Lupus and recurrent pregnancy loss: the role of female sex hormones and B cells.

Systemic lupus erythematosus is a debilitating autoimmune disease characterized by uncontrolled activation of adaptive immunity, particularly B cells, which predominantly affects women in a 9 to 1 ratio compared to men. This stark sex disparity strongly suggests a role for female sex hormones in the disease's onset and progression. Indeed, it is widely recognized that estradiol not only enhances the survival of autoreactive B cells but also stimulates the production of autoantibodies associated with systemic lupus erythematosus, such as anti-nuclear antibodies and anti-dsDNA antibodies. Clinical manifestations of systemic lupus erythematosus typically emerge after puberty and persist throughout reproductive life. Furthermore, symptoms often exacerbate during the premenstrual period and pregnancy, as increased levels of estradiol can contribute to disease flares. Despite being fertile, women with lupus face a heightened risk of pregnancy-related complications, including pregnancy loss and stillbirth, which significantly surpass the rates observed in the healthy population. Therefore, this review aims to summarize and discuss the existing literature on the influence of female sex hormones on B-cell activation in patients with systemic lupus erythematosus, with a particular emphasis on their impact on pregnancy loss.

Splenic B1 B Cells Acquire a Proliferative and Anti-Inflamatory Profile During Pregnancy in Mice.

B cells are a heterogeneous cell population with differential ontogeny, anatomical location, and functions. B1 B cells are a distinct subpopulation characterized by their unique capacity of self-renewal, the production of large quantities of IL-10, and the ability to secrete protective, anti-inflammatory natural antibodies (NAbs), presumably upon down-regulation of CD1d expression. Although natural antibodies are thought to be protective, due to their polyreactivity, their participation in certain autoimmune diseases has been suggested. In the context of pregnancy, the role of B1 B cells has been discussed controversially. While in human pregnancies B1 B cells and natural/polyreactive antibodies they produce are involved in the development of preeclampsia, in mice they promote healthy gestation and fetal protection. In this work, we aimed to functionally characterize the splenic B1 B cell population during pregnancy in mice. Functional enrichment analysis using only up-regulated transcripts from a transcriptomic profile performed on total splenic B cells from pregnant compared to non-pregnant mice showed augmented cell cycle and DNA replication pathways. Proliferation studies by flow cytometry showed augmented Ki-67 proliferation marker expression and percentages of B1 B cells. Furthermore, B1 B cells produced higher levels of IL-10 and lower levels of TNF-α leading to an increased IL-10/TNF-α ratio and showing an immunoregulatory phenotype. Finally, we observed lower expression of CD1d on B1 B cells, suggesting a higher capacity to produce NAbs in the context of pregnancy. In summary, our results showed not only an expanded and proliferative splenic B1 B cell population during pregnancy but also the acquisition of immunomodulatory capacities suggesting its critical role in the intricate process of pregnancy tolerance.

An overview of the role of probiotics in pregnancy-associated pathologies with a special focus on preterm birth.

Probiotics can modulate the host immune system and keep a healthy microbiota thus enhancing the integrity of the mucosal epithelium. They were proven to be useful as therapeutic strategies for a variety of inflammatory diseases. Preterm birth is a multicausal complication where the early inflammatory cascade activation affects both the mother and the fetus and may have an irreversibly impact on infant development. Prevention of preterm birth is a challenge that calls for different strategies targeting its diverse etiologies. For the past decade, novel and numerous studies investigated the role of probiotics for the prevention of preterm birth given their above-mentioned abilities. This allows a wider approach to the multifactorial causes of preterm birth. In this sense, probiotic administration was shown to be inversely related to the rate of preterm birth. In this review, we discuss the latest reports involving probiotics and preterm birth, and the chances of probiotics becoming a therapeutic strategy for the prevention of pregnancy-associated pathologies like preterm birth shortly.

B cells acquire a unique and differential transcriptomic profile during pregnancy.

Pregnancy alters B cell development and function. B cell activation is initiated by antigens binding to the BCR leading to B cell survival, proliferation, antigen presentation and antibody production. We performed a genome-wide transcriptome profiling of splenic B cells from pregnant (P) and non-pregnant (NP) mice and identified 1136 genes exhibiting differential expression in B cells from P mice (625 up- and 511 down-regulated) compared to NP animals. In silico analysis showed that B cell activation through BCR seems to be lowered during pregnancy. RT-qPCR analysis confirmed these data. Additionally, B cells from pregnant women stimulated in vitro through BCR produced lower levels of inflammatory cytokines compared to non-pregnant women. Our results suggest that B cells acquire a state of hypo-responsiveness during gestation, probably as part of the maternal immune strategy for fetal tolerance but also open new avenues to understand why pregnant women are at highest risk for infections.

