Integrating tumor and healthy epithelium in a micro-physiology multi-compartment approach to study renal cell carcinoma pathophysiology.

The advent of micro-physiological systems (MPS) in biomedical research has enabled the introduction of more complex and relevant physiological into in vitro models. The recreation of complex morphological features in three-dimensional environments can recapitulate otherwise absent dynamic interactions in conventional models. In this study we developed an advanced in vitro Renal Cell Carcinoma (RCC) that mimics the interplay between healthy and malignant renal tissue. Based on the TissUse Humimic platform our model combines healthy renal proximal tubule epithelial cells (RPTEC) and RCC. Co-culturing reconstructed RPTEC tubules with RCC spheroids in a closed micro-perfused circuit resulted in significant phenotypical changes to the tubules. Expression of immune factors revealed that interleukin-8 (IL-8) and tumor necrosis factor-alfa (TNF-α) were upregulated in the non-malignant cells while neutrophil gelatinase-associated lipocalin (NGAL) was downregulated in both RCC and RPTEC. Metabolic analysis showed that RCC prompted a shift in the energy production of RPTEC tubules, inducing glycolysis, in a metabolic adaptation that likely supports RCC growth and immunogenicity. In contrast, RCC maintained stable metabolic activity, emphasizing their resilience to external factors. RNA-seq and biological process analysis of primary RTPTEC tubules demonstrated that the 3D tubular architecture and MPS conditions reverted cells to a predominant oxidative phosphorylate state, a departure from the glycolytic metabolism observed in 2D culture. This dynamic RCC co-culture model, approximates the physiology of healthy renal tubules to that of RCC, providing new insights into tumor-host interactions. Our approach can show that an RCC-MPS can expand the complexity and scope of pathophysiology and biomarker studies in kidney cancer research.

Microfluidic-based prostate cancer model for investigating the secretion of prostate-specific antigen and microRNAs in vitro.

The study of prostate cancer in vitro relies on established cell lines that lack important physiological characteristics, such as proper polarization and expression of relevant biomarkers. Microphysiological systems (MPS) can replicate cancer microenvironments and lead to cellular phenotypic changes that better represent organ physiology in vitro. In this study, we developed an MPS model comprising conventional prostate cancer cells to evaluate their activity under dynamic culture conditions. Androgen-sensitive (LNCaP) and androgen-insensitive (PC3) cells were grown in conventional and 3D cultures, both static and dynamic. Cell morphology, the secretion of prostate-specific antigen, and the expression of key prostate markers and microRNAs were analyzed. LNCaP formed spheroids in 3D and MPS cultures, with morphological changes supported by the upregulation of cytokeratins and adhesion proteins. LNCaP also maintained a constant prostate-specific antigen secretion in MPS. PC3 cells did not develop complex structures in 3D and MPS cultures. PSA expression at the gene level was downregulated in LNCaP-MPS and considerably upregulated in PC3-MPS. MicroRNA expression was altered by the 3D static and dynamic culture, both intra- and extracellularly. MicroRNAs associated with prostate cancer progression were mostly upregulated in LNCaP-MPS. Overall dynamic cell culture substantially altered the morphology and expression of LNCaP cells, arguably augmenting their prostate cancer phenotype. This novel approach demonstrates that microRNA expression in prostate cancer cells is sensitive to external stimuli and that MPS can effectively promote important physiological changes in conventional prostate cancer models.

Oxygen regulates ILC3 antigen presentation potential and pregnancy-related hormone actions.

Early pregnancy is marked by placentation and embryogenesis, which take place under physiological low oxygen concentrations. This oxygen condition is crucial for many aspects of placentation, trophoblast function, vascularization and immune function. Recently, a new family of innate lymphoid cells has been found to be expressed at the fetomaternal interface. Among these, type 3 innate lymphoid cells (ILC3) are important antigen presenting cells in the context of MHC-II. The expression of MHC-II on ILC3s during pregnancy is reduced. We tested the hypothesis that low oxygen concentrations reduce the potential of ILC3s to present antigens promoting fetal tolerance.Using an in vitro approach, NCR+ ILC3s generated from cord blood stem cell precursors were incubated under different O2 concentrations in the presence or absence of the pregnancy-related hormones hCG and TGF-ß1. The expression of MHC-II, accessory molecules and an activation marker were assessed by flow cytometry. We observed that 1% O2 reduced the expression of the MHC-II molecule HLA-DR as compared to 21% O2 and modulated the relative effects of hCG and TGF-ß1.Our data indicate that low oxygen concentrations reduce the antigen presentation potential of NCR+ ILC3s and suggest that it may promote fetal tolerance during the first trimester of pregnancy.

