Ayelet Zauberman

Stress activated protein kinase p38 is involved in IL-6 induced transcriptional activation of STAT3.

The pleiotropic cytokine interleukin-6 (IL-6) induces acute phase protein expression in HepG2 human hepatoma cells and promotes the growth of mouse B9 hybridoma. The signaling cascades leading to these biological functions are only partially known. We analysed the involvement of MAPK homologues in IL-6 transduction pathways and found that interleukin-6 triggered activation of p38 stress-activated protein kinase (p38) but not of jun kinase. p38 activity was required for biological functions including acute phase protein secretion from HepG2 hepatoma and proliferation of B9 hybridoma cells. Using a reporter gene construct containing a 190 bp promoter fragment of the acute phase protein haptoglobin we found that p38 is involved in transcriptional activation of the haptoglobin promoter by STAT3 but not by NF-IL6. Thus, we present evidence for a role of p38 in IL-6 induced functions and a possible cross-talk between this MAPK homologue and the STAT pathway.

Involvement of p21 WAF1/Cip1 , CDK4 and Rb in activin A mediated signaling leading to hepatoma cell growth inhibition

Cytokines are growth inhibitory in a target cell specific manner. The signaling pathways that characterize each cell type play a crucial role in determining the responsiveness to cytokine triggering. Activin A has been shown to suppress the growth of primary hepatocytes. Similarly, the human HepG2 hepatoma cell line was growth arrested by activin A as judged by lack of cell proliferation and suppression of DNA synthesis. In HepG2 cells activin A further induced accumulation of retinoblastoma protein in the hypophosphorylated form known to prevent entrance into S phase. This finding implies the involvement of cyclin dependent kinases and CDK inhibitors. Examination of HepG2 cells following addition of activin A revealed reduced expression of CDK4 and conversely, an increase in the CKI p21WAF1/Cip1. This accumulation of p21WAF1/Cip1 protein was partly due to increased transcriptional activity. Functional inactivation of p53, using a miniprotein that oligomerizes with p53 and abrogates DNA binding, abolished the ability of activin A to induce transcriptional activation from the p21WAF1/Cip1 promoter. Thus, activin A, like transforming growth factor β, seems to suppress cell growth through the downstream target Rb. However, each of these cytokines seem to operate through a distinct pathway.

Interaction of Yersinia pestis with Macrophages: Limitations in YopJ-Dependent Apoptosis

ABSTRACT The enteropathogenic Yersinia strains are known to downregulate signaling pathways in macrophages by effectors of the type III secretion system, in which YopJ/YopP plays a crucial role. The adverse effects of Yersinia pestis, the causative agent of plague, were examined by infecting J774A.1 cells, RAW264.7 cells, and primary murine macrophages with the EV76 strain and with the fully virulent Kimberley53 strain. Y. pestis exerts YopJ-dependent suppression of tumor necrosis factor alpha secretion and phosphorylation of mitogen-activated protein kinases and thus resembles enteropathogenic Yersinia. However, Y. pestis is less able to activate caspases, to suppress NF-κB activation, and to induce apoptosis in macrophages than the high-virulence Y. enterocolitica WA O:8 strain. These differences appear to be related to lower efficiency of YopJ effector translocation by Y. pestis. The efficiencies of effector translocation and of apoptosis induction can be enhanced either by using a high bacterial load in a synchronized infection or by overexpressing exogenous YopJ in Y. pestis. Replacing YopJ with the homologous Y. enterocolitica effector YopP can further enhance these effects. Overexpression of YopP in a yopJ-deleted Y. pestis background leads to rapid and effective translocation into target cells, providing Y. pestis with the high cytotoxic potential of Y. enterocolitica WA O:8. We suggest that the relative inferiority of Y. pestis in triggering cell death in macrophages may be advantageous for its in vivo propagation in the early stages of infection.

Smad Proteins Suppress CCAAT/Enhancer-binding Protein (C/EBP) β- and STAT3-mediated Transcriptional Activation of the Haptoglobin Promoter

Activin A, a member of the transforming growth factor β (TGFβ) superfamily, blocks interleukin (IL)-6 biological functions. The molecular basis of the influence of this TGFβ signaling on the IL-6 receptor triggered cascade is unknown. We studied IL-6-induced secretion of the acute phase protein haptoglobin by hepatoma cells. Overexpression of the C/EBPβ gene, a downstream effector in the IL-6 pathway, activated transcription from the haptoglobin promoter. This was abolished by either a constitutively active form of activin A type IB receptor (CAactRIB) or by a combination of Smad3 and Smad4. Similarly, Smads abolished transcriptional activation by co-stimulation with IL-6 and STAT3. The transcription co-activator p300 partially overcame the suppressive effect of Smads. Electrophoretic mobility shift assays indicated that C/EBPβ binding to haptoglobin promoter DNA was reduced by over-expression of CAactRIB and Smad4. We thus show that Smad proteins operate as transcription inhibitors on target genes of the IL-6 induced pathway. The effect of Smads is exerted on components of the transcription activation complex and may also involve interference with DNA binding. This study thus depicts molecular sites of interaction between the TGFβ superfamily and the IL-6 signaling cascades.

