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Immunomodulation during human pregnancy: placental exosomes as vehicles of immune suppression.
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. (Lucia Mincheva-Nilsson)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The mammalian pregnancy comprises a challenge to the maternal immune system since the fetus is semi-allogeneic and could thus be rejected. Pregnancy success is associated with the placenta that is not only essential for oxygen supply, nourishment and pregnancy hormones but also plays a role in the protection of the fetus against maternal immunologic attack. The aim of the current studies was to elucidate the role of human placenta as an immunomodulatory organ with a special focus on placental exosomes as vehicles for establishment of maternal tolerance to the fetus.

We discovered that the syncytiotrophoblast in human normal pregnancy constitutively produces and secretes exosomes. Exosomes are 30-100 nanometer-sized membrane vesicles of endosomal origin that convey intercellular communication. Exosomes are produced and released through the endosomal compartment and reflect the type and the activation state of the cells that produce and secrete them. They carry cytosolic and membrane-bound proteins and nucleic acids and can influence and re-program recipient cells. Depending on their interactions with cells of the immune system they can be divided into immunostimulatory or immunosuppressive.

We developed methods for isolation and culture of trophoblast and placental explants from human normal first trimester pregnancy and isolated exosomes from the culture supernatants.  These exosomes were characterized biochemically and functionally regarding mechanisms with potential importance in the establishment of maternal tolerance towards the fetus. The following aspects were studied: 1) exosomal modulation of the NKG2D receptor-ligand system, a major cytotoxic pathway for NK- and cytotoxic T cells and thus potentially dangerous to the fetus; 2) placental exosome-mediated apoptosis of activated immune effector cells; and 3) Foxp3-expressing T regulatory cells in human pregnant uterine mucosa, the decidua.

Using immuno electron microscopy we show that human early syncytiotrophoblast constitutively expresses the stress-inducible NKG2D ligands MICA/B and ULBP1-5, and the apoptosis inducing molecules FasL and TRAIL. While MICA/B were expressed both on the cell surface and intracellularly on the limiting membrane of multivesicular bodies (MVB) and on exosomes, the ULBP1-5, FasL and TRAIL  were solely  processed through the MVB of the endosomal compartment and secreted on exosomes. The NKG2D ligand-expressing placental exosomes were able to internalize the cognate receptor from the cell surface of activated NK- and T cells thus down regulating their cytotoxic function. In our studies of apoptosis we found that placental exosomes carry the proapoptotic ligands FasL and TRAIL in their active form as a hexameric complex of two homotrimeric molecules, required for triggering of the apoptotic signaling pathways. This finding was supported by the ability of isolated placental FasL/TRAIL expressing exosomes to induce apoptosis in activated peripheral blood mononuclear cells (PBMC) and Jurkat T cells. Additionally, we studied Foxp3-expressing T regulatory (Treg) cells in paired human decidual and blood samples from pregnant women compared to non-pregnant controls. The CD4+CD25+Foxp3+ Treg cells were 10 fold enriched in the decidual mucosa compared to peripheral blood of pregnant women and non-pregnant controls. We discovered a pool of Foxp3-expressing, CD4+CD25- cells in human decidua, a phenotype consistent with naïve/precursor Foxp3+ Treg cells. These results suggest local enrichment of Treg cells in decidua of normal pregnancy. Furthermore, we have results indicating that the exosomes, isolated from placental explant cultures, carry PD-L1 and TGFβ on their surface, molecules known to promote induction of Treg cells. Taken together, our results provide evidence that placental exosomes are immunosuppressive and underline their role in the maternal immune modulation during pregnancy. The constitutive production and secretion of immunosuppressive placental exosomes create a protective exosomal gradient in the blood surrounding the feto-placental unit. This “cloud of immunosuppressive exosomes” conveys immunologic privilege to the developing fetus and thus contributes to the solution of the immunological challenge of mammalian pregnancy. 