Probiotic Lactobacillus kefiri prevents endotoxin-induced preterm birth and stillbirth in mice.

Preterm birth (PTB), defined as birth occurring before 37 weeks of pregnancy, affects 5-18% of pregnancies and is the leading cause of neonatal morbidity and mortality worldwide. Although PTB is considered a syndrome, infection-induced inflammation accounts for up to 50% of all cases. Despite the effort to reduce the incidence of PTB, it continues to rise worldwide and current approaches for preventing or treating PTB are largely unsatisfactory. Probiotics are live microorganisms which, when administered in adequate amounts, confer a health benefit on the host. It is well known that probiotics can modulate the host immune system exerting a potent anti-inflammatory activity. The main aim of this work was to evaluate the capacity of the probiotic Lactobacillus kefiri (Lk48) to prevent preterm birth in mice. C57BL/6 female mice were treated with Lk48 or vehicle a week before and during pregnancy and were challenged with LPS (10 µg), a dose known to induce PTB on gestational day 16. Percentages of PTB as well as stillbirth were evaluated. We observed that oral administration of Lk48 significantly reduced the occurrence of LPS-induced PTB and stillbirth as well as improved post-natal development. This protective effect was associated with a reduction in leucocyte infiltration and reduced inflammation-induced damage in reproductive tissue. Besides, Lk48 treatment also modulated the diversity of vaginal microbiota. Our results demonstrated that prophylactic consumption of probiotic L. kefiri prevented LPS-induced PTB and still birth in mice and opens new avenues for exploring novel and promising strategies for preventing PTB in humans.

Expression of IL-33 Receptor Is Significantly Up-Regulated in B Cells During Pregnancy and in the Acute Phase of Preterm Birth in Mice.

Interleukin-33 (IL-33) is a mucosal alarmin belonging to the IL-1 cytokine family and is now recognized to have a key role in innate and adaptive immunity, contributing to tissue homeostasis and response to environmental stresses. In addition, IL-33 has also been shown to work as a positive regulator that initiates and maintains a Th2 immune response. In the context of pregnancy, it has been recently demonstrated that upon certain stress conditions, such as an infection induced inflammation, IL-33 is released from the uterine mucosa and triggers decidual B cells to produce anti-inflammatory molecules, which in turn restore immune homeostasis and prevents the development of preterm birth. In this study we therefore performed a detailed characterization of IL-33 receptor (Il1rl1 or ST2) expression in B cells during normal pregnancy, as well as in a mouse model of preterm birth. We observed that splenic B cells significantly up-regulate the expression of Il1rl1 during pregnancy and identified the B1 B cell population as the main ST2-expressing B cell subset. A further kinetic analysis showed that percentages of ST2-expressing B1 B cells are significantly augmented on days 12 and 14 of pregnancy, both in the spleen and peritoneal cavity of pregnant mice, and then drop toward the end of pregnancy to the levels observed in non-pregnant animals. Furthermore, using a mouse model of LPS-induced preterm birth, we demonstrated that not only are the percentages of ST2-expressing B1 B cells significantly enlarged in the spleen during the acute phase of preterm birth, but decidual B cells also significantly up-regulate ST2 expression as compared to term-pregnant mice. Overall, our results suggest a functional role of ST2 expression in B cells during pregnancy and reinforce the importance of the IL-33/ST2 axis in B cells as a critical mechanism to control inflammation-induced preterm birth.

The enrichment of maternal environment prevents pre-term birth in a mice model.

Maternal lifestyle affects both mother health and pregnancy outcome in humans. Several studies have demonstrated that interventions oriented toward reducing stress and anxiety have positive effects on pregnancy complications such as preeclampsia, excessive gestational weight, gestational diabetes and preterm birth. In this work, we showed that the environmental enrichment (EE), defined as a noninvasive and biologically significant stimulus of the sensory pathway combined with voluntary physical activity, prevented preterm birth (PTB) rate by 40% in an inflammatory mouse model induced by the systemic administration of bacterial lipopolysaccharide (LPS). Furthermore, we found that EE modulates maternal metabolism and produces an anti-inflammatory environment that contributes to pregnancy maintenance. In pregnant mice uterus, EE reduces the expression of TLR4 and CD14 (the LPS receptor and its coactivator protein), preventing the LPS-induced increase in PGE2 and PGF2α release and nitric oxide synthase (NOS) activity. In cervical tissue, EE inhibits cervical ripening events, such as PGE2 release, matrix metalloproteinase (MMP)-9 increased activity and neutrophil recruitment, therefore conserving cervical function. It seems that EE exposure could mimic the stress and anxiety-reducing techniques mentioned above, explaining, at least partially, the beneficial effects of having a healthy lifestyle before and during gestation. Furthermore, we propose that designing an EE protocol for humans could be a noninvasive and preventive therapy for pregnancy complications, averting pre-term birth occurrence and dreaded sequelae that are present in the offspring born too soon.

