Esteban Grasso

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.

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.

Trophoblast cells induce a tolerogenic profile in dendritic cells.

BACKGROUND Dendritic cells (DCs), which are biased toward a tolerogenic profile, play a pivotal role in tissue-remodeling processes and angiogenesis at the maternal-fetal interface. Here, we analyzed the effect of trophoblast cells on the functional profile of DCs to gain insight on the tolerogenic mechanisms underlying the human placental-maternal dialog at early stages of gestation. METHODS DCs were differentiated from peripheral blood monocytes obtained from fertile women (n = 21), in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor during 5 days in culture. Then, DCs were cultured with trophoblast cells (Swan-71 cell line obtained from normal cytotrophoblast, at 7 weeks) for 24 h and for an additional 24 h in the absence or presence of lipopolysaccharide (LPS) from Escherichia coli. DCs were recovered and used for flow cytometry, enzyme-linked immunosorbent assay, RT-PCR and suppression and migration assays. RESULTS Trophoblast cells significantly prevented the increase in CD83 expression induced by LPS without affecting the expression of CD86, CD40 and human leukocyte antigen-DR (P < 0.05). Trophoblast cells significantly decreased the production of IL-12p70 and tumor necrosis factor-α, while it increased the production of IL-10 (P < 0.05). No changes were observed in the production of IL-6 and monocyte chemotactic protein-1. The culture of DCs with trophoblast cells, also suppressed the stimulation of the allogeneic response triggered by LPS (P < 0.05). Conditioned DCs were able to increase the frequency of CD4 + CD25 + Foxp3 cells and this effect was accompanied by an increase in indoleamine 2, 3-dioxygenase expression in DCs (P < 0.05). CONCLUSIONS The interaction of DCs with trophoblast cells promotes the differentiation of DCs into cells with a predominantly tolerogenic profile that could contribute to a tolerogenic microenvironment at the maternal-fetal interface.

Modulation of maternal LIF producers T cells by trophoblast and paternal antigens.

Citation Fraccaroli L, Grasso E, Zeitler E, Lombardi E, Gogorza S, Etchepareborda JJ, Nagle C, Cortelezzi M, Leirós CP, Ramhorst R. Modulation of maternal LIF producers T cells by trophoblast and paternal antigens. Am J Reprod Immunol 2011; 65: 133–145

VIP boosts regulatory T cell induction by trophoblast cells in an in vitro model of trophoblast-maternal leukocyte interaction.

Inducible regulatory T cells (Tregs) exert a timely and efficient immunosuppressive action at the critical peri‐implantation stage essential for maternal tolerance to the conceptus. Vasoactive intestinal peptide (VIP) promotes anti‐inflammatory and tolerogenic profiles through binding to VIP receptors on immune cells. We evaluated whether VIP produced by trophoblast cells induces Tregs during the early interaction of maternal leukocytes with trophoblast cells, thus contributing to maternal tolerance. We used an in vitro model of maternal leukocyte–trophoblast cell interaction represented by cocultures of fertile womens PBMCs with a human trophoblast cell line (Swan‐71) and evaluated the effect of VIP added exogenously and of the endogenous polypeptide. VIP increased the frequency of CD4+CD25+FoxP3+ cells after coculture, and these cells were able to suppress the maternal alloresponse. VIP also increased the frequency of CD4+IL10+ and CD4+TGFβ+ cells, but it did not modulate IFN‐γ or IL‐17 production. Swan‐71 secreted VIP, and their coculture with maternal PBMCs significantly increased the frequency of Tregs. This effect was even more pronounced if the trophoblast cells had been pretreated with VIP. In both situations, the VIP antagonist prevented the increase in the frequency of CD4+Foxp3+ cells, reflecting a specific effect of the polypeptide after the interaction with Swan‐71 cells. Finally, the increase in CD4+CD25+FoxP3+ frequency was prevented by an anti–TGF‐β Ab and a VIP antagonist. These results suggest that VIP could have an active role in the immunoregulatory processes operating in the maternal–placental interface by contributing to the induction of Tregs through a mechanism involving TGF‐β1.

