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MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection.

ABSTRACT: Milk exosomes are a rich source of microRNAs (miRNAs) that are protected from degradation. Ingestion of milk and subsequent absorption of miRNAs into recipient cells by endocytosis may play a role in the regulation of neonatal innate and adaptive immunity. In contrast, the miRNA content of milk exosomes may also be indicative of a lactating animal's health; whereby, the presence or absence of specific miRNAs could serve as biomarkers for early detection of bacterial infection that can lead to mastitis. In the present study, we therefore analyzed and compared miRNA expression profiles of milk exosomes from four Holstein cows obtained during mid-lactation prior to and after infection (48 h) of the mammary gland with Staphylococcus aureus.Milk exosomes, purified from control and S. aureus infected cows, were extracted for RNA. Following preparation indexed libraries from both groups the samples were subjected to next generation sequencing.Next generation sequencing of eight, unpooled small RNA libraries derived from milk exosomes produced about 60.5 million high-quality, bovine-specific sequence reads for comparison of miRNA expression between treatments. Sequence identity analysis showed the miRNAs make up about 13 % of the average RNA content of these exosomes. Although 417 known bovine miRNAs were identified, miRNAs represented the least diverse class of RNA accounting for only 1 % of all unique sequences. The 20 most prevalent unique sequences within this class accounted for about 90 % of the total miRNA-associated reads across samples. Non-annotated, unique reads provided evidence for another 303 previously unknown bovine miRNAs. Expression analyses found 14 known bovine microRNAs significantly differed in frequency between exosomes from infected and control animals.Our survey of miRNA expression from uninfected milk exosomes and those produced in response to infection provides new and comprehensive information supporting a role for delivery into milk of specific miRNAs involved in immune response. In particular, bta-miR-142-5p, and -223 are potential biomarkers for early detection of bacterial infection of the mammary gland. Additionally, 22 mammary-expressed genes involved in regulation of host immune processes and response to inflammation were identified as potential binding targets of the differentially expressed miRNAs.

SUBMITTER: Sun J

PROVIDER: S-EPMC4609085 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection.

Sun Jiajie J Aswath Kshama K Schroeder Steven G SG Lippolis John D JD Reinhardt Timothy A TA Sonstegard Tad S TS

BMC genomics 20151016

<h4>Background</h4>Milk exosomes are a rich source of microRNAs (miRNAs) that are protected from degradation. Ingestion of milk and subsequent absorption of miRNAs into recipient cells by endocytosis may play a role in the regulation of neonatal innate and adaptive immunity. In contrast, the miRNA content of milk exosomes may also be indicative of a lactating animal's health; whereby, the presence or absence of specific miRNAs could serve as biomarkers for early detection of bacterial infection ...[more]

PMID: 26475455

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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:BackgroundMastitis is an economically important disease of dairy cows with Staphylococcus aureus a major cause worldwide. Challenge of Holstein-Friesian cows demonstrated that S. aureus strain MOK124, which belongs to Clonal Complex (CC)151, caused clinical mastitis, while strain MOK023, belonging to CC97, caused mild or subclinical mastitis. The aim of this study was to elucidate the molecular mechanisms of the host immune response utilising a transcriptomic approach. Milk somatic cells were collected from cows infected with either S. aureus MOK023 or MOK124 at 0, 24, 48, 72 and 168 h post-infection (hpi) and analysed for differentially expressed (DE) genes in response to each strain.ResultsIn response to MOK023, 1278, 2278, 1986 and 1750 DE genes were found at 24, 48, 72 and 168 hpi, respectively, while 2293, 1979, 1428 and 1544 DE genes were found in response to MOK124 at those time points. Genes involved in milk production (CSN1, CSN10, CSN1S2, CSN2, a-LACTA and PRLR) were downregulated in response to both strains, with a more pronounced decrease in the MOK124 group. Immune response pathways such as NF-κB and TNF signalling were overrepresented in response to both strains at 24 hpi. These immune pathways continued to be overrepresented in the MOK023 group at 48 and 72 hpi, while the Hippo signalling, extracellular matrix interaction (ECM) and tight junction pathways were overrepresented in the MOK124 group between 48 and 168 hpi. Cellular composition analysis demonstrated that a neutrophil response was predominant in response to MOK124, while M1 macrophages were the main milk cell type post-infection in the MOK023 group.ConclusionsA switch from immune response pathways to pathways involved in maintaining the integrity of the epithelial cell layer was observed in the MOK124 group from 48 hpi, which coincided with the occurrence of clinical signs in the infected animals. The higher proportion of M1 macrophages in the MOK023 group and lack of substantial neutrophil recruitment in response to MOK023 may indicate immune evasion by this strain. The results of this study highlight that the somatic cell transcriptomic response to S. aureus is dependent on the genotype of the infecting strain.

Dataset Information

MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection.

Publications

MicroRNA expression profiles of bovine milk exosomes in response to Staphylococcus aureus infection.

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