A role for the endocannabinoid system in premature luteal regression and progesterone withdrawal in lipopolysaccharide-induced early pregnancy loss model.

STUDY QUESTION: What is the role of the endocannabinoid system (eCS) in the alterations of the endocrine system in a murine model of lipopolysaccharide (LPS)-induced miscarriage? SUMMARY ANSWER: In 7-days pregnant wild type, but not cannabinoid receptor type 1 knockout (CB1-KO) mice, LPS increased COX-2 expression and prostaglandin F2α (PGF2α) production in the uterus leading to lower expression of prolactin receptor in the ovary and a marked regression of corpora lutea (CL), suggesting that the eCS mediates the deleterious effects of LPS on reproductive events. WHAT IS KNOWN ALREADY: Appropriate systemic progesterone levels are critical for a successful pregnancy outcome. Precocious loss of luteal progesterone (P4) secretion leads to miscarriage in rodents. We have previously shown that LPS administration to pregnant mice induces embryonic resorption accompanied by a dramatic decrease in systemic progesterone levels in a murine model of inflammatory miscarriage, with the eCS mediating these LPS-induced deleterious effects. STUDY DESIGN SAMPLES/MATERIALS, METHODS: CD1 wild-type (WT) and CB1-KO mice were randomly allocated to Vehicle (saline; i.p.) or LPS (0.5 µg/g body weight; i.p.) treated groups: (WT-Vehicle; WT-LPS; CB1-KO-Vehicle and CB1-KO-LPS). A single injection was given on day 7 of pregnancy and tissues (blood, ovary, uterus) were collected 6, 12, 24 and 48 h later. P4 and PGF2α plasma levels were determined by radioimmunoassay. Cyclooxygenase-2 (COX-2) mRNA (RT-PCR) and protein (Western blot) content in uterus was assayed. COX-2 and prolactin receptor (PrlR) mRNA levels in the ovary were assayed by RT-PCR. Tissue morphology of the CL was assessed by haematoxylin-eosin staining. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of 7-day pregnant WT mice with LPS induced a P4 withdrawal (p < 0.05), increased in uterine COX-2 mRNA and protein expression (p < 0.05) as well as an increase in uterine PGF2α production (p < 0.05). These changes were absent in LPS-treated 7-day pregnant CB1-KO mice. In ovarian tissues, LPS treatment to 7-day pregnant WT mice induced a downregulation of PrlR mRNA expression (p < 0.05) together with an increase in COX-2 mRNA expression (p < 0.05) and PGF2α content (p < 0.05). These effects were absent in the CB1-KO mice. Collectively, our results suggest a role for the eCS mediating LPS-induced deleterious effects on reproductive tissues. LIMITATIONS, REASONS FOR CAUTION: An important caveat of this study is the endocrine differences between mice and humans during pregnancy (e.g. P4 is produced by the CL throughout pregnancy in mice, whereas this is not the case in humans), which limits the extrapolation of the results presented here. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide new insights in the role of the endocannabinoid system in the physiopathology of reproduction as well as the role of this endogenous system as a mediator of LPS deleterious effects on reproductive tissues. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2010/0813 and PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). The authors have no competing interests.

Study of the uterine local immune response in a murine model of embryonic death due to Tritrichomonas foetus.

PROBLEM: Bovine tritrichomonosis is a sexually transmitted disease caused by Tritrichomonas foetus, characterized by conceptus loss. We developed a mouse model of tritrichomonosis to study the mechanisms involved in the embryonic death. We hypothesized that embryonic death may be due to an exacerbated maternal response to the pathogen that then affects embryo development. METHOD OF STUDY: We infected BALB/c mice with Tritrichomonas foetus and paired them after confirming active infection. We studied the expression of pro- and anti-inflammatory cytokines, markers for T regulatory and T helper 17 cells as well as haem-oxygenase-1 expression in uterine tissue by real-time RT-PCR. RESULTS: As expected, TNF-α was augmented in infected animals. IL-10 and IL-4 were also up-regulated. Treg-associated genes were higher expressed in uteri of infected group. In mice that have lost their conceptus after the infection, haem-oxygenase-1 (HO-1) mRNA levels were strongly decreased, while RORγt mRNA, a reliable marker for Th17, was augmented in uterus. CONCLUSION: A T effector response of type 1 and 17 may be involved in tritrichomonosis-related embryonic death. This alters protective mechanisms as HO-1. Increased regulatory T cells may facilitate embryonic death by promoting the persistence of infection.

