Matthias Husmann

Vascular Leakage in Severe Dengue Virus Infections: A Potential Role for the Nonstructural Viral Protein NS1 and Complement

BACKGROUND Vascular leakage and shock are the major causes of death in patients with dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Thirty years ago, complement activation was proposed to be a key underlying event, but the cause of complement activation has remained unknown. METHODS The major nonstructural dengue virus (DV) protein NS1 was tested for its capacity to activate human complement in its membrane-associated and soluble forms. Plasma samples from 163 patients with DV infection and from 19 patients with other febrile illnesses were prospectively analyzed for viral load and for levels of NS1 and complement-activation products. Blood and pleural fluids from 9 patients with DSS were also analyzed. RESULTS Soluble NS1 activated complement to completion, and activation was enhanced by polyclonal and monoclonal antibodies against NS1. Complement was also activated by cell-associated NS1 in the presence of specific antibodies. Plasma levels of NS1 and terminal SC5b-9 complexes correlated with disease severity. Large amounts of NS1, complement anaphylatoxin C5a, and the terminal complement complex SC5b-9 were present in pleural fluids from patients with DSS. CONCLUSIONS Complement activation mediated by NS1 leads to local and systemic generation of anaphylatoxins and SC5b-9, which may contribute to the pathogenesis of the vascular leakage that occurs in patients with DHF/DSS.

Clathrin- and Caveolin-Independent Entry of Human Papillomavirus Type 16—Involvement of Tetraspanin-Enriched Microdomains (TEMs)

Background Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway. Methodology/Principal Findings Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16. Conclusions/Significance Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

Possible Protective Role for C-Reactive Protein in Atherogenesis Complement Activation by Modified Lipoproteins Halts Before Detrimental Terminal Sequence

Background—Previous work indicated that enzymatically remodeled LDL (E-LDL) might activate complement in atherosclerotic lesions via a C-reactive protein (CRP)–dependent and CRP-independent pathway. We sought to substantiate this contention and determine whether both pathways drive the sequence to completion. Methods and Results—E-LDL was prepared by sequential treatment of LDL with a protease and cholesteryl esterase. Trypsin, proteinase K, cathepsin H, or plasmin was used with similar results. Functional tests were used to assess total complement hemolytic activity, and immunoassays were used to demonstrate C3 cleavage and to quantify C3a, C4a, C5a, and C5b-9. E-LDL preparations activated complement to completion, independent of CRP, when present above a threshold concentration (100 to 200 μg/mL in 5% serum). Below the threshold, all E-LDL preparations activated complement in dependence of CRP, but the pathway then halted before the terminal sequence. Native LDL and oxidized LDL did not activate complement under any circumstances tested. Immunohistological analyses corroborated the concept that CRP-dependent complement activation inefficiently generates C5b-9. Conclusions—Binding of CRP to E-LDL is the first trigger for complement activation in the atherosclerotic lesion, but the terminal sequence is thereby spared. This putatively protective function of CRP is overrun at higher E-LDL concentrations, so that potentially harmful C5b-9 complexes are generated.

Global functional analyses of cellular responses to pore-forming toxins.

Here we present the first global functional analysis of cellular responses to pore-forming toxins (PFTs). PFTs are uniquely important bacterial virulence factors, comprising the single largest class of bacterial protein toxins and being important for the pathogenesis in humans of many Gram positive and Gram negative bacteria. Their mode of action is deceptively simple, poking holes in the plasma membrane of cells. The scattered studies to date of PFT-host cell interactions indicate a handful of genes are involved in cellular defenses to PFTs. How many genes are involved in cellular defenses against PFTs and how cellular defenses are coordinated are unknown. To address these questions, we performed the first genome-wide RNA interference (RNAi) screen for genes that, when knocked down, result in hypersensitivity to a PFT. This screen identifies 106 genes (∼0.5% of genome) in seven functional groups that protect Caenorhabditis elegans from PFT attack. Interactome analyses of these 106 genes suggest that two previously identified mitogen-activated protein kinase (MAPK) pathways, one (p38) studied in detail and the other (JNK) not, form a core PFT defense network. Additional microarray, real-time PCR, and functional studies reveal that the JNK MAPK pathway, but not the p38 MAPK pathway, is a key central regulator of PFT-induced transcriptional and functional responses. We find C. elegans activator protein 1 (AP-1; c-jun, c-fos) is a downstream target of the JNK-mediated PFT protection pathway, protects C. elegans against both small-pore and large-pore PFTs and protects human cells against a large-pore PFT. This in vivo RNAi genomic study of PFT responses proves that cellular commitment to PFT defenses is enormous, demonstrates the JNK MAPK pathway as a key regulator of transcriptionally-induced PFT defenses, and identifies AP-1 as the first cellular component broadly important for defense against large- and small-pore PFTs.

Role of C-Reactive Protein in Atherogenesis. Can the Apolipoprotein E Knockout Mouse Provide the Answer?