VIP contribution to the decidualization program: regulatory T cell recruitment

During early pregnancy, the human uterus undergoes profound tissue remodeling characterized by leukocyte invasion and production of proinflammatory cytokines, followed by tissue repair and tolerance maintenance induction. Vasoactive intestinal peptide (VIP) is produced by trophoblast cells and modulates the maternal immune response toward a tolerogenic profile. Here, we evaluated the contribution of the VIP/VPAC to endometrial renewal, inducing decidualization and the recruitment of induced regulatory T cells (iTregs) that accompany the implantation period. For that purpose, we used an in vitro model of decidualization with a human endometrial stromal cell line (HESC) stimulated with progesterone (P4) and lipopolysaccharide (LPS) simulating the inflammatory response during implantation and human iTregs (CD4(+)CD25(+)FOXP3(+)) differentiated from naïve T cells obtained from peripheral blood mononuclear cells of fertile women. We observed that VIP and its receptor VPAC1 are constitutively expressed in HESCs and that P4 increased VIP expression. Moreover, in HESC VIP induced expression of RANTES (CCL5), one of the main chemokines involved in T cell recruitment, and this effect is enhanced by the presence of P4 and LPS. Finally, assays of the migration of iTregs toward conditioned media from HESCs revealed that endogenous VIP production induced by P4 and LPS and RANTES production were involved, as anti-RANTES neutralizing Ab or VIP antagonist prevented their migration. We conclude that VIP may have an active role in the decidualization process, thus contributing to recruitment of iTregs toward endometrial stromal cells by increasing RANTES expression in a P4-dependent manner.

Decoding the chemokine network that links leukocytes with decidual cells and the trophoblast during early implantation

ABSTRACT Chemokine network is central to the innate and adaptive immunity and entails a variety of proteins and membrane receptors that control physiological processes such as wound healing, angiogenesis, embryo growth and development. During early pregnancy, the chemokine network coordinates not only the recruitment of different leukocyte populations to generate the maternal-placental interface, but also constitutes an additional checkpoint for tissue homeostasis maintenance. The normal switch from a pro-inflammatory to an anti-inflammatory predominant microenvironment characteristic of the post-implantation stage requires redundant immune tolerance circuits triggered by key master regulators. In this review we will focus on the recruitment and conditioning of maternal immune cells to the uterus at the early implantation period with special interest on high plasticity macrophages and dendritic cells and their ability to induce regulatory T cells. We will also point to putative immunomodulatory polypeptides involved in immune homeostasis maintenance at the maternal-placental interface.

Defects in the vasoactive intestinal peptide (VIP)/VPAC system during early stages of the placental-maternal leucocyte interaction impair the maternal tolerogenic response.

Successful embryo implantation occurs followed by a local inflammatory/T helper type 1 (Th1) response, subsequently redirected towards a tolerogenic predominant profile. The lack of control of this initial local inflammatory response may be an underlying cause of early pregnancy complications as recurrent spontaneous abortions (RSA). Considering that vasoactive intestinal peptide (VIP) mediates anti‐inflammatory and tolerogenic effects in several conditions we hypothesized that VIP might contribute to tolerance towards trophoblast antigens during the early interaction of maternal leucocytes and trophoblast cells. In this study we investigated VIP/VPAC system activity and expression on maternal peripheral blood mononuclear cells (PBMCs) after interaction with immortalized trophoblast cells (Swan‐71 cell line) as an in‐vitro model of feto–maternal interaction, and we analysed whether it modulates maternal regulatory T cell (Treg)/Th1 responses. We also investigated the contribution of the endogenous VIP/VPAC system to RSA pathogenesis. VIP decreased T‐bet expression significantly, reduced monocyte chemotactic protein‐1 (MCP‐1) and nitrite production in co‐cultures of PBMCs from fertile women with trophoblast cells; while it increased the frequency of CD4+CD25+ forkhead box protein 3 (Foxp3)+ cells, transforming growth factor (TGF)‐β expression and interleukin (IL)‐10 secretion. These effects were prevented by VIP‐specific antagonist. Interestingly, PBMCs from RSA patients displayed significantly higher T‐bet expression, lower Treg frequency and lower frequency of VIP‐producer CD4 lymphocytes after the interaction with trophoblast cells. Moreover, the patients displayed a significantly lower frequency of endometrial CD4+VIP+ cells in comparison with fertile women. VIP showed a Th1‐limiting and Treg‐promoting response in vitro that would favour early pregnancy outcome. Because RSA patients displayed defects in the VIP/VPAC system, this neuropeptide could be a promising candidate for diagnostic biomarker or surrogate biomarker for recurrent spontaneous abortions.

