miRNAs associated with endoplasmic reticulum stress and unfolded protein response during decidualization.

RESEARCH QUESTION: Do microRNAs (miRNAs) play a role in regulating endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) in decidualized cells and endometrium associated with reproductive failures? DESIGN: Endometrial stromal cell line St-T1b was decidualized in vitro with 8-Br-cAMP over 5 days, or treated with the ERS inducer thapsigargin. Expression of ERS sensors, UPR markers and potential miRNA regulators was analysed by quantitative PCR. Endometrial biopsies from patients with recurrent pregnancy loss (RPL) and recurrent implantation failure (RIF) were investigated for the location of miRNA expression. RESULTS: Decidualization of St-T1b cells resulted in increased expression of ERS sensors including ATF6α, PERK and IRE1α, and the UPR marker, CHOP. TXNIP, which serves as a link between the ERS pathway and inflammation, as well as inflammasome NLRP3 and interleukin 1ß expression increased in decidualized cells. An in-silico analysis identified miR-17-5p, miR-21-5p and miR-193b-3p as miRNAs potentially involved in regulation of the ERS/UPR pathways and inflammation associated with embryo implantation. Their expression decreased significantly (P ≤ 0.0391) in non-decidualized cells in the presence of thapsigargin. Finally, expression of the selected miRNAs was localized by in-situ hybridization in stromal and glandular epithelial cells in endometrial samples from patients with RPL and RIF. Expression in stroma cells from patients with RPL was lower in comparison with stroma cells from patients with RIF. CONCLUSIONS: Decidualization in St-T1b cells is accompanied by ERS/UPR processes, associated with an inflammatory response that is potentially influenced by miR-17-5p, miR-21-5p and miR-193b-3p. These miRNAs are expressed differentially in stromal cells from patients with RPL and RIF, indicating an alteration in regulation of the ERS/UPR pathways.

Addressing microchimerism in pregnancy by ex vivo human placenta perfusion.

The physical connection of mother and offspring during pregnancy allows the bi-directional exchange of a small number of cells through the placenta. These cells, which can persist long-term in the recipient individual are genetically foreign to it and therefore fulfill the principle of microchimerism. Over the last years, pioneer research on microchimeric cells revealed their role in immune adaptation during pregnancy and priming of tolerogenic responses in the progeny. However, the mechanisms involved in cell transfer across the placenta barrier remain poorly investigated. In this review, we summarize the evidence of fetomaternal microchimerism, propose a mechanism for cell trafficking through the placenta and discuss the different models and techniques available for its analysis. Likewise, we aim to generate interest in the use of ex vivo placenta perfusion to investigate microchimerism in physiological and pathological settings.

Immunomodulatory properties of extracellular vesicles in the dialogue between placental and immune cells.

Extracellular vesicle (EV)-mediated communication has been implicated in the cooperative alliance between trophoblast and immune cells toward maternal tolerance and placentation. Syncytiotrophoblast cells secrete EVs directly into the maternal circulation, which are taken up by immune cells, endothelial cells, and other cell types. Initial evidence also shows that EVs produced by immune cells are, in turn, incorporated by trophoblast cells and modulate placental responses. Non-coding RNAs (ncRNAs), proteins, and lipid mediators transported in EVs are able to influence proliferation, differentiation, cytokine production, and immunological responses of recipient cells. The molecular alphabet and cellular targets involved in this dialogue are being revealed. Nevertheless, several questions regarding the whole content, surface markers, and biological functions of EVs still remain to be investigated in both physiological and pathological conditions. Analysis of circulating EVs in maternal blood has the potential to serve as a minimally invasive approach to monitoring placental functions and immunological features of pregnancy, aiding in the diagnostics of complications. This review addresses the immunomodulatory properties of EVs and their tasks in the communication between placental and immune cells.

IκBNS and IL-6 expression is differentially established in the uterus of pregnant healthy and infected mice.

