Marcia Arenas-Hernandez

Immune cells in term and preterm labor

Labor resembles an inflammatory response that includes secretion of cytokines/chemokines by resident and infiltrating immune cells into reproductive tissues and the maternal/fetal interface. Untimely activation of these inflammatory pathways leads to preterm labor, which can result in preterm birth. Preterm birth is a major determinant of neonatal mortality and morbidity; therefore, the elucidation of the process of labor at a cellular and molecular level is essential for understanding the pathophysiology of preterm labor. Here, we summarize the role of innate and adaptive immune cells in the physiological or pathological activation of labor. We review published literature regarding the role of innate and adaptive immune cells in the cervix, myometrium, fetal membranes, decidua and the fetus in late pregnancy and labor at term and preterm. Accumulating evidence suggests that innate immune cells (neutrophils, macrophages and mast cells) mediate the process of labor by releasing pro-inflammatory factors such as cytokines, chemokines and matrix metalloproteinases. Adaptive immune cells (T-cell subsets and B cells) participate in the maintenance of fetomaternal tolerance during pregnancy, and an alteration in their function or abundance may lead to labor at term or preterm. Also, immune cells that bridge the innate and adaptive immune systems (natural killer T (NKT) cells and dendritic cells (DCs)) seem to participate in the pathophysiology of preterm labor. In conclusion, a balance between innate and adaptive immune cells is required in order to sustain pregnancy; an alteration of this balance will lead to labor at term or preterm.

An imbalance between innate and adaptive immune cells at the maternal–fetal interface occurs prior to endotoxin-induced preterm birth

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality worldwide. A transition from an anti-inflammatory state to a pro-inflammatory state in the mother and at the maternal–fetal interface has been implicated in the pathophysiology of microbial-induced preterm labor. However, it is unclear which immune cells mediate this transition. We hypothesized that an imbalance between innate and adaptive immune cells at the maternal–fetal interface will occur prior to microbial-induced preterm labor. Using an established murine model of endotoxin-induced PTB, our results demonstrate that prior to delivery there is a reduction of CD4+ regulatory T cells (Tregs) in the uterine tissues. This reduction is neither linked to a diminished number of Tregs in the spleen, nor to an impaired production of IL10, CCL17, or CCL22 by the uterine tissues. Endotoxin administration to pregnant mice does not alter effector CD4+ T cells at the maternal–fetal interface. However, it causes an imbalance between Tregs (CD4+ and CD8+), effector CD8+ T cells, and Th17 cells in the spleen. In addition, endotoxin administration to pregnant mice leads to an excessive production of CCL2, CCL3, CCL17, and CCL22 by the uterine tissues as well as abundant neutrophils. This imbalance in the uterine microenvironment is accompanied by scarce APC-like cells such as macrophages and MHC II+ neutrophils. Collectively, these results demonstrate that endotoxin administration to pregnant mice causes an imbalance between innate and adaptive immune cells at the maternal–fetal interface.

Invariant NKT cell activation induces late preterm birth that is attenuated by rosiglitazone

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality worldwide. Although intra-amniotic infection is a recognized cause of spontaneous preterm labor, the noninfection-related etiologies are poorly understood. In this article, we demonstrated that the expansion of activated CD1d-restricted invariant NKT (iNKT) cells in the third trimester by administration of α-galactosylceramide (α-GalCer) induced late PTB and neonatal mortality. In vivo imaging revealed that fetuses from mice that underwent α-GalCer–induced late PTB had bradycardia and died shortly after delivery. Yet, administration of α-GalCer in the second trimester did not cause pregnancy loss. Peroxisome proliferator–activated receptor (PPAR)γ activation, through rosiglitazone treatment, reduced the rate of α-GalCer–induced late PTB and improved neonatal survival. Administration of α-GalCer in the third trimester suppressed PPARγ activation, as shown by the downregulation of Fabp4 and Fatp4 in myometrial and decidual tissues, respectively; this suppression was rescued by rosiglitazone treatment. Administration of α-GalCer in the third trimester induced an increase in the activation of conventional CD4+ T cells in myometrial tissues and the infiltration of activated macrophages, neutrophils, and mature dendritic cells to myometrial and/or decidual tissues. All of these effects were blunted after rosiglitazone treatment. Administration of α-GalCer also upregulated the expression of inflammatory genes at the maternal–fetal interface and systemically, and rosiglitazone treatment partially attenuated these responses. Finally, an increased infiltration of activated iNKT-like cells in human decidual tissues is associated with noninfection-related preterm labor/birth. Collectively, these results demonstrate that iNKT cell activation in vivo leads to late PTB by initiating innate and adaptive immune responses and suggest that the PPARγ pathway has potential as a target for prevention of this syndrome.

