Yong Q. Koh

Differential response to lipopolysaccharide by JEG-3 and BeWo human choriocarcinoma cell lines

OBJECTIVE To determine the effect of lipopolysaccharide (LPS) on NF-κB gene expression and proinflammatory cytokine release from trophoblast cell models, JEG-3 and BeWo human choriocarcinoma cells. STUDY DESIGN Serum-starved JEG-3 and BeWo cells were treated with LPS (from Escherichia coli serotype 0111:B4) for 24 or 48h. Cell culture medium was collected and assayed for interleukin (IL)-1β, IL-6, IL-8, and transforming necrosis factor (TNF)-α cytokine release using enzyme-linked immunosorbent assays. RNA was extracted from the cells and real-time PCR was performed to measure NF-κB mRNA expression. All results were analyzed by one-way analysis of variance tests followed by Sidaks post hoc analysis. p<0.05 was considered statistically significant. RESULTS LPS triggered an inflammatory response in JEG-3 cells by inducing a 1.5-fold increase in NF-κB mRNA expression and TNF-α release (0μg/mL: 15.13±2.14, 1μg/mL: 14.94±0.75, 10μg/mL: 23.05±4.50, p<0.05) and a 2-fold elevation in IL-6 secretion (0μg/mL: 12.54±5.44, 1μg/mL: 25.54±0.91, 10μg/mL: 24.28±4.43, p<0.05). In contrast, BeWo cells were not as sensitive to LPS exposure; NF-κB mRNA expression was unchanged between LPS-treated and control cells, whereas a small but significant 1.3-fold increase in TNF-α release was found (TNF-α: 15.45±1.53pg/mL, control: 12.24±1.00pg/mL, p<0.05). The inflammatory pathways in BeWo cells were found to be active given that treatment of these cells with IL-1β and TNF-α induced IL-6 secretion. Interestingly, 1μg/mL LPS appeared to decrease IL-6 and TNF-α release from BeWo cells. IL-1β and IL-8 secretion were not detected from either cell lines. CONCLUSION LPS activates the NF-κB pathway in JEG-3 but not BeWo human choriocarcinoma cells and this may be the reason for their differential inflammatory response to LPS exposure.

Review: Eicosanoids in preterm labor and delivery: potential roles of exosomes in eicosanoid functions

Preterm delivery is a major obstetric health problem contributing to poor neonatal outcome including low birth weight, respiratory distress syndrome, gastrointestinal, immunologic, central nervous system, hearing, and vision problems. Worldwide, approximately 15 million babies are born prematurely each year. The critical question which remains is how to identify women destined to deliver preterm from those who will achieve a term delivery. Prostaglandins, in all mammals, are important in the parturient process. Increased intrauterine prostaglandin production is associated with labor and in fact prostaglandin E2 (PGE2) or analogs are widely used clinically for cervical ripening and labor induction. Measurements of circulating eicosanoids have been problematic because of the rapid and major clearance by the lungs and then kidneys resulting in very low concentrations in plasma. Moreover, since eicosanoids are produced by all mammalian tissues, the sources of the measured eicosanoids are unknown. Our understanding of how cells communicate has undergone a paradigm shift with the recognition of the role of exosomes in intercellular signaling. Recent publications have identified enzymes and products of arachidonic acid metabolism (eicosanoids) within exosomes. This review will explore the potential roles of exosomes in eicosanoid functions that are critical in preterm labor and delivery.

A method for the isolation and enrichment of purified bovine milk exosomes

Exosomes are nanovesicles that play important roles in intercellular communication as they carry information to target cells. Isolation of high purity exosomes will aid in studying the exosomal cargo and quantity as well as how cell-specific messages are carried. We describe a new method incorporating size exclusion chromatography (SEC) to enrich milk-derived exosomes from extracellular vesicles (EVs). This involved the initial isolation of EVs from bovine milk via milk processing and ultracentrifugation; followed by a new method to enrich exosomes using SEC. This method was compared to buoyant density gradient centrifugation, a widely used method of enrichment. Exosomes were characterised by particle concentration and size (nanoparticle tracking analysis, NTA), morphology (transmission electron microscopy, TEM), presence of exosomal markers (immunoblotting) and protein concentration (bicinchoninic acid assay, BCA). Proteomic profiles of exosomal fractions were analyzed by mass spectrometry using Information Dependant Acquisition. Milk exosomal fractions were shown to contain exosomal markers flotillin-1 (FLOT-1) and tumor susceptibility gene-101 (TSG-101). The new method produced a higher yield of exosomes compared to buoyant density gradient centrifugation. Pooled exosomal fractions exhibited intact morphology by TEM. The use of SEC confirmed the fractionation of exosomes based on size while minimizing the interference with proteins. Tetraspanins CD9 and CD81 were observed via mass spectrometry in exosomal fractions. This new and efficient method confirmed the signatures for exosomes derived from unpasteurized bovine milk. Purification of exosomes is a foundational technique in the study of biomarkers for pathological conditions and effective drug delivery systems.

