In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

Essential Role of CRIM1 on Endometrial Receptivity in Goat.

In domestic ruminants, endometrial receptivity is related to successful pregnancy and economic efficiency. Despite several molecules having been reported in the past regarding endometrial receptivity regulation, much regarding the mechanism of endometrial receptivity regulation remains unknown due to the complex nature of the trait. In this work, we demonstrated that the cysteine-rich transmembrane bone morphogenetic protein (BMP) regulator 1 (CRIM1) served as a novel regulator in the regulation of goat endometrial receptivity in vitro. Our results showed that hormones and IFN-τ increased the expression of CRIM1 in goat endometrial epithelial cells (EECs). Knockdown of CRIM1 via specific shRNA hindered cell proliferation, cell adhesion and prostaglandins (PGs) secretion and thus derailed normal endometrial receptivity. We further confirmed that receptivity defect phenotypes due to CRIM1 interference were restored by ATG7 overexpression in EECs while a loss of ATG7 further impaired receptivity phenotypes. Moreover, our results showed that changing the expression of ATG7 affected the reactive oxygen species (ROS) production. Moreover, mR-143-5p was shown to be a potential upstream factor of CRIM1-regulated endometrial receptivity in EECs. Overall, these results suggest that CRIM1, as the downstream target of miR-143-5p, has effects on ATG7-dependent autophagy, regulating cell proliferation, cell adhesion and PG secretion, and provides a new target for the diagnosis and treatment of early pregnancy failure and for improving the success rates of artificial reproduction.

Neutrophils recognize and amplify IFNT signals derived from day 7 bovine embryo for stimulation of ISGs expression in vitro: A possible implication for the early maternal recognition of pregnancy.

Previously, we reported that the presence of multiple day 7 (D7) bovine embryos in the uterus induces systemic immune responses in circulating polymorphonuclear neutrophils (PMNs), but with unknown mechanism. Thus, this study aimed to investigate the direct impact of D7 bovine embryo on PMNs' immune responses in vitro and whether these PMNs can amplify and transfer embryo signals further to another PMN population. PMNs were directly stimulated by embryo culture media (ECM) or interferon tau (IFNT) (10 ng/ml) followed by evaluating mRNA expression by real-time PCR and phenotypic analysis by flow cytometry. To test whether PMNs can transfer embryo signals to a new PMN population, PMNs triggered by ECM or IFNT, were thoroughly washed and diluted to remove any media components, and again were incubated in fresh culture media for 3 h, from which culture supernatants were collected and used as PMN conditioned media (CM) to stimulate a new PMN population. Similar to ECM, IFNT directly stimulated expressions of IFNs (IFNA, IFNG), interferon-stimulated genes (ISGs; OAS1, ISG15, MX1), STAT1, TGFB and IL8, and downregulated TNFA in PMNs. Flow cytometrical analyses demonstrated that IFNT stimulated expressions of pregnancy-related phenotypic markers, CD16 and arginase-1 (ARG1), in PMNs. Most importantly, PMN CM induced ISGs and STAT1 mRNA in fresh PMNs. Since IFNT directly upregulated IFNA expression in PMNs, the impact of IFNA on PMNs' immune responses was further tested. Stimulation of PMNs with IFNA, especially at a low level (1 pg/ml), induced IFNT-like immune responses comparable to those induced by PMN CM. Together, these findings indicated that D7 bovine embryos induce direct anti-inflammatory responses with upregulation of ISGs expressions in PMNs mainly via IFNT. Additionally, PMNs can amplify and transfer embryo signals to a new PMN population in a cell-to-cell communication mechanism possibly mediated in part by IFNA. Such a novel immunological crosstalk might contribute to embryo tolerance and pregnancy establishment in cattle.

In vitro effects of Type I interferons (IFNτ and IFNα) on bovine hepatocytes cultured with or without Kupffer cells.