Progesterone and VIP cross-talk enhances phagocytosis and anti-inflammatory profile in trophoblast-derived cells

Trophoblast cells produce several inmmuneregulators like the Vasoactive Intestinal Peptide (VIP) and P4 targeting multiple circuits, and also display an intese phagocytic ability allowing embryo implantation in a tolerogenic context. Here, we explored whether P4 and VIP- crosstalk modulates trophoblast cell function, focus on the phagocytic ability and the immune homeostasis maintenance. P4 enhanced the phagocytosis in trophoblast-derived cells quantified by the engulfment of latex-beads or eryptotic erythrocytes. P4 and VIP modulated the balance of anti/pro-inflammatory mediators, increasing TGF-β expression, with no changes in IL-1, IL-6, or nitrites production. This modulation was accompained by transcription factor expression changes that could turn on tolerogenic programs represented by increased PPAR-γ and decreased IRF-5 expression. Finally, P4 stimulated VPAC2 expression in trophoblast cells and VPAC2 over-expression enhanced phagocytosis mimicking P4-effect. Therefore, P4 and VIP network enhances the phagocytic ability of trophoblast-derived cells, through a mechanism involving VPAC2 accompained with an anti-inflammatory context.

Trophoblast cells primed with vasoactive intestinal peptide enhance monocyte migration and apoptotic cell clearance through αvβ3 integrin portal formation in a model of maternal–placental interaction

STUDY HYPOTHESIS Is apoptotic cell phagocytosis by monocytes modulated by pathways elicited by vasoactive intestinal peptide (VIP) action on trophoblast? STUDY FINDING Targeting trophoblast cells with VIP induces monocyte migration, polarization to anti-inflammatory phenotypes and apoptotic trophoblast cell clearance which involves increased αvβ3 integrin expression on phagocytic cells and binding to thrombospondin 1. WHAT IS KNOWN ALREADY Monocytes recruited to the maternal-placental interface interact with trophoblast cells and differentiate to alternatively activated macrophages involved in the silent clearance of apoptotic cells. Vasoactive intestinal peptide (VIP) is an immunomodulatory polypeptide synthesized at the human placenta that can target both trophoblast cells and monocytes/macrophages. Integrin αvβ3 and thrombospondin 1 are involved in the formation of a phagocytic portal for the immunosuppressant clearance of apoptotic cells. STUDY DESIGN, SAMPLES/MATERIALS, METHODS This is a laboratory-based study studying monocytes isolated from peripheral blood of healthy women (n = 33) and their interaction in vitro with first trimester trophoblast cell lines. Peripheral blood monocytes were isolated from healthy volunteers by Percoll gradient and tested in co-culture settings with first trimester trophoblast cell lines (Swan 71 and HTR8) or with trophoblast cell conditioned media obtained in the presence or absence of 10 or 100 nM VIP. The effect of VIP-conditioned media on monocyte migration was assessed through transwell systems and monocyte/macrophage phenotype was determined by flow cytometry. Phagocytosis of apoptotic cells and the mechanisms involved in phagocytic portal formation were assessed by flow cytometry, confocal microscopy, immunological blockade and RT-PCR. MAIN RESULTS AND THE ROLE OF CHANCE Exposing cells to 100 nM VIP increased the migration of monocytes toward trophoblast cell conditioned media (VIP conditioned medium) (P < 0.05 versus conditioned media from cells not exposed to VIP) and contributed to the monocytes acquiring an anti-inflammatory profile with increased CD39 and IL-10 expression (P < 0.05). Phagocytosis of apoptotic trophoblast cells by monocytes and monocyte-differentiated macrophages was increased by VIP conditioned medium (P < 0.05 versus media conditioned in the absence of VIP or direct addition of 100 nM VIP). The boosting effect of VIP conditioned medium on phagocytosis involved increased expression and re-localization of αvβ3 integrin on phagocytic cells along with enhanced expression of thrombospondin 1 on trophoblast cells. LIMITATIONS, REASONS FOR CAUTION The conclusions are based on in vitro experiments with monocytes drawn from peripheral blood of healthy individuals and trophoblast cell lines and we were unable to ascertain that these mechanisms operate similarly in vivo. We cannot rule out a differential behavior of either trophoblast cells targeted in vivo with VIP, or primary cultures of first trimester trophoblast cells assayed in vitro. WIDER IMPLICATIONS OF THE FINDINGS The results presented provide new clues for immune and trophoblast cell pharmacological targeting in pregnancy complications of immunopathologic nature. STUDY FUNDING/COMPETING INTERESTS This work was funded by the National Agency of Sciences and Technology ANPCyT (PICT 2011-0144), National Research Council CONICET (PIP 602/2012) and University of Buenos Aires (UBACyT 20020130100040BA) to C.P.L. The authors have no conflicts of interest to disclose.