The developing human ovary: immunohistochemical analysis of germ-cell-specific VASA protein, BCL-2/BAX expression balance and apoptosis.

BACKGROUND: Germ cell number during ovarian organogenesis is regulated through programmed cell death. We investigated the expression of germ-cell-specific VASA protein, apoptosis-related proteins BAX and BCL-2 and DNA fragmentation in developing human ovaries from gestation week 12 to term. METHODS: Human fetal ovaries from 13 women undergoing spontaneous abortion were fixed, paraffin-embedded and processed for immunohistochemistry to analyse temporal and cellular localization of VASA, BCL-2 and BAX, and to detect apoptosis by TUNEL assay. RESULTS: VASA showed a differential pattern of expression throughout the differentiation and proliferative phase and prophase I to finally associate with Balbiani's body in primordial and primary follicles. BCL-2 was detected from week 12 to 17 and became undetectable thereafter. Strong BAX signal was detected in oogonia and oocytes from week 12 to term. Low levels (

The ovary of the gestating South American plains vizcacha (Lagostomus maximus): suppressed apoptosis and corpora lutea persistence.

The South American plains vizcacha, Lagostomus maximus, displays an exceptional ovulation rate of up to 800 eggs per cycle, the highest rate recorded for a mammal. Massive polyovulation arises from the overexpression of the apoptosis-inhibiting BCL2 gene leading to a suppression of apoptotic pathways responsible for follicular atresia in mammals. We analyzed the ovarian histology, ovarian apoptosis, and apoptosis-related protein expression with special emphasis in corpora lutea throughout the 5-mo-long gestation period, at parturition day and early postpartum, in L. maximus. Corpora lutea were abundant throughout gestation with no sign of structural regression even at the end of gestation. Both immunohistochemistry and Western blot analysis showed strong signals for apoptosis-inhibiting BCL2 protein, whereas the proapoptotic BAX protein was just detected in isolated luteal cells in gestating females and postpartum females. Apoptosis-associated DNA fragmentation detected by TUNEL was very scarce and occasional and correlated with BAX detection in luteal cells. Marked expression of progesterone and alpha-estrogen receptors in luteal cells was found at early, mid-, and late gestation as well as at parturition day and early postpartum samples. Additionally, serum level of progesterone increased markedly to reach maximal values at late gestation and decreasing at parturition to levels found at early gestation, suggesting that corpora lutea remained functional throughout gestation. These results point out that the unusual ovarian environment of L. maximus in which germ cell demise is abolished through antiapoptotic BCL2 gene overexpression also preserves structural integrity and functionality of corpora lutea during the whole gestation. Overexpression of antiapoptotic BCL2 gene may represent a strategy for an essential need of ovary and corpora lutea in order to maintain pregnancy until term.

Naturally suppressed apoptosis prevents follicular atresia and oocyte reserve decline in the adult ovary of Lagostomus maximus (Rodentia, Caviomorpha).

It has been widely accepted that mammalian females are born with a non-renewing, finite pool of oocytes that will be continuously cleared by atresia, with only a small proportion of them reaching ovulation. Apoptosis regulates this mass germ cell death, especially through the balance between pro- and anti-apoptotic proteins encoded by the BCL-2 gene family. The caviomorph rodent Lagostomus maximus, the South American plains viscacha, displays the highest ovulation rate known for a mammal releasing 400-800 eggs per cycle. We tested the hypothesis that in L. maximus massive polyovulation is a consequence of reduced apoptosis resulting in suppressed follicular atresia. We found that anti-apoptotic BCL-2 gene is markedly expressed in all kind of follicles from primordial to fully mature antral stages in the adult ovary of L. maximus. On the other hand, pro-apoptotic BAX gene showed weak signals or was undetectable by immunohistochemical examination. Western blot against both proteins confirmed immunohistochemical results. Screening for DNA fragmentation by TUNEL assay was conspicuously negative in ovaries from both pregnant and non-pregnant females. In addition, alpha-oestrogen receptor also showed an enhanced expression from primordial stage to fully mature antral follicles. Our results show that natural preferential expression of BCL-2 and restricted BAX expression greatly suppresses apoptosis in the ovary of L. maximus. This prevents the decline of the oocyte reserve by abolishing follicular atresia and enables the highest ovulation rate known for a mammal, 400-800 or more eggs per cycle.