Decidualized human decidual stromal cells inhibit chemotaxis of activated T cells: a potential mechanism of maternal-fetal immune tolerance.

Background: Numerous lines of evidence confirm that decidual stromal cells (DSCs) play a key role in maternal-fetal immune tolerance. Under the influence of progesterone and other hormones, the DSCs go through a process of differentiation (decidualization) during normal pregnancy. In mice, DSCs inhibit the expression of chemokines that attract abortigenic Th1 and Tc cells to the decidua. We have studied this phenomenon in humans. Methods: We established human DSC lines and decidualized these cells in vitro with progesterone and cAMP. We determined the expression of the chemokines CXCL9, CXCL10 and CXCL11, whose receptor CXCR3 is expressed by Th1 and Tc cells, in undifferentiated DSCs and decidualized DSCs by qRT-PCR. Activated CD3+CXCR3+ cells, including CD4+ Th1 cells and CD8+ Tc cells, were induced in vitro. The migration capacity of these activated lymphocytes was investigated in Transwell chambers with conditioned media from undifferentiated and decidualized DSCs. Results: We demonstrated that CXCL9 was not expressed by DSCs, whereas the expression of CXCL10 and CXCL11 was inhibited in decidualized cells. Conditioned media from decidualized cells significantly inhibited the migration of Th1 and Tc cells. We found that decidualized cells secrete factors of MW less than 6000-8000 Da, which actively inhibit the chemotaxis of these lymphocytes. Discussion: These results confirm in humans that decidualization of DSCs inhibits the expression by these cells of chemokines that attract Th1 and Tc cells and induces the secretion by DSCs of factors that inhibit the chemotaxis of these lymphocytes, thus preventing the arrival of abortigenic T cells in the decidua.

Influence of the ectopic location on the antigen expression and functional characteristics of endometrioma stromal cells.

RESEARCH QUESTION: Are the alterations observed in the endometriotic cells, such as progesterone resistance, already present in the eutopic endometrium or acquired in the ectopic location? DESIGN: The response to decidualization with progesterone and cyclic AMP for up to 28 days was compared in different endometrial stromal cell (EnSC) lines established from samples of endometriomas (eEnSC), eutopic endometrium from women with endometriosis (eBEnSC), endometrial tissue from healthy women (BEnSC) and menstrual blood from healthy donors (mEnSC). RESULTS: Usual features of decidualized cells, such as changes in cell morphology and expression of prolactin, were similarly observed in the three types of eutopic EnSC studied, but not in the ectopic cells upon decidualization. Among the phenotypic markers analysed, CD105 was down-regulated under decidualization in all cell types (mEnSC, P = 0.005; BEnSC, P = 0.029; eBEnSC, P = 0.022) except eEnSC. mEnSC and BEnSC underwent apoptosis during decidualization, whereas eBEnSC and eEnSC were resistant to the induction of cell death. Lastly, migration studies revealed that mEnSC secreted undetermined factors during decidualization that inhibited cell motility, whereas eEnSC showed a significantly lower ability to produce those migration-regulating factors (P < 0.0001, P  < 0.001 and P = 0.0013 for the migration of mEnSC at 24, 48 and 72 h, respectively; P  < 0.0001 for the migration of eEnSC at all times studied). CONCLUSIONS: This study provides novel insights into the differences between endometriotic and eutopic endometrial cells and reinforces the idea that the microenvironment in the ectopic location plays additional roles in the acquisition of the alterations that characterize the cells of the endometriotic foci.

Stromal cells of the endometrium and decidua: in search of a name and an identity†.

Human endometrial and decidual stromal cells are the same cells in different environments (nonpregnancy and pregnancy, respectively). Although some authors consider decidual stromal cells to arise solely from the differentiation of endometrial stromal cells, this is a debatable issue given that decidualization processes do not end with the formation of the decidua, as shown by the presence of stromal cells from both the endometrium and decidua in both undifferentiated (nondecidualized) and decidualized states. Furthermore, recent functional and transcriptomic results have shown that there are differences in the decidualization process of endometrial and decidual stromal cells, with the latter having a greater decidualization capacity than the former. These differences suggest that in the terminology and study of their characteristics, endometrial and decidual stromal cells should be clearly distinguished, as should their undifferentiated or decidualized status. There is, however, considerable confusion in the designation and identification of uterine stromal cells. This confusion may impede a judicious understanding of the functional processes in normal and pathological situations. In this article, we analyze the different terms used in the literature for different types of uterine stromal cells, and propose that a combination of differentiation status (undifferentiated, decidualized) and localization (endometrium, decidua) criteria should be used to arrive at a set of accurate, unambiguous terms. The cell identity of uterine stromal cells is also a debatable issue: phenotypic, functional, and transcriptomic studies in recent decades have related these cells to different established cells. We discuss the relevance of these associations in normal and pathological situations.

