Jean-Paul Klein

NF-kappaB and the MAP kinases/AP-1 pathways are both involved in interleukin-6 and interleukin-8 expression in fibroblast-like synoviocytes stimulated by protein I/II, a modulin from oral streptococci.

As in rheumatoid arthritis (RA), it was demonstrated recently that bacterial fragments of DNA or rRNA are present in the joint and therefore could play a role in inducing or perpetuating the disease, this work was initiated to define mechanisms that account for the stimulatory activities of the oral streptococcal modulin, protein I/II, on fibroblast‐like synoviocytes (FLSs) from RA patients. FLSs from RA patients were stimulated with protein I/II, and expression of interleukin (IL)‐6 and IL‐8 mRNA was evaluated by reverse transcription–polymerase chain reaction (RT–PCR). Immunoblotting by antibodies specific for activated forms of MAPKs and electrophoretic mobility shift assays (EMSAs) were performed to study downstream signalling, which allowed the synthesis of IL‐6 and IL‐8. We reported that protein I/II interactions with FLSs from RA patients trigger the synthesis and release of IL‐6 and IL‐8. We also demonstrated that protein I/II enhances the phosphorylation of ERK 1/2, p38 and JNKs and that ERK 1/2 and JNK MAPKs seem to play a more important role than p38 in protein I/II‐mediated synthesis of IL‐6 and IL‐8. Our experiments also indicated that stimulation of FLSs with protein I/II induces nuclear translocation of NF‐κB, AP‐1‐binding activity and that NF‐κB plays a major role in IL‐6 and IL‐8 secretion from activated cells.

Vaccination with Toxoplasma gondii SAG-1 Protein Is Protective against Congenital Toxoplasmosis in BALB/c Mice but Not in CBA/J Mice

ABSTRACT We evaluated the effect of vaccination with the SAG1 protein of Toxoplasma gondii against congenital toxoplasmosis in mice with different genetic backgrounds. In BALB/c mice (H-2d), vaccination reduced the number of infected fetuses by 50% and was associated with a mixed type 1 and type 2 immunity. In CBA/J mice (H-2k), vaccination increased the number of infected fetuses by 50% and was associated with a predominant type 2 response. Our results indicate that the effect of vaccination with SAG1 is controlled by the genetic background of the mouse.

Cross Talk between MyD88 and Focal Adhesion Kinase Pathways

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in signaling downstream of integrins, linking bacterial detection, cell entry, and initiation of proinflammatory response through MAPKs and NF-κB activation. In this study, using protein I/II from Streptococcus mutans as a model activator of FAK, we investigated the potential link between FAK and TLR pathways. Using macrophages from TLR- or MyD88-deficient mice, we report that MyD88 plays a major role in FAK-dependent protein I/II-induced cytokine release. However, response to protein I/II stimulation was independent of TLR4, TLR2, and TLR6. The data suggest that there is a cross talk between FAK and MyD88 signaling pathways. Moreover, MyD88-dependent, LPS-induced IL-6 secretion by human and murine fibroblasts required the presence of FAK, confirming that MyD88 and FAK pathways are interlinked.

Role of NK Cells and Gamma Interferon in Transplacental Passage of Toxoplasma gondii in a Mouse Model of Primary Infection

ABSTRACT Protective immunity in mice infected with Toxoplasma gondii is mainly mediated by NK cells, CD4 and CD8 T cells, and type 1 cytokines, such as gamma interferon (IFN-γ). To clarify the roles of NK cells and IFN-γ in protection against primary congenital toxoplasmosis, we used recombination activating gene 2 knockout (RAG-2−/−) mice, which lack T and B lymphocytes, in comparison with the wild-type BALB/c model. RAG-2−/− mice had a significantly lower risk of fetal toxoplasmosis than BALB/c mice (25 versus 63.9%; P = 0.003). This protection was associated with an increased number of maternal NK cells, IFN-γ secretion by spleen cells, and decreased parasitemia. In the RAG-2−/− mice, NK cell depletion increased both the rate of fetal infection, to 56.5% (P = 0.02), and the blood parasite burden. Conversely, in the BALB/c mice, this treatment did not modify maternofetal transmission or the blood parasite burden. Neutralization of IFN-γ in both infected RAG-2−/− and BALB/c mice decreased congenital Toxoplasma transmission, contrasting with an exacerbation of maternal infection. These data suggest that a partially protective immunity against congenital toxoplasmosis is achieved due to the increased number of NK cells in RAG-2−/− mice. However, it seems that IFN-γ enhances, directly or indirectly, the transplacental transmission.

