Jens Ehrhardt

B Cell Development Undergoes Profound Modifications and Adaptations During Pregnancy in Mice

ABSTRACT Pregnancy hides an immunological riddle combining two antagonistic characteristics of immunology: the existence of a tolerance that allows the gestation of a semiallogeneic fetus and proper protection against pathogens threatening the health of the immunocompromised mother. Despite the fundamental role that B cells play in orchestrating an immune response, their behavior in the context of pregnancy has been barely investigated. Here we demonstrate that numbers of pre/pro and immature B cells were progressively diminished in the bone marrow (BM) of pregnant mice, leading to a reduced influx of B cells in blood and spleen. Correspondingly, lower levels of B cell-activating factor of the TNF family were observed in serum of pregnant mice. In contrast to immature B cells, mature B cells were accumulated in the BM during pregnancy. Accordingly, higher numbers of mature B cells were observed in the lymph nodes draining the uterus as well as in the peritoneal cavity of pregnant mice, both tissues in close contact with the fetuses. Despite an increase in spleen size, pregnant mice showed lower numbers of splenic B cells, which was mirrored by lower numbers of immature and FO B cells. However, marginal zone B cells in the spleen increased during pregnancy. Additionally, serum IgM, IgA, and IgG3 titers were elevated in pregnant mice. Collectively, our data show how the B cell compartment adapts to the presence of the semiallogeneic fetus during gravidity.

Cytokine IL-6 secretion by trophoblasts regulated via Sphingosine-1-phosphate receptor 2 involving Rho/Rho-kinase and Rac1 signaling pathways

Various cytokines derived from placental cells are essential for normal placenta development and successful pregnancy. Interleukin-6 (IL-6) is a multifunctional cytokine produced by extravillous and cytotrophoblasts regulating the functions of these cells, e.g. migration, invasion, trophoblast differentiation and proliferation. In macrophages, newly synthesized IL-6 accumulates in the Golgi complex and exits in tubulovesicular carriers fused with recycling endosomes and secreted as a soluble protein. Sphingosine-1-phosphate (S1P) induces various cytokine secretions including IL-6 in different cell types. The signaling mechanisms regulating the IL-6 secretion are unknown. In this study, we found that S1PR2 was the major S1P receptor being expressed in BeWo cells. S1P regulated IL-6 protein secretion in early phase (6 h) and gene expression in later phase (24 h). IL-6 secretion was completely inhibited via inhibitor of transcription (Actinomycin D) or protein synthesis (Cycloheximide) confirming that IL-6 releases constitutively from BeWo cells. By using specific S1PR2 inhibitor JTE-013 and S1PR2 gene silencing, we found that S1PR2 was the main receptor that regulates IL-6 secretion. Furthermore, S1P induced RhoGTPases-dependent pathways that are required for IL-6 secretion. Pretreatment of cells with specific Rho-kinase inhibitor (Y27632) and Rac1 inhibitor (NSC23766) drastically inhibited S1P-induced IL-6 secretion. By using a specific Phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), we found that basal activity of PI3K was required for secretion but was independent of S1P/S1PR2 axis activation. In summary, we report first time that binding of S1P to S1PR2 activates multiple RhoGTPases-dependent pathways that coordinate with PI3K pathway for secretion of IL-6 in BeWo cells.

Non-apoptotic Fas-induced regulation of cytokines in undifferentiated and decidualized human endometrial stromal cells depends on caspase-activity