SLAMF8 Downregulates Mouse Macrophage Microbicidal Mechanisms via PI3K Pathways.

Signaling lymphocytic activation molecule family 8 (SLAMF8) is involved in the negative modulation of NADPH oxidase activation. However, the impact of SLAMF8 downregulation on macrophage functionality and the microbicide mechanism remains elusive. To study this in depth, we first analyzed NADPH oxidase activation pathways in wild-type and SLAMF8-deficient macrophages upon different stimulus. Herein, we describe increased phosphorylation of the Erk1/2 and p38 MAP kinases, as well as increased phosphorylation of NADPH oxidase subunits in SLAMF8-deficient macrophages. Furthermore, using specific inhibitors, we observed that specific PI3K inhibition decreased the differences observed between wild-type and SLAMF8-deficient macrophages, stimulated with either PMA, LPS, or Salmonella typhimurium infection. Consequently, SLAMF8-deficient macrophages also showed increased recruitment of small GTPases such as Rab5 and Rab7, and the p47phox subunit to cytoplasmic Salmonella, suggesting an impairment of Salmonella-containing vacuole (SCV) progression in SLAMF8-deficient macrophages. Enhanced iNOS activation, NO production, and IL-6 expression were also observed in the absence of SLAMF8 upon Salmonella infection, either in vivo or in vitro, while overexpression of SLAMF8 in RAW264.7 macrophages showed the opposite phenotype. In addition, SLAMF8-deficient macrophages showed increased activation of Src kinases and reduced SHP-1 phosphate levels upon IFNγ and Salmonella stimuli in comparison to wild-type macrophages. In agreement with in vitro results, Salmonella clearance was augmented in SLAMF8-deficient mice compared to that in wild-type mice. Therefore, in conclusion, SLAMF8 intervention upon bacterial infection downregulates mouse macrophage activation, and confirmed that SLAMF8 receptor could be a potential therapeutic target for the treatment of severe or unresolved inflammatory conditions.

Decidualization modulates the mesenchymal stromal/stem cell and pericyte characteristics of human decidual stromal cells. Effects on antigen expression, chemotactic activity on monocytes and antitumoral activity.

Decidual stromal cells (DSCs) are the most abundant cellular component of human decidua and play a central role in maternal-fetal immune tolerance. Antigen phenotyping and functional studies recently confirmed the relationship of DSCs with mesenchymal stem/stromal cells (MSCs) and pericytes, the latter two cell types being closely related or identical. The present study investigated the effect of decidualization, a process of cell differentiation driven by progesterone (P4) and other pregnancy hormones, on the MSC/pericyte characteristics of DSCs. To this end we isolated undifferentiated DSC (preDSC) lines that were decidualized in vitro (dDSC) by the effect of P4 and cAMP. Using flow cytometry, we found significant downmodulation of the expression of the MSC/pericyte markers α-smooth muscle actin, nestin, CD140b, CD146 and SUSD2 in dDSCs. The dDSCs did not differ, compared to preDSCs, in the expression of angiogenic factors (characteristic of pericytes) HGF, FGF2, ANGPT1 or VEGF according to RT-PCR results, but had significantly increased PGF expression. In migration assays, preDSC-conditioned media had a chemotactic effect on the THP-1 monocytic line (characteristic of pericytes), and this effect was significantly greater in dDSC-conditioned media. Media conditioned with dDSC, but not with preDSC, induced apoptosis in 4 out of 6 different tumor cell lines (characteristic of MSCs) according to propidium iodide staining and flow cytometry results. Our findings show that decidualization induces phenotypic and functional changes in the MSC/pericyte properties of DSCs that may have a role in the normal development of pregnancy.

Endometrial and decidual stromal precursors show a different decidualization capacity.

