Rosanna Ramhorst

Modulation and recruitment of inducible regulatory T cells by first trimester trophoblast cells.

Citation Ramhorst R, Fraccaroli L, Aldo P, Alvero AB, Cardenas I, Leirós CP, Mor G. Modulation and recruitment of inducible regulatory T cells by first trimester trophoblast cells. Am J Reprod Immunol 2012; 67: 17–27

A potential tolerogenic immune mechanism in a trophoblast cell line through the activation of chemokine-induced T cell death and regulatory T cell modulation

BACKGROUND Successful implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by Th2. Regulated upon activation, normal T cell expressed and secreted (RANTES) promotes a Th1 response and is implicated as a physiologic tolerogenic factor; therefore, we studied its potential role in the trophoblast-maternal leukocyte dialog. METHODS We performed co-cultures of immortalized trophoblast cell line (Swan 71) and peripheral blood mononuclear cells (PBMCs) from fertile women (n = 23) or with recurrent spontaneous abortions (n = 18, RSA). After 24 and 48 h, supernatant and cells were analyzed by enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, Western blot and apoptosis assay. To investigate the physiological effects at peripheral level, we co-cultured maternal and paternal PBMCs with conditioned media from Swan cells and progesterone. RESULTS Following interaction of maternal PBMCs and trophoblast cells, RANTES production increased (P < 0.05) and was accompanied by low levels of interferon gamma, interleukin-12 p70 and high levels of tumor necrosis factor-alpha, nitrites and leukemia-inhibitory factor. RANTES production resulted in elevated apoptosis of potentially deleterious maternal CD3+ lymphocytes, accompanied by a decrease in the proliferative maternal response. During fetal-maternal dialog, the anti-RANTES antibody significantly reduced the frequency of CD4+CD25+Foxp3+ cells (P < 0.05) and was associated with trophoblast cell survival. However, co-cultures of Swan cells and RSA-PBMCs displayed a differential RANTES kinetics, lower levels of regulatory T cells (Tregs) and CD3+annexin-V+cells, accompanied by higher levels of apoptotic trophoblast cells. CONCLUSIONS RANTES promotes an adequate pro-implantatory microenvironment that influences trophoblast cell survival and modulates the balance of maternal Treg/T effector lymphocytes in favor of maternal tolerance.

VIP modulates the pro‐inflammatory maternal response, inducing tolerance to trophoblast cells

Background and purpose:  Successful embryo implantation is followed by a local pro‐inflammatory and Th1 response, subsequently controlled by a Th2 response. Vasoactive intestinal peptide (VIP) has anti‐inflammatory effects and promotes tolerogenic/Th2 responses while favouring embryonic development. We investigated the potential regulatory role of VIP on human trophoblast cells, maternal pro‐inflammatory responses and trophoblast‐maternal leukocyte interactions.

Galectin-1 confers immune privilege to human trophoblast: implications in recurrent fetal loss

Mechanisms accounting for the protection of the fetal semi-allograft from maternal immune cells remain incompletely understood. In previous studies, we showed that galectin-1 (Gal1), an immunoregulatory glycan-binding protein, hierarchically triggers a cascade of tolerogenic events at the mouse fetomaternal interface. Here, we show that Gal1 confers immune privilege to human trophoblast cells through the modulation of a number of regulatory mechanisms. Gal1 was mainly expressed in invasive extravillous trophoblast cells of human first trimester and term placenta in direct contact with maternal tissue. Expression of Gal1 by the human trophoblast cell line JEG-3 was primarily controlled by progesterone and pro-inflammatory cytokines and impaired T-cell responses by limiting T cell viability, suppressing the secretion of Th1-type cytokines and favoring the expansion of CD4(+)CD25(+)FoxP3(+) regulatory T (T(reg)) cells. Targeted inhibition of Gal1 expression through antibody (Ab)-mediated blockade, addition of the specific disaccharide lactose or retroviral-mediated siRNA strategies prevented these immunoregulatory effects. Consistent with a homeostatic role of endogenous Gal1, patients with recurrent pregnancy loss showed considerably lower levels of circulating Gal1 and had higher frequency of anti-Gal1 auto-Abs in their sera compared with fertile women. Thus, endogenous Gal1 confers immune privilege to human trophoblast cells by triggering a broad tolerogenic program with potential implications in threatened pregnancies.

Induction of maternal tolerance to fetal alloantigens by RANTES production.

Problem  Previous studies have demonstrated a requirement for RANTES (regulated on activated normal T‐cell expressed, and secreted) at immune privileged sites; we have investigated the role of RANTES in the induction of maternal–fetal tolerance.

Implication of RANTES in the Modulation of Alloimmune Response By Progesterone During Pregnancy

Several studies indicate that RANTES (regulated on activation, normal T cell expressed and secreted) is able to downregulate T‐cell responses which suggest it might be relevant for fetal tolerance induction. However, the role of RANTES in pregnancy had not been established. Here we investigate RANTES regulation during early pregnancy and potential failures leading to losses of pregnancies.

