LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function

- LPS-treatment of bovine endometrial epithelial cells causes differential DNA methylation of genes associated with inflammation and endometrial function.
- Background: Lipopolysaccharide (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..
- 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.
- Integration of DNA methylation data obtained here with our previously published gene expression data obtained from the same samples showed a negative correlation ( r.
- Gene ontology and pathway analyses showed that most of the differentially methylated genes (DMGs) were associated with cell proliferation and apoptotic processes.
- Conclusions: The 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..
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- The Toll-like receptor (TLR) signaling pathway is a central component of the primary innate immune response to pathogenic challenge.
- Epigenetic modifications, such as DNA methylation, have been shown to be associated with changes in gene expression in the endometrium during early pregnancy [11], and may regulate the uterine response to embryo implantation [12, 13].
- Several studies on gene expression and DNA methylation mainly performed in the human species have highlighted important genes and pathways affecting reproductive function during early or late preg- nancy stages [14–19].
- Other studies addressed the im- pact of infection and LPS on the DNA methylation status of immune cells.
- Endometrial epithelial cells (EECs) are key players in the defense of the uterus against most inflammatory dis- eases by triggering immune responses [21–23].
- Despite this, in the cow, the in- formation related to epigenetic regulation of the above pathways and of the immune response in EECs in case of uterine infection is scarce, to the best of our know- ledge.
- However, genome-wide epigenetic approaches have not been used so far to investigate changes in DNA methylation of the.
- The aim of the present study was to identify genomic regions presenting differential DNA methylation in bEECs following exposure to LPS, by using reduced rep- resentation bisulfite sequencing (RRBS).
- Thus, providing insights in alterations of DNA methylation induced by LPS possibly influencing endometrial function..
- RRBS and DNA methylation profile.
- For the sake of consistency when studying correlation between DNA methylation and gene expression results, the same biological material was used (same cells ex- posed to same LPS dosages and time point) as in our former RNAseq study [28].
- After quality filtering, 60–62% of the reads were successfully aligned to the bo- vine reference genome sequence (bosTau8), whereas 40–50% of the reads were uniquely mapped.
- In total, we identified 2.1–2.3 million CpG sites per sample, of which 1.93 million were covered in all samples, representing 7.1% of the total number of CpGs.
- 27 M) in the Bos taurus genome.
- the DNA methylation pattern.
- However, differential DNA methylation analyses detected 511 and 469 significant DMRs (q-value.
- The combined- LPS analysis detected 803 DMRs, sharing many DMRs identified in the two LPS groups (Fig.
- Finally, to avoid omission of functionally important methylated re- gions, we included in the analysis, those DMRs that did not withstand the q-value threshold in combined-LPS comparison but were significantly differentially methyl- ated in either 2 μg or 8 μg LPS-treated samples.
- LPS treatment induced a higher proportion of hypo- methylation when compared to control DMRs identified in the comparison between time 24 h and 0 h in controls (Fig.
- The chromosomal distribution of the DMRs was deter- mined to assess whether or not DMRs were associated to specific chromosomal features.
- The distribution of total targeted regions (n was not associated with telomeric regions (20 kb) of the chromosomes.
- 1 LPS effects on DNA methylation in bovine endometrial epithelial cells (bEECs).
- d Bar plot showing distribution of the percent of hyper and hypomethylated DMRs when comparing time 0 h and 24 h in controls, and 24 h control with 2 μ g or 8 μ g, and 2 μ g + 8 μ g combined LPS groups.
- When analyzing the distribution of DMRs in relation to genes and CpG islands 46% of the total number of identified DMRs were located in CpG islands, 31% at the shores, while 23% were located in other genomic regions (Fig.
- n = 613), whereas 4.8% (n = 63) were located in promoter regions (2 kb 5′ of the transcription start site) (Fig.
- Among genes that contained at least three DMRs in the gene body and promoter regions, NSG1 had the highest number with five DMRs.
- Colour intensity shows CG dinucleotide occurrence (million as unit) in the chromosomes.
- Correlation between DNA methylation and gene expression.
- The transcriptome- wide association between gene expression and DNA methylation within promoter regions and gene bodies was examined.