Endometrial stromal cells (EnSCs) and decidual stromal cells (DSCs) originate from fibroblastic precursors located around the vessels of the human nonpregnant endometrium and the pregnant endometrium (decidua), respectively. Under the effect of ovarian or pregnancy hormones, these precursors differentiate (decidualize), changing their morphology and secreting factors that appear to be essential for the normal development of pregnancy. However, the different physiological context - that is, non-pregnancy vs pregnancy - of those precursors (preEnSCs, preDSCs) might affect their phenotype and functions. In the present study, we established preEnSC and preDSC lines and compared the antigen phenotype and responses to decidualization factors in these two types of stromal cell line. Analyses with flow cytometry showed that preEnSCs and preDSCs exhibited a similar antigen phenotype compatible with that of bone marrow mesenchymal stem/stromal cells. The response to decidualization in cultures with progesterone and cAMP was evaluated by analyzing changes in cell morphology by microscopy, prolactin and IL-15 secretion by enzyme immunoassay and the induction of apoptosis by flow cytometry. In all four analyses, preDSCs showed a significantly higher response than preEnSCs. The expression of progesterone receptor (PR), protein kinase A (PKA) and FOXO1 was studied with Western blotting. Both types of cells showed similar levels of PR and PKA, but the increase in PKA RI subunit expression in response to decidualization was again significantly greater in preDSCs. We conclude that preEnSCs and preDSCs are equivalent cells but differ in their ability to decidualize. Functional differences between them probably derive from factors in their different milieus.

A simple approximation for the evaluation of the photon iso-effective dose in Boron Neutron Capture Therapy based on dose-independent weighting factors.

The current methodology for determining the biological effect of Boron Neutron Capture Therapy (BNCT) has recently been questioned, and a more accurate framework based in the photon iso-effective dose has been proposed. In this work we derive a first order approximation to this quantity. The new approach removes the main drawbacks of the current method, being based on new weighting factors which are true constants (dose independent) but which can be evaluated from published data on the existing (dose-dependent) weighting factors. In addition to this, we apply the formalism to allow the comparison to a fractionated conventional radiotherapy treatment, for which there is a lot of knowledge from clinical practice. As an application, the photon iso-effective dose of a BNCT treatment for a brain tumor is estimated. An excel sheet used for these calculations is also provided as supplementary material and can be used also with user-provided input data for the estimation of the photon iso-effective dose for comparison with conventional radiotherapy, both to single and fractionated treatments.

Human predecidual stromal cells are mesenchymal stromal/stem cells and have a therapeutic effect in an immune-based mouse model of recurrent spontaneous abortion.

BACKGROUND: Human decidual stromal cells (DSCs) are involved in the maintenance and development of pregnancy, in which they play a key role in the induction of immunological maternal-fetal tolerance. Precursors of DSCs (preDSCs) are located around the vessels, and based on their antigen phenotype, previous studies suggested a relationship between preDSCs and mesenchymal stromal/stem cells (MSCs). This work aimed to further elucidate the MSC characteristics of preDSCs. METHODS: We established 15 human preDSC lines and 3 preDSC clones. Physiological differentiation (decidualization) of these cell lines and clones was carried out by in vitro culture with progesterone (P4) and cAMP. Decidualization was confirmed by the change in cellular morphology and prolactin (PRL) secretion, which was determined by enzyme immunoassay of the culture supernatants. We also studied MSC characteristics: (1) In mesenchymal differentiation, under appropriate culture conditions, these preDSC lines and clones differentiated into adipocytes, osteoblasts, and chondrocytes, and differentiation was confirmed by cytochemical assays and RT-PCR. (2) The expression of stem cell markers was determined by RT-PCR. (3) Cloning efficiency was evaluated by limited dilution. (4) Immunoregulatory activity in vivo was estimated in DBA/2-mated CBA/J female mice, a murine model of immune-based recurrent abortion. (5) Survival of preDSC in immunocompetent mice was analyzed by RT-PCR and flow cytometry. RESULTS: Under the effect of P4 and cAMP, the preDSC lines and clones decidualized in vitro: the cells became rounder and secreted PRL, a marker of physiological decidualization. PreDSC lines and clones also exhibited MSC characteristics. They differentiated into adipocytes, osteoblasts, and chondrocytes, and preDSC lines expressed stem cell markers OCT-4, NANOG, and ABCG2; exhibited a cloning efficiency of 4 to 15%; significantly reduced the embryo resorption rate (P < 0.001) in the mouse model of abortion; and survived for prolonged periods in immunocompetent mice. The fact that 3 preDSC clones underwent both decidualization and mesenchymal differentiation shows that the same type of cell exhibited both DSC and MSC characteristics. CONCLUSIONS: Together, our results confirm that preDSCs are decidual MSCs and suggest that these cells are involved in the mechanisms of maternal-fetal immune tolerance.