Low Abundance Fusobacterium Nucleatum Supports Early Pregnancy Development - An In Vitro Study.

Pregnancy success depends greatly on a balanced immune homeostasis. The detection of bacterial components in the upper reproductive tract in non-pregnant and pregnant women raised questions on its possible beneficial role in reproductive health. The local conditions that allow the presence of bacteria to harmonize with the establishment of pregnancy are still unknown. Among the described bacterial species in endometrial and placental samples, Fusobacterium nucleatum was found. It has been observed that F. nucleatum can induce tumorigenesis in colon carcinoma, a process that shares several features with embryo implantation. We propose that low concentrations of F. nucleatum may improve trophoblast function without exerting destructive responses. Inactivated F. nucleatum and E. coli were incubated with the trophoblastic cell lines HTR8/SVneo, BeWo, and JEG-3. Viability, proliferation, migratory capacity, invasiveness and the secretion of chemokines, other cytokines and matrix metalloproteinases were assessed. The presence of F. nucleatum significantly induced HTR8/SVneo invasion, accompanied by the secretion of soluble mediators (CXCL1, IL-6 and IL-8) and metalloproteinases (MMP-2 and MMP-9). However, as concentrations of F. nucleatum increased, these did not improve invasiveness, hindered migration, reduced cell viability and induced alterations in the cell cycle. Part of the F. nucleatum effects on cytokine release were reverted with the addition of a TLR4 blocking antibody. Other effects correlated with the level of expression of E-cadherin on the different cell lines tested. Low amounts of F. nucleatum promote invasion of HTR8/SVneo cells and induce the secretion of important mediators for pregnancy establishment. Some effects were independent of LPS and correlated with the expression of E-cadherin on trophoblasts.

Thrombin impairs the angiogenic activity of extravillous trophoblast cells via monocyte chemotactic protein-1 (MCP-1): A possible link with preeclampsia.

Cytokines' secretion from the decidua and trophoblast cells has been known to regulate trophoblast cell functions, such as Extravillous trophoblasts (EVTs) cell migration and invasion and remodeling of spiral arteries. Defective angiogenesis and spiral arteries transformation are mainly caused by proinflammatory cytokines and excessive thrombin generation during preeclampsia. Monocyte chemotactic protein-1 (MCP-1), a crucial cytokine, has a role in maintaining normal pregnancy. In this study, we explored whether thrombin regulates the secretion of MCP-1 in HTR-8/SVneo cells; if yes, what is its function? We used HTR-8/SVneo cells, developed from first trimester villous explants of early pregnancy, as the model of EVTs. MCP-1 gene silencing was performed using gene-specific siRNA. qPCR and ELISA were performed to estimate the expression and secretion of MCP-1. Here, we found that thrombin enhanced the secretion of MCP-1 in HTR-8/SVneo cells. Proteinase-activated receptor-1 (PAR-1) was found as the primary receptor, regulating MCP-1 secretion in these cells. Furthermore, MCP-1 secretion is modulated via protein kinase C (PKC) α, ß, and Rho/Rho-kinase-dependent pathways. Thrombin negatively regulates HTR-8/SVneo cells' ability to mimic tube formation in an MCP-1 dependent manner. In conclusion, we propose that thrombin-controlled MCP-1 secretion may play an essential role in normal placental development and successful pregnancy maintenance. Improper thrombin production and MCP-1 secretion during pregnancy might cause inadequate vascular formation and transformation of spiral arteries, which may contribute to pregnancy disorders, such as preeclampsia.

Hormonally controlled ILC antigen presentation potential is reduced during pregnancy.

