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Transcriptome Profiling of m⁶A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

ABSTRACT: Mastitis is a common disease in dairy cows that is mostly caused by E. coli, and it brings massive losses to the dairy industry. N⁶-Methyladenosine (m⁶A), a methylation at the N⁶ position of RNA adenine, is a type of modification strongly associated with many diseases. However, the role of m⁶A in mastitis has not been investigated. In this study, we used MeRIP-seq to sequence the RNA of bovine mammary epithelial cells treated with inactivated E. coli for 24 h. In this in vitro infection model, there were 16,691 m⁶A peaks within 7066 mRNA transcripts in the Con group and 10,029 peaks within 4891 transcripts in the E. coli group. Compared with the Con group, 474 mRNAs were hypermethylated and 2101 mRNAs were hypomethylated in the E. coli group. Biological function analyses revealed differential m⁶A-modified genes mainly enriched in the MAPK, NF-κB, and TGF-β signaling pathways. In order to explore the relationship between m⁶A and mRNA expression, combined MeRIP-seq and mRNA-seq analyses revealed 212 genes with concomitant changes in the mRNA expression and m⁶A modification. This study is the first to present a map of RNA m⁶A modification in mastitis treated with E. coli, providing a basis for future research.

SUBMITTER: Li T

PROVIDER: S-EPMC8230414 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHeat stress has a substantial negative economic impact on the dairy industry. N6-methyladenosine (m6A) is the most common internal RNA modification in eukaryotes and plays a key role in regulating heat stress response in animals. In dairy cows, however, this modification remains largely unexplored. Therefore, we examined the effects of heat stress on the m6A modification and gene expression in bovine mammary epithelial cells to elucidate the mechanism of heat stress response. In this study, Mammary alveolar cells-large T antigen (MAC-T) cells were incubated at 37 °C (non-heat stress group, NH) and 40 °C (heat stress group, H) for 2 hours, respectively. HSP70, HSF1, BAX and CASP3 were up regulated in H group compared with those in the NH group.ResultsMethylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted to identify m6A peaks and to produce gene expression data of MAC-T cells in the H and NH groups. In total, we identified 17,927 m6A peaks within 9355 genes in the H group, and 18,974 peaks within 9660 genes in the NH groups using MeRIP-seq. Compared with the NH group, 3005 significantly differentially enriched m6A peaks were identified, among which 1131 were up-regulated and 1874 were down-regulated. In addition, 1502 significantly differentially expressed genes were identified using RNA-seq, among which 796 were up-regulated and 706 were down-regulated in the H group compared to the NH group. Furthermore, 199 differentially expressed and synchronously differentially methylated genes were identified by conjoint analysis of the MeRIP-seq and RNA-seq data, which were subsequently divided into four groups: 47 hyper-up, 53 hyper-down, 59 hypo-up and 40 hypo-down genes. In addition, GO enrichment and KEGG analyses were used to analyzed the potential functions of the genes in each section.ConclusionThe comparisons of m6A modification patterns and conjoint analyses of m6A modification and gene expression profiles suggest that m6A modification plays a critical role in the heat stress response by regulating gene expression.

Project description:BackgroundMicroRNAs (miRNAs) can post-transcriptionally regulate gene expression and have been shown to be critical regulators to the fine-tuning of epithelial immune responses. However, the role of miRNAs in bovine responses to E. coli and S. aureus, two mastitis causing pathogens, is not well understood.ResultsThe global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with and without heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria at 0, 6, 12, 24, and 48 hr was profiled using RNA-Seq. A total of 231 known bovine miRNAs were identified with more than 10 counts per million in at least one of 13 libraries and 5 miRNAs including bta-miR-21-5p, miR-27b, miR-22-3p, miR-184 and let-7f represented more than 50% of the abundance. One hundred and thirteen novel miRNAs were also identified and more than one third of them belong to the bta-miR-2284 family. Seventeen miRNAs were significantly (P < 0.05) differentially regulated by the presence of pathogens. E. coli initiated an earlier regulation of miRNAs (6 miRNAs differentially regulated within the first 6 hrs post challenge as compared to 1 miRNA for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (bta-miR-184, miR-24-3p, miR-148, miR-486 and let-7a-5p) were unique to E. coli while four (bta-miR-2339, miR-499, miR-23a and miR-99b) were unique to S. aureus. In addition, our study revealed a temporal differential regulation of five miRNAs (bta-miR-193a-3p, miR-423-5p, miR-30b-5p, miR-29c and miR-un116) in unchallenged cells. Target gene predictions of pathogen differentially expressed miRNAs indicate a significant enrichment in gene ontology functional categories in development/cellular processes, biological regulation as well as cell growth and death. Furthermore, target genes were significantly enriched in several KEGG pathways including immune system, signal transduction, cellular process, nervous system, development and human diseases.ConclusionUsing next-generation sequencing, our study identified a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development. Our study provides a further confirmation of the involvement of mammary epithelia cells in contributing to the immune response to infecting pathogens and suggests the potential of miRNAs to serve as biomarkers for diagnosis and development of control measures.