Fas has originally been described as a member of the death-receptor family, mediating apoptosis upon stimulation by Fas-ligand (FasL). However, Fas expressing human endometrial stromal cells (ESCs) are resistant to Fas-mediated apoptosis. Since the implanting embryo secretes FasL, we examined whether Fas mediates non-apoptotic effects in human ESCs in vitro. ESCs were isolated from hysterectomy specimens, decidualized using progesterone and 17β-estradiol and incubated with an activating anti-Fas antibody, recombinant FasL and a caspase-inhibitor. Leukemia inhibitory factor (LIF), interleukin (IL)-11, -6, -8, monocyte chemoattractant protein (MCP)-1 and RANTES (Regulated on Activation Normal T cell Expressed and Secreted) were measured using ELISA and real-time RT-PCR. Viability of ESCs was determined using an MTT assay. Caspase-activity was measured by luminescent assays. Activation of nuclear factor (NF)-κB was detected by in-cell western and transcription factor assays. LIF and IL-11 in undifferentiated and IL-8 in decidualized ESCs were up-regulated by non-apoptotic Fas-signaling. In contrast, IL-6, MCP-1 and RANTES were not regulated by Fas. Caspases were activated upon Fas-stimulation and the Fas-mediated effects on LIF, IL-11 and -8 were reversed by caspase-inhibition. The transcription factor NF-κB was not activated in ESCs after stimulation of Fas. These results suggest a differential regulatory role of caspase-dependent Fas-signaling at the feto-maternal interface during early implantation. Remarkably, this typical death machinery mediates non-apoptotic effects in the human endometrium rather than inducing apoptosis.

Marginal zone B cells emerge as a critical component of pregnancy well-being

The success of eutherian mammal evolution was certainly supported by the ability of the already existing immune system to adapt to the presence of the semi-allogeneic fetus without losing the capability to defend the mother against infections. This required the acquisition of highly regulated and coordinated immunological mechanisms. Failures in the development of these strategies not only lead to the interruption of pregnancy but also compromise maternal health. Alongside changes on the cytokine profile - expansion of tolerogenic dendritic and regulatory T cells - a profound adaptation of the B cell compartment during pregnancy was recently described. Among others, the suppression of B cell lymphopoiesis and B cell lymphopenia were proposed to be protective mechanisms tending to reduce the occurrence of autoreactive B cells that might recognize fetal structures and put pregnancy on risk. On the other hand, expansion of the pre-activated marginal zone (MZ) B cell phenotype was described as a compensatory strategy launched to overcome B cell lymphopenia thus ensuring a proper defense. In this work, using an animal model of pregnancy disturbances, we demonstrated that the suppression of B cell lymphopoiesis as well as splenic B cell lymphopenia occur independently of pregnancy outcome. However, only animals undergoing normal pregnancies, but not those suffering from pregnancy disturbances, could induce an expansion and activation of the MZ B cells. Hence, our results clearly show that MZ B cells, probably due to the production of natural protective antibodies, participate in the fine balance of immune activation required for pregnancy well-being.

The molecular charge and size of heparins determine their impact on the decidualization of human endometrial stromal cells.

Heparin modulates the decidualization of human endometrial stromal cells (ESCs), but the molecular mechanisms behind these effects are still unknown. In the present study, we further specified this biological effect of heparin in human ESCs in vitro. ESCs were isolated from hysterectomy specimens, decidualized over 12 days using progesterone and 17β-estradiol and incubated with thrombin, factor Xa (FXa), unfractionated heparin, dextran sulfate, danaparoid or different low-molecular-weight heparins (LMWHs). Production of insulin-like growth factor (IGF)-I, prolactin (PRL) and IGF-binding protein (IGFBP)-1 by ESCs was measured using ELISAs. Like heparin, thrombin and FXa cause an increase in IGF-I in ESCs, suggesting an action of heparin independent from its anticoagulatory effects. This was supported by demonstrating the induction of the same effects on IGF-I, PRL and IGFBP-1 as heparin by dextran sulfate, a polysaccharide of similar size and charge as heparin, but without anticoagulatory properties. LMWHs with the same anti-FXa activity as heparin showed less pronounced effects on ESCs than heparin, whereas the very short pentasaccharide fondaparinux (17 kDa) had barely any effect, further supporting the primary role of molecular size and charge mediating these biological effects of heparin on ESCs. In conclusion, the effects of heparin on the decidualization of human ESCs seem to be independent of its anticoagulatory function, but rather depend on the charge and the size of this polysulfated glycosaminoglycan. Therefore, highly sulfated polysaccharides with a molecular weight >17 kDa might be an interesting pharmacological approach for the therapy of endometrial pathologies, e.g. the treatment of women suffering from recurrent miscarriage or repeated implantation failure.