Strategically located in mucosal barriers, innate lymphoid cells (ILCs) are relevant in local containment and tolerance of commensal microflora. ILCs have been recently described at the fetomaternal interface, where the development of a semi-allogeneic fetus can only succeed in a well-controlled immune environment. We postulate that ILCs adapt their antigen presentation capacity to protect pregnancy from excessive immune responses. Human ILCs were studied in deciduae of term pregnancies, peripheral blood and in in vitro generated ILCs. Fresh isolated lymphocytes or cells treated with pregnancy-related factors were investigated. The fetal antigen rejection-based CBA/J × DBA/2J mouse model (poor outcome pregnant mice; POPM) was used to characterize ILC antigen presentation potential in normal and immunologically disturbed pregnancies. ILC antigen presentation potential was characterized by flow cytometry and qPCR. We discovered that the distribution of ILC subsets changed during both human and murine pregnancy. Moreover, the pregnancy was accompanied by reduced MHCII expression in splenic ILCs during normal pregnancy (CBA/J × BALB/c; good outcome pregnant mice; GOPM) but increased in splenic and intestinal ILCs of CBA/J × DBA/2J mice. In vitro, splenic ILCs from pregnant mice increased MHCII expression after stimulation with IL-1ß and IL-23. In contrast, uterine ILCs displayed lower MHCII expression, which remained unchanged after stimulation. Finally, pregnancy-related factors and hormones present in the uterine environment reduced antigen presentation potential of human ILCs in vitro. Together, these data indicate that, during pregnancy, peripheral and especially uterine ILCs adapt their antigen presenting potential to maintain a level of tolerance and support pregnancy.

Pregnancy status alters IL-21-mediated effects on murine B lymphocytes.

A favorable outcome of pregnancy depends greatly on an adequate balance of immune protection and fetal tolerance at the fetomaternal interface. IL-21 is a pro-inflammatory cytokine associated with altering immune responses in autoimmune diseases. IL-21 has pleiotropic functions, including induction of Th17 T cells, inhibition of Treg development, and modulation of antibody responses of B lymphocytes. Genetic polymorphisms of IL21 have been associated to poor pregnancy outcomes. However, the mechanism of IL-21 actions needs further evaluation. Here, we postulate that IL-21 affects splenic B cell function during pregnancy and shapes immune responses. We show that splenic B cells from CBA/J × BALB/c mice with favorable pregnancy outcome expressed lower IL21R levels than in CBA/J × DBA/2J mice, a mouse model for immune-induced bad pregnancy outcome. As a consequence, B cells from CBA/J × BALB/c mice reacted less sensitively to IL-21 than B cells from non-pregnant mice (NPM) or from CBA/J × DBA/2J mice. Also, LPS-induced apoptotic rates were altered in NPM and CBA/J × DBA/2J but not in CBA/J × BALB/c mice. This is accompanied by improved survival of B cells that produce the anti-inflammatory cytokine IL-10 upon stimulation with LPS. We also observed lower numbers of CD4+CXCR5+Bcl-6+ follicular T-helper cells (Tfh) in normal pregnant mice, compared to non-pregnant and mice with disturbed pregnancies. Our data indicate that alterations of the Tfh/IL-21/IL-10 axis may have important influence on pregnancy outcome.

Characterization of murine amniotic fluid B cells in normal pregnancy and in preterm birth.

The amniotic fluid provides mechanical protection and immune defense against pathogens to the fetus. Indeed, components of the innate and adaptive immunity, including B cells, have been described in the amniotic fluid. However, limited information concerning phenotype and functionality of amniotic fluid B cells is available. Hence, we aimed to perform a full phenotypical and functional characterization of amniotic fluid B cells in normal pregnancy and in a mouse model of preterm birth. Phenotypic analysis depicted the presence of two populations of amniotic fluid B cells: an immature population, resembling B1 progenitor cells and a more mature population. Further isolation and in vitro co-culture with a bone marrow stroma cell line demonstrated the capacity of the immature B cells to mature. This was further supported by spontaneous production of IgM, a feature of the B1 B cell sub-population. An additional in vitro stimulation with lipopolysaccharide induced the activation of amniotic fluid B cells as well as the production of pro and anti-inflammatory cytokines. Furthermore, amniotic fluid B cells were expanded in the acute phase of LPS-induced preterm birth. Overall our data add new insight not only on the phenotype and developmental stage of the amniotic fluid B1 B cells but especially on their functionality. This provides important information for a better understanding of their role within the amniotic fluid as immunological protective barrier, especially with regard to intraamniotic infection and preterm birth.