In cattle, maternal recognition of early pregnancy depends on the effects of the embryonic signal interferon (IFN)-τ. IFN-stimulated genes have been upregulated in the maternal liver during early pregnancy. In this study, primary hepatocyte cell culture models were evaluated for their suitability to test Type I IFN effects invitro. The expression of target genes (interferon-stimulated gene 15 (ISG-15), interferon-induced GTP-binding protein (MX-1), C-X-C motif chemokine 10 (CXCL-10), CXCL-5, insulin-like growth factor 1 (IGF-1), IGF binding protein 2 (IGFBP-2)) was measured using reverse transcription-quantitative polymerase chain reaction in hepatocytes from monoculture or in indirect coculture with Kupffer cells (HKCid) on Days 1, 2, 3 and 4 of culture (n=21 donor cows). Gene expression was also measured on Day 4 after challenging the cultures with recombinant IFNτ, IFNα, progesterone (P4), IFNτ+IFNα or IFNτ+P4 for 6h. A significant increase in the mRNA expression of target genes in hepatocytes was shown in response to stimulation with IFNτ. The Kupffer cells in coculture did not influence the effects of IFNτ in hepatocytes. In conclusion, primary bovine hepatocyte cultures are suitable for stimulation experiments with Type I IFNs and as an extrauterine model for embryo-maternal communication. The proposed endocrine action of IFNτ in the liver may affect maternal metabolism and immune function in the liver.

IFN-τ Attenuates LPS-Induced Endometritis by Restraining HMGB1/NF-κB Activation in bEECs.

Endometritis is a common inflammatory disease in uterine tissues that leads to animal infertility. Among the causes, Escherichia coli infection is one of the main reasons. Interferon-tau (IFN-τ) is the initial pregnancy signal for ruminant embryos and can induce immune tolerance in humans and other species. However, there are scarce reports on whether IFN-τ has a regulatory effect on endometrial inflammatory damage through HMGB1-NF-κB signalling. The purpose of this study was to investigate the regulatory mechanism of IFN-τ in HMGB1-NF-κB signalling in LPS-induced endometritis. ELISA and qPCR were used to detect the expression of LPS-induced pro-inflammatory cytokines in bovine endometrial epithelial cells (bEECs or BEND) under IFN-τ intervention, and the levels of HMGB1, p-IKK and p-p65 were detected by Western blotting. The nuclear translocation of NF-κB p65 was determined through immunofluorescence. In addition, bEECs were transfected with si-HMGB1 to elucidate the key role of HMGB1 and IFN-τ in the endometrial inflammatory cascade. The results indicated that IFN-τ inhibits the expression of related pro-inflammatory cytokines in an inflammatory injury model of bovine endometrial epithelial cells induced by LPS. Furthermore, experiments have proven that IFN-τ has protective effects on E. coli endotoxin-induced endometritis in mice in vivo. IFN-τ inhibited the HMGB1-NF-κB axis and significantly reduced the secretion of pro-inflammatory cytokines, the expression of HMGB1 protein and the levels of IKK and NF-κB p65 phosphorylation. In summary, our results showed that IFN-τ resists E. coli endotoxin-induced endometritis by attenuating HMGB1/NF-κB signalling.

Endometrial extracellular matrix rigidity and IFNτ ensure the establishment of early pregnancy through activation of YAP.

BACKGROUND: In mammals, early pregnancy is a critical vulnerable period during which complications may arise, including pregnancy failure. Establishment of a maternal endometrial acceptance phenotype is a prerequisite for semiheterogeneous embryo implantation, comprising the rate-limiting step of early pregnancy. METHODS: Confocal fluorescence, immunohistochemistry and western blot for nuclear and cytoplasmic protein were used to examine the activation of yes-associated protein (YAP) in uterine tissue and primary endometrial cells. The target binding between miR16a and YAP was verified by dual-luciferase reporter gene assay. The mouse pregnancy model and pseudopregnancy model were used to investigate the role of YAP in the maternal uterus during early pregnancy in vivo. RESULTS: We showed that YAP translocates into the nucleus in the endometrium of cattle and mice during early pregnancy. Mechanistically, YAP acts as a mediator of ECM rigidity and cell density, which requires the actomyosin cytoskeleton and is partially dependent on the Hippo pathway. Furthermore, we found that the soluble factor IFNτ, which is a ruminant pregnancy recognition factor, also induced activation of YAP by reducing the expression of miR-16a. CONCLUSIONS: This study revealed that activation of YAP is necessary for early pregnancy in bovines because it induced cell proliferation and established an immunosuppressive local environment that allowed conceptus implantation into the uterine epithelium.

Placenta-specific protein 1 enhances liver metastatic potential and is associated with the PI3K/AKT/NF-κB signaling pathway in colorectal cancer.

