Primary Uterine Epithelial Cells Research Articles

Expression of pattern recognition receptors in porcine uterine epithelial cells in vivo and in culture

Preservation of a pathogen free uterine environment is critical for maintaining healthy swine herds with high reproductive performance. Considering that uterine epithelial cells are the most numerous and thus likely point of cellular contact for pathogens in the uterus, we hypothesize that these cells may be critical for activating the immune system to clear uterine infections. Although uterine epithelial cells have not been well characterized in pigs, studies in several other species have shown that these cells express several pattern recognition receptors (PRR) and thus may act as sentinels for the uterine immune response. To characterize PRR expression in the porcine uterine epithelia, we used laser-capture microdissection to isolate epithelial cells lining the porcine uterus to quantify in vivo mRNA expression levels for select PRRs. As well, primary uterine epithelial cells (UECs) were isolated, cultured, polarized and PRR expression was quantified. Immunohistofluorescence and immunofluorescence were used to determine subcellular localization of TLR3, TLR4 and TLR9 in both uterine tissue and in polarized primary UECs. Finally, polarized primary UECs were stimulated with ligands for TLR3, TLR4, TLR9 and NOD2 to determine their functional innate immune response. Uterine epithelial cells (in vivo and in vitro) were shown to express TLR1-7, TLR9, NOD1, NOD2, NLRP3, NLRP6, NLRX1, RIG1, MDA5 and LGP2. Subcellular localization of in vivo and polarized primary UECs exhibited TLR3 and TLR9 localized to the apical cell surface whereas TLR4 was localized to the intracellular space. Polarized primary UECs stimulated with TLR3, TLR4 and TLR9 ligands showed induced secretion of IL-6, IL-13 and IL-10, respectively indicating that these receptors were functional. These results indicate that pig uterine epithelial cells are functional innate immune cells that may act as sentinels to protect against uterine infection.

TLR4 Signaling Is a Major Mediator of the Female Tract Response to Seminal Fluid in Mice.

Seminal fluid interacts with epithelial cells lining the female reproductive tract to induce expression of proinflammatory cytokines and chemokines, initiating immune tolerance mechanisms to facilitate pregnancy. TGFB cytokines are key signaling agents in seminal plasma but do not fully account for the female response to seminal fluid. We hypothesized that additional molecular pathways are utilized in seminal fluid signaling. Affymetrix microarray was employed to compare gene expression in the endometrium of mice 8 h after mating with either intact males or seminal fluid deficient (SVX/VAS) males. Bioinformatics analysis revealed TLR4 signaling as a strongly predicted upstream regulator activated by the differentially expressed genes and implicated TGFB signaling as a second key pathway. Quantitative PCR and microbead data confirmed that seminal fluid induces endometrial synthesis of several TLR4-regulated cytokines and chemokines, including CSF3, CXCL1, CXCL2, IL1A, IL6, LIF, and TNF. In primary uterine epithelial cells, CSF3, CXCL1, and CXCL2 were strongly induced by the TLR4 ligand LPS but suppressed by TGFB, while IL1A, TNF, and CSF2 were induced by both ligands. TLR4 was confirmed as essential for the full endometrial cytokine response using mice with a null mutation in Tlr4, where seminal fluid failed to induce endometrial Csf3, Cxcl2, Il6, and Tnf expression. This study provides evidence that TLR4 contributes to seminal fluid modulation of the periconception immune environment. Activation of TLR4 signaling by microbial or endogenous components of seminal fluid is thus implicated as a key element of the female tract response to seminal fluid at the outset of pregnancy in mice.

Entosis allows timely elimination of the luminal epithelial barrier for embryo implantation.

SUMMARYDuring implantation, uterine luminal epithelial (LE) cells first interact with the blastocyst trophectoderm. Within 30 hr after the initiation of attachment, LE cells surrounding the blastocyst in the implantation chamber (crypt) disappear, allowing trophoblast cells to make direct physical contact with the underneath stroma for successful implantation. The mechanism for the extraction of LE cells was thought to be mediated by apoptosis. Here, we show that LE cells in direct contact with the blastocyst are endocytosed by trophoblast cells by adopting the nonapoptotic cell-in-cell invasion process (entosis) in the absence of caspase 3 activation. Our in vivo observations were reinforced by the results of co-culture experiments with primary uterine epithelial cells with trophoblast stem cells or blastocysts showing internalization of epithelial cells by trophoblasts. We have identified entosis as a mechanism to remove LE cells by trophoblast cells in implantation, conferring a role for entosis in an important physiological process.