Heparin inhibits interferon-γ signaling in human endometrial stromal cells by interference with the cellular binding of interferon-γ

OBJECTIVE To examine the impact of heparins on interferon-γ (IFN-γ) signaling in human endometrial stromal cells (ESCs) in vitro. DESIGN In vitro experiment. SETTING Research laboratory at a medical university center. PATIENT(S) Premenopausal women undergoing hysterectomy for benign reasons. INTERVENTION(S) The ESCs were isolated from hysterectomy specimens, decidualized in vitro using P and 17β-E(2), and incubated with recombinant IFN-γ, unfractionated heparin, and low molecular weight heparins (LMWHs). MAIN OUTCOME MEASURE(S) Interferon response factor 1 (IRF-1) and N-myc interactor (Nmi) messenger RNA (mRNA) were measured using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphorylation of signal transducer and activator of transcription 1 (STAT-1) was detected by an in-cell Western assay, expression of the IFN-γ receptor by flow cytometry. Cell-bound IFN-γ was determined in lysates by an ELISA. RESULT(S) Heparin and LMWHs inhibit the IFN-γ-mediated induction of IRF-1, but not Nmi in undifferentiated and decidualized ESCs. The phosphorylation of signal transducer and activator of transcription 1 STAT-1 upon IFN-γ stimulation is inhibited as well. Heparin has no effect on the IFN-γ receptor in ESCs, but inhibits the binding of IFN-γ to the cells. CONCLUSION(S) Unfractionated heparin, as well as LMWHs, are able to inhibit IFN-γ signaling in human ESCs and therefore might be clinically interesting agents to modulate the actions of this proinflammatory cytokine at the implantation site.

Sphingosine 1-phosphate regulates IL-8 expression and secretion via S1PR1 and S1PR2 receptors-mediated signaling in extravillous trophoblast derived HTR-8/SVneo cells.

INTRODUCTION Both villous and extravillous trophoblast (EVT) cells produce a wide range of cytokines and also respond to them in autocrine and paracrine manner. Deregulation of cytokine secretion may lead to various pathologic conditions including preeclampsia. IL-8, a pro-inflammatory cytokine, regulates various cellular functions such as neutrophil trafficking, cell adhesion, tumor growth and has a role in placental development. IL-8 also promotes trophoblast cell migration and invasion, and stimulates the secretion of progesterone. The induction and mechanism of IL-8 secretion by EVT is still unknown. METHODS IL-8 mRNA expression and secretion was determined using real-time PCR and ELISA respectively. To identify the mechanism of IL-8 expression and secretion, selective antagonists and agonist of S1P receptor subtypes, Rac1 and Rho-kinase inhibitors were used. RESULTS We found that S1P induces IL-8 gene expression and protein secretion in EVT derived HTR-8/SVneo cells but not in BeWo cells. SEW2781, the selective agonist of S1PR(1), induced IL-8 gene expression but not protein secretion. The specific S1PR(2) inhibitor JTE-013 could drastically inhibit IL-8 secretion. Furthermore, pre-treatment of cells with the selective S1PR(1)/S1PR(3) antagonist VPC23019 inhibited IL-8 secretion by ∼45%. Selective Rho-kinase inhibitor Y27632 and Rac1 inhibitor NSC23766 could block IL-8 secretion in these cells. DISCUSSION In this study, we could show for the first time that S1P induces IL-8 mRNA expression and protein secretion in EVT cell line. S1P-induced IL-8 gene expression is mainly regulated via S1PR(1) and its secretion is regulated through S1PR(2) receptor subtype. Rho GTPases signaling is essential for S1P-induced IL-8 secretion.

