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RNA-seq analysis reveals RNA expression profiles following LPS-induced inflammation in bovine mammary epithelial cells

ABSTRACT: Purpose: to detect expression profile of RNA (lncRNA and circRNA) and elucidate differentially expressed RNAs (DElncRNAs and DEcircRNAs) with potential roles during lipopolysaccharide (LPS)-induced inflammation models of bovine mammary epithelial cells MAC-T in vitro. Methods: bovine mammary epithelial cells MAC-T were exposed to LPS for 0, 6 and 12 hours to assess the expression profiles of RNA (lncRNA and circRNA) using RNA-seq. Results: totally 112 DElncRNAs and 71 DEcircRNAs were screened out at different time points. Functional enrichment analysis on target genes of lncRNAs and host genes of circRNAs indicated that these genes were involve in regulating inflammation-related signaling pathways, including Notch, NF-κB, MAPK, PI3K-Akt, mTOR, MAPK and NOD-like receptor signaling pathway. Conclusion: these differentially expressed RNAs (DElncRNAs and DEcircRNAs) may be involved in the regulation of a variety of immune-related processes including inflammatory responses bovine mammary epithelial cells exposed to LPS via some vital signaling pathways. This study lays a foundation for further research on molecular regulation of bovine mastitis, and also provides a reference for breeding strategies based on molecular markers for mastitis resistance in dairy cows.

ORGANISM(S): Bos taurus

PROVIDER: GSE181658 | GEO | 2021/11/17

REPOSITORIES: GEO

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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.

Project description:Purpose: The goal of this study is to explore the role of miRNAs in dairy cow response to E. coli and S. aureus, mastitis causing pathogens, is not well understood. Results: The global expression of miRNAs in bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing. A total of 231 known bovine miRNAs were identified with more than 10 counts per million (CPM) 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 total reads of known bovine miRNAs. One hundred and fifty novel miRNAs were identified and half 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 one for S. aureus) while S. aureus presented a delayed response. Five differentially expressed miRNAs (Bta-miR184, miR-24-3p, miR-148, miR-486 and bta-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 (Kyoto encyclopedia of genes and genomes) pathways of the immune system, signal transduction, cellular process, nervous system, development and pathways in human diseases, especially cancer. Conclusion: Using next-generation sequencing, our study identified 150 novel bovine miRNAs and revealed a pathogen directed differential regulation of miRNAs in MAC-T cells with roles in immunity and development. E. coli elicited an earlier differential regulation of miRNAs as opposed to a delayed regulation by S. aureus. Furthermore, target gene prediction showed significant enrichments for functions in different biological and cellular processes as well as KEGG pathways in immunity, development and human diseases. 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 of mastitis and development of control measures. Bovine mammary epithelial cells (MAC-T cells) challenged with heat-inactivated Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) bacteria (treatments: 6, 12, 24 and 48 hr) and without challenge (control: 0, 6, 12, 24 and 48 hr) was profiled using next-generation-sequencing, no replicates, using illumina HiScanSQ platform.

Project description:Differential response of bovine mammary epithelial cells to Staphylococcus aureus or Escherichia coli agonists of the innate immune system. 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 (3 and 6 h of exposure to agonists). By using HEK 293 cells transfected with human 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 alpha-hemolysin were all present in SaS, and bMEC were found to be responsive to each of these molecules. Transcriptome profiling by a microarray and confirmation by RT-qPCR revealed an innate immune response which was common to both LPS and SaS. However, LPS induced expression of a significant higher number of genes and the fold changes were of greated magnitude than those induced by SaS. Overall, the analysis of microarray data suggests that the activation pathways and the early chemokine and cytokine production preceded the defense and stress responses. Chemokines were among the most up-regulated genes, in particular ELRCXC chemokines that target neutrophils. A major differential response was the activation of the type I IFN pathway by LPS but not by SaS. This was in accordance with the much stronger up-regulation of Cxcl10, Ccl5 and Nos2 by LPS than by SaS. The higher upregulation of chemokines that target mononuclear leucocytes (CXCL10, CCL2, CCL5 and CCL20) 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. It is noteworthy that at the protein level, secretion of TNF-alpha and IL-1-beta was not induced by either stimulus. These results suggest that the response of MEC to diffusible bacterial stimuli is able to contribute to the onset of the response with differential leucocyte recruitment but does not account directly for the differential production of major pro-inflammatory cytokines. Transcriptional profiling of bovine mammary epithelial cells (Holstein breed) comparing untreated control with mammary epithelial cells (MECs) stimulated by Staphylococcus aureus (SA) versus Escherichia coli lipopolysaccharide (LPS). 16 microarrays (2 stimuli * 2 times * quadruplicate) in a two-color dye-swap experimental design. Untreated cells served as the control. One replicate per array. Log2-intensity of each dye was analyzed separately => 32 samples.

Project description:Bovine mastitis, an inflammatory reaction of the mammary gland, is commonly caused by Staphylococcus aureus or Escherichia coli infections in high-yielding dairy cows. Immunotranscriptomic responses of bovine mammary epithelial cell (bMEC) are pivotal for the understanding of the defense against invading pathogens, and for the fate of udder inflammation. The intracellular chemotaxic protein Cyclophilin A (CyPA), secreted by bMEC during inflammatory reaction is being considered as a marker for the early diagnosis of mastitis. Taken together, the present study aimed to investigate the microarray-based transcriptome alterations in bMEC after in vitro infection with two S. aureus strains (S. aureus JE2 and S. aureus SA003) or E. coli crude lipopolysaccharide (LPS). In addition, the CyPA-induced expression changes of selected cytokine and chemokines were evaluated by qRT-PCR. Differential expression analysis identified 447, 465 and 520 differentially expressed genes (DEGs) in bMEC after LPS, S. aureus JE2 and S. aureus SA003 stimulation, respectively. LPS-induced DEGs are involved in the activation of cytokine-cytokine receptors interaction, NOD-like receptor signaling, chemokine signaling, interleukin-1 signaling and Toll-like receptor signaling pathway; while S. aureus induced DEGs were involved in the enrichment of neuroactive ligand-receptor interaction, GPCR signaling, cytokine-cytokine receptor interaction, and JAK-STAT pathway. A major differential inflammatory response to LPS and S. aureus treatments was the activation of the chemokine signaling pathway by LPS but not either by S. aureus JE2 or SA003. Unlike the S. aureus strains, LPS stimulation resulted significant upregulation of CCL2, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, IL1A and IL1B that are known to target mononuclear leukocytes, and could induce systemic inflammation. CyPA stimulation was able to upregulate chemokines (CXCL2, CXCL3, CXCL8, IL1A and IL1B) expression in bMEC indicating its ability to promote inflammation. Several of the LPS-induced DEGs were found to have transcription factor binding sites, and their expressions were also predicted to be regulated by sets of microRNAs. The transcriptional alterations associated with inflammatory response in this study may reflect immunomodulatory mechanisms underlying the interaction of bMEC with E. coli and S. aureus during mastitis.

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RNA-seq analysis reveals RNA expression profiles following LPS-induced inflammation in bovine mammary epithelial cells

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