Matthias Krüll

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.

Adrenomedullin Reduces Endothelial Hyperpermeability

Abstract— Endothelial hyperpermeability induced by inflammatory mediators is a hallmark of sepsis and adult respiratory distress syndrome. Increased levels of the regulatory peptide adrenomedullin (ADM) have been found in patients with systemic inflammatory response. We analyzed the effect of ADM on the permeability of cultured human umbilical vein endothelial cell (HUVEC) and porcine pulmonary artery endothelial cell monolayers. ADM dose-dependently reduced endothelial hyperpermeability induced by hydrogen peroxide (H2O2), thrombin, and Escherichia coli hemolysin. Moreover, ADM pretreatment blocked H2O2-related edema formation in isolated perfused rabbit lungs and increased cAMP levels in lung perfusate. ADM bound specifically to HUVECs and porcine pulmonary artery endothelial cells and increased cellular cAMP levels. Simultaneous inhibition of cAMP-degrading phosphodiesterase isoenzymes 3 and 4 potentiated ADM-dependent cAMP accumulation and synergistically enhanced ADM-dependent reduction of thrombin-induced hyperpermeability. However, ADM showed no effect on endothelial cGMP content, basal intracellular Ca2+ levels, or the H2O2-stimulated, thrombin-stimulated, or Escherichia coli hemolysin–stimulated Ca2+ increase. ADM diminished thrombin- and H2O2-related myosin light chain phosphorylation as well as stimulus-dependent stress fiber formation and gap formation in HUVECs, suggesting that ADM may stabilize the barrier function by cAMP-dependent relaxation of the microfilament system. These findings identify a new function of ADM and point to ADM as a potential interventional agent for the reduction of vascular leakage in sepsis and adult respiratory distress syndrome.

Bartonella henselae Induces NF-κB-Dependent Upregulation of Adhesion Molecules in Cultured Human Endothelial Cells: Possible Role of Outer Membrane Proteins as Pathogenic Factors

ABSTRACT The endothelium is a specific target for Bartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis. Mechanisms of Bartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed. B. henselae strain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-κB activation and enhanced adhesion molecule expression. These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay. Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion. In our hands, B. henselae Berlin-1 was substantially more active than the typing strain B. henselae ATCC 49882. E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay. Induction of adhesion molecules was mediated via NF-κB activation and could be blocked by a specific NF-κB inhibitor. Additional studies indicated that B. henselae-induced effects did not require living bacteria or Bartonella lipopolysaccharides. Exposure of HUVEC to purified B. henselae outer membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation. In conclusion, B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells. B. henselae OMPs are sufficient to induce NF-κB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes. These observations identify important new properties of B. henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.

β-PIX and Rac1 GTPase Mediate Trafficking and Negative Regulation of NOD2

The nucleotide-binding domain and leucine-rich repeat containing protein NOD2 serves as a cytoplasmic pattern recognition molecule sensing bacterial muramyl dipeptide (MDP), whereas TLR2 mediates cell surface recognition of bacterial lipopeptides. In this study, we show that NOD2 stimulation activated Rac1 in human THP-1 cells and primary human monocytes. Rac1 inhibition or knock-down, or actin cytoskeleton disruption increased MDP-stimulated IL-8 secretion and NF-κB activation, whereas TLR2-dependent cell activation was suppressed by Rac1 inhibition. p21-activated kinase [Pak]-interacting exchange factor (β-PIX) plays a role in this negative regulation, because knock-down of β-PIX also led to increased NOD2-mediated but not TLR2-mediated IL-8 secretion, and coimmunoprecipitation experiments demonstrated that NOD2 interacted with β-PIX as well as Rac1 upon MDP stimulation. Moreover, knock-down of β-PIX or Rac1 abrogated membrane recruitment of NOD2, and interaction of NOD2 with its negative regulator Erbin. Overall, our data indicate that β-PIX and Rac1 mediate trafficking and negative regulation of NOD2-dependent signaling which is different from Rac1’s positive regulatory role in TLR2 signaling.

Porphyromonas gingivalis Strain-Dependent Activation of Human Endothelial Cells

ABSTRACT Porphyromonas gingivalis is an important bacterium involved in periodontal diseases. Colonization by periodontopathogens has been associated with severe local inflammatory reactions in the connective tissue. In this study we characterized P. gingivalis-mediated infection and activation of human umbilical vein endothelial cells by using two strains of different virulence capacities, strains ATCC 53977 and DSMZ 20709. Both strains were able to adhere to and infect endothelial cells with an infection rate of 0.48% for ATCC 53977 and 0.007% for DSMZ 20709. The triggering of two signal transduction pathways in P. gingivalis-infected endothelial cells was demonstrated for both strains, with a rapid increase of p38 mitogen-activated protein kinase phosphorylation and a more delayed degradation of IκBα, followed by nuclear translocation of NF-κB. In addition, both strains induced enhanced expression of endothelial adhesion molecules E-selectin and intracellular adhesion molecule 1 (ICAM-1). Target cell activation was independent of bacterial fimbriae expression since the fimA knockout strain A7436 ΔfimA induced the same level of ICAM-1 as the corresponding wild type (A7436-WT). Thus, two P. gingivalis strains, ATCC 53799 and DSMZ 20709, infect endothelial cells and trigger signaling cascades leading to endothelial activation, which in turn may result in or promote severe local and systemic inflammation.