CD83 is locally regulated and differentially expressed in disturbed murine pregnancy.

Alterations in the immunologic balance during pregnancy have been associated with poor pregnancy outcomes. The underlying mechanisms are complex and mouse models delivered valuable information on inflammatory imbalance in disturbed pregnancies and served as model to test potential anti-inflammatory therapies. CD83 is a transmembrane protein (mCD83) with a soluble form (sCD83) which possesses strong anti-inflammatory properties. During murine pregnancy, upregulated mCD83 expression induces sCD83 release after in vitro stimulation with LPS, phorbol myristate acetate (PMA) and ionomycin. The release mechanism of sCD83 and its control are yet to be elucidated. In this study, the expression of mCD83 and sCD83 has been extensively studied in the CBA/J × DBA/2J mouse model of pro-inflammatory-mediated pregnancy disturbances. mCD83 was higher expressed on splenic B cells, uterus-draining lymph nodes T cells and dendritic cells from mice with poor pregnancy outcome (PPOM) compared to mice with good pregnancy outcome (GPOM). PPOM, however, was accompanied by lower sCD83 serum levels. In vitro treatment of splenic B cells with progesterone led to a reduction of TIMP1 expression, mCD83 expression and sCD83 release, while TIMP1 treatment had a positive effect on sCD83 availability. These results suggest that tissue and matrix components are involved in the regulation of CD83 in murine pro-inflammatory pregnancies.

Thrombin stimulates gene expression and secretion of IL-11 via protease-activated receptor-1 and regulates extravillous trophoblast cell migration.

Extravillous trophoblast (EVT) migration and invasion is the crucial step for normal placental development. IL-11 is a cytokine regulating cell migration and invasion in cells and is a critical factor for successful implantation of an embryo. Higher expression of thrombin receptor PAR-1 was reported in early pregnancy. The precise role of thrombin in trophoblast functions is not well understood. In this study, we asked whether thrombin can induce IL-11 secretion in trophoblasts if yes, which physiological cell functions are possibly affected? In this study, HTR-8/SVneo cells, which were originally derived from first-trimester villous explants of early pregnancy were used as the extravillous trophoblast (EVT) model. BeWo cells were used as the cytotrophoblast model. For gene silencing, qPCR and ELISA, each experiment was performed in triplicates for minimum three times. Here, we found that thrombin stimulates IL-11 gene expression and protein secretion in HTR-8/SVneo cells but not in BeWo cells. PAR-1 was the only receptor which was highly expressed in HTR-8/SVneo cells. Thrombin-mediated expression and secretion of IL-11 were mainly activated via PAR-1 receptor. Rac1, but not Rho-kinase activation is required for thrombin-induced IL-11 secretion. We also found that thrombin stimulation significantly enhanced cell migration that was inhibited after silencing the IL-11 gene. In conclusion, this study demonstrates the role of thrombin in regulating human EVT migration via IL-11 secretion. We propose that thrombin might regulate EVT migration through the decidua and spiral artery remodeling. Failure of thrombin-dependent EVT migration results in pregnancy disorder, such as preeclampsia.

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.

Autonomic outcome is better after endarterectomy than after stenting in patients with asymptomatic carotid stenosis.

