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Acute mammary and liver transcriptome response after an intra-mammary E. coli lipopolysaccharide challenge in postpartal dairy cows

ABSTRACT: The study investigated the acute and simultaneous response of the mammary and liver transcriptome to an intra-mammary lipopolysaccharide (LPS) challenge in early-lactating cows, and its consequences on metabolic biomarkers and liver composition. At 7 days of lactation, 7 cows served as controls (CTR) and 7 cows (LPS) received an intra-mammary E. coli LPS challenge. The mammary and liver tissues were sampled at 2.5 h from challenge for transcriptomic profiling. Liver composition was evaluated at 2.5 h and 7 d after challenge and blood biomarkers were analized at -2, 3, 7 and 14 d from challenge. In mammary tissue, the LPS challenge resulted in 189 differentially expressed gene (DEG), with 20 downregulated and 169 upregulated, while in the liver were found 107 DEG with 42 downregulated and 65 upregulated in LPS vs CTR. In the udder, the Dynamic Impact Approach (DIA) highlighted the activation of NOD-like receptor signaling, Toll-like receptor signaling, RIG-I-like receptor signaling and Apoptosis pathways, while in the liver were inhibited the Fatty acid elongation in mitochondria and activated the p53 signaling pathway. The LPS induced the alteration of liver lipid metabolism (rise of total lipid and triglyceride concentration), a systemic inflammation (rise of blood ceruloplasmin and bilirubin) and an increase of body fat mobilization. In cows subjected to intra-mammary LPS, the mammary gland responds activating mechanisms of pathogen recognition. In the liver the response likely depends by mediators coming from udder and affects the liver functionality and mainly the fatty acid metabolism.

ORGANISM(S): Bos taurus

PROVIDER: GSE64225 | GEO | 2014/12/17

SECONDARY ACCESSION(S): PRJNA270483

REPOSITORIES: GEO

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Project description:The study investigated the acute and simultaneous response of the mammary and liver transcriptome to an intra-mammary lipopolysaccharide (LPS) challenge in early-lactating cows, and its consequences on metabolic biomarkers and liver composition. At 7 days of lactation, 7 cows served as controls (CTR) and 7 cows (LPS) received an intra-mammary E. coli LPS challenge. The mammary and liver tissues were sampled at 2.5 h from challenge for transcriptomic profiling. Liver composition was evaluated at 2.5 h and 7 d after challenge and blood biomarkers were analized at -2, 3, 7 and 14 d from challenge. In mammary tissue, the LPS challenge resulted in 189 differentially expressed gene (DEG), with 20 downregulated and 169 upregulated, while in the liver were found 107 DEG with 42 downregulated and 65 upregulated in LPS vs CTR. In the udder, the Dynamic Impact Approach (DIA) highlighted the activation of NOD-like receptor signaling, Toll-like receptor signaling, RIG-I-like receptor signaling and Apoptosis pathways, while in the liver were inhibited the Fatty acid elongation in mitochondria and activated the p53 signaling pathway. The LPS induced the alteration of liver lipid metabolism (rise of total lipid and triglyceride concentration), a systemic inflammation (rise of blood ceruloplasmin and bilirubin) and an increase of body fat mobilization. In cows subjected to intra-mammary LPS, the mammary gland responds activating mechanisms of pathogen recognition. In the liver the response likely depends by mediators coming from udder and affects the liver functionality and mainly the fatty acid metabolism. Fourteen Holstein cows entering their second or greater lactation were used. Cows were housed in a ventilated tie-stall barn and were fed a common lactation diet (Net energy of lactation = 1.69 Mcal/kg DM) as a total mixed ration once daily (0600 h) and milked twice daily (0400 and 1600 h). A bovine oligonucleotide (70-mers) microarray with >13,000 annotated sequences developed at the University of Illinois, was used for transcript profiling. Details on the development, annotation, and use of this microarray have been reported previously by Loor et al., 2007 (http://physiolgenomics.physiology.org/content/32/1/105.abstract). Methods for microarray hybridization and scanning were as reported by Loor et al. (2007). Briefly, slides were hydrated, dried, and placed in a UV Stratalinker 1800 (Stratagene, La Joya, CA) for ~5 min. Slides were washed with 0.2% SDS solution, rinsed with MilliQ (Millipore) H2O, and placed in warm prehybridization soln for 45 min at 42 C. The same amount of Cy3- or Cy5-labelled cDNA from mammary or liver and a reference standard RNA pool (made of different bovine tissues) were co-hybridized using a dye-swap design (i.e., two microarrays per sample). Slides were incubated for 48 h at 45 C prior to scanning. Criteria for evaluation of slide quality included: identification of number of spots with a minimum median signal intensity of 3 SD above background; keeping slides with a minimum of 20,000 spots with minimum median signal intensity of 3 SD above background in both Cy3 and Cy5 channels; and keeping slides with a minimum mean intensity of 400 relative fluorescent units in both Cy3 and Cy5 channels across the entire slide. Data from a total of 112 microarrays were normalized for dye and microarray effects (i.e., Lowess normalization and microarray centering) and used for statistical analysis. Data were analyzed using the Proc MIXED procedure of SAS (SAS, SAS Inst. Inc., Cary, NC). Fixed effects were treatment (LPS challenge, control (no infsuion)) and dye. Random effects included cow and microarray. Raw P values were adjusted using Benjamini and HochbergM-bM-^@M-^Ys false discovery rate (FDR). Differences in relative expression due to treatment were considered significant at an FDR-adjusted P < 0.05. For a more stringent characterization between the two treatments, a 1.5-fold difference in mRNA expression was set as threshold among differentially expressed genes.