Differences in Cell Activation by Chlamydophila pneumoniae and Chlamydia trachomatis Infection in Human Endothelial Cells

ABSTRACT Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.

Chlamydophila pneumoniae Mechanisms of target cell infection and activation

Chlamydophila (Chlamydia) pneumoniae, a gram-negative obligate intracellular bacterium, is a widespread respiratory pathogen. Chronic C. pneumoniae infection has been suggested as a trigger/promoter of inflammation that may result in vascular lesions. Although the genome of C. pneumoniae has been sequenced completely this information has not yet led to an understanding of the mechanisms of acute infection and target cell activation nor to the identification of potential chlamydial virulence factors. Intriguingly, current antibiotic treatment options for acute chlamydial infection were proven to be ineffective with respect to clinical outcome in different groups of atherosclerotic patients. The reason might be that primary infection of vascular smooth muscle cells and blood monocytes with C. pneumoniae resembles rather a persistent, antibiotic-resistant, than an active infection. In this review we will focus on the importance of putative host cell receptors for C. pneumoniae and subsequently activated signal transduction pathways.

Essential Role of Mitochondrial Antiviral Signaling, IFN Regulatory Factor (IRF)3, and IRF7 in Chlamydophila pneumoniae-Mediated IFN-β Response and Control of Bacterial Replication in Human Endothelial Cells

Chlamydophila pneumoniae infection of the vascular wall as well as activation of the transcription factor IFN regulatory factor (IRF)3 have been linked to development of chronic vascular lesions and atherosclerosis. The innate immune system detects invading pathogens by use of pattern recognition receptors, some of which are able to stimulate IRF3/7 activation and subsequent type I IFN production (e. g., IFN-β). In this study, we show that infection of human endothelial cells with C. pneumoniae-induced production of IFN-β, a cytokine that so far has been mainly associated with antiviral immunity. Moreover, C. pneumoniae infection led to IRF3 and IRF7 nuclear translocation in HUVECs and RNA interference experiments showed that IRF3 and IRF7 as well as the mitochondrial antiviral signaling (MAVS) were essential for IFN-β induction. Finally, C. pneumoniae replication was enhanced in endothelial cells in which IRF3, IRF7, or MAVS expression was inhibited by small interfering RNA and attenuated by IFN-β treatment. In conclusion, C. pneumoniae infection of endothelial cells activates an MAVS-, IRF3-, and IRF7-dependent signaling, which controls bacterial growth and might modulate development of vascular lesions.

Simvastatin Reduces Chlamydophila pneumoniae–Mediated Histone Modifications and Gene Expression in Cultured Human Endothelial Cells

Inflammatory activation of the endothelium by Chlamydophila pneumoniae infection has been implicated in the development of chronic vascular lesions and coronary heart disease by seroepidemiological and animal studies. We tested the hypothesis that C pneumoniae induced inflammatory gene expression is regulated by Rho-GTPase–related histone modifications. C pneumoniae infection induced the liberation of proinflammatory interleukin-6, interleukin-8, granulocyte colony-stimulating factor, macrophage inflammatory protein-1β, granulocyte/macrophage colony-stimulating factor, and interferon-γ by human endothelial cells. Cytokine secretion was reduced by simvastatin and the specific Rac1 inhibitor NSC23766 but was synergistically enhanced by inhibitors of histone deacetylases trichostatin A and suberoylanilide hydroxamic acid. Infection of endothelial cells with viable C pneumoniae, but not exposure to heat-inactivated C pneumoniae or infection with C trachomatis, induced acetylation of histone H4 and phosphorylation and acetylation of histone H3. Pretreatment of C pneumoniae–infected cells with simvastatin or NSC23766 reduced global histone modifications as well as specific modifications at the il8 gene promoter, as shown by chromatin immunoprecipitation. Reduced recruitment of nuclear factor &kgr;B p65/RelA as well as of RNA polymerase II was observed in statin-treated cells. Taken together, Rac1-mediated histone modifications seem to play an important role in C pneumoniae–induced cytokine production by human endothelial cells.

Effects of propofol and taurine on intracellular free amino acid profiles and immune function markers in neutrophils in vitro.

Abstract We have examined the effects of propofol, taurine, and the combination of propofol and taurine on amino acid profiles and the immune function markers superoxide anion (O2 −), hydrogen peroxide (H2O2), and released myeloperoxidase (MPO) activity in neutrophils (PMN). Propofol led to significant changes in the dynamic PMN-free amino acid pool. Exogenous taurine significantly reduced PMN neutral amino acid and α-aminobutyrate (α-aba) as intracellular taurine increased. Incubation with propofol plus taurine resulted in lower intracellular taurine levels and elevated α-aba and neutral amino acid concentrations compared to propofol alone. Concerning PMN immune function markers, propofol significantly decreased O2 −-and H formation and released MPO. Taurine led to an increased release of MPO and concomitant significantly reduced O2 − and H2O2 levels. When propofol and taurine were applied together they appeared to act additively with regard to superoxide and hydrogen peroxide formation. In the case of MPO, taurine neutralized propofols effects, supporting the idea that MPO activity may be regulated by taurine. We believe therefore that taurine is important for strengthening PMN host defense capability, although the mechanisms are not yet clear. Moreover, taurine appears to act primarily by altering the PMN osmotic balance, while propofol seems to affect PMN amino acid metabolism and/or uptake and release.