To better explore the underlying mechanism of liver metastatic formation by placenta-specific protein 1 (PLAC1) in human colorectal cancer, we investigated the proliferation, invasion and angiogenic capabilities of human colorectal cancer cells with different liver metastatic potentials as well as the mechanism of action of PLAC1 in the metastatic process. The expression of PLAC1 was detected by reverse transcriptase PCR, western blot, and real-time PCR. The effect of PLAC1 on metastatic potential was determined by proliferation, invasion, and angiogenesis assays, including an in-vitro coculture system consisting of cancer cells and vascular endothelial cells that were used to detect the relationship between cancer cells and angiogenesis. In addition, we also determined PLAC1 downstream targets that preferentially contribute to the metastatic process. PLAC1 was expressed in HT-29, WiDr, and CaCo-2 colorectal cancer cells but not in Colo320 colorectal cancer cells. PLAC1 not only enhanced significantly the proliferation of CoLo320 and human umbilical vein endothelial cells (HUVECs) but also promoted the invasion of CoLo320 cells. The angiogenesis of HUVECs was enhanced by PLAC1 in a dose-dependent manner. In cocultured systems, angiogenesis was significantly increased by coculture with HT-29 cells. In addition, PLAC1 could promote angiogenesis in coculture with HT-29 cells. Furthermore, PLAC1-enhanced metastatic potential of colorectal cancer cells was dependent on the activation of the PI3K/Akt/NF-κB pathway. The activation of PI3K/Akt/NF-κB signaling by PLAC1 may be critical for metastasis of colorectal cancer cells. According to our results, we suggest that modification of PLAC1 function might be a promising new therapeutic approach to inhibit the aggressive spread of colorectal cancer.

Magnetically Responsive Polymeric Microparticles for the Triggered Delivery of a Complex Mixture of Human Placental Proteins.

Bone loss through traumatic injury is a significant clinical issue. Researchers have created many scaffold types to mimic an extracellular matrix to provide structural support for the formation of new bone, however functional regeneration of larger scaffolds has not been fully achieved. Newer scaffolds aim to deliver bioactive molecules to improve tissue regeneration. To achieve a more comprehensive regenerative response, a magnetically triggerable polymeric microparticle platform is developed for the on-demand release of a complex mixture of isolated human placental proteins. This system is composed of polycaprolactone (PCL) microparticles, encapsulating magnetic nanoparticles (MNPs), and placental proteins. When subjected to an alternating magnetic field (AMF), the MNPs heat and melt the PCL, enhancing the diffusion of proteins from microparticles. When the field is off, the PCL re-solidifies. This potentially allows for cyclic drug delivery. Here the design, synthesis, and proof-of-concept experiments for this system are reported. In addition, it is shown that the proteins retain function after being magnetically released. The ability to trigger the release of complex protein mixtures on-demand may provide a significant advantage with wounds where stagnation of healing processes can occur (e.g., large segmented bone defects).

Peptidoglycan disrupts early embryo-maternal crosstalk via suppression of ISGs expression induced by interferon-tau in the bovine endometrium.

Uterine infection with bacteria and the release of peptidoglycan (PGN), antigenic cell wall components of both Gram-negative and Gram-positive bacteria, can cause early pregnancy losses in ruminants, but the associated mechanisms remain unsolved. Day 7 blastocyst starts to secrete a minute amount of interferon-tau (IFNT) in the uterine horn which is required for early stage of maternal recognition of pregnancy (MRP) in ruminants, and it induces interferon stimulated genes (ISGs) for driving uterine receptivity in cows. This study investigated if PGN disrupts IFNT response through modulation of endometrial ISGs expressions. Cultured bovine endometrial epithelial cells (BEECs) were treated with embryo culture medium (ECM) or IFNT (1 ng/ml) in the presence or absence of a low level of PGN (10 pg/ml) for 24 h. A real-time PCR analyses revealed that the presence of PGN suppressed IFNT-induced ISGs (OAS1 and ISG15) and STAT1 expressions in BEECs. To visualize the impact of PGN in an ex-vivo model that resembles the in vivo status, endometrial explants were treated by IFNT (1 ng/ml) with or without PGN (10 pg/ml) for 12 h. PGN suppressed IFNT-induced gene expressions of the above factors, but not for IFNA receptor type1 (IFNAR1) or type2 (IFNAR2) in explants. Immunofluorescence analysis illustrated that PGN completely suppressed the IFNT-triggered OAS1 protein expression in the luminal epithelium of explants. Of note, PGN did not stimulate pro-inflammatory cytokines (TNFA and IL1B) or TLR2 mRNA expression in both models. These findings indicate that the presence of low levels of PGN suppresses ISGs expression induced by IFNT secreted from early embryo, at the luminal epithelium of the bovine endometrium. This could severely interfere with early stage of MRP processes in cows, leading to pregnancy failure.