In vivo activation of invariant natural killer T cells induces systemic and local alterations in T-cell subsets prior to preterm birth

Preterm birth, the leading cause of neonatal morbidity and mortality worldwide, is frequently preceded by spontaneous preterm labour, a syndrome of multiple aetiologies. Pathological inflammation is causally linked to spontaneous preterm labour. Indeed, direct activation of invariant natural killer T (iNKT) cells via α‐galactosylceramide induces preterm labour/birth largely by initiating systemic and local (i.e. decidua and myometrium) innate immune responses. Herein, we investigated whether iNKT‐cell activation altered local and systemic T‐cell subsets. Administration of α‐galactosylceramide induced an expansion of activated CD1d‐restricted iNKT cells in the decidua and a reduction in the number of: (1) total T cells (conventional CD4+ and CD8+ T cells) through the down‐regulation of the CD3ɛ molecule in the peripheral circulation, spleen, uterine‐draining lymph nodes (ULNs), decidua and/or myometrium; (2) CD4+ regulatory T cells in the spleen, ULNs and decidua; (3) T helper type 17 (Th17) cells in the ULNs but an increase in the number of decidual Th17 cells; (4) CD8+ regulatory T cells in the spleen and ULNs; and (5) CD4+ and CD8+ forkhead box protein 3 negative (Foxp3–) responder T cells in the spleen and ULNs. As treatment with rosiglitazone prevents iNKT‐cell activation‐induced preterm labour/birth, we also explored whether the administration of this peroxisome proliferator‐activated receptor gamma (PPARγ) agonist would restore the number of T cells. Treating α‐galactosylceramide‐injected mice with rosiglitazone partially restored the number of T cells in the spleen but not in the decidua. In summary, iNKT‐cell activation altered the systemic and local T‐cell subsets prior to preterm labour/birth; however, treatment with rosiglitazone partially reversed such effects.

Isolation of leukocytes from the murine tissues at the maternal-fetal interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (>70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Chemotactic Activity of Gestational Tissues Through Late Pregnancy, Term Labor, and RU486‐Induced Preterm Labor in Guinea Pigs

Is increased leukocyte chemotactic activity (CA) from gestational tissues necessary for term or preterm labor in guinea pigs?

In vivo T-cell activation by a monoclonal αCD3ε antibody induces preterm labor and birth.

Activated/effector T cells seem to play a role in the pathological inflammation associated with preterm labor. The aim of this study was to determine whether in vivo T‐cell activation by a monoclonal αCD3ε antibody induces preterm labor and birth.

Housekeeping gene expression stability in reproductive tissues after mitogen stimulation