The NlpD Lipoprotein Is a Novel Yersinia pestis Virulence Factor Essential for the Development of Plague

Yersinia pestis is the causative agent of plague. Previously we have isolated an attenuated Y. pestis transposon insertion mutant in which the pcm gene was disrupted. In the present study, we investigated the expression and the role of pcm locus genes in Y. pestis pathogenesis using a set of isogenic surE, pcm, nlpD and rpoS mutants of the fully virulent Kimberley53 strain. We show that in Y. pestis, nlpD expression is controlled from elements residing within the upstream genes surE and pcm. The NlpD lipoprotein is the only factor encoded from the pcm locus that is essential for Y. pestis virulence. A chromosomal deletion of the nlpD gene sequence resulted in a drastic reduction in virulence to an LD50 of at least 107 cfu for subcutaneous and airway routes of infection. The mutant was unable to colonize mouse organs following infection. The filamented morphology of the nlpD mutant indicates that NlpD is involved in cell separation; however, deletion of nlpD did not affect in vitro growth rate. Trans-complementation experiments with the Y. pestis nlpD gene restored virulence and all other phenotypic defects. Finally, we demonstrated that subcutaneous administration of the nlpD mutant could protect animals against bubonic and primary pneumonic plague. Taken together, these results demonstrate that Y. pestis NlpD is a novel virulence factor essential for the development of bubonic and pneumonic plague. Further, the nlpD mutant is superior to the EV76 prototype live vaccine strain in immunogenicity and in conferring effective protective immunity. Thus it could serve as a basis for a very potent live vaccine against bubonic and pneumonic plague.

Yersinia pestis Endowed with Increased Cytotoxicity Is Avirulent in a Bubonic Plague Model and Induces Rapid Protection against Pneumonic Plague

An important virulence strategy evolved by bacterial pathogens to overcome host defenses is the modulation of host cell death. Previous observations have indicated that Yersinia pestis, the causative agent of plague disease, exhibits restricted capacity to induce cell death in macrophages due to ineffective translocation of the type III secretion effector YopJ, as opposed to the readily translocated YopP, the YopJ homologue of the enteropathogen Yersinia enterocolitica O∶8. This led us to suggest that reduced cytotoxic potency may allow pathogen propagation within a shielded niche, leading to increased virulence. To test the relationship between cytotoxic potential and virulence, we replaced Y. pestis YopJ with YopP. The YopP-expressing Y. pestis strain exhibited high cytotoxic activity against macrophages in vitro. Following subcutaneous infection, this strain had reduced ability to colonize internal organs, was unable to induce septicemia and exhibited at least a 107-fold reduction in virulence. Yet, upon intravenous or intranasal infection, it was still as virulent as the wild-type strain. The subcutaneous administration of the cytotoxic Y. pestis strain appears to activate a rapid and potent systemic, CTL-independent, immunoprotective response, allowing the organism to overcome simultaneous coinfection with 10,000 LD50 of virulent Y. pestis. Moreover, three days after subcutaneous administration of this strain, animals were also protected against septicemic or primary pneumonic plague. Our findings indicate that an inverse relationship exists between the cytotoxic potential of Y. pestis and its virulence following subcutaneous infection. This appears to be associated with the ability of the engineered cytotoxic Y. pestis strain to induce very rapid, effective and long-lasting protection against bubonic and pneumonic plague. These observations have novel implications for the development of vaccines/therapies against Y. pestis and shed new light on the virulence strategies of Y. pestis in nature.

Discordance in the Effects of Yersinia pestis on the Dendritic Cell Functions Manifested by Induction of Maturation and Paralysis of Migration

ABSTRACT The encounter between invading microorganisms and dendritic cells (DC) triggers a series of events which include uptake and degradation of the microorganism, induction of a maturation process, and enhancement of DC migration to the draining lymph nodes. Various pathogens have developed strategies to counteract these events as a measure to evade the host defense. In the present study we found that interaction of the Yersinia pestis EV76 strain with DC has no effect on cell viability and is characterized by compliance with effective maturation, which is manifested by surface display of major histocompatibility complex class II, of costimulatory markers, and of the chemokine receptor CCR7. This is in contrast to maturation inhibition and cell death induction exerted by the related species Yersinia enterocolitica WA O:8. Y. pestis interactions with DC were found, however, to impair functions related to cytoskeleton rearrangement. DC pulsed with Y. pestis failed to adhere to solid surfaces and to migrate toward the chemokine CCL19 in an in vitro transmembrane assay. Both effects were dependent on the presence of the pCD1 virulence plasmid and on a bacterial growth shift to 37°C prior to infection. Moreover, while instillation of a pCD1-cured Y. pestis strain into mouse airways triggered effective transport of alveolar DC to the mediastinal lymph node, instillation of Y. pestis harboring the plasmid failed to do so. Taken together, these results suggest that virulence plasmid-dependent impairment of DC migration is the major mechanism utilized by Y. pestis to subvert DC function.