Objective—Human C-reactive protein (CRP) was reported to accelerate atherosclerotic lesion development in male but not in female apolipoprotein E (apoE) knockout mice. Here, mice expressing rabbit CRP (rbCRP) were crossbred onto apoE knockout animals, and the effect on atherogenesis was studied. Methods and Results—Hemolytic complement activity could not be detected in apoE knockout mice. Furthermore, in contrast to human complement, neither rabbit nor human CRP complexed to modified low-density lipoprotein–activated murine complement. At 52 weeks, rbCRP levels were similar in male and female transgenic animals. Serum cholesterol levels were equivalent in female animals irrespective of rbCRP expression, whereas rbCRP–positive males had significantly higher serum cholesterol levels than the rbCRP-negative counterparts. All mice exhibited extensive atherosclerotic lesions, as studied en face, and no differences were noted between rbCRP-negative and rbCRP-positive animals. Atherosclerotic luminal obstruction of aortic arch and first-order neck branches did not differ significantly between rbCRP-positive and rbCRP-negative mice. There was no correlation between rbCRP levels and atherosclerotic lesion formation. Conclusions—No marked effect of rbCRP on the formation of moderately advanced atherosclerotic lesions could be discerned in the apoE knockout mouse. Because of the oddities of the mouse complement system, however, this may not be a good model to investigate the role of CRP in human atherosclerosis.

Elimination of a bacterial pore‐forming toxin by sequential endocytosis and exocytosis

Staphylococcus aureus α‐toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small β‐barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium‐independent mechanisms. Here we show that cells can internalize α‐toxin, that uptake is essential for cellular survival, and that pore‐complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome‐like structures, which we term toxosomes.

Atherogenic Properties of Enzymatically Degraded LDL: Selective Induction of MCP-1 and Cytotoxic Effects on Human Macrophages

The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor-dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic protein 1 (MCP-1) from macrophages and that expression of interleukin 8 or RANTES remains unchanged. Release of MCP-1 was preceded by a rapid expression of MCP-1 mRNA, which was detectable after 15 minutes, reached maximum levels after 1 hour, and remained detectable for 12 hours after exposure to concentrations as low as 10 microg/mL E-LDL. MCP-1 mRNA induction and protein release by E-LDL exceeded that evoked by oxidized LDL. Release of MCP-1 was dependent on de novo protein synthesis and on the activity of tyrosine kinases. At higher concentrations, E-LDL, but not oxidized LDL, exerted toxic effects on macrophages that in part appeared to be due to apoptosis. The results show that E-LDL possesses major properties of an atherogenic lipoprotein.

Subcytocidal Attack by Staphylococcal Alpha-Toxin Activates NF-κB and Induces Interleukin-8 Production

ABSTRACT Formation of transmembrane pores by staphylococcal alpha-toxin can provoke a spectrum of events depending on target cell species and toxin dose, and in certain cases, repair of the lesions has been observed. Here, we report that transcriptional processes are activated as a response of cells to low toxin doses. Exposure of monocytic (THP-1) or epithelial (ECV304) cells to 40 to 160 ng/ml alpha-toxin provoked a drop in cellular ATP level that was followed by secretion of substantial amounts of interleukin-8 (IL-8). Cells transfected with constructs comprising the proximal IL-8 promoter fused to luciferase or to green fluorescent protein cDNA exhibited enhanced reporter gene expression following toxin treatment. Electrophoretic mobility shift and immunofluorescence assays demonstrated that IL-8 secretion was preceded by activation of NF-κB. Transfection experiments conducted with p65/p50 double-deficient cells showed that activation of the IL-8 promoter/reporter by toxin was absolutely dependent on NF-κB. In contrast, this transcription factor was not required for lesion repair. Attack of cells by low doses of a pore-forming toxin can lead to transcriptional gene activation, which is followed by production of mediators that may contribute to the initiation and propagation of inflammatory lesions.

Potassium Regulates IL-1β Processing Via Calcium-Independent Phospholipase A2

We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1β by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1β and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1β processing was not due to perturbation of the export machinery for pro-IL-1β and IL-1β or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1β processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1β maturation.

Pore-forming toxins activate MAPK p38 by causing loss of cellular potassium.

Mitogen activated protein kinase (MAPK) p38 has emerged as a survival protein in cells that are attacked by bacterial toxins forming small membrane pores. Activation of p38 by pore forming toxins (PFT) has been attributed to osmotic stress, but here we show that loss of K+ is likely to be the critical parameter. Several lines of evidence support this conclusion: first, osmoprotection did not prevent p38-phosphorylation in alpha-toxin-loaded cells. Second, treatment of cells with a K+ ionophore, or simple incubation in K+-free medium sufficed to cause robust p38-phosphorylation. Third, media containing high [K+] prevented p38-activation by Staphylococcus aureus alpha-toxin, Vibrio cholerae cytolysin (VCC), Streptolysin O (SLO), or Escherichia coli hemolysin (HlyA), but did not impair activation by H2O2. Fourth, potential roles of LPS, TLR4, or calcium-influx were ruled out. Therefore, we propose that PFT trigger the p38 MAPK-pathway by causing loss of cellular K+.