Andreas C. Hocke

Illustration of Pneumococcal Polysaccharide Capsule during Adherence and Invasion of Epithelial Cells

ABSTRACT The capsular polysaccharide of Streptococcus pneumoniae represents an important virulence factor and protects against phagocytosis. In this study the amount of capsular polysaccharide present on the bacterial surface during the infection process was illustrated by electron microscopic studies. After infection of A549 cells (type II pneumocytes) and HEp-2 epithelial cells a modified fixation method was used that allowed visualization of the state of capsule expression. This modified fixation procedure did not require the use of capsule-specific antibodies. Visualization of pneumococci in intimate contact and invading cells demonstrated that pneumococci were devoid of capsular polysaccharide. Pneumococci not in contact with the cells did not show alterations in capsular polysaccharide. After infection of the cells, invasive pneumococci of different strains and serotypes were recovered. Single colonies of these recovered pneumococci exhibited an up-to-105-fold-enhanced capacity to adhere and an up-to-104-fold-enhanced capacity to invade epithelial cells. Electron microscopic studies using a lysine-ruthenium red (LRR) fixation procedure or cryo-field emission scanning electron microscopy revealed a reduction in capsular material, as determined in detail for a serotype 3 pneumococcal strain. The amount of polysaccharide in the serotype 3 capsule was also determined after intranasal infection of mice. This study illustrates for the first time the phenotypic variation of the polysaccharide capsule in the initial phase of pneumococcal infections. The modified LRR fixation allowed monitoring of the state of capsule expression of pathogens during the infectious process.

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.

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.

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.

Streptococcus pneumoniae-induced p38 MAPK-dependent Phosphorylation of RelA at the Interleukin-8 Promotor

Streptococcus pneumoniae is the major cause of community-acquired pneumonia and one of the most common causes of death by infectious disease in industrialized countries. Little is known concerning the mechanisms of target cell activation in this disease. The present study shows that NF-κB and p38 MAPK signaling pathways contribute to chemokine synthesis by lung epithelial cells in response to pneumococci. In infected lungs of mice pneumococci stimulate expression of the interleukin (IL)-8 homolog keratinocyte-derived chemokine and granulocyte-macrophage colony-stimulating factor, as well as activate p38 MAPK. Human bronchial epithelium was chosen as a cellular model, because it establishes the first barrier against pathogens, and little is known about its function in innate immunity. Pneumococci infection induces expression of IL-8 and granulocyte-macrophage colony-stimulating factor as well as activation of p38 MAPK in human bronchial epithelial cells (BEAS-2B). Inhibition of p38 MAPK activity by SB202190 and SB203580 blocks pneumococci-induced cytokine release. In mouse lungs in vivo as well as in cultured cells, pneumococci activate NF-κBinanIκB kinase-dependent manner. Inhibition of p38 MAPK by chemical inhibitors or by RNA interference targeting p38α reduces pneumococci-induced NF-κB-dependent gene transcription. Blockade of p38 activity did not affect inducible nuclear translocation and recruitment of NF-κB/RelA to the IL-8 promotor but did reduce the level of phosphorylated RelA (serine 536) at IL-8 promotor and inhibited pneumococci-mediated recruitment of RNA polymerase II to IL-8 promotor. Thus, p38 MAPK contributes to pneumococci-induced chemokine transcription by modulating p65 NF-κB-mediated transactivation.

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

In mice, different alleles of the mNAIP5 (murine neuronal apoptosis inhibitory protein-5)/mBirc1e gene determine whether macrophages restrict or support intracellular replication of Legionella pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection. In the resistant mice strains, the nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member mNAIP5/mBirc1e, as well as the NLR protein mIpaf (murine ICE protease-activating factor), are involved in recognition of Legionella flagellin and in restriction of bacterial replication. Human macrophages and lung epithelial cells support L. pneumophila growth, and humans can develop severe pneumonia (Legionnaires disease) after Legionella infection. The role of human orthologs to mNAIP5/mBirc1e and mIpaf in this bacterial infection has not been elucidated. Herein we demonstrate that flagellin-deficient L. pneumophila replicate more efficiently in human THP-1 macrophages, primary monocyte-derived macrophages, and alveolar macrophages, and in A549 lung epithelial cells compared with wild-type bacteria. Additionally, we note expression of the mNAIP5 ortholog hNAIP in all cell types examined, and expression of hIpaf in human macrophages. Gene silencing of hNAIP or hIpaf in macrophages or of hNAIP in lung epithelial cells leads to an enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication. In contrast to experiments with wild-type L. pneumophila, hNAIP or hIpaf knock-down affects the (enhanced) replication of flagellin-deficient Legionella only marginally. In conclusion, hNAIP and hIpaf mediate innate intracellular defense against flagellated Legionella in human cells.

CEACAM1 inhibits Toll-like receptor 2-triggered antibacterial responses of human pulmonary epithelial cells.