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2014. , 72 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1640
Keyword [en]
exosomes, microvesicles, pregnancy, placenta, syncytiotrophoblast, immune privilege, apoptosis, cytotoxicity, T regulatory cells, NKG2D, MICA/B, FasL, TRAIL
National Category
Immunology in the medical area
Research subject
Immunology
Identifiers
URN: urn:nbn:se:umu:diva-87566ISBN: 978-91-7601-034-1 (print)OAI: oai:DiVA.org:umu-87566DiVA: diva2:709924
Public defence
2014-04-25, A103 - Astrid Fagraeus-salen, byggnad 6A, Norrlands universitetssjukhus, Umeå, 13:00 (English)
Opponent
Supervisors
Available from: 2014-04-04 Created: 2014-04-03 Last updated: 2014-04-04Bibliographically approved
List of papers
1. An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies
Open this publication in new window or tab >>An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies
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2008 (English)In: American Journal of Reproductive Immunology and Microbiology, ISSN 8755-8920, Vol. 60, no 1, 33-42 p.Article in journal (Refereed) Published
Abstract [en]

PROBLEM: The uniqueness of the human placenta cannot be replaced by animal models. In vitro studies are compulsory to elucidate the biology of human placenta and require isolation and purification of villous trophoblasts, which can be used in molecular and functional studies. Constant improvement in the isolation technique is required to obtain a high yield of pure trophoblast cells with high viability and well preserved morphology.

METHOD OF STUDY: Optimized isolation procedure for human villous trophoblasts based on mild enzymatic treatment, Percoll gradient centrifugation and additional purification step involving positive or negative immunoselection on magnetic beads is described.

RESULTS: A simple and effective isolation protocol gave a reasonably high yield of villous trophoblast cells with high purity and viability, and excellent morphology as assessed by flow cytometry and electron microscopy.

CONCLUSION: This protocol provides an efficient, optimized method for isolation and enrichment of villous trophoblast cells, suitable for phenotypic, ultrastructural, molecular and functional analyses and for establishment of primary cultures.

Keyword
Human placenta;isolation method;Percoll gradient;villous trophoblast
National Category
Immunology in the medical area Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-21496 (URN)10.1111/j.1600-0897.2008.00588.x (DOI)18593436 (PubMedID)
Available from: 2009-04-14 Created: 2009-04-14 Last updated: 2014-04-04Bibliographically approved
2. Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function
Open this publication in new window or tab >>Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function
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2009 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 183, no 1, 340-351 p.Article in journal (Refereed) Published
Abstract [en]

During mammalian pregnancy maternal-fetal tolerance involves a number of immunosuppressive factors produced by placenta. Recently, placenta-derived exosomes have emerged as new immune regulators in the maternal immune tolerance. Exosomes are membrane nanovesicles with defined morphology, which are secreted from endosomal multivesicular bodies (MVB) upon fusion with the plasma membrane. Previously, we reported that the MHC class I chain-related (MIC) proteins A and B, human ligands of the activating NK cell receptor NKG2D, are expressed by placenta, sorted to MVB of syncytiotrophoblast and probably released via MIC-bearing exosomes. In this report, we show that the second family of human NKG2D ligands, the UL-16 binding proteins (ULBP), is also expressed by placenta. Importantly, this expression was not due to placental CMV infection. Immunoelectron microscopy disclosed that ULBP1-5 are produced and retained in MVB of the syncytiotrophoblast on microvesicles/exosomes. Using human placenta explant cultures and different assays, we demonstrate that exosomes bearing NKG2D ligands are released by human placenta. Isolated placental exosomes carried ULBP1-5 and MIC on their surface and induced down-regulation of the NKG2D receptor on NK, CD8(+), and gammadelta T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathway. Release of placental NKG2D ligands via exosomes is an alternative mechanism for generation of bioactive soluble form of these ligands. These findings highlight a role for NKG2D ligand-bearing placental exosomes in the fetal immune escape and support the view of placenta as a unique immunosuppressive organ.

National Category
Immunology in the medical area Obstetrics, Gynecology and Reproductive Medicine
Identifiers
urn:nbn:se:umu:diva-36070 (URN)10.4049/jimmunol.0803477 (DOI)19542445 (PubMedID)
Available from: 2010-09-15 Created: 2010-09-15 Last updated: 2014-04-04Bibliographically approved
3. Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus
Open this publication in new window or tab >>Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus
2013 (English)In: Journal of Immunology, ISSN 0022-1767, E-ISSN 1550-6606, Vol. 191, no 11, 5515-5523 p.Article in journal (Refereed) Published
Abstract [en]