Expression analysis of cannabinoid receptors 1 and 2 in B cells during pregnancy and their role on cytokine production.

The endocannabinoid system consists in a family of lipids that binds to and activates cannabinoid receptors. There are two receptors so far described, the cannabinoid receptor 1 (CB1) and 2 (CB2). In the context of pregnancy, the endocannabinoid system was shown participates in different key aspects of reproductive events. B-lymphocytes are pleiotropic cells belonging to the adaptive arm of the immune system. Besides immunoglobulin production, B-lymphocytes were recently shown to be actively involved in antigen presentation as well as cytokine production, thus playing a central role in immunity. In this study we first aimed to characterize the expression of CB1 and CB2 receptors in B cells during pregnancy and then analyze the impact of their activation in term of cytokine production by B cells from pregnant and non-pregnant mice. We observed that the expression of CB1 and CB2 receptors in B-lymphocytes is differentially regulated during pregnancy. While CB2 expression is down regulated CB1 is augmented in B-lymphocytes of pregnant mice. Additionally, the treatment of activated B-lymphocytes with specific CB1 and CB2 agonists, showed a different response in term of cytokine production. Particularly, CB1 against boosted the production of the anti-inflammatory cytokine IL-10 by activated B-lymphocytes from pregnant mice.

A Kinetic Study of CD83 Reveals an Upregulation and Higher Production of sCD83 in Lymphocytes from Pregnant Mice

For the normal development of pregnancy, a balance between immune tolerance and defense is crucial. However, the mechanisms mediating such a balance are not fully understood. CD83 is a transmembrane protein whose expression has been linked to anti-inflammatory functions of T and B cells. The soluble form of CD83, released by cleavage of the membrane-bound protein, has strong anti-inflammatory properties and was successfully tested in different mouse models. It is assumed that this molecule contributes to the establishment of immune tolerance. Therefore, we postulated that the expression of CD83 is crucial for immune tolerance during pregnancy in mice. Here, we demonstrated that the membrane-bound form of CD83 was upregulated in T and B cells during allogeneic murine pregnancies. An upregulation was also evident in the main splenic B cell subtypes: marginal zone, follicular zone, and transitional B cells. We also showed that there was an augmentation in the number of CD83+ cells toward the end of pregnancy within splenic B and CD4+ T cells, while CD83+ dendritic cells were reduced in spleen and inguinal lymph nodes of pregnant mice. Additionally, B lymphocytes in late-pregnancy presented a markedly higher sensitivity to LPS in terms of CD83 expression and sCD83 release. Progesterone induced a dosis-dependent upregulation of CD83 on T cells. Our data suggest that the regulation of CD83 expression represents a novel pathway of fetal tolerance and protection against inflammatory threats during pregnancy.

Human pregnancy is accompanied by modifications in B cell development and immunoglobulin profile

Though human pregnancy success has been classically linked with a shift into a Th2 immunoglobulin producing cell response, a clear picture concerning B cell development and immunoglobulin profile during human pregnancy is missing. We analyzed in this work the dynamic of different B cell populations in peripheral blood of pregnant women on the first, second and third trimester of pregnancy. As control, age-matched non-pregnant fertile women were included. Additionally, we quantified the levels of immunoglobulin (IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE) in the serum of pregnant and non-pregnant women. We observed a significant decrease in the percentages of transitional B cells in peripheral blood of pregnant women as compared to non-pregnant control women. Besides, percentages of naïve as well as switched and non-switched memory B cells in peripheral blood of pregnant women were similar to those in non-pregnant control women. Interestingly, although we did not observe differences in the activation status of B cells as well as in the percentages of plasma cells between pregnant and non-pregnant women, we observed significantly higher levels of IgM, IgA, IgG3, more likely natural antibodies, as well IgG4 in serum of pregnant women compared to non-pregnant age matched control women.