Project description:The objective was to evaluate the effect of nutrient restriction and intramammary lipopolysaccharide (LPS) challenge on mammary gland (MG) gene expression in early lactation cows. At 24 ± 3 d in milk, multiparous cows were either allowed to continue ad libitum intake of a lactation diet (CON, n = 6), or the ration was diluted with barley straw (48% DM) for 4 d (RES, n = 6). On d 3, one healthy rear mammary quarter was infused with 50 µg of LPS. Mammary biopsies were performed 24 h after LPS challenge. RNA and proteins analyzed using bovine 44K microarrays (Agilent Technologies) and micro-LC-MS/MS, respectively. Transcriptomic data were analyzed using GeneSpring (moderated-t-test with Westfall-Young correction, P < 0.05). Proteins were analyzed with Progenesis LC-MS software v4.1 (Nonlinear Dynamics). Production and energy balance did not differ prior to diet change. Negative energy balance was aggravated in RES (41 vs 97 ± 15 % of requirements, mean ± SD; P < 0.001). A total of 87 differentially expressed genes (DEG) were highlighted through the comparison of RES vs CON group. Among the 33 DEG identified in the transcriptomic analyses, 11 and 22 were down- and upregulated by restriction, respectively. Among the upregulated DEG, there were PDK4 and CPT1A which are involved in the regulation of fatty acid, ketone, and glucose metabolism. CPT1A is the key enzyme in the carnitine dependent fatty acid transport, promoting fatty acid oxidation. Genes involved in immune response such as PGLYRP3 and TRIB2 were upregulated, suggesting a higher inflammatory response in RES than CON. Proteomic analysis identified 54 proteins with 14 up- and 40 downregulated in RES cows. Upregulated proteins were mostly involved in gene expression mechanisms such as translation, RNA splicing and cellular protein modification. The downregulated proteins (e.g., EIF3H, RS27A, RS15) take part in protein metabolism. This is coherent with transcriptomic results, namely the downregulation of RPL 37A, a component of ribosomal complex, which catalyzes protein synthesis and may partially explain the lower milk protein yield in RES (834 vs 1163 g/d; P = 0.02). Proteins involved in antigen processing and presentation were downregulated in RES compared to CON, suggesting an impaired ability to counteract inflammation in RES MG. Preliminary transcriptomics and proteomics analyses show that undernutrition may influence the MG response to inflammation at each level of gene expression.

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Acute mammary and liver transcriptome response after an intra-mammary E. coli lipopolysaccharide challenge in postpartal dairy cows

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