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Exploration of bovine milk exosomal microRNAs during Streptococcus uberis induced mastitis

ABSTRACT: Milk is an indispensable source of infant nutrition in all mammals, made up of complex constituents needed for infant nourishment and immunity. Comparison of miRNA profiles between infected and non-infected/control Holstein cattle could provide important information on the differences in composition of milk at the level of miRNA, if any, and broaden our perspective and understanding of the effect of pathology on cellular compositions and functions. In the present study we therefore analyzed the expression profiles of bovine milk exosomal miRNAs during S. uberis infection and identified 328 known miRNAs and 82 high-confidence miRNA candidates by deep sequencing. The top 10 miRNAs in both control and infected replicates accounted for approximately 80% of total counts, which were predicted to target 605 genes using two computational approaches. In addition, 15 significantly differentially expressed miRNAs were identified between control and infected replicates during S. uberis infection, and a total of 1,852 unique genes were predicted to be targeted by these miRNAs. Ingenuity Canonical Pathways and Diseases and Biological Function analyses using IPA indicated that the identified miRNAs targets mainly enriched in regulating the innate and adaptive immune responses in newborns, as well as infant growth and development. The characterization of these miRNAs could contribute to a better understanding of the molecular mechanisms involved in lactation physiology and milk imparted immune function in the dairy cattle.

ORGANISM(S): Bos taurus

PROVIDER: GSE59794 | GEO | 2017/07/25

SECONDARY ACCESSION(S): PRJNA256214

REPOSITORIES: GEO

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Project description:Human milk (breastmilk) is much more than nutrition for the infant, containing an array of regulatory agents with immunoprotective and developmental functions. Amongst those, microRNAs (miRNAs) have recently been identified, with their properties, roles, origin and distribution in breastmilk as well as in the mammary gland being still undetermined. In this study, we examined the miRNA profile of different fractions of human milk (cells and lipids) using the OpenArray system (Applied Biosystems, 770 miRNA species measured per sample) and compared it with maternal peripheral blood mononuclear cells (PBMCs) and plasma. Although PBMCs were the richest group in miRNA species, plasma showed very low expression pattern. Thus, the human milk fractions (cells, lipid) and skim milk (not being investigated in this study) were found to conserve higher levels of miRNAs than blood in general. Specifically, human milk cell miRNA quantity was found relatively close to PBMCs, and higher than milk lipids. Correlation and clustering analyses indicated that miRNA expression and types of milk cells were highly similar to those in lipids. Milk miRNAs showed a slight correlation to PBMCs, so PBMCs potentially are not contributing to milk miRNAs. Plasma was different to all other three groups in miRNA content and expression pattern. Further, two infant formulae (a plant-based and a cow milk-based) were compared to human milk and found to contain significantly fewer miRNA species than human milk cells and lipids (p>0.001). Taken together with previous studies on miRNAs, our findings demonstrate that human milk is one of the richest sources of miRNAs among human body fluids. As a non-invasive and plentiful source of miRNAs, human milk could be used as a disease biomarker for the mammary gland, with potential in assessing lactation performance. Finally, gene target and pathways analyses identified several target mRNAs regulated by miRNAs found to be abundant in breastmilk. Given the recently identified stability and function of food-derived miRNAs in regulating mammalian genes, we propose that breastmilk is a rich source of miRNA ingested by the infant during the first months of life, and which potentially contribute to early infant development. 10 exclusively breastfeeding dyads were recruited. 10 whole milk and 10 whole blood samples were collected and fractionated to obtain 10 milk cells, 10 milk lipid, 10 mononeucleoted blood cells (PBMCs), and 10 plasma. In addition to the above 40 samples, 2 infant formula were profiled. 4 different extraction kits were used, miRNeasy mini Kit for human milk cell and PBMC samples. miRCURY RNA Isolation-Biofluids Kit for human milk lipid samples and both infant formulae. mirVana PARIS Kit for plasma samples. NanoDrop 2000 and Bioanalyzer 2100 were used to determine concentration and purity of the extracted miRNA from all samples (n=42). miRNA OpenArray panel system (Life Technologies, CA, USA) was used to profile 754 human mature miRNAs in samples. RNU48, RNU44 and U6 rRNA were used as housekeeping controls for normalisation. ath-miR159a was used as a negative control for human samples. GeneGO and Ingenuity Pathway Analysis were used to determine biological pathways. Please note that normalization of miRNAs was done in R but without generating deltaCT values, thus [1] only the list of normalized miRNA with Ct vlaue between 8 and 29 and that detected in at least 4 samples out of 10 analysed in each group is provided ('normalized_miRNAs_list.txt') [2] the sample data tables contain raw data.