Effect of exosomes from plasma of dairy cows with or without an infected uterus on prostaglandin production by endometrial cell lines

A contributing factor to declining fertility in dairy cows is an activated inflammatory system associated with uterine infection. Detecting uterine disease using biomarkers may allow earlier diagnosis and intervention with resultant improvements in fertility. Exosomes are known to participate in intercellular communication, paracrine, and endocrine signaling. Exosomes carry a cargo of proteins, lipids, and nucleic acids that represent specific cellular sources. Prostaglandins are lipids that are critical determinants of bovine fertility. In this study exosomes were isolated from the plasma of cows before (d 0) and during (d 10) the study in healthy animals or those with an induced uterine infection in a 2 × 2 factorial design. Exosomes were characterized for size and number (nanoparticle tracking analysis), exosomal marker expression (Western blot), and morphology (transmission electron microscopy). No significant differences were observed in exosome size or number. The abundance of exosome-enriched markers was confirmed in noninfected and infected animals. Transmission electron microscopy confirmed the morphology of the exosomes. These exosomes were co-incubated with bovine endometrial epithelial and stromal cells. Exosomes from d-10-infected animal plasma decreased PGF2α production in endometrial epithelial but not stromal cells. For future research, the identification of effectors in the cargo may provide a useful basis for early diagnosis of uterine infection using an exosomal characterization approach.

Effect of circulating exosomes from transition cows on Madin-Darby bovine kidney cell function

The greatest risk of metabolic and infectious disease in dairy cows is during the transition from pregnancy to lactating (i.e., the transition period). The objective of this experiment was to determine the effects of extracellular vesicles (microvesicles involved in cell-to-cell signaling) isolated from transition cows on target cell function. We previously identified differences in the protein profiles of exosomes isolated from cows divergent in metabolic health status. Therefore, we hypothesized that these exosomes would affect target tissues differently. To investigate this, 2 groups of cows (n = 5/group) were selected based on the concentration of β-hydroxybutyrate and fatty acids in plasma and triacylglycerol concentration in liver at wk 1 and 2 postcalving. Cows with high concentrations of β-hydroxybutyrate, fatty acids, and triacylglycerol were considered at increased risk of clinical disease during the transition period (high-risk group; n = 5) and were compared with cows that had low concentrations of the selected health indicators (low-risk group; n = 5). At 2 time points during the transition period (postcalving at wk 1 and 4), blood was sampled and plasma exosomes were isolated from the high-risk and low-risk cows. The exosomes were applied at concentrations of 10 and 1 µg/mL to 5 × 103 Madin-Darby bovine kidney cells grown to 50% confluence in 96-well plates. Results indicate a numerical increase in cell proliferation when exosomes from high-risk cows were applied compared with those from low-risk cows. Consistent with an effect on cell proliferation, quantitative reverse transcriptase PCR indicated a trend for upregulation of 3 proinflammatory genes (granulocyte colony-stimulating factor, ciliary neurotrophic factor, and CD27 ligand) with the application of high-risk exosomes, which are involved in cellular growth and survival. Proteomic analysis indicated 2 proteins in the low-risk group that were not identified in the high-risk group (endoplasmin and catalase), which may also be indicative of the metabolic state of origin. It is likely that the metabolic state of the transition cow affects cellular function through exosomal messaging; however, more in-depth research into cross-talk between exosomes and target cells is required to determine whether exosomes influence Madin-Darby bovine kidney cells in this manner.

