk patients across CRB classes 0–4. In conclusion, procalcitonin levels on admission predict the severity and outcome of community-acquired pneumonia with a similar prognostic accuracy as the CRB-65 score and a higher prognostic accuracy compared with C-reactive protein and leukocyte count. Procalcitonin levels can provide independent identification of patients at low risk of death within CRB-65 (confusion, respiratory rate ≥30 breaths·min−1, low blood pressure (systolic value <90 mmHg or diastolic value ≤60 mmHg) and age ≥65 yrs) risk classes.

Nod1-Mediated Endothelial Cell Activation by Chlamydophila pneumoniae

Seroepidemiological and animal studies, as well as demonstration of viable bacteria in atherosclerotic plaques, have linked Chlamydophila pneumoniae infection to development of chronic vascular lesions and coronary heart disease. Inflammation and immune responses are dependent on host recognition of invading pathogens. The recently identified cytosolic Nod proteins are candidates for intracellular recognition of bacteria, such as the obligate intracellular chlamydia. In the present study, mechanisms of endothelial cell activation by C. pneumoniae via Nod proteins were examined. Viable, but not heat-inactivated, chlamydia activated human endothelial cells, suggesting that invasion of these cells is necessary for their profound activation. Endothelial cells express Nod1. Nod1 gene silencing by small interfering RNA reduced C pneumoniae–induced IL-8 release markedly. Moreover, in HEK293 cells, overexpressed Nod1 or Nod2 amplified the capacity of C pneumoniae to induce nuclear factor &kgr;B (NF-&kgr;B) activation. Interestingly, heat-inactivated bacteria were still able to induced a NF-&kgr;B reporter gene activity via Nod proteins when transfected intracellularly, but not when provided from the extracellular side. In contrast, TLR2 sensed extracellular heat-inactivated chlamydia. In conclusion, we demonstrated that C pneumoniae induced a Nod1-mediated and Nod2-mediated NF-&kgr;B activation in HEK293 cells. In endothelial cells, Nod1 played a dominant role in triggering a chlamydia-mediated inflammatory process.

The NLRP3 Inflammasome Is Differentially Activated by Pneumolysin Variants and Contributes to Host Defense in Pneumococcal Pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

VEGF induces hyperpermeability by a direct action on endothelial cells

Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.

Listeria monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide-binding oligomerization domain 1-dependent manner in endothelial cells.

Nucleotide-binding oligomerization domain (Nod) proteins serve as intracellular pattern recognition molecules recognizing peptidoglycans. To further examine intracellular immune recognition, we used Listeria monocytogenes as an organism particularly amenable for studying innate immunity to intracellular pathogens. In contrast to wild-type L. monocytogenes, the nonpathogenic Listeria innocua, or L. monocytogenes mutants lacking internalin B or listeriolysin O, poorly invaded host cells and escaped into host cell cytoplasm, respectively, and were therefore used as controls. In this study, we show that only the invasive wild-type L. monocytogenes, but not the listeriolysin O- or internalin B-negative L. monocytogenes mutants or L. innocua, substantially induced IL-8 production in HUVEC. RNA interference and Nod1-overexpression experiments demonstrated that Nod1 is critically involved in chemokine secretion and NF-κB activation initiated by L. monocytogenes in human endothelial cells. Moreover, we show for the first time that Nod1 mediated activation of p38 MAPK signaling induced by L. monocytogenes. Finally, L. monocytogenes- and Nod1-induced IL-8 production was blocked by a specific p38 inhibitor. In conclusion, L. monocytogenes induced a Nod1-dependent activation of p38 MAPK signaling and NF-κB which resulted in IL-8 production in endothelial cells. Thus, Nod1 is an important component of a cytoplasmic surveillance pathway.

p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability

Vascular endothelial growth factor (VEGF) is not only essential for vasculogenesis and angiogenesis but also is a potent inducer of vascular permeability. Although a dissection of the molecular pathways between angiogenesis‐ and vascular permeability‐inducing properties would be desirable for the development of angiogenic and anti‐angiogenic therapies, such mechanisms have not been identified yet. Here we provide evidence for a role of the p38 MAPK as the signaling molecule that separates these two processes. Inhibition of p38 MAPK activity enhances VEGF‐induced angiogenesis in vitro and in vivo, a finding that was accompanied by prolonged Erk1/2 MAPK activation, increased endothelial survival, and plasminogen activation. Conversely, the same inhibitors abrogate VEGF‐induced vascular permeability in vitro and in vivo. These dualistic properties of p38 MAPK are relevant not only for therapeutic angiogenesis but also for reducing edema formation and enhancing tissue repair in ischemic diseases.

