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Mammary Gland transcriptome modifications by nutrient restriction in early lactation Holstein cows challenged with intramammary lipopolysaccharide

ABSTRACT: 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.

ORGANISM(S): Bos taurus

PROVIDER: GSE114975 | GEO | 2019/03/27

REPOSITORIES: GEO

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Project description:The objective was to evaluate the effects of nutrient restriction on the liver transcriptomic response after 24 h challenge to intramammary lipopolysaccharide (LPS) in early lactation cows. At 24 ± 3 days of lactation, multiparous cows were fed either an ad libitum lactation diet (CON, n = 6), or a ration diluted with barley straw (48% DM) for 4 days (RES, n = 6). On day 3, one healthy rear mammary quarter from each cow was infused with 50 µg of LPS. Blood and liver biopsies were collected on day 4, corresponding to 24 h after LPS challenge. The liver transcriptome was analyzed using 44K bovine microarrays (Agilent Technologies). Blood and transcriptomic data were analyzed using SAS mixed models and GeneSpring (moderate t-test with Westfall-Young correction, P < 0.05), respectively, and data mining was performed using Panther and Pathway Studio software. The energy balance was no different before the diet change. By experimental design, energy intake was 41 and 97 ± 15 % of NEL requirements in RES and CON, respectively. Plasma NEFA and BHBA were higher, and glucose was lower in RES than in CON, which is consistent with a 4-day nutrient deficit in RES. Seventy seven differentially expressed genes (DEGs) between CON and RES were identified, with 29 genes downregulated and 48 upregulated in RES. Genes involved in fatty acid synthesis (ACAT2, FASN, SCD), lactate metabolism (LDHC), and cortisol binding (SERPINA6) were downregulated in RES, while those involved in fatty acid oxidation, detoxification, cholesterol synthesis, lipoprotein lipid secretion, and gluconeogenesis (ACADVL, CPT1A, CPT1B, ANGPTL4, CYP4A11, HMGCSA, APOA1, APOA4, GK, PC and PCK2) were upregulated in RES. Overall, DEGs were in agreement with the negative energy balance and plasma metabolite profile, and reflect a state of intense lipomobilization, glucose deficit and ketogenesis in RES. Preliminary results suggest that nutrient restriction did not alter hepatic expression of genes directly involved in immune function 24 h after an intramammary LPS challenge.

Project description:Purpouse: The aim was evaluated the impact of estradiol cypionate (ECP) supplementation at the onset of a proestrus [i.e. at progesterone (P4) device removal of the synchronization of ovulation protocol] on the global endometrial gene expression of suckled anestrous beef cows Methods: A total of 12 suckled cows presenting absence of corpus luteum detected by ultrasound received 2 mg of estradiol benzoate im and an intravaginal P4 device. Eight days later, P4 devices were removed, and the cows received 500 mg of sodium cloprostenol IM. Cows were blocked according to body condition score and the diameter of largest follicle (LF) at P4 device removal and were randomly assigned to receive an im treatment with 1mg of ECP (ECP, n=6) or not (CON, n=6) at the P4 device removal. All cows received 10μg of buserelin acetate 48 h after the P4 device removal and were timed artificially inseminated immediately after. Cows presenting a new corpus luteum formed 6 d after the GnRH treatment had an endometrial fragment collected by transcervical biopsy Results: The integrated analysis was performed using DAVID database. A total of 135 transcripts were differentially expressed between ECP and CON groups, of which 73 genes were up regulated by ECP suplmentation and 62 genes in the CON cows. Two pathways were overrepresented by the ECP-induced transcripts: pathways in cancer (n=5 genes) and small cell lung cancer (n=3 genes). On the other hand, ECP-inhibited transcripts indicated the enrichment of three pathways: Parkinson’s disease (n=3 genes), oxidative phosphorylation (n=3 genes) and Alzheimer’s disease (n=3 genes). More specifically, ECP-induced transcripts associated with pathways in cancer [gene symbol (fold change); respectively] were LAMC3 (1.55), PTCH1 (1.51), PTCH2 (1.52), PIK3R3 (1.22) and PIAS1 (1.18), whereas ECP-inhibited transcripts associated with oxidative phosphorylation were ATP5F1 (1.18), ATP5J (1.24) and NDUFB3 (1.37). Conclusion: The ECP supplementation at onset of the synchronized proestrous slightly alters the uterine transcriptome.

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:Segmental instillation of lipopolysaccharide (LPS) by bronchoscopy can be used as a model to safely induce transient airway inflammation in the human lung. The LPS challