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Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions.

ABSTRACT: Lipopolysaccharide (LPS) expressed on the surface of Gram-negative bacteria activates pro-inflammatory pathways, dys-regulates the function of endometrial cells and is a key player in the mechanisms involved in endometritis. This study aimed to investigate the effects of LPS on bovine endometrial epithelial cells (bEEC) from whole transcriptome with a special focus on genes involved in embryo-maternal interactions. Following in vitro culture, bEEC from three cows were exposed to 0, 2, and 8 ?g/mL LPS for 24h. RNA samples extracted at 0 and 24 hours were analyzed by RNA sequencing (RNA-seq). At 24h, 2035 differentially expressed genes (DEGs) were identified between controls and samples treated with 2 ?g/mL LPS. Gene ontology analysis showed that over-expressed DEGs were associated to immune response, response to stress and external stimuli, catalytic activity, and cell cycle. Genes associated with cell membrane and cell adhesion pathways were under-expressed. LPS induced changes in expression of specific genes related to embryo-maternal interactions including under-expression of eight members of the cadherin superfamily, over-expression of six members of the mucin family, and differential expression of a large set of genes binding the above molecules and of more than 20 transcripts coding for cytokines and their receptors. Type I interferon-? dependent genes were also over-expressed. From a sub-set of 19 genes, (biological replicates of bEEC from cows taken at time 6 (n = 3), 24 (n = 6) and 48 hours (n = 3), and 2 technical replicates per sample) differential gene expression was confirmed by RT2-qPCR (r2 between fold changes at 24 hours by RT2-qPCR and RNA-seq = 0.97). These results indicate that LPS affects the function of bEEC in many ways by differential transcription, glycolytic metabolism and oxidative stress. Many transcriptomic signatures related to implantation and embryo maternal interactions were strongly affected by LPS. These results pave the way for further studies to investigate the duration of these changes and their possible impact on endometrial function and fertility.

SUBMITTER: Guo Y

PROVIDER: S-EPMC6728075 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions.

Guo Yongzhi Y van Schaik Tom T Jhamat Naveed N Niazi Adnan A Chanrot Metasu M Charpigny Gilles G Valarcher Jean Francois JF Bongcam-Rudloff Erik E Andersson Göran G Humblot Patrice P

PloS one 20190905 9

Lipopolysaccharide (LPS) expressed on the surface of Gram-negative bacteria activates pro-inflammatory pathways, dys-regulates the function of endometrial cells and is a key player in the mechanisms involved in endometritis. This study aimed to investigate the effects of LPS on bovine endometrial epithelial cells (bEEC) from whole transcriptome with a special focus on genes involved in embryo-maternal interactions. Following in vitro culture, bEEC from three cows were exposed to 0, 2, and 8 μg/m ...[more]

PMID: 31487323

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Project description:BackgroundLipopolysaccharide (LPS) endotoxin stimulates pro-inflammatory pathways and is a key player in the pathological mechanisms involved in the development of endometritis. This study aimed to investigate LPS-induced DNA methylation changes in bovine endometrial epithelial cells (bEECs), which may affect endometrial function. Following in vitro culture, bEECs from three cows were either untreated (0) or exposed to 2 and 8??g/mL LPS for 24?h.ResultsDNA samples extracted at 0?h and 24?h were sequenced using reduced representation bisulfite sequencing (RRBS). When comparing DNA methylation results at 24?h to time 0?h, a larger proportion of hypomethylated regions were identified in the LPS-treated groups, whereas the trend was opposite in controls. When comparing LPS groups to controls at 24?h, a total of 1291 differentially methylated regions (DMRs) were identified (55% hypomethylated and 45% hypermethylated). Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation (r?=?-?0.41 for gene promoter, r?=?-?0.22 for gene body regions, p?<?0.05). Differential methylation analysis revealed that effects of LPS treatment were associated with methylation changes for genes involved in regulation of immune and inflammatory responses, cell adhesion, and external stimuli. Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes; and pathways such as calcium-, oxytocin- and MAPK-signaling pathways with recognized roles in innate immunity. Several DMGs were related to systemic inflammation and tissue re-modelling including HDAC4, IRAK1, AKT1, MAP3K6, Wnt7A and ADAMTS17.ConclusionsThe present results show that LPS altered the DNA methylation patterns of bovine endometrial epithelial cells. This information, combined with our previously reported changes in gene expression related to endometrial function, confirm that LPS activates pro-inflammatory mechanisms leading to perturbed immune balance and cell adhesion processes in the endometrium.

Project description:Meloxicam is a non-steroidal anti-inflammatory drug and has been used to relieve pain and control inflammation in cows with metritis and endometritis. Meloxicam has been found to be effective in inhibiting tissue or cell growth when it is used as an anti-inflammatory therapy. However, the influence of meloxicam on bovine endometrial regeneration has not been reported. This study was to research the effect of meloxicam (0.5 and 5 μM) on the proliferation of primary bovine endometrial epithelial cells (BEECs) stimulated by Escherichia coli lipopolysaccharide. The cell viability, cell cycle, and cell proliferation were evaluated by Cell Counting Kit-8, flow cytometry, and cell scratch test, respectively. The mRNA transcriptions of prostaglandin-endoperoxide synthase 1 (PTGS1) and PTGS2, Toll-like receptor 4, and proliferation factors were detected using quantitative reverse-transcription polymerase chain reaction. The activations of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and Wnt/β-catenin pathways were determined using western blot and immunofluorescence. As a result, co-treatment of meloxicam and lipopolysaccharide inhibited (P < 0.05) the cell cycle progression and reduced (P < 0.05) the cell healing rate and the mRNA level of proliferation factors as compared with the cells treated with lipopolysaccharide alone. Meloxicam decreased (P < 0.05) the lipopolysaccharide-induced PTGS2 gene expression. Neither lipopolysaccharide nor meloxicam changed PTGS1 mRNA abundance (P > 0.05). Meloxicam inhibited (P < 0.05) the lipopolysaccharide-activated Wnt/β-catenin pathway by reducing (P < 0.05) the protein levels of β-catenin, c-Myc, cyclin D1, and glycogen synthase kinase-3β and prevented the lipopolysaccharide-induced β-catenin from entering the nucleus. Meloxicam suppressed (P < 0.05) the phosphorylation of PI3K and AKT. In conclusion, meloxicam alone did not influence the cell cycle progression or the cell proliferation in BEEC but caused cell cycle arrest and inhibited cell proliferation in lipopolysaccharide-stimulated BEEC. This inhibitory effect of meloxicam was probably mediated by Wnt/β-catenin and PI3K/AKT pathways.

Project description:Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C. fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C. fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C. fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C. fetus. The presence of C. fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C. fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C. fetus, a gentamicin protection assay was performed. Although C. fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C. fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Dataset Information

Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions.

Publications

Differential gene expression in bovine endometrial epithelial cells after challenge with LPS; specific implications for genes involved in embryo maternal interactions.

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