Design of highly immunogenic liposomal constructs combining structurally independent B cell and T helper cell peptide epitopes

We have designed liposomal diepitope constructs that allow the physical combination, within the same vesicle, of B and Th epitopes as structurally separate entities. The immune response against such constructs was explored using TPEDPTDPTDPQDPSS (TPE), a B cell epitope originating from a Streptococcus mutans surface adhesin and QYIKANSKFIGITEL (QYI), a “universal” Th epitope from tetanus toxin. The two peptides were linked to the outer surface of small (diameter approximately 100 nm) unilamellar liposomes by covalent conjugation to two different anchors. To that end we have developed a strategy that allows the controlled chemical coupling of TPE and QYI, functionalized at their N terminus with a thiol, to preformed liposomes containing thiol‐reactive derivatives of phosphatidylethanolamine and the lipopeptide S‐[2,3‐bis (palmitoyloxy)‐(2‐RS)‐propyl]‐N‐palmitoyl‐(R)‐cysteinyl‐alanyl‐glycine (Pam3CAG), respectively. This synthetic construct (administered i.  p. to BALB/c mice) induced highly intense (titers > 20 000), anamnestic and long‐lasting (over 2 years) immune responses, indicating that this strategy is successful. Two parameters were of prime importance to elicit this response with our liposomal diepitope constructs: (1) the simultaneous expression of B and Th epitopes on the same vesicle, and (2) the lipopeptide Pam3CAG anchor of the Th epitope QYI could not be replaced by a phosphatidylethanol‐amine anchor (a lesser immune response was observed). Analysis of the antibody response revealed a complex pattern; thus, besides the humoral response (production of IgG1, IgG2a, IgG2b) a superposition of a T‐independent (TI‐2 type) response was also found (IgM and IgG3). These results indicate that liposomal diepitope constructs could be attractive in the development of synthetic peptide‐based vaccines.

Role of gamma interferon and T cells in congenital Toxoplasma transmission

In the BALB/c mouse model, primary infection with Toxoplasma gondii during the second third of gestation leads to a high percentage of infected foetuses. However, immunity induced by infection contracted before pregnancy prevents parasites from crossing the placenta and completely protects the foetuses, as well as the pregnant women. In order to clarify the roles of CD4+, CD8+T lymphocytes and IFN‐γ in this protection, pregnant BALB/c mice were treated with depleting monoclonal antibodies against CD4, CD8, IFN‐γ, or control antibody. Only the foetuses of the groups treated with anti‐CD8 and anti‐IFN‐γ antibodies developed congenital toxoplasmosis. The maternal production of IFN‐γ was depressed in the mice depleted of CD4 and CD8 cells (P < 0·001). Determination of the blood parasite load demonstrated that materno‐foetal transmission of T. gondii correlates with maternal parasitaemia. Together, these results show that CD8+T lymphocytes and IFN‐γ play an important role in protection against congenital toxoplasmosis during reinfection.

Toxoplasma gondii-induced foetal resorption in mice involves interferon-γ-induced apoptosis and spiral artery dilation at the maternofoetal interface

The severity of congenital toxoplasmosis depends on the stage of the pregnancy at which infection takes place. Infection during the first trimester generally leads to miscarriage, through an unknown mechanism. Toxoplasma gondii infection is normally controlled by a strong Th1-type response with IFN-gamma production. To investigate the mechanisms of foetal resorption induced by T. gondii, pregnant Swiss-Webster mice were infected 1 day post coïtum with the avirulent Me49 strain. Mated recipients were examined at mid-gestation. Few parasites and no cytolytic effects were detected 10 days post coïtum in implantation sites undergoing resorption. Resorption was accompanied by haemorrhage, spiral artery dilation, hypocellularity of the decidua basalis, apoptosis of placental cells, a decline in uterine mature natural killer cell numbers, increased indoleamine 2,3-dioxygenase mRNA levels and reduced IL-15 mRNA levels. Given the role of IFN-gammaR(-/-) in non-infectious abortive processes, IFN-gammaR(-/-) mice were used to investigate its local role in T. gondii-induced foetal resorption. IFN-gammaR(-/-) mice showed 50% less foetal resorption than their wild-type counterparts, and spiral artery dilation and placental cell apoptosis were both abolished. These results strongly suggest that, at least in mice, T. gondii-induced abortion in early gestation is not due to a direct action of the parasite at the maternofoetal interface but rather to massive IFN-gamma release.