Project description:Background Our objective was to compare mammary tissue gene expression profiles during a Streptococcus uberis (S. uberis) mastitis challenge between lactating cows subjected to dietary-induced negative energy balance (NEB; n = 5) and cows fed ad libitum to maintain positive energy balance (PEB; n = 5). The NEB cows were feed-restricted to 60% of calculated net energy for lactation requirements for 7 d, and cows assigned to PEB were fed the same diet for ad libitum intake. Five days after feed restriction, one rear mammary quarter of each cow was inoculated with 5,000 cfu of S. uberis (O140J). At 20 h post-inoculation, both the S. uberis-infected mammary quarters (i.e., YES) and non-infected rear quarters (i.e., NO) from all cows were biopsied for RNA extraction. Effect of S. uberis Intramammary Infection (IMI; YES vs NO) S. uberis IMI resulted in 2,102 oligos (1,939 annotated genes) differentially expressed genes (DEG; YES versus NO). Of these, 1,082 genes were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA1. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Pathway analysis of all DEG indicated that IL-10 Signaling, IL-6 Signaling, and Glucocorticoid Receptor Signaling were among the top canonical pathways affected by infection. Interestingly, LXR/RXR Signaling was also a primary affected pathway, indicating a relationship between immune system response and metabolism during an IMI. Network analysis indicated that TNF had positive relationships (i.e. up-regulation) with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships (i.e., down-regulation) with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1). Results provided information into the early response factors associated with the innate immune response to S. uberis infection. Our study indicated that IMI challenge with S. uberis (strain O140J) elicited a strong transcriptomic response, leading to an overall up-regulation of genes involved in the innate immune response and a down-regulation of genes involved with lipid metabolism. Effect of NEB Within Infected Quarters (YES) Energy balance (NEB vs. PEB) resulted in 285 DEG (FDR ≤ 0.05), with 85 DEG up-regulated and 200 DEG down-regulated. Canonical pathways most affected by NEB were IL-8 Signaling (10 genes), Glucocorticoid Receptor Signaling (13), and NRF2-mediated Oxidative Stress Response (10). Among genes differentially expressed by NEB, Cell Growth and Proliferation (48) and Cellular Development (36) were the most enriched functions. Up-regulated genes included ANXA1, HMOX1, and HLAA; down-regulated genes included NOTCH1 and CCND1. Regarding immune response, HLAA was up-regulated due to NEB, whereas the majority of genes involved in immune response were down-regulated (e.g., AKT1, IRAK1, MAPK9, and TRAF6). Results helped identify mechanisms affected by dietary-induced NEB that may be associated with the impairment of immune function and increased risk of mastitis in cows experiencing postpartal NEB. Effect of NEB Within Non-Infected Quarters (NO) Energy balance (NEB vs. PEB) resulted in 278 DEG within non-infected rear mammary quarters. Up-regulated DEG most affected by NEB (genes = 180) included genes involved in lipid metabolism and amino acid metabolism. The down-regulated DEG most affected by NEB (n = 98) included genes involved in carbohydrate metabolism, anti-inflammatory response, and apoptosis. Among genes up-regulated, Ingenuity Pathway Analysis® identified Lipid Metabolism (8), Molecular Transport (14), and Small Molecule Biochemistry (15) as some of the most enriched molecular functions. Genes down-regulated by NEB were associated with Cell Growth and Proliferation (21) and Cell Death (18). Results indicate that dietary induced-NEB alters genes primarily associated with cellular metabolism, proliferation, and tumorigenesis but no candidate genes were identified as being associated with risk of disease.

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Exploration of bovine milk exosomal microRNAs during Streptococcus uberis induced mastitis

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