Project description:Mastitis caused by Escherichia coli and Staphylococcus aureus is a major pathology of dairy cows. To better understand the differential response of the mammary gland to these two pathogens, we stimulated bovine mammary epithelial cells (bMEC) with either E. coli crude lipopolysaccharide (LPS) or with S. aureus culture supernatant (SaS) to compare the transcriptomic profiles of the initial bMEC response. By using HEK 293 reporter cells for pattern recognition receptors, the LPS preparation was found to stimulate TLR2 and TLR4 but not TLR5, Nod1 or Nod2, whereas SaS stimulated TLR2. Biochemical analysis revealed that lipoteichoic acid, protein A and ?-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling revealed a core innate immune response partly shared by LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greater magnitude than those induced by SaS. Microarray data analysis suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. The higher upregulation of chemokines (Cxcl10, Ccl2, Ccl5 and Ccl20) that target mononuclear leucocytes by LPS than by SaS is likely to be related to the differential activation of the type I IFN pathway, and could induce a different profile of the initial recruitment of leucocytes. The MEC responses to the two stimuli were different, as LPS was associated with NF-?B and Fas signaling pathways, whereas SaS was associated with AP-1 and IL-17A signaling pathways. It is noteworthy that at the protein level secretion of TNF-? and IL-1? was not induced by either stimulus. These results suggest that the response of MEC to diffusible stimuli from E. coli and S. aureus contributes to the onset of the response with differential leucocyte recruitment and distinct inflammatory and innate immune reactions of the mammary gland to infection.

Project description:Bovine mastitis is the inflammatory reaction of the mammary gland and is commonly caused by bacterial infections in high-yielding dairy cows. The detailed investigation of the immunotranscriptomic response of bovine mammary epithelial (BME) cells to pattern recognition receptors (PRRs) activation by microbial-associated molecular patterns (MAMPs) can be of great importance for understanding the innate immune defense mechanisms, and for exploring the immunomodulatory candidate genes. In this work, we investigated the transcriptome modifications of BME cells after the in vitro stimulation with Escherichia coli derived lipopolysaccharide (LPS) and heat-killed Staphylococcus aureus JE2 and S. aureus SA003. In addition, the effect of Pam3CSK4 (a synthetic triacylated lipopeptide that activates Toll-like receptor 2 (TLR2)), and the intracellular chemotactic protein cyclophilin A (CyPA), which is secreted by BME cells during mastitis, in the expression changes of selected cytokines and chemokines were evaluated by qPCR. Microarray analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in the BME cells after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. A major differential response in the inflammatory gene expression was noticed between the stimulation of LPS and S. aureus strains. Unlike the S. aureus strains, LPS stimulation resulted in significant upregulation of CCL2, CXCL2, CXCL3, CXCL8, IL1 and IL1, which were confirmed by qPCR analysis. Pam3CSK4 was not able to induce significant changes in the expression of cytokines and chemokines in challenged BME cells. The exogenous CyPA administration was able to upregulate CXCL2, CXCL3, CXCL8, IL1 and IL1 expression in BME cells indicating its ability to promote inflammation. The identification of transcriptional markers of mastitis specific for individual inflammatory factors such as LPS, Pam3CSK4 or CyPA, which can be evaluated in vitro in BME cells, may enable the development of novel diagnostics and/or immunomodulatory treatments, providing new tools for the effective management of mastitis in dairy cows. The results of this work are an advance in this regard.

Project description:Mastitis is a common disease that hinders the development of dairy industry and animal husbandry. It leads to the abuse of antibiotics, the emergence of super drug-resistant bacteria, and poses a great threat to human food health and safety. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) are the most common pathogens of mastitis in dairy cows and usually cause subclinical or clinical mastitis. CircRNAs and N6-methyladenosine (m6A) play important roles in immunological diseases. However, the mechanisms by which m6A modifies circRNA in bovine mammary epithelial cells remain poorly understood. The aim of our study was to investigate m6A-modified circRNAs in bovine mammary epithelial cells (MAC-T cells) injured by S. aureus and E. coli. The profile of m6A-modified circRNA showed a total of 1599 m6A peaks within 1035 circRNAs in the control group, 35 peaks within 32 circRNAs in the S. aureus group, and 1016 peaks within 728 circRNAs in the E. coli group. Compared with the control group, 67 peaks within 63 circRNAs were significantly different in the S. aureus group, and 192 peaks within 137 circRNAs were significantly different in the E. coli group. Furthermore, we found the source genes of these differentially m6A-modified circRNAs in the S. aureus and E. coli groups with similar functions according to GO and KEGG analyses, which were mainly associated with cells injury, such as inflammation, apoptosis, and autophagy. CircRNA-miRNA-mRNA interaction networks predicted the potential circRNA regulation mechanism in S. aureus- and E. coli-induced cell injury. We found that the mRNAs in the networks, such as BCL2, MIF and TNFAIP8L2, greatly participated in the MAPK, WNT, and inflammation pathways. This is the first report on m6A-modified circRNA regulation of cells under S. aureus and E. coli treatment, and sheds new light on potential mechanisms and targets from the perspective of epigenetic modification in mastitis and other inflammatory diseases.

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Transcriptome Profiling of m⁶A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

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Transcriptome Profiling of m6A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.

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Transcriptome Profiling of m⁶A mRNA Modification in Bovine Mammary Epithelial Cells Treated with Escherichia coli.