Estrogen up-regulates MMP2/9 expression in endometrial epithelial cell via VEGF-ERK1/2 pathway

ObjectiveTo study the effect of estrogen on anovulatory dysfunctional uterine bleeding (ADUB). MethodsPrimary endometrial epithelial cells of Hainan Lizu female was cultured and hydrolytic activity of gelatinase was determined by gelatin zymography analysis. Cellular mRNA and protein synthesis was blocked respectively to determine whether the increased expression of MMP-2/9 was induced by estrogen. The expression of VEGF was blocked by siRNA. After treatment with various factors, MMP-9, VEGF, total Erk and phosphorylated Erk expression in primary uterine epithelial cells was detected by Western blotting analysis. Cell MMP-2/9mRNA levels was measured by real-time RT-PCR. ResultsThe activity and expression of MMP2/9 was increased in the endometrium of patients with ADUB. Estrogen could up-regulate the expression of VEGF and activate Erk 1/2-Elk1 signal path. After interference by siRNA, ERK1/2 pathway was blocked in cells, and the expression of MMP-2/9 was down-regulated. ERK1/2 specific blocker U0126 blocked ERK phosphorylation, and it could down-regulate the expression of MMP-2/9. ConclusionsThe results showed that the estrogen can increase the expression of VEGF, and thus activate ERK1/2 pathway to induce MMP-2/9 expression.

Uterine Epithelial Cells Specifically Induce Interferon-Stimulated Genes in Response to Polyinosinic-Polycytidylic Acid Independently of Estradiol

Interferon β (IFNβ) is an antiviral cytokine secreted in response to pathogenic exposure that creates a restrictive intracellular environment through the action of downstream interferon-stimulated genes (ISG). The objective of this study was to examine the expression of IFNβ and ISG in both human uterine epithelial cells (UEC) and the ECC-1 uterine epithelial cell line and determine if expression changes with TLR stimulation and hormone exposure. Stimulation of primary uterine epithelial cells and ECC-1 cells with the TLR3 agonist poly (I∶C) induced the mRNA expression of IFNβ, MxA, OAS2 and PKR. Other TLR agonists including imiquimod and CpG had no effect on either IFNβ or ISG expression. In contrast to ECC-1 cell responses which were slower, maximal IFNβ upregulation in UEC occurred 3 hours post-stimulation and preceded the ISG response which peaked approximately 12 hours after poly (I∶C) exposure. Unexpectedly, estradiol, either alone or prior to treatment with poly (I∶C), had no effect on IFNβ or ISG expression. Blockade of the IFN receptor abrogated the upregulation of MxA, OAS2 and PKR. Furthermore, neutralizing antibodies against IFNβ partially inhibited the upregulation of all three ISG. Estradiol, directly and in the presence of poly (I∶C) had no effect on IFNβ and ISG expression. These results indicate that uterine epithelial cells are important sentinels of the innate immune system and demonstrate that uterine epithelial cells are capable of mounting a rapid IFN-mediated antiviral response that is independent of estradiol and is therefore potentially sustained throughout the menstrual cycle to aid in the defense of the uterus against potential pathogens.

Effect of Toll‐Like Receptor (TLR) Agonists on TLR and Microbicide Expression in Uterine and Vaginal Tissues of the Mouse