Apoptosis is crucially important in mediating immune privilege of the fetus during pregnancy. We investigated the expression and in vitro apoptotic activity of two physiologically relevant death messengers, the TNF family members Fas ligand (FasL) and TRAIL in human early and term placentas. Both molecules were intracellularly expressed, confined to the late endosomal compartment of the syncytiotrophoblast, and tightly associated to the generation and secretion of placental exosomes. Using immunoelectron microscopy, we show that FasL and TRAIL are expressed on the limiting membrane of multivesicular bodies where, by membrane invagination, intraluminal microvesicles carrying membranal bioactive FasL and TRAIL are formed and released in the extracellular space as exosomes. Analyzing exosomes secreted from placental explant cultures, to our knowledge, we demonstrate for the first time that FasL and TRAIL are clustered on the exosomal membrane as oligomerized aggregates ready to form death-inducing signaling complex. Consistently, placental FasL-and TRAIL-carrying exosomes triggered apoptosis in Jurkat T cells and activated PBMC in a dose-dependent manner. Limiting the expression of functional FasL and TRAIL to exosomes comprise a dual benefit: 1) storage of exosomal FasL and TRAIL in multivesicular bodies is protected from proteolytic cleavage and 2) upon secretion, delivery of preformed membranal death molecules by exosomes rapidly triggers apoptosis. Our results suggest that bioactive FasL-and TRAIL-carrying exosomes, able to convey apoptosis, are secreted by the placenta and tie up the immunomodulatory and protective role of human placenta to its exosome-secreting ability.

Place, publisher, year, edition, pages
The American Association of Immunologists, 2013
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-84107 (URN)10.4049/jimmunol.1301885 (DOI)000327180600019 ()
Available from: 2013-12-18 Created: 2013-12-16 Last updated: 2017-12-06Bibliographically approved
4. Maternal Foxp3 expressing CD4+ CD25+ and CD4+ CD25- regulatory T-cell populations are enriched in human early normal pregnancy decidua: a phenotypic study of paired decidual and peripheral blood samples.
Open this publication in new window or tab >>Maternal Foxp3 expressing CD4+ CD25+ and CD4+ CD25- regulatory T-cell populations are enriched in human early normal pregnancy decidua: a phenotypic study of paired decidual and peripheral blood samples.
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2011 (English)In: American Journal of Reproductive Immunology and Microbiology, ISSN 8755-8920, Vol. 66, no Suppl 1, 44-56 p.Article in journal (Refereed) Published
Abstract [en]

PROBLEM: Regulatory T cells (Treg cells), a small subset of CD4(+) T cells maintaining tolerance by immunosuppression, are proposed contributors to the survival of the fetal semiallograft. We investigated Treg cells in paired decidual and peripheral blood (PB) samples from healthy women in early pregnancy and PB samples from non-pregnant women.

METHOD OF STUDY: Distribution, location, cytokine mRNA, and phenotype were assessed in CD4(+) CD25(+) Treg cells from paired samples using immunohistochemistry, immunofluorescence, flow cytometry, and real-time quantitative RT-PCR.

RESULTS: The presence and in situ distribution of CD4(+) Foxp3(+) Treg cells in decidua are hereby demonstrated for the first time. Three Foxp3(+) cell populations, CD4(+) CD25(++) Foxp3(+), CD4(+) CD25(+) Foxp3(+), and CD4(+) CD25(-) Foxp3(+), were enriched locally in decidua. In contrast, no statistically significant difference in numbers of circulating Treg cells between pregnant and non-pregnant women was found. The Foxp3(+) cells expressed the surface molecules CD45RO, CTLA-4, CD103, Neuropilin-1, LAG-3, CD62L, and TGFβ1 mRNA consistent with Treg phenotype. The population of CD4(+) CD25(-) Foxp3(+) cells, not described in human decidua before, was enriched 10-fold compared with PB in paired samples. Their cytokine expression was often similar to Th3 profile, and the Foxp3 mRNA expression level in CD4(+) CD25(-) cells was stable and comparable to that of CD4(+) CD25(+) Treg cells implying that the majority of CD4(+) CD25(-) Foxp3(+) cells might be naïve Treg cells.

CONCLUSION: (i) There is a local enrichment of Treg cells in decidua (ii) The exclusive accumulation of decidual CD4(+) CD25(-) Foxp3(+) cells suggests an additional reservoir of Foxp3(+) naïve Treg cells that can be converted to 'classical' Treg cells in uterus.

Place, publisher, year, edition, pages
John Wiley & Sons, 2011
Keyword
CD4+ CD25+ Foxp3+ cells, CD4+ CD25++ Foxp3+ cells, CD4+ CD25− Foxp3+ cells, Foxp3, human decidua, pregnancy, Treg cells
National Category
Obstetrics, Gynecology and Reproductive Medicine Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-52220 (URN)10.1111/j.1600-0897.2011.01046.x (DOI)21726337 (PubMedID)
Available from: 2012-02-14 Created: 2012-02-14 Last updated: 2017-12-07Bibliographically approved

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Stenqvist, Ann-Christin

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