- However, 39 genes (49.9%) showed an inverse relationship between their de- gree of DNA methylation (in promoter or body) and gene expression (Fig.
- a Scatterplot showing mean gene expression and boxplot showing mean DNA methylation in differentially methylated regions (DMRs) in treated group for DMRs in promoters (a) and DMRs in gene bodies (b), with lines representing a linear trend.
- Bars in the box plot correspond to the median.
- Pro-inflammatory mechanisms may be favored by epi- genetic changes in the HDAC4 gene (two hypomethy- lated DMRs in intron 1 and one hypermethylated DMR in intron 2) and hypermethylation of two DMRs associ- ated to AKT1 gene (in AKT1 intron 1).
- We observed also the hypomethylation of one DMR in each of the promoters of MAP3K6 and BCL2 genes that regulates apoptosis.
- Differentially methylated regions (DMRs) were identified here after controlling for the individual cow effects in the analysis and using combined results for 2 and 8 μg/ml LPS.
- The remaining significant DMRs were fur- ther scrutinized for changes reported in the literature es- pecially for those relating to differential gene expression shown in our RNAseq study from the same cells [28]..
- Despite differences in the LPS dos- age and time of observation used, our results are consist- ent with previous studies in human and mouse showing that bacterial and viral infection induces hypomethyla- tion of host cell DNA [40–42] and the decrease in methylation observed in other bovine cell types follow- ing LPS challenge [27, 38].
- Globally, DNA methylation changes were enriched in sub-telomeric regions.
- Enrich- ment of DMRs and aberrant DNA hypomethylation of the X chromosome genes have also been reported in uterine leiomyoma [45] and in ovarian, cervical and breast cancers [46, 47]..
- The analysis of individual DMRs revealed that a number of them mapped to genes involved in the control of endo- metrial function and/or were related to endometrial dys- function and infertility as documented mainly in humans and mice.
- The three main pathways associated specifically with the control of endometrial function, namely i) prolif- eration and differentiation, ii) cell migration, cell adhesion and extracellular matrix remodeling and iii) immune re- sponses will be subsequently discussed especially in the light of corresponding changes in gene [28], protein ex- pression [32], and phenotypic response to LPS [31] from the same cells..
- impacts of LPS on HDAC4 methylation associated with a lower expression of several members of the HDAC’s family [28] on the proliferation of bovine endometrial cells would need specific investigations..
- Wnt signaling pathway is also involved in cell pro- liferation and differentiation in the endometrium..
- Further studies would be needed to demonstrate their specific role as part of the mechanisms explaining the strong proliferative phenotype observed in this model [31] and in different cell types [58]..
- Some of the roles ADAMTS1 on endometrial function have been described whereas less information exists for ADAMTS17.
- ADAMTS1 participates in the bovine endometrial remodeling at time of implantation and placental development [59], promotes epithelial cell invasion [60], and favors migration and alter adhesion [61, 62].
- However, DNA methylation changes found here concerned ADAMTS2, ADAMTS14 and ADAMTS17 .
- DNA methylation changes in genes from the cortico- tropin signaling network could be of biological signifi- cance due to the possible involvement of the proteins encoded by these genes in trophoblast invasion ( TFAP2A and vascularization ( PRKCA and PRKCG .
- This change in chromatin structure in transcriptional regulatory regions results in the consequential changes of mRNA expres- sion.
- However, it may be hypothe- sized from these results that in LPS-treated cells, the strong activation of the TLR-NF-kβ pathway, and the over expres- sion of TNF receptor associated factors (TRAFs) acting as NF-kβ activators [28] may result from the hypermethyla- tion of AKT1, which normally represses the above pathway and the hypomethylation of IRAK1, which activates TLR signaling..
- We observed also a differential methylation of the per- oxisome proliferator activated receptor alpha (PPARA, hypomethylation in intron 1), which could be of interest due to its possible role in enhancement of inflammation and restoration of physiological conditions in the endo- metrium following LPS challenge [74]..
- Future studies, based especially on sys- tems allowing long term cell culture and combining the different types of endometrial cells, would deserve further investigations to fully demonstrate the bio- logical significance of these methylation changes in the context of disease..
- LPS induces changes in DNA methylation patterns of bovine endometrial epithelial cells, towards mainly hypo- methylation that correlates with overall increased gene expression.
- The purity of the epithelial cell culture was estimated by morphological observation and confirmed by anti- cytokeratin 18 antibody (Abcam, Cambridge, UK) and anti-vimentin V9 antibody (Abcam) immunofluores- cence staining.
- These concentrations of LPS, which may mimic those during days after acute infection, are in the lower range of those previously re- ported in cow uterine fluid in case of clinical endometri- tis and/or in vivo experimental infection [29, 30].
- For all samples, the ratios of the absorbance at 260 nm and 280 nm (A260/280) and at 260 nm and 230 nm (A260/230) were between 1.8–2.0 and 2.1–2.4, respectively..
- Libraries for RRBS were generated by MspI digestion of the DNA followed by end-repair/A-tailing and 5mC adaptor ligation.
- Ensembl gene annotation version 84 was used in the methylation analysis of genic regions, whereas CpG islands and repetitive element annotations for bosTau8 genome were obtained from the UCSC gen- ome browser database (https://genome.ucsc.edu/)..
- Genomic distribution of DMRs in LPS treated bEECs and annotated genes in the vicinity.
- We thank SLU Bioinformatics Infrastructure (SLUBI) for management and processing of the sequencing data.
- and a grant from Rajamangala University of Technology Srivijaya (RMUTSV), Thailand, covering most of running expenses for collection and preparation of biological material used and the scholarship of the PhD student Metasu Chanrot.
- The funding bodies did not participate in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
- All RRBS raw sequencing data were deposited in the European Nucleotide Archive (ENA) under accession number PRJEB36023.
- Raw RNAseq sequencing data are also available in the ENA database under accession PRJEB34011.
- GCA CpG islands and repetitive element annotations for bosTau8 are available in the UCSC genome browser database (https://genome.ucsc.edu/.
- 3 Department of Information Technology, University of the Punjab, Gujranwala Campus, Gujranwala, Pakistan.
- Defining postpartum uterine disease and the mechanisms of infection and immunity in the female reproductive tract in cattle.
- Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium.
- Expression and function of toll-like receptor 4 in the endometrial cells of the uterus.
- Toll-like receptor 4 mediates the response of epithelial and stromal cells to lipopolysaccharide in the endometrium.
- DNA methylation is correlated with gene expression during early pregnancy in Bos taurus..
- Is DNA methylation an epigenetic contribution to transcriptional regulation of the bovine endometrium during the estrous cycle and early pregnancy? Mol Cell Endocrinol.
- The effect of DNA methylation inhibitor 5-Aza-2 ′ -deoxycytidine on human endometrial stromal cells.
- Impaired down- regulation of E-cadherin and beta-catenin protein expression in endometrial epithelial cells in the mid-secretory endometrium of infertile patients with endometriosis.
- Aberrant methylation at HOXA10 may be responsible for its aberrant expression in the endometrium of patients with endometriosis.
- The immune status of the bovine uterus during the peripartum period.
- Interactions of metabolism, inflammation, and reproductive tract health in the postpartum period in dairy cattle.
- Modulation of the immune system during postpartum uterine inflammation.
- Modulation of the maternal immune system by the pre-implantation embryo.
- LPS-induces IL-6 and IL-8 gene expression in bovine endometrial cells "through DNA methylation".
- The relationship between uterine pathogen growth density and ovarian function in the postpartum dairy cow.
- Bacterial Lipopolysaccharide Induced Alterations of Genome-Wide DNA Methylation and Promoter Methylation of Lactation-Related Genes in Bovine Mammary Epithelial Cells.
- Bacterial infection remodels the DNA methylation landscape of human dendritic cells.
- The relationship between DNA methylation and telomere length in dyskeratosis congenita..
- Loss of the inactive X chromosome and replication of the active X in BRCA1-defective and wild-type breast cancer cells.
- Dynamic regulation of Wnt7a expression in the primate endometrium:.
- Activation of cytosolic phospholipase A2 downstream of the Src-phospholipase D1 (PLD1)-protein kinase C gamma (PKCgamma) signaling axis is required for hypoxia-induced pathological retinal angiogenesis.
- The influence of experimentally induced endometritis on the PPAR expression profile in the bovine endometrium.
- methylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles

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