Epithelial cells lining the uterine lumen are the first line of defense against pathogenic microbes. The objective of this study was to examine the expression of Toll-like receptors (TLRs), defensins and secretory leukocyte protease inhibitor (SLPI) in the mouse uterus and vagina and in primary uterine epithelial cells and to determine whether TLR agonists induce TLR and defensin expression. The mRNA expression of alpha- and beta-defensins (AD1, 2 and 5 and BD1, 2 and 4) and SLPI was examined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) along with the secretion of macrophage chemotactic protein-1 (MCP-1), measured by enzyme-linked immunosorbent assay. Expression of TLR1-9 as well as beta-defensins 1, 2 and 4 and SLPI by uterine and vaginal tissues was demonstrated by RT-PCR. beta-Defensins and SLPI expression was greater in the vagina than in the uterus. Comparison of fresh and polarized uterine epithelial cells indicated that TLR2-6 expression was unaffected by culture. Incubation of polarized epithelial cells with TLR agonists [lipopolysaccharide (LPS), Pam3Cys, Poly (I:C) or PGN] induced TLR5 and TLR9 expression but had no effect on TLR4, defensins or SLPI. Furthermore, exposure to LPS, Pam3Cys, Poly (I:C) or PGN, induced MCP-1 secretion by polarized epithelial cells in culture. These results indicate that the uterus and vagina as well as uterine epithelial cells are responsive to bacterial and viral pathogens. Not only do epithelial cells respond to TLR agonists by releasing MCP-1, which mediates inflammatory responses, but they also influence the expression of selected TLR genes to further enhance innate immune protection.

Innate Immunity in the Human Female Reproductive Tract: Antiviral Response of Uterine Epithelial Cells to the TLR3 Agonist Poly(I:C)

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.

CCL20/Macrophage Inflammatory Protein 3α and Tumor Necrosis Factor Alpha Production by Primary Uterine Epithelial Cells in Response to Treatment with Lipopolysaccharide or Pam3Cys

Having previously shown that CCL20/macrophage inflammatory protein 3alpha and tumor necrosis factor alpha (TNF-alpha) are released by polarized primary rat uterine epithelial cells (UEC) in response to Escherichia coli but not to Lactobacillus rhamnosus, we sought to determine if epithelial cells are responsive to pathogen-associated molecular patterns (PAMP), including lipopolysaccharide (LPS), lipoteichoic acid (LTA), and Pam(3)Cys, a bacterial lipoprotein analog. Epithelial cells were grown to confluence on Nunc cell culture inserts prior to apical treatment with PAMPs. In response to LPS, LTA, and Pam(3)Cys (EMC Microcollection GmbH, Tubingen, Germany), CCL20 levels increased (4- to 10-fold) while PAMPs caused increased TNF-alpha (1- to 4-fold) in the medium collected after 24 h of incubation. Both apical and basolateral secretion of CCL20 and TNF-alpha increased in response to PAMPs, but treatments had no effect on cell viability and integrity, as measured by transepithelial resistance. Time course studies of CCL20 and TNF-alpha release in response to Pam(3)Cys and LPS indicated that CCL20 release peaked between 2 and 4 h after treatment, whereas TNF-alpha release was gradual over the length of the incubation. Freeze-thaw and cell lysis experiments, along with actinomycin D studies, suggested that CCL20 and TNF-alpha are synthesized in response to PAMP stimulation. Taken together, these studies demonstrate that E. coli and selected PAMPs have direct effects on the production of CCL20 and TNF-alpha without affecting cell integrity. Since CCL20 is known to be both chemotactic and antimicrobial, the increase in apical and basolateral release by UEC in response to PAMPs suggests a new mechanism of innate immune protection in the female reproductive tract.

Differential effects of interferon-tau on the prostaglandin synthetic pathway in bovine endometrial cells treated with phorbol ester.