OBJECTIVE: Carotid endarterectomy and stenting have comparable efficacy in stroke prevention in asymptomatic carotid stenosis. In patients with carotid stenosis, cardiac events have a more than threefold higher incidence than cerebrovascular events. Autonomic dysfunction predicts cardiovascular morbidity and mortality, and carotid stenosis interferes with baroreceptor and chemoreceptor function. We assessed the effect of elective carotid revascularization (endarterectomy vs stenting) on autonomic function as a major prognostic factor of cardiovascular health. METHODS: In 42 patients with ≥70% asymptomatic extracranial carotid stenosis, autonomic function was determined by analysis of heart rate variability (total band power [TP], high frequency band power [HF], low-frequency band power [LF], very low frequency band power [VLF]), baroreflex sensitivity (αHF, αLF), respiratory chemoreflex sensitivity (central apnea-hypopnea index), and cardiac chemoreflex sensitivity (hyperoxic TP, HF, LF, and VLF ratios) before and 30 days after revascularization. RESULTS: Patients with endarterectomy were older than patients with stenting (69 ± 7 vs 62 ± 7 years; P ≤ .008) but did not differ in gender distribution and preintervention autonomic function. Compared with stenting, postintervention heart rate variability was higher (ln TP, 6.7 [95% confidence interval (CI), 6.3-7.0] vs 6.1 [95% CI, 5.8-6.5; P ≤ .009]; ln HF, 4.5 [95% CI, 4.1-5.0] vs 4.0 [95% CI, 3.4-4.5; P ≤ .05]; ln VLF, 6.0 [95% CI, 5.7-6.4] vs 5.5 [95% CI, 5.2-5.9; P ≤ .02]); respiratory chemoreflex sensitivity (central apnea-hypopnea index, 5.5 [95% CI, 2.8-8.2] vs 10.0 [95% CI, 6.9-13.1; P ≤. 01]) and cardiac chemoreflex sensitivity (TP ratio, 1.2 [95% CI, 1.1-1.3] vs 1.0 [95% CI, 0.9-1.0; P ≤ .0001]; HF ratio, 1.4 [95% CI, 1.2-1.5] vs 0.9 [95% CI, 0.8-1.1; P ≤ .001]; LF ratio, 1.5 [95% CI, 1.3-1.6] vs 1.0 [95% CI, 0.8-1.1; P ≤ .0001]; VLF ratio, 1.2 [95% CI, 1.1-1.3) vs 1.0 [95% CI, 0.9-1.1; P ≤ .002]) were lower after endarterectomy. Postintervention baroreflex sensitivity did not differ after endarterectomy and stenting. CONCLUSIONS: Autonomic function was better after endarterectomy than after stenting. Better autonomic function after endarterectomy was based on restoration of chemoreceptor but not baroreceptor function and may improve cardiovascular long-term outcome.

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.

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.

Sleep apnea and asymptomatic carotid stenosis: a complex interaction.

BACKGROUND: Carotid arteriosclerosis and sleep apnea are considered as independent risk factors for stroke. Whether sleep apnea mediates severity of carotid stenosis remains unclear. Sleep apnea comprises two pathophysiologic conditions: OSA and central sleep apnea (CSA). Although OSA results from upper airway occlusion, CSA reflects enhanced ventilatory drive mainly due to carotid chemoreceptor dysfunction. METHODS: Ninety-six patients with asymptomatic extracranial carotid stenosis of ≥ 50% underwent polysomnography to (1) determine prevalence and severity of sleep apnea for different degrees of carotid stenosis and (2) analyze associations between OSA and CSA, carotid stenosis severity, and other arteriosclerotic risk factors. RESULTS: Sleep apnea was present in 68.8% of patients with carotid stenosis. Prevalence and severity of sleep apnea increased with degree of stenosis (P ≤ .05) because of a rise in CSA (P ≤ .01) but not in OSA. Sleep apnea (OR, 3.8; P ≤ .03) and arterial hypertension (OR, 4.1; P ≤ .05) were associated with stenosis severity, whereas diabetes, smoking, dyslipidemia, BMI, age, and sex were not. Stenosis severity was related to CSA (P ≤ .06) but not to OSA. In addition, CSA but not OSA showed a strong association with arterial hypertension (OR, 12.5; P ≤ .02) and diabetes (OR, 4.5; P ≤ .04). CONCLUSIONS: Sleep apnea is highly prevalent in asymptomatic carotid stenosis. Further, it is associated with arteriosclerotic disease severity as well as presence of hypertension and diabetes. This vascular risk constellation seems to be more strongly connected with CSA than with OSA, possibly attributable to carotid chemoreceptor dysfunction. Because sleep apnea is well treatable, screening should be embedded in stroke prevention strategies.

B cell development undergoes profound modifications and adaptations during pregnancy in mice.

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.

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 17ß-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.

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 17ß-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.