Although Moraxella catarrhalis and Neisseria meningitidis are important human pathogens, they often colonize the human respiratory tract without causing overt clinical symptoms. Both pathogens express structurally unrelated proteins that share the ability to stimulate the adhesion molecule CEACAM1 expressed on human cells. Here we demonstrate that the interaction of CEACAM1 with ubiquitous surface protein A1 expressed on M. catarrhalis or with opacity-associated proteins on N. meningitidis resulted in reduced Toll-like receptor 2–initiated transcription factor NF-κB–dependent inflammatory responses of primary pulmonary epithelial cells. These inhibitory effects were mediated by tyrosine phosphorylation of the immunoreceptor tyrosine-based inhibitory motif of CEACAM1 and by recruitment of the phosphatase SHP-1, which negatively regulated Toll-like receptor 2–dependent activation of the phosphatidylinositol 3-OH kinase–Akt kinase pathway. Our results identify a CEACAM1-dependent immune-evasion strategy.

Role of pneumolysin for the development of acute lung injury in pneumococcal pneumonia.

Objective:Acute respiratory failure is a significant complication of severe pneumococcal pneumonia. In a mouse model, we observed early-onset lung microvascular leakage after pulmonary infection with Streptococcus pneumoniae, and we hypothesized that the important virulence factor pneumolysin may be the direct causative agent. Design:Controlled, in vivo, ex vivo, and in vitro laboratory study. Setting:Laboratory. Subjects:Female mice, 8–12 wks old. Interventions:Ventilated and blood-free perfused murine lungs were challenged with recombinant pneumolysin via the airways as well as via the vascular bed. In addition, we analyzed the impact of pneumolysin on electrical cell impedance and hydraulic conductivity of human umbilical vein endothelial cell (HUVEC) and alveolar epithelial cell (A549) monolayers. Measurements and Main Results:Aerosolized pneumolysin dose-dependently increased capillary permeability with formation of severe lung edema but did not affect pulmonary vascular resistance. Intravascular pneumolysin caused an impressive dose-dependent increase in pulmonary vascular resistance and in lung microvascular permeability. By immunohistochemistry, pneumolysin was detected mainly in endothelial cells of pulmonary arterial vessels, which concomitantly displayed strong vasoconstriction. Moreover, pneumolysin increased permeability of HUVEC and A549 monolayers. Interestingly, immunofluorescence of endothelial cell monolayers exposed to pneumolysin showed gap formation and moderate stress fiber generation. Conclusions:Pneumolysin may play a central role for early-onset acute lung injury due to severe pneumococcal pneumonia by causing impairment of pulmonary microvascular barrier function and severe pulmonary hypertension.

Statins Control Oxidized LDL-Mediated Histone Modifications and Gene Expression in Cultured Human Endothelial Cells

Objective—Activation of the endothelium by oxidized low-density lipoprotein (oxLDL) has been implicated in the development of atherosclerosis. Histone modifications impact on the transcriptional activity state of genes. We tested the hypothesis that oxLDL-induced inflammatory gene expression is regulated by histone modifications and experienced the effect of statins on these alterations. Methods and Results—OxLDL-related interleukin-8 (IL-8) and monocyte-chemoattractant protein-1 (MCP-1) secretion in endothelial cells was reduced by statins but enhanced by histone deacetylase inhibitors. OxLDL induced lectin-like oxidized LDL receptor-1 (LOX-1) and extracellular regulated kinases (ERK1/2)-dependent acetylation of histone H3 and H4 as well as phosphorylation of histone H3, both globally and on the promoters of il8 and mcp1. Pretreatment of oxLDL-exposed cells with statins reduced the above mentioned histone modification, as well as recruitment of CREB binding protein (CBP) 300, NF-&kgr;B, and of RNA polymerase II but prevented loss of binding of histone deacetylase (HDAC)-1 and -2 at the il8 and mcp1 gene promoters. OxLDL reduced HDAC1 and 2 expression, and statins partly restored global HDAC-activity. Statin-related effects were reverted with mevalonate. In situ experiments indicated decreased expression of HDAC2 in endothelial cells in atherosclerotic plaques of human coronary arteries. Conclusions—Histone modifications seem to play an important role in atherosclerosis.

Moraxella catarrhalis is internalized in respiratory epithelial cells by a trigger-like mechanism and initiates a TLR2- and partly NOD1-dependent inflammatory immune response

Moraxella catarrhalis is an important pathogen in patients with chronic obstructive lung disease (COPD). While M. catarrhalis has been categorized as an extracellular bacterium so far, the potential to invade human respiratory epithelium has not yet been explored. Our results obtained by electron and confocal microscopy demonstrated a considerable potential of M. catarrhalis to invade bronchial epithelial (BEAS‐2B) cells, type II pneumocytes (A549) and primary small airway epithelial cells (SAEC). Moraxella invasion was dependent on cellular microfilament as well as on bacterial viability, and characterized by macropinocytosis leading to the formation of lamellipodia and engulfment of the invading organism into macropinosomes, thus indicating a trigger‐like uptake mechanism. In addition, the cells examined expressed TLR2 as well as NOD1, a recently found cytosolic protein implicated in the intracellular recognition of bacterial cell wall components. Importantly, inhibition of TLR2 or NOD1 expression by RNAi significantly reduced the M. catarrhalis‐induced IL‐8 secretion. The role of TLR2 and NOD1 was further confirmed by overexpression assays in HEK293 cells. Overall, M. catarrhalis may employ lung epithelial cell invasion to colonize and to infect the respiratory tract, nonetheless, the bacteria are recognized by cell surface TLR2 and the intracellular surveillance molecule NOD1.