Pentraxin-3 mediates prosurvival actions of interferon tau in bovine luteinized granulosa cells.

Pentraxin 3 (PTX3), a multimeric glycoprotein, is implicated in various biological functions. PTX3 was shown to be elevated in the corpus luteum (CL) of early pregnant ewes; however, its role in sheep or other ruminants' CL during this reproductive stage or how it is regulated remain unknown. Here we explored the role of PTX3 and its relationship with interferon-tau (IFNT; the pregnancy recognition signaling molecule during early pregnancy in domestic ruminants) in bovine luteinized granulosa cells (LGCs). IFNT robustly elevated PTX3 expression in bovine LGCs, and significantly stimulated its expression in luteal endothelial cells, along with CL slices; yet, LGCs were the most responsive and sensitive among these luteal models. ALK2/ALK3/ALK6 kinase inhibitor, dorsomorphin, dose-dependently inhibited basal and IFNT-elevated PTX3 expression in LGCs. In contrast, ALK4/5/7 inhibitor, SB431542, did not alter basal and TGFB1-induced PTX3. We found that recombinant human PTX3 itself moderately but significantly increases LGC numbers. Because PTX3 is highly expressed in bovine LGCs, we next examined the impact of lowering endogenous PTX3 levels with siRNA. PTX3 silencing decreased the viable cell numbers and reversed IFNT actions on cell viability, percentage of proliferating cells, and on two key survival/death genes: BIRC5 encoding surviving protein, and FASL - a death-inducing signal. Interestingly, thrombospondin-1, a known luteal proapoptotic factor, was inversely related to PTX3 in LGCs. Together, these findings suggest a novel role for PTX3 during early pregnancy, as mediator of IFNT prosurvival actions supporting CL maintenance during this reproductive stage.

Applied use of interferon-tau stimulated genes expression in polymorphonuclear cells to detect pregnancy compared to other early predictors in beef cattle.

We aimed to evaluate the accuracy of Interferon-tau stimulated genes (ISG) abundance in peripheral blood polymorphonuclear cells (PMNs) on D20 after fixed-time artificial insemination (FTAI; D0) as a pregnancy diagnosis method against CL evaluation by Doppler ultrasonography and progesterone (P4) concentrations on D20, as well as Pregnancy Associated Glycoproteins (PAG) concentrations on D25. Additionally, we evaluated the potential of ISG abundance in PMNs as pregnancy loss predictors. Nelore heifers (n = 103) and cows (n = 144) underwent estrous synchronization and were artificially inseminated on D0. Pregnancy was diagnosed by B-mode ultrasonography on D30 and D70, and after the final diagnosis, females were classified in four groups: Pregnant; Non-pregnant; Functional CL on D20 but non-pregnant (CL-NP) and Pregnancy loss between D30 and D70 (PL). After determining cutoff values, the Sensitivity (SE), Specificity (SP), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and Accuracy (ACC) were determined for each method. All methods were classified as significant (P < 0.05) predictors of pregnancy. Both ISG expression and PAG concentrations were greater (P < 0.05) in pregnant females than in non-pregnant and CL-NP females but did not differ (P > 0.05) from the PL group. ISG15 expression was greater (P < 0.05) in heifers than in cows, but this difference was not found in OAS1 expression and PAG concentrations. All the methods evaluated were proven to be adequate predictors of pregnancy, but greater accuracies were obtained through PAG concentrations and Doppler-US, due to the decreased number of false positive and false negative results.

Placental Protein 13 (Galectin-13) Polarizes Neutrophils Toward an Immune Regulatory Phenotype.