Cardiovascular and Inflammatory Biomarkers to Predict Short- and Long-Term Survival in Community-acquired Pneumonia: Results from the German Competence Network, CAPNETZ

RATIONALE Several new biomarkers are related to mortality in community-acquired pneumonia (CAP). OBJECTIVES Aim of this study was to compare new biomarkers for the prediction of short- and long-term all-cause mortality in CAP. METHODS We enrolled 728 patients (59.0 ± 18.2 yr) with CAP. Midregional proadrenomedullin (MR-proADM), midregional proatrial natriuretic peptide (MR-proANP), proarginin-vasopressin (copeptin), proendothelin-1 (CT-proET-1), procalcitonin (PCT), C-reactive protein, white blood cell (WBC) count, and clinical confusion, respiratory rate, blood pressure, and age over 65 years (CRB-65) score were determined on admission. Patients were followed up for 180 days. MEASUREMENTS AND MAIN RESULTS In patients who died of any cause within 28 and 180 days (2.5 and 5.1%, respectively), MR-proADM, MR-proANP, copeptin, CT-proET-1 and PCT as well as CRB-65 were significantly higher compared with survivors. MR-proADM had the best performance for 28 days (HR 3.67) and 180 days (HR 2.84) survival. The C index of MR-proADM for 28-day survival (0.85) was superior to MR-proANP (0.81), copeptin (0.78), CT-proET-1 (0.79), and CRB-65 (0.72) for the prediction of mortality. For prediction of mortality at 180 days, the C index of MR-proADM (0.78) was higher than that for MR-proANP (0.74), copeptin (0.73), CT-proET-1 (0.76), PCT, C-reactive protein, and white blood cells. MR-proADM was independent of CRB-65, and added prognostic information for short- and long-term mortality. MR-proADM was an independent and strong predictor of short- and long-term mortality. CONCLUSIONS All new biomarkers were good predictors of short- and long-term all-cause mortality, superior to inflammatory markers, and at least comparable to CRB-65 score. MR-proADM showed the best performance. A combination of CRB-65 with MR-proADM might be the best predictor for mortality.

Listeria monocytogenes-infected human peripheral blood mononuclear cells produce IL-1beta, depending on listeriolysin O and NLRP3.

Different NOD-like receptors, including NLRP1, NLRP3, and NLRC4, as well as the recently identified HIN-200 protein, AIM2, form multiprotein complexes called inflammasomes, which mediate caspase-1–dependent processing of pro-IL-1β. Listeria monocytogenes is an intracellular pathogen that is actively phagocytosed by monocytes/macrophages and subsequently escapes from the phagosome into the host cell cytosol, depending on its pore-forming toxin listeriolysin O (LLO). In this study, we demonstrate that human PBMCs produced mature IL-1β when infected with wild-type L. monocytogenes or when treated with purified LLO. L. monocytogenes mutants lacking LLO or expressing a noncytolytic LLO as well as the avirulent Listeria innocua induced strongly impaired IL-1β production. RNA interference and inhibitor experiments in human PBMCs as well as experiments in Nlrp3 and Rip2 knockout bone marrow-derived macrophages demonstrated that the Listeria-induced IL-1β release was dependent on ASC, caspase-1, and NLRP3, whereas NOD2, Rip2, NLRP1, NLRP6, NLRP12, NLRC4, and AIM2 appeared to be dispensable. We found that L. monocytogenes-induced IL-1β production was largely dependent on phagosomal acidification and cathepsin B release, whereas purified LLO activated an IL-1β production independently of these mechanisms. Our results indicate that L. monocytogenes-infected human PBMCs produced IL-1β, largely depending on an LLO-mediated phagosomal rupture and cathepsin B release, which is sensed by Nlrp3. In addition, an LLO-dependent but cathepsin B-independent NLRP3 activation might contribute to some extent to the IL-1β production in L. monocytogenes-infected cells.