BackgroundIntrauterine infection during pregnancy can trigger a local inflammatory response leading to several complications, such as preterm labor. Many studies have used in vitro and in vivo models employing mitogens to induce the expression of the characteristic proinflammatory mediators triggered by infection. However, relative expression assays depend on the stability of housekeeping gene expression, which can vary depending on certain stimuli. In this study, we analyzed the stability and pairwise variation in the expression of GAPDH, ACTB and RNA18S1 in cultured reproductive tissues under mitogen stimulation. We used fetal membranes, placental villous and umbilical cord explants from patients with normal term pregnancies (>37 weeks of gestation), as well as myometrium and cervix explants from patients undergoing hysterectomies. Tissues were stimulated with lipopolysaccharide or phytohemagglutinin for 24 hours. We then analyzed the expression stability and the pairwise variation of GAPDH, ACTB and RNA18S1 from real time quantitative RT-PCR absolute threshold cycles (Cp) using geNorm software.ResultsIn all of the tissues, the three housekeeping genes showed great stability under our experimental conditions. Pairwise variation analyses showed that only two reference genes are required for adequate normalization, GAPDH and ACTB being optimal in the cervix, fetal membranes and umbilical cord, while GAPDH and RNA18S1 are best for normalization in the placenta and myometrium.ConclusionOur results show that GAPDH, ACTB and RNA18S1 are adequate references for gene expression normalization in reproductive tissues stimulated with mitogens in culture.

Hypoxic Stress Forces Irreversible Differentiation of a Majority of Mouse Trophoblast Stem Cells Despite FGF4

ABSTRACT Hypoxic, hyperosmotic, and genotoxic stress slow mouse trophoblast stem cell (mTSC) proliferation, decrease potency/stemness, and increase differentiation. Previous reports suggest a period of reversibility in stress-induced mTSC differentiation. Here we show that hypoxic stress at 0.5% O2 decreased potency factor protein by ∼60%–90% and reduced growth to nil. Hypoxia caused a 35-fold increase in apoptosis at Day 3 and a 2-fold increase at Day 6 above baseline. The baseline apoptosis rate was only 0.3%. Total protein was never less than baseline during hypoxic treatment, suggesting 0.5% O2 is a robust, nonmorbid stressor. Hypoxic stress induced ∼50% of trophoblast giant cell (TGC) differentiation with a simultaneous 5- to 6-fold increase in the TGC product antiluteolytic prolactin family 3, subfamily d, member 1 (PRL3D1), despite the presence of fibroblast growth factor 4 (FGF4). Hypoxia-induced TGC differentiation was also supported by potency and differentiation mRNA marker analysis. FGF4 removal at 20% O2 committed cell fate towards irreversible differentiation at 2 days, with similar TGC percentages after an additional 3 days of culture under potency conditions when FGF4 was readded or under differentiation conditions without FGF4. However, hypoxic stress required 4 days to irreversibly differentiate cells. Runted stem cell growth, forced differentiation of fewer cells, and irreversible differentiation limit total available stem cell population. Were mTSCs to respond to stress in a similar mode in vivo, miscarriage might occur as a result, which should be tested in the future.

Evaluation of reference genes for expression studies in leukocytes from term human pregnancy.

INTRODUCTION Human labor is considered an inflammatory process modulated by systemic and local leukocytes that infiltrate into the maternal-fetal interface. The putative roles of these leukocytes are currently being studied with gene expression assays. Such assays are normalized by the expression of housekeeping genes. However, expression of housekeeping genes may vary depending on the cell type and/or the experimental conditions. The aim of this study was to analyze the expression stability of several housekeeping genes in leukocytes from term human pregnancies, considering both anatomical origin and presence of labor. METHODS We analyzed the gene expression of ACTB, B2M, GAPDH, GUSB, PGK1, RN18S1, TBP and UBC in leukocytes from maternal peripheral blood, placental blood and choriodecidua in women delivering at term with or without the presence of labor through real-time qPCR. Then we used geNorm to evaluate expression stability and pairwise variation. RESULTS The expression of all tested genes showed to be stable independent of the anatomical compartment and the absence or presence of labor. However, PGK1, GUSB and TBP showed to be the most stable and RN18S1 the least stable. Pairwise variation analyses showed that only two genes are needed for normalization yet the inclusion of a third improves its accuracy. DISCUSSION PGK1, GUSB and TBP are the most adequate reference genes for gene expression normalization in leukocytes from term pregnancies, regardless of their anatomical origin (maternal peripheral blood, placental blood or choriodecidua) or the presence or absence of labor. Our study is the first report on housekeeping gene stability in leukocytes from healthy pregnant women.