Termed as galectin-13, placental protein 13 (PP13) is exclusively expressed in the placenta of anthropoid primates. Research on PP13 in normal and pathologic pregnancies show alteration of PP13 concentrations in pregnancy affected by preeclampsia or gestational diabetes. Galectins are also described as potent immunomodulators, and PP13 regulates T cell function in the placenta. Therefore, this study aims to investigate the effects of PP13 on neutrophils; a cell type often ignored in pregnancy, but present in the uterus and placenta from the early stages of pregnancy. Since neutrophil function is dysregulated during pathologic pregnancies, a link between PP13 and neutrophil activity is possible. We determined that PP13 reduces the apoptosis rate in neutrophils. Also, PP13 increases the expression of PD-L1 and production of HGF, TNF-α, reactive oxygen species (ROS), and MMP-9 in these cells. This phenotype resembles one observed in permissive tumor neutrophils; able to sustain tissue and vessel growth, and inhibit T cell activation. At the same time, PP13 does not alter all neutrophil functions, i.e., extrusion of neutrophil extracellular traps, degranulation, phagocytosis, and ROS production following bacterial exposure. PP13 seems to play an essential role in regulating the activity of neutrophils in the placenta by polarizing them toward a placental-growth-permissive phenotype.

Receptor expression by JEG-3 trophoblast cells in the presence of placenta secreted factors.

Preeclampsia still remains one of the most severe pregnancy complications and is an actual problem in the obstetrics practice. At present, the joint impact of cytokines and other placenta secreted factors on trophoblast cell functional activity during preeclampsia complicated pregnancy remains unclear. The aim of the study is to estimate the surface receptors expression by trophoblast cells in the presence of placenta secreted factors during physiological pregnancy and at preeclampsia. Trophoblast cells of the JEG-3 line were incubated in the presence of supernatants obtained by cultivation of placentas from women with physiological pregnancy and with preeclampsia. Surface receptors expression by trophoblast cells was estimated by FACS Canto II flow cytometer. It was established that in the third trimester both under normal and pathological conditions, the placenta secreted factors impact on the cytokine receptor expression by trophoblast differs while the trophoblast response capacity to the migration and proliferation stimulating and inhibiting signals remains stable. JEG-3 line cells enhanced the expression of CD186, CD140a, Integrin ß6, VE-cadherin, CD29, and CD140a in the case of incubation in the presence of placenta supernatants from the third-trimester pregnancy complicated with preeclampsia compared to incubation in the presence of placenta supernatants form the third trimester of physiological pregnancy.

Newly identified interferon tau-responsive Hes family BHLH transcription factor 4 and cytidine/uridine monophosphate kinase 2 genes in peripheral blood granulocytes during early pregnancy in cows.

In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.

Ephrin A1 promotes proliferation of bovine endometrial cells with abundant expression of proliferating cell nuclear antigen and cyclin D1 changing the cell population at each stage of the cell cycle.

Ephrin A1 has a role in a variety of biological events, including cell proliferation, differentiation, migration, and angiogenesis. Ephrin A1 expression is abundant in trophoblasts and endometrial cells during the implantation period; however, its intracellular activities have not yet been reported in bovine endometrial (BEND) epithelial cells. The aim of this study was to identify the functional role of ephrin A1 in BEND cells, which have served as a good model system for investigating the regulation of signal transduction following treatment with interferon-τ (IFNT) in vitro. Supplementation of ephrin A1 to BEND cells increased cell proliferation and increased levels of proliferating cell nuclear antigen and cyclin D1 protein in BEND cell nuclei. To investigate intracellular mechanisms regulated by ephrin A1, we performed Western blot analysis focused on mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) signaling, which are significantly involved in the successful maintenance of pregnancy. Ephrin A1 dose-dependently increased phosphorylation of extracellular signal-regulated kinases (ERK)1/2, c-Jun N-terminal kinases (JNK), P38, protein kinase B (AKT), P70S6K, S6, and cyclin D1, and the activated proteins were suppressed by pharmacological inhibitors including wortmannin (a PI3K inhibitor), U0126 (an ERK1/2 inhibitor), and SP600125 (a JNK inhibitor). Among ephrin A1 receptors, abundant expression of EPHA2 and EPHA4 messenger RNA was detected in BEND cells by reverse transcription polymerase chain reaction analysis. Furthermore, tunicamycin-induced endoplasmic reticulum (ER) stress was inactivated by ephrin A1 treatment of BEND cells. Our findings suggest that ephrin A1 promotes the development of BEND cells and likely enhances uterine capacity and maintenance of pregnancy by activating MAPK and PI3K signaling cascades and by restoring ER stress.