Neutralization of Yersinia pestis-mediated macrophage cytotoxicity by anti-LcrV antibodies and its correlation with protective immunity in a mouse model of bubonic plague

Plague is a life-threatening disease caused by Yersinia pestis, for which effective-licensed vaccines and reliable predictors of in vivo immunity are lacking. V antigen (LcrV) is a major Y. pestis virulence factor that mediates translocation of the cytotoxic Yersinia protein effectors (Yops). It is a well-established protective antigen and a part of currently tested plague subunit vaccines. We have developed a highly sensitive in vitro macrophage cytotoxicity neutralization assay which is mediated by anti-LcrV antibodies; and studied the potential use of these neutralizing antibodies as an in vitro correlate of plague immunity in mice. The assay is based on a Y. pestis strain with enhanced cytotoxicity to macrophages in which endogenous yopJ was replaced by the more effectively translocated yopP of Y. enterocolitica O:8. Mice passively immunized with rabbit anti-LcrV IgG or actively immunized with recombinant LcrV were protected against lethal doses of a virulent Y. pestis strain, in a mouse model of bubonic plague. This protection significantly correlated with the in vitro neutralizing activity of the antisera but not with their corresponding ELISA titers. In actively immunized mice, a cutoff value for serum neutralizing activity, above which survival was assured with high degree of confidence, could be established for different vaccination regimes. The impact of overall findings on the potential use of serum neutralizing activity as a correlate of protective immunity is discussed.

The mesenchyme expresses T cell receptor mRNAs: Relevance to cell growth control

The mesenchyme plays a crucial regulatory role in organ formation and maintenance. However, comprehensive molecular characterization of these cells is lacking. We found unexpectedly that primary mesenchyme, as well as mesenchymal cell clones, express T cell receptor (TCR)αβ mRNAs, lacking the variable region. Immunological and genetic evidence support the expression of a corresponding TCRβ protein. Additionally, mRNAs encoding TCR complex components including CD3 and ζ chain are present. A relatively higher expression of the mesenchymal TCRβ mRNA by cultured mesenchymal cell clones correlates with fast growth, whereas poorly expressing cells are slow growers and are contact inhibited. The clones that express relatively higher amount of the TCR mRNA exhibit an increased capacity to form tumors in nude mice. However, the expression of this mRNA in the mesenchyme is not per se leading to tumorigenesis, as demonstrated by primary mesenchyme that does not form tumors in mice while expressing moderate amounts of the TCR transcripts. The expression of mesencymal TCRβ was confined to the G2/M phases of the cell cycle in the MBA-13 mesenchymal cell line. This cell cycle dependent expression, considered together with the correlation between growth properties and the level of TCR expression by cell clones, implies association of mesenchymal TCR with cell growth control.

Circumventing Y. pestis Virulence by Early Recruitment of Neutrophils to the Lungs during Pneumonic Plague

Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated with a delayed immune response in the lungs. Because neutrophils are the first immune cells recruited to sites of infection, we investigated the mechanisms responsible for their delayed homing to the lung. During the first 24 hr after pulmonary infection with a fully virulent Y. pestis strain, no significant changes were observed in the lungs in the levels of neutrophils infiltrate, expression of adhesion molecules, or the expression of the major neutrophil chemoattractants keratinocyte cell-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor (G-CSF). In contrast, early induction of chemokines, rapid neutrophil infiltration and a reduced bacterial burden were observed in the lungs of mice infected with an avirulent Y. pestis strain. In vitro infection of lung-derived cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition of chemoattractants expression. However, the recruitment of neutrophils to the lungs of mice infected with the mutant was still delayed and associated with rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and G-CSF mRNA levels in the lungs were up-regulated early after infection with the mutant, their protein levels remained constant, suggesting that Y. pestis may employ additional mechanisms to suppress early chemoattractants induction in the lung. It therefore seems that prevention of the early influx of neutrophils to the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led to rapid homing of neutrophils to the lung followed by a reduction in bacterial counts at 24 hr post-infection and improved survival rates. These observations shed new light on the virulence mechanisms of Y. pestis during pneumonic plague, and have implications for the development of novel therapies against this pathogen.