Rationale for these experiments was to evaluate the dose effects of bovine interferon-tau (IFN-tau) on the prostaglandin secretory pathway of immortalized bovine endometrial (BEND) cells in response to phorbol 12, 13-dibutyrate (PdBu) and to characterize similar responses in primary bovine uterine epithelial cells as a biomonitor of embryo-induced antiluteolytic effects on the endometrium. The BEND cells were treated with PdBu (0 or 100 ng/mL) and IFN-tau (0 or 50 ng/mL) for 6 h. The PdBu stimulated secretions of PGF2alpha and prostaglandin E2 (PGE2). Co-treatment of cells with IFN-tau blocked PdBu-induced secretion of both PGF2alpha and PGE2. Treatment with PdBu for 6 h induced expression of prostaglandin H synthase-2 mRNA, prostaglandin H synthase-2 protein, and prostaglandin E synthase mRNA, which were blocked with concurrent addition of IFN-tau. Doses of IFN-tau (0.05, 0.5, 1, 5, 10, and 20 microg/mL) were used with PdBu (0 and 100 ng/mL). The IFN-tau alone failed to stimulate secretion of PGF2alpha and PGE2, whereas IFN-tau doses <5 microg/mL suppressed PdBu-stimulated secretions of PGF2alpha and PGE2. Uterine epithelial cells were isolated from cows at d 17 after estrus and were cultured to confluence in serum-free medium. Cells were treated with IFN-tau (0, 50, or 500 ng/mL) and PdBu (0 or 100 ng/mL) before media were collected after 24 h for PGF2alpha and PGE2 analyses. Treatment of primary uterine epithelial cells with PdBu induced PGF2alpha secretion, and IFN-tau (50 and 500 ng/mL) caused a reduction in PGF2alpha secretion induced by PdBu. In the absence of PdBu, IFN-tau increased basal secretion of PGF2alpha. Concentrations of PGE2 increased in response to PdBu, and the 50-ng/mL dose of IFN-tau had a stimulatory effect on PGE2 concentrations compared with the 500-ng/mL dose in the absence of PdBu. Phorbol ester-induced gene transcription as related to prostaglandin synthesis is regulated by IFN-tau in vitro.

Insulin-like growth factor-1 regulates VEGF expression in primary human uterine epithelial cells

Objective: Angiogenesis, the formation of new blood vessels from preexisting endothelial cells, is important for the normal functioning of many organs and tissues, including the female reproductive tract. In addition, the process of angiogenesis is understood to play a requisite role in the growth and metastasis of tumor cells. Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells, necessary for the normal process of angiogenesis during events such as cyclic endometrial regeneration, corpus luteum formation and normal wound healing. In addition, VEGF has been implicated in the growth of many tumors. Therefore, the dysregulation of the VEGF signaling pathway may play a role in the development and/or progression of cancers and other pathologies of the female reproductive tract that require vascularization. Previous studies of uterine cells have shown that VEGF expression can be regulated by the ovarian steroid hormones estradiol (E) and progesterone (P). Other studies from our laboratory and others have shown that uterine insulin-like growth factor-1 (IGF-1) expression is increased by estrogens and that IGF-1 alone can stimulate many of the same uterine events as E. Because IGF-1 is also implicated in the growth of many cancers, the experiments herein were designed to determine whether IGF-1 also stimulates VEGF expression in uterine cells. Design: Tissue culture based research Materials and Methods: Primary human uterine epithelial cells were isolated from endometrial tissue, cultured on collagen-coated tissue culture dishes, and treated with either vehicle or IGF-1. Conditioned medium was then assayed for VEGF content, and cells were examined for the expression of VEGF mRNA. To investigate the mechanism through which IGF-1 stimulates an increase in VEGF expression by primary uterine epithelial cells, inhibitors to the phosphatidylinositol 3-kinase (PI3-K) and MAP kinase (MAPK) signaling pathways were used to investigate the signaling pathway responsible for IGF-1 stimulation of VEGF in uterine cells. Results: In a time-dependent manner, IGF-1 increased the level of VEGF secreted by the primary uterine epithelial cells. Furthermore, IGF-1 stimulated an increase in the levels of VEGF mRNA expressed by the cells. Inhibition of PI3-K signaling, but not MAPK signaling, decreased the amount of VEGF secreted by the uterine cells in response to IGF-1, indicating that PI3-K may be the primary pathway through which IGF-1 regulates VEGF expression in uterine cells. Conclusion: Taken together, these results demonstrate that IGF-1 can increase VEGF production by primary uterine epithelial cells in a PI3-K dependent manner. Elevated IGF-1 levels, in addition to or in response to, elevated E levels may contribute to the vascularization and, thus, growth of endometrial pathologies.

Production and characterization of WEG-1, an epidermal growth factor/transforming growth factor-alpha-responsive mouse uterine epithelial cell line.