Toxoplasma gondii regulates ICAM-1 mediated monocyte adhesion to trophoblasts

Materno‐foetal transmission causes one of the most serious forms of infection with the intracellular protozoan parasite Toxoplasma gondii. In the placenta, trophoblast cells constitute the barrier between maternal circulation and foetal tissue. We looked at the factors that determine the extent of cell adhesion to human BeWo trophoblast cells during T. gondii infection. BeWo monolayers stimulated with the supernatant of T. gondii‐infected PBMC showed a large increase in THP‐1 cell adhesion and upregulation of the intercellular adhesion molecule (ICAM)‐1. Neutralization of cytokines by corresponding antibodies demonstrated that anti‐IFN‐γ, but not anti‐TNF‐α or anti‐IL‐1β, led to a significant reduction of THP‐1 adhesion to a BeWo monolayer. Treatment of BeWo cells with single cytokines failed to induce upregulation of adhesion. In contrast, simultaneous treatment with IFN‐γ and either TNF‐α or IL‐1β mimicked strongly the effect of infected cell supernatant. The results suggest that IFN‐γ plays a pivotal role in the cell adhesion process through upregulation of ICAM‐1 and in the process of congenital transmission of T. gondii.

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor alpha in the monocyte cell line THP-1.

The induction of tumour necrosis factor (TNF)‐α from the monocytic cell line THP‐1 by the streptococcal antigen I/II from Streptococcus mutans serotype f (protein I/IIf) was studied by use of recombinant polypeptides containing discrete domains of the protein. The derivatives carrying the N‐terminal alanine‐rich region (A region) and the adjacent variable region (extended V region) of the protein bound to THP‐1 cell extracts in a saturable fashion, and one derivative lacking both the A and the extended V regions was not able to bind monocyte cell extracts, suggesting that the domains responsible for the binding of protein I/IIf to monocytes were the A and the extended V regions. Sodium metaperiodate pretreatment of THP‐1 cell extracts, tunicamycin pretreatment of monocyte cells or competition with N‐acetyl neuraminic acid (NANA) and fucose resulted in a 45–70% reduction in binding activity of the derivatives carrying the extended V region, demonstrating the lectin‐like mode of recognition of the monocytic receptor by the extended V region and the role of NANA and fucose in this recognition process. Besides, the stimulation of monocytes to release TNF‐α by the derivatives containing the A region and the extended V region was effective and was not affected by the addition of polymyxin B or vitamin D3, suggesting that CD14 does not play the role of receptor in stimulation of monocytes by protein I/IIf to release TNF‐α.

Involvement of alpha5beta1 integrins in interleukin 8 production induced by oral viridans streptococcal protein I/IIf in cultured endothelial cells.

Using human endothelial cells, we define a mechanism that accounts for the induction of interleukin 8 (IL‐8) by protein I/IIf, an adhesin from Streptococcus mutans serotype f. We report that protein I/IIf interactions with endothelial cells increased the tyrosine phosphorylation of three cellular components with relative mass of 145 000, 125 000 and 70 000 in endothelial cells. These proteins were identified as phospholipase Cγ (PLCγ), focal adhesion kinase (FAK) and paxillin after immunoprecipitation with monoclonal antibodies (mAbs) and immunoblotting with antiphosphotyrosine mAbs. These results suggested that β1 integrins could be one of the components implicated in the modulin activity of protein I/IIf. By incubating protein I/IIf with either purified α5β1 integrins or with α5β1 integrins overexpressing CHO cells, we demonstrated that α5β1 integrins act as cell receptors for protein I/IIf. We also showed that protein I/IIf interactions with α5β1 integrins lead to IL‐8 secretion. Using specific inhibitors, we demonstrated that protein I/IIf‐induced IL‐8 release involves mitogen‐activated protein kinases (MAPKs), and that PLCγ and PKC also seem to contribute to protein I/IIf stimulation. However, PI‐3K activation is not involved in IL‐8 release. Altogether, these results indicate that, after binding to α5β1 integrins, protein I/IIf induces IL‐8 release by activating the MAPKs signalling pathways.