Eicosanoid pathway expression in bovine endometrial epithelial and stromal cells in response to lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha

During endometrial inflammation, bovine endometrium responds by increasing the production of pro-inflammatory mediators, such as interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and eicosanoids. The purpose of this study was to establish and characterize an in vitro model of endometrial inflammation using bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. We evaluated the effects of the infectious agent (bacterial lipopolysaccharide; LPS) and pro-inflammatory mediators (IL-1β and TNFα) on eicosanoid biosynthesis pathway gene expression and production by bEEL and bCSC cells. Based on concentration-response experiments, the optimal concentrations for responses were 1 μg/mL LPS, 10 ng/mL IL-1β and 50 ng/mL TNFα. Real-time PCR results show that there was an upregulation of relative mRNA expression of PTGS2 when bEEL and bCSC were treated with LPS, IL-1β and TNFα. An increase in PTGES3 expression was observed when bEEL cells were treated with LPS and IL-1β and PTGES2 when treated with IL-1β. In bCSC cells, FAAH relative mRNA was decreased upon treatments. Rate of production of PGE2, PGF2α, PGE2-EA and PGF2α-EA were also determined using liquid chromatography tandem mass spectrometry. Our results show that eicosanoid production was increased in both cell lines in response to LPS, IL-1β, and TNFα. We suggest that the characteristics of bEEL and bCSC cell lines mimic the physiological responses found in mammals with endometrial infection, making them excellent in vitro models for intrauterine environment studies.

Regulation of inflammatory mediator expression in bovine endometrial cells: effects of lipopolysaccharide, interleukin 1 beta, and tumor necrosis factor alpha

An abnormal uterine environment can influence maternal–fetal communication, conception rate and disrupt normal embryo development, thereby affecting fertility and the reproductive performance of dairy cows. Animal variability means that development of endometrial cell lines with appropriate characteristic are required. We evaluated the effect of an infectious agent (i.e., bacterial lipopolysaccharide; LPS) and proinflammatory mediators (i.e., Interleukin 1 beta; IL‐1β, and tumor necrosis factor alpha; TNFα) on inflammatory mediator gene expression and production by bovine endometrial epithelial (bEEL) and stromal (bCSC) cell lines. Expression of CXCL8/IL8, IL1A, IL1B, and IL6 cytokine genes was significantly upregulated in both epithelial and stromal cells when treated with LPS and IL‐1β. LPS treatment of epithelial cells (compared with treatment by IL‐1β and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Whereas, in stromal cells, IL‐1β treatment (compared with LPS and TNFα) exhibited greater CXCL8/IL8, IL1A, IL1B, and IL6 cytokine gene expression. Interestingly, bEEL and bCSC cells treated with IL‐1β increased IL1B gene expression, suggesting that IL‐1β may act unusually in an autocrine‐positive feedback loop. Cytokine production was stimulated by these agents in both cell types. We suggest that the characteristics of these two cell lines make them excellent tools for the study of intrauterine environment.

Proteome profiling of exosomes derived from plasma of heifers with divergent genetic merit for fertility

The current study evaluated exosomes isolated from plasma of heifers bred to have high or low fertility through developing extreme diversity in fertility breeding values, however, key animal traits (e.g., body weight, milk production, and percentage of North American genetics) remained similar between the 2 groups. The exosomes were isolated by a combined ultracentrifugation and size exclusion chromatography approach and characterized by their size distribution (nanoparticle tracking analysis), morphology (transmission electron microscopy), and presence of exosomal markers (immunoblotting). In addition, a targeted mass spectrometry approach was used to confirm the presence of 2 exosomal markers, tumor susceptibility gene 101 and flotillin 1. The number of exosomes from plasma of high fertility heifers was greater compared with low fertility heifers. Interestingly, the exosomal proteomic profile, evaluated using mass spectrometry, identified 89 and 116 proteins in the high and low fertility heifers respectively, of which 4 and 31 were unique, respectively. These include proteins associated with specific biological processes and molecular functions of fertility. Most notably, the tetratricopeptide repeat protein 41-related, glycodelin, and kelch-like protein 8 were identified in plasma exosomes unique to the low fertility heifers. These proteins are suggested to play a role in reproduction; however, the role of these proteins in dairy cow reproduction remains to be elucidated. Their identification underscores the potential for proteins within exosomes to provide information on the fertility status and physiological condition of the cow. This may potentially lead to the development of prognostic tools and interventions to improving dairy cow fertility.