Differential Migration and Activation Profile of Monocytes after Trophoblast Interaction

Macrophages at the maternal-placental interface coordinate opposite demands under the control of trophoblast cells such as the response against pathogens on one hand, and apoptotic cell clearance and wound healing with the production of suppressor cytokines. Here, we investigated whether trophoblast cells induce maternal monocyte activation towards an alternative activated macrophage profile and whether bacterial or viral stimuli modulate their migratory properties. We used an in vitro model of the maternal-placental interface represented by co-cultures of CD14+ cells isolated from fertile women with first trimester trophoblast cell line (Swan-71 cells) in the presence or absence of pathogen associated molecular pattern (PAMP) stimuli lipopolysaccharide (LPS), peptidoglycan (PGN) or poly [I:C]). Maternal CD14+ cells showed increased CD16 and CD39 expression, both markers associated to an alternative activation profile, with no changes in CD80 expression after trophoblast cell interaction. These changes were accompanied by increased IL-10 and decreased IL-12 production by CD14+ cells. After stimulation with LPS, PGN or poly [I:C], monocytes co-cultured with trophoblast cells had lower production of TNF-α and IL-1β compared with non co-cultured monocytes. Interestingly, monocyte migration towards trophoblast cells was prevented in the presence of LPS or PGN but not after 24h of stimulation with poly [I:C]. LPS or PGN also decreased CCR5, CXCL-8 and CCL5 expression. Finally, trophoblast cells co-cultured with monocytes in the presence of pathological stimuli failed to increase chemokine expression, indicating a bidirectional effect. In conclusion, trophoblast might ‘instruct’ maternal monocytes to express an alternative activation profile and restrain their early recruitment under pathological threats as one of the first strategies to avoid potential tissue damage at the maternal-placental interface.

Potential immunomodulatory role of VIP in the implantation sites of prediabetic nonobese diabetic mice

Among several factors known to modulate embryo implantation and survival, uterine quiescence and neovascularization, maternal immunotolerance through the Th1/Th2 cytokine balance towards a Th2 profile, local regulatory T-cell (Treg) activation, and high levels of progesterone were assigned a prominent role. Vasoactive intestinal peptide (VIP) is a neuroimmunopeptide that has anti-inflammatory effects, promotes Th2 cytokines and CD4(+)CD25(+)FOXP3(+) Treg activation, and stimulates exocrine secretion, smooth muscle relaxation, and vasodilatation favoring uterus quiescence. The goal of the present work was to explore the participation of VIP in the implantation sites of normal and pregnant prediabetic nonobese diabetic (NOD) females, a mouse strain that spontaneously develops an autoimmune exocrinopathy similar to Sjögrens syndrome. Our results indicate a reduction in litter size from the third parturition onwards in the NOD female lifespan with increased resorption rates. Progesterone systemic levels were significantly decreased in pregnant NOD mice compared with BALB/c mice, although the allogeneic response to progesterone by spleen cells was not impaired. VIP receptors, Vipr1 and Vipr2 (Vpac1 and Vpac2), were expressed at the implantation sites and VIP induced leukemia inhibitory factor (LIF) and Treg marker expression in both strains; however, a reduced Vip expression was found in NOD implantation sites. We conclude that the reduced birth rate at 16-week-old NOD mice with a Th1 systemic cytokine profile involves resorption processes with a lower expression of VIP at the sites of implantation, which acts as a local inducer of pro-implantatory LIF and Treg activation.

Intracellular Expression of CD69 in Endometrial and Peripheral T cells Represents a Useful Marker in Women with Recurrent Miscarriage: Modulation After Allogeneic Leukocyte Immunotherapy

PROBLEM:  To characterize in fertile women and women with recurrent spontaneous abortions (RSA) the expression and functional status of T cells expressing the CD69 molecule.

Monocytes from Sjögren's syndrome patients display increased vasoactive intestinal peptide receptor 2 expression and impaired apoptotic cell phagocytosis

Sjögrens syndrome (SS) is a chronic autoimmune disease characterized by salivary and lacrimal gland dysfunction. Clinical observations and results from animal models of SS support the role of aberrant epithelial cell apoptosis and immune homeostasis loss in the glands as triggering factors for the autoimmune response. Vasoactive intestinal peptide (VIP) promotes potent anti‐inflammatory effects in several inflammatory and autoimmune disease models, including the non‐obese diabetic (NOD) mouse model of SS. With the knowledge that VIP modulates monocyte function through vasoactive intestinal peptide receptors (VPAC) and that immune homeostasis maintenance depends strongly upon a rapid and immunosuppressant apoptotic cell clearance by monocytes/macrophages, in this study we explored VPAC expression on monocytes from primary SS (pSS) patients and the ability of VIP to modulate apoptotic cell phagocytic function and cytokine profile. Monocytes isolated from individual pSS patients showed an increased expression of VPAC2 subtype of VIP receptors, absent in monocytes from control subjects, with no changes in VPAC1 expression. VPAC2 receptor expression could be induced further with lipopolysaccharide (LPS) in pSS monocytes and VIP inhibited the effect. Moreover, monocytes from pSS patients showed an impaired phagocytosis of apoptotic epithelial cells, as evidenced by reduced engulfment ability and the failure to promote an immunosuppressant cytokine profile. However, VIP neither modulated monocyte/macrophage phagocytic function nor did it reverse their inflammatory profile. We conclude that monocytes from pSS patients express high levels of VPAC2 and display a deficient clearance of apoptotic cells that is not modulated by VIP.