Involvement of interferon-tau in the induction of apoptotic, pyroptotic, and autophagic cell death-related signaling pathways in the bovine uterine endometrium during early pregnancy.

Interferon-tau (IFNT), a type I interferon (IFN), is known as pregnancy recognition signaling molecule secreted from the ruminant conceptus during the preimplantation period. Type I IFNs, such as IFN-alpha and IFN-beta, are known to activate cell-death pathways as well as induce apoptosis. In cows, induction of apoptosis with DNA fragmentation is induced by IFNT in cultured bovine endometrial epithelial cells. However, the status of cell-death pathways in the bovine endometrium during the preimplantation period still remains unclear. In the present study, we investigated the different cell-death pathways, including apoptosis, pyroptosis, and autophagy, in uterine tissue obtained from pregnant cows and in vitro cultured endometrial epithelial cells with IFNT stimulation. The expression of CASP7, 8, and FADD (apoptosis-related genes) was significantly higher in pregnant day 18 uterine tissue in comparison to non-pregnant day 18 tissue. The expression of CASP4, 11, and NLRP3 (pyroptosis-related genes) was significantly higher in the pregnant uterus in comparison to non-pregnant uterus. In contrast, autophagy-related genes were not affected by pregnancy. We also investigated the effect of IFNT on the expression of cell-death pathway-related genes, as well as DNA fragmentation in cultured endometrial epithelial cells. Similar to its effects in pregnant uterine tissue, IFNT affected the increase of apoptosis-related (CASP8) and pyroptosis-related genes (CASP11), but did not affect autophagy-related gene expression. IFNT also increased γH2AX-positive cells, which is a marker of DNA fragmentation. These results suggest that apoptosis- and pyroptosis-related genes are induced by IFNT in the pregnant bovine endometrial epithelial cells.

The newly established bovine endometrial gland cell line (BEGC) forms gland acini in vitro and is only IFNτ-responsive (MAPK42/44 activation) after E2 and P4-pre-incubation.

INTRODUCTION: Uterine glands (UG) are crucial for the establishment of ruminant pregnancy and influenced (orchestrated manner) by estrogen (E2), progesterone (P4) and interferon tau (IFNτ). In the study we established a bovine endometrial glandular cell line (BGEC) and tested its functional reactivity (signaling) to IFNτ. METHODS: BGEC was characterized by light microscopy (LM), epithelial markers (ezrin, CK18) [immunofluorescence (IF)/immunohistochemistry (IHC)] and ultrastructure (TEM/SEM) (apical microvilli). In vitro formation of gland acini and transepithelial-electric-resistance (TEER) measurements (EVOM) were done. The expression of mRNA-transcripts (RT-PCR) of steroid receptors (PR, PGRMC1/2, ESR1/2) and the IFNτ-system (IFNAR1/2, IRF1, 2, 9) was checked. BEGC was stimulated with IFNτ (10 ng/ml;1000 ng/ml) (15 min) after steroid pre-treatment [10 pg/ml E2 (two days)/20 ng/ml P4 (two days)]. Activation of MAPK42/44;STAT1 was evaluated (densitometrical Western Blot). RESULTS: BGEC cells expressed epithelial markers and possessed apical microvilli. High TEER-values could be measured (2320-2620 ohm/cm2). The assembled BEGC acini (25 days) were similar to UG in vivo (markers/ultrastructure). All transcripts (steroid receptors/IFNτ-system) could be detected in BEGC (mRNA). MAPK42/44 were significantly activated after E2/P4 pre-treatment and IFNτ stimulation (10 ng/ml) (p < 0.05), whilst 1000 ng/ml IFNτ did not activate MAPK42/44. Neither a STAT1 (by IFNτ) nor an activation (MAPK42/44;STAT1) by IFNτ-only was observed. DISCUSSION: BGEC retains its epithelial phenotype in culture and forms gland acini in vitro thereby confirming its glandular character. Cells were only reactive to (low) IFNτ concentrations when pre-treated with steroids thereby closely resembling implantation physiology in vivo. BEGC can be used as a bovine implantation model to study embryo-maternal communication during early pregnancy in cattle.