During pregnancy, NF-κB plays an important role for embryo implantation and the onset of labor. Regulated IL-6 production, under transcriptional control of NF-κB, is essential for a successful pregnancy outcome and the atypical regulator IκBNS is involved in this process. Previously, we showed that IκBNS negatively regulates IL-6 in uterine tissues during mouse estrous cycle. In this work, we analyzed if IκBNS and IL-6 expression in pregnant mice under physiological or L. monocytogenes-infected conditions would remain as observed in estrous cycle. In the healthy pregnancy IL-6 was highly expressed during implantation/placentation and labor stages but decreased during fetal development and post-partum stages. In contrast, in mice infected before pregnancy, IL-6 expression was not increased in the implantation stage, and its regulator IκBNS increased more in the infected condition rather than in the healthy pregnancy. IκBNS expression was reduced in post-implantation infection, allowing for IL-6 overexpression. The IκBNS-unrelated cytokine IL-36γ, used as inflammatory cytokine marker, was severely increased in the infected uterine tissues. When we analyzed the effect of infection over the fetuses, we found that pre-implantation infection caused the resorption (rejection) of some products, while the post-implantation infection restricted the intrauterine growth of fetuses. The results suggest that in the uterine tissue of pregnant mice the regulatory effect of IκBNS over IL-6 is more evident in an infection status rather than in a healthy condition.

Doxorubicin induces cytotoxicity and miR-132 expression in granulosa cells.

Doxorubicin (DOX) is one of the most commonly used drugs for the treatment of childhood cancers, including leukemia and lymphomas. Despite the high survival rate, female leukemia survivors are at higher risk of ovarian failure and infertility later in life. Treatment with chemotherapeutic drugs like DOX is associated with damage in ovarian follicles, but the affectation grade of granulosa cells remains unclear. To assess and avoid the possible side-effects of DOX, early biomarkers of ovarian injury and chemotherapy-induced ovarian toxicity should be identified. MicroRNAs (miRNAs) have emerged in recent years as a promising new class of biomarkers for drug-induced tissue toxicity. In this study, the effects of DOX on cell viability, steroidogenesis, and miRNA expression were studied in primary granulosa cells (GCs) and in two cellular models (COV434 and KGN cells). We report that compared to other chemotherapeutic drugs, DOX treatment is more detrimental to granulosa cells as observed by decrease of cell viability. Treatment with DOX changes the expression of the aromatase gene (CYP19A1) and the secretion of 17ß-estradiol (E2) in a cell-specific manner. miR-132-3p is dose-dependently increased by DOX in all cellular models. In absence of DOX, miR-132-3p overexpression in COV434 cells has no effect on E2 secretion or CYP19A1 expression. Altogether, these findings contribute to understanding the hormonal disbalance caused by DOX in human ovarian cells and suggest miR-132 as a putative sensor to predict DOX-induced ovarian toxicity.

Identification of miRNAs and associated pathways regulated by Leukemia Inhibitory Factor in trophoblastic cell lines.

INTRODUCTION: Leukemia Inhibitory Factor (LIF) regulates behavior of trophoblast cells and their interaction with immune and endothelial cells. In vitro, trophoblast cell response to LIF may vary depending on the cell model. Reported differences in the miRNA profile of trophoblastic cells may be responsible for these observations. Therefore, miRNA expression was investigated in four trophoblastic cell lines under LIF stimulation followed by in silico analysis of altered miRNAs and their associated pathways. METHODS: Low density TaqMan miRNA assays were used to quantify levels of 762 mature miRNAs under LIF stimulation in three choriocarcinoma-derived (JEG-3, ACH-3P and AC1-M59) and a trophoblast immortalized (HTR-8/SVneo) cell lines. Expression of selected miRNAs was confirmed in primary trophoblast cells and cell lines by qPCR. Targets and associated pathways of the differentially expressed miRNAs were inferred from the miRTarBase followed by a KEGG Pathway Enrichment Analysis. HTR-8/SVneo and JEG-3 cells were transfected with miR-21-mimics and expression of miR-21 targets was assessed by qPCR. RESULTS: A similar number of miRNAs changed in each tested cell line upon LIF stimulation, however, low coincidence of individual miRNA species was observed and occurred more often among choriocarcinoma-derived cells (complete data set at http://www.ncbi.nlm.nih.gov/geo/ under GEO accession number GSE130489). Altered miRNAs were categorized into pathways involved in human diseases, cellular processes and signal transduction. Six cascades were identified as significantly enriched, including JAK/STAT and TGFB-SMAD. Upregulation of miR-21-3p was validated in all cell lines and primary cells and STAT3 was confirmed as its target. DISCUSSION: Dissimilar miRNA responses may be involved in differences of LIF effects on trophoblastic cell lines.