Human predecidual stromal cells have distinctive characteristics of pericytes: Cell contractility, chemotactic activity, and expression of pericyte markers and angiogenic factors.

INTRODUCTION: Human decidual stromal cells (DSCs) play a key role in maternal-fetal interactions. Precursors of DSCs (preDSCs) localize around vessels in both the endometrium and decidua. Previous studies suggested a relationship between preDSCs and pericytes because these cells share a perivascular location, alpha smooth muscle actin (α-SM actin) expression and the ability to contract under the effects of cytokines. METHODS: To further study this relationship, we established 15 human preDSC lines and 3 preDSC clones. The preDSC lines and clones were tested by flow cytometry with a panel of 29 monoclonal antibodies, 14 of which are pericyte markers. The expression of angiogenic factors was determined by RT-PCR, chemotactic activity was studied with the migration assay, and cell contractility was evaluated with the collagen cell contraction assay. Confocal microscopy was used to study decidual sections. RESULTS: Under the effect of progesterone and cAMP, these lines decidualized in vitro: the cells became rounder and secreted prolactin, a marker of physiological DSC differentiation (decidualization). The antigen phenotype of these preDSC lines and clones was fully compatible with that reported for pericytes. PreDSC lines displayed pericyte characteristics: they expressed angiogenic factors and showed chemotactic and cytokine-induced contractile activity. Confocal microscopic examination of decidual sections revealed the expression of antigens detected in preDSC lines: α-SM actin colocalized with CD146, CD140b, MFG-E8, nestin, and STRO-1 (all of which are pericyte markers) in cells located around the vessels, a distinctive location of preDSCs and pericytes. DISCUSSION: Taken together, our results show that preDSCs are pericyte-like cells.

ROS-induced DNA damage and PARP-1 are required for optimal induction of starvation-induced autophagy.

In response to nutrient stress, cells start an autophagy program that can lead to adaptation or death. The mechanisms underlying the signaling from starvation to the initiation of autophagy are not fully understood. In the current study we show that the absence or inactivation of PARP-1 strongly delays starvation-induced autophagy. We have found that DNA damage is an early event of starvation-induced autophagy as measured by γ-H2AX accumulation and comet assay, with PARP-1 knockout cells displaying a reduction in both parameters. During starvation, ROS-induced DNA damage activates PARP-1, leading to ATP depletion (an early event after nutrient deprivation). The absence of PARP-1 blunted AMPK activation and prevented the complete loss of mTOR activity, leading to a delay in autophagy. PARP-1 depletion favors apoptosis in starved cells, suggesting a pro-survival role of autophagy and PARP-1 activation after nutrient deprivation. In vivo results show that neonates of PARP-1 mutant mice subjected to acute starvation, also display deficient liver autophagy, implying a physiological role for PARP-1 in starvation-induced autophagy. Thus, the PARP signaling pathway is a key regulator of the initial steps of autophagy commitment following starvation.

Regulation of the resistance to TRAIL-induced apoptosis in human primary T lymphocytes: role of NF-kappaB inhibition.

Several combined strategies have been recently proposed to overcome the resistance to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) showed by some tumor cells, thus improving the use of this death ligand in antitumor therapy. However, the molecular mechanisms of the tumor selective activity of TRAIL are not completely understood and hence the effects of the combined therapy on normal cells are unknown. Here, we have studied the resistance of primary T lymphocytes to TRAIL-mediated apoptosis. No significant differences were found in the expression of proteins involved in TRAIL-mediated apoptosis between resting and activated T cells. The low expression of death receptors TRAIL-R1/-R2 as well as the high levels of the antiapoptotic proteins TRAIL-R4 and cellular Fas-associated death domain-like IL-1beta-converting enzyme-inhibitory protein (c-FLIP) may explain the lack of caspase-8 activation observed upon TRAIL treatment in both cell types. We have also analyzed the effect of different sensitizing agents such as genotoxic drugs, phosphatidylinositol-3 kinase (PI3K) inhibitors, proteasome inhibitors, microtubule depolymerizing agents, histone deacetylase inhibitors (HDACi), and NF-kappaB inhibitors. Although some of them induced T cell death, only NF-kappaB inhibitors sensitized activated T cells to TRAIL-induced apoptosis, maybe through the regulation of the antiapoptotic proteins TRAIL-R4, c-FLIP(S) and members of the inhibitors of apoptosis proteins (IAP) family. These results question the safety of the combined treatments with TRAIL and NF-kappaB inhibitors against tumors.