Uterine epithelial cells (UEC) isolated from 6-week-old CF-1 mice were immortalized using retroviral-mediated transfection of SV40 large T-antigen. One line, WEG-1, retained epithelial morphology and reacted with antibodies to cytokeratins 18, 19, laminin, fibronectin, and beta-catenin. In addition, WEG-1 cells displayed strong nuclear immunoreactivity to SV40 large T-antigen, confirming integration of the retrovirus vector and expression of this gene. WEG-1 cells were negative for nonepithelial markers such as desmin and factor 8. WEG-1 cells did not proliferate in serum-free medium; however, addition of 0.5% FBS supported proliferation to the same extent as 10% FBS. Addition of 50 ng/ml epidermal growth factor to medium containing 0.5% charcoal-stripped FBS restored proliferation comparable with 0.5% whole FBS. Epidermal growth factor or transforming growth factor-alpha (50 ng/ml), but not transforming growth factor-beta, leukemia-inhibiting factor, or fibroblast growth factor, induced the secretion of three proteins (M(r) approximately to 158K, 148K, and 36K). Comparison of protein secretions of WEG-1 cells and UEC showed shared as well as distinct bands. Like UEC, WEG-1 cells secreted PGF2a and PGE2 and expressed PG GH synthase-2. Unlike UEC, WEG-1 cells showed no apical/basal preference for either uptake of methionine or secretion of proteins. The absence of immunoreactive E-cadherin or zona occludens-1 was consistent with the absence of cell polarity in WEG-1 cells. Primary UEC, which polarize in vitro, do not support blastocyst attachment. WEG-1 cells, although not polarized in vitro, also exhibited delayed blastocyst attachment compared with nonuterine cell lines, suggesting that WEG-1 cells partially retained some aspects of UEC function relevant to embryo attachment. WEG-1 cells expressed messenger RNA for Muc-1, an UEC mucin suggested to have antiadhesive properties. Furthermore, WEG-1 cells did not display high affinity heparin binding sites, an activity associated with embryo attachment. WEG-1 cells may provide a model for studying various aspects of UEC function and murine embryo attachment.

Production and characterization of WEG-1, an epidermal growth factor/transforming growth factor-alpha-responsive mouse uterine epithelial cell line

Uterine epithelial cells (UEC) isolated from 6-week-old CF-1 mice were immortalized using retroviral-mediated transfection of SV40 large T-antigen. One line, WEG-1, retained epithelial morphology and reacted with antibodies to cytokeratins 18, 19, laminin, fibronectin, and beta-catenin. In addition, WEG-1 cells displayed strong nuclear immunoreactivity to SV40 large T-antigen, confirming integration of the retrovirus vector and expression of this gene. WEG-1 cells were negative for nonepithelial markers such as desmin and factor 8. WEG-1 cells did not proliferate in serum-free medium; however, addition of 0.5% FBS supported proliferation to the same extent as 10% FBS. Addition of 50 ng/ml epidermal growth factor to medium containing 0.5% charcoal-stripped FBS restored proliferation comparable with 0.5% whole FBS. Epidermal growth factor or transforming growth factor-alpha (50 ng/ml), but not transforming growth factor-beta, leukemia-inhibiting factor, or fibroblast growth factor, induced the secretion of three proteins (M(r) approximately to 158K, 148K, and 36K). Comparison of protein secretions of WEG-1 cells and UEC showed shared as well as distinct bands. Like UEC, WEG-1 cells secreted PGF2a and PGE2 and expressed PG GH synthase-2. Unlike UEC, WEG-1 cells showed no apical/basal preference for either uptake of methionine or secretion of proteins. The absence of immunoreactive E-cadherin or zona occludens-1 was consistent with the absence of cell polarity in WEG-1 cells. Primary UEC, which polarize in vitro, do not support blastocyst attachment. WEG-1 cells, although not polarized in vitro, also exhibited delayed blastocyst attachment compared with nonuterine cell lines, suggesting that WEG-1 cells partially retained some aspects of UEC function relevant to embryo attachment. WEG-1 cells expressed messenger RNA for Muc-1, an UEC mucin suggested to have antiadhesive properties. Furthermore, WEG-1 cells did not display high affinity heparin binding sites, an activity associated with embryo attachment. WEG-1 cells may provide a model for studying various aspects of UEC function and murine embryo attachment.