IL-36 Cytokines: Regulators of Inflammatory Responses and Their Emerging Role in Immunology of Reproduction.

The IL-36 subfamily of cytokines has been recently described as part of the IL-1 superfamily. It comprises three pro-inflammatory agonists (IL-36α, IL-36ß, and IL-36γ), their receptor (IL-36R), and one antagonist (IL-36Ra). Although expressed in a variety of cells, the biological relevance of IL-36 cytokines is most evident in the communication between epithelial cells, dendritic cells, and neutrophils, which constitute the common triad responsible for the initiation, maintenance, and expansion of inflammation. The immunological role of IL-36 cytokines was initially described in studies of psoriasis, but novel evidence demonstrates their involvement in further immune and inflammatory processes in physiological and pathological situations. Preliminary studies have reported a dynamic expression of IL-36 cytokines in the female reproductive tract throughout the menstrual cycle, as well as their association with the production of immune mediators and cellular recruitment in the vaginal microenvironment contributing to host defense. In pregnancy, alteration of the placental IL-36 axis has been reported upon infection and pre-eclampsia suggesting its pivotal role in the regulation of maternal immune responses. In this review, we summarize current knowledge regarding the regulatory mechanisms and biological actions of IL-36 cytokines, their participation in different inflammatory conditions, and the emerging data on their potential role in normal and complicated pregnancies.

Competition/antagonism associations of biofilm formation among Staphylococcus epidermidis Agr groups I, II, and III.

Staphylococci have quorum-sensing (QS) systems that enable cell-to-cell communication, as well as the regulation of numerous colonization and virulence factors. The accessory gene regulator (Agr) operon is one of the Staphylococcus genus QS systems. Three groups (I, II, and III) are present in Staphylococcus epidermidis Agr operon. To date, it is unknown whether Agr groups can interact symbiotically during biofilm development. This study analyzed a symbiotic association among Agr groups during biofilm formation in clinical and commensal isolates. Different combinations among Agr group isolates was used to study biofilm formation in vitro and in vivo (using a mouse catheter-infection model). The analysis of Agr groups were also performed from samples of human skin (head, armpits, and nostrils). Different predominant coexistence was found within biofilms, suggesting symbiosis type. In vitro, Agr I had a competition with Agr II and Agr III. Agr II had a competition with Agr III, and Agr II was an antagonist to Agr I and III when the three strains were combined. In vivo, Agr II had a competition to Agr I, but Agr I and II were antagonists to Agr III. The associations found in vitro and in vivo were also found in different sites of the skin. Besides, other associations were observed: Agr III antagonized Agr I and II, and Agr III competed with Agr I and Agr II. These results suggest that, in S. epidermidis, a symbiotic association of competition and antagonism occurs among different Agr groups during biofilm formation.

Estrous Cycle and Gestational Age-Dependent Expression of Members of the Interleukin-36 Subfamily in a Semi-Allogeneic Model of Infected and Non-Infected Murine Pregnancy.

The IL-36 subfamily is a recently described group of cytokines with pro-inflammatory behavior, comprising three agonists (α, ß, and γ), its receptor (R), and one antagonist (Ra). The expression and function of IL-36 subfamily members in the estrous cycle in healthy and infected pregnancy has not been described. We evaluated mRNA and protein expression of IL-36 family members during the estrous cycle, implantation, fetal development, and post-labor periods in a model of allogenic pregnancy in mice. We also explored the ability of Listeria monocytogenes to modulate the expression of IL-36 subfamily members during pregnancy. Expression of IL-36 subfamily members showed different expression during the estrous cycle and pregnancy but was induced at estrous, 16.5 days post coitum (dpc), 18.5 dpc, and labor. IL-36 subfamily members showed a characteristic distribution in the glandular epithelium, perimetrium, myometrium, and stratum vasculare. Infection with L. monocytogenes during pregnancy induced strong production of IL-36 subfamily members, an observation that correlated with an increasing prevalence of fetal loss. In conclusion, IL-36 agonists showed specific patterns of mRNA and protein expression that might suggest functional specialization or specific target cells. Infection with L. monocytogenes during pregnancy induced strong production of IL-36 subfamily members.