Oviduct epithelium induces interferon-tau in bovine Day-4 embryos, which generates an anti-inflammatory response in immune cells.

Recent studies indicate that communication between the bovine embryo and the mother begins in the oviduct. Here, we aimed to investigate the effect of embryos on bovine oviducts for their immune responses using an in vitro model. First, zygotes were cultured with or without bovine oviduct epithelial cells (BOECs) for 4 days, when embryos had reached the 16-cell stage. At that time, we detected interferon-tau (IFNT) in embryos co-cultured with BOECs, but not in embryos cultured alone. Next, peripheral blood mononuclear cells (PBMCs) were incubated either in media from embryo alone cultures or from co-cultures of embryos with BOECs. The medium from embryo alone cultures did not modulate PBMCs gene expression; whereas the embryo-BOEC co-culture medium increased interferon-stimulated genes (ISGs: ISG15, OAS1, MX2), STAT1, PTGES and TGFB1 but suppressed IL17 expression in PBMCs. Both IFNT-treated BOEC culture medium and IFNT-supplemented fresh medium alone without BOEC, modulated PBMCs gene expressions similar to those by the embryo-BOEC co-culture medium. Further, specific antibody to IFNT neutralized the effect of embryo-BOEC co-culture medium on PBMCs gene expression. Our results indicate that BOECs stimulate embryos to produce IFNT, which then acts on immune cells to promote an anti-inflammatory response in the oviduct.

Specific interferon tau gene-regulation networks in bovine endometrial luminal epithelial cells.

Interferon tau (IFNT) plays a critical role as a pregnancy recognition factor in early pregnancy by regulating uterine epithelial gene expression. Illuminating the relation between IFNT and pregnancy will contribute significantly to early pregnancy research in ruminants. Therefore, in this study, we treated primary bovine endometrial luminal epithelial cells (bELECs) without or with IFNT (200 ng/mL) for 6 or12 h. Subsequently, RNA sequencing (RNA-seq) technology was used to evaluate differences in gene expression. In total, 707 differentially expressed genes (DEGs) were detected. These DEGs were significantly enriched in immune-related categories or pathways, including immune system process, MHC class I protein complex, antigen processing and presentation, and graft-versus-host disease. Furthermore, an integrated regulatory network was constructed to elucidate the interactions among these DEGs. A set of candidate genes (RAC2, DVL3, PSMB9, STAT1, ISG15, JAK1, and MUC1) was identified. Upon integration of these node genes, we speculated that IFNT might upregulate MHC molecules via a JAK1-STAT1-ISG15/PSMB9 axis involved in the maintenance of a tolerant environment during early pregnancy. Our results forma foundation for dissecting the molecular mechanism of IFNT in the uterus; future studies will use these data to identify and characterize new IFNT regulatory mechanisms in the endometrium.

JAK3 and PI3K mediate bovine Interferon-tau stimulated gene expression in the blood neutrophils.

Interferon tau, a 23 kDa trophoblast derived protein diffuses out from the uterus into the circulation and leads to the expression of IFNτ stimulated genes viz. ISG15 and OAS1 in blood neutrophils. The IFNτ pathway is species as well as tissue specific. To unsnarl the IFNτ downstream signaling pathway, the blood neutrophils were incubated simultaneously with 10 ng/ml of recombinant bovine interferon tau and the inhibitors of JAK2 (AG490), JAK3 (CP690550), p38 (SB202190), PI3K/Akt (LY294002), and MAPK/Erk (U0126) at specific doses for 4-hr duration. The IFNτ pathway was determined through real-time gene expression of ISG15 and OAS1; immunocytochemistry of ISG15; and Western blotting of ISG15, OAS1, pJAK3 and PI3K. The ISG15 and OAS1 expression decreased significantly (p < 0.001) in the presence of pJAK3 and PI3K inhibitors as compared to a positive control where only interferon tau was used. Immunocytochemistry revealed an attenuated ISG15 response while stimulating blood neutrophils with pJAK3 inhibitor (CP690550) and PI3K inhibitor (LY294002). Similarly, Western blot analysis of neutrophil protein fraction showed weak signals of ISG15, OAS1, pJAK3 and PI3K in the presence of pJAK3 and PI3K inhibitors. The expression profile, immunocytochemistry and western blot analysis revealed a JAK3 and PI3K mediated interferon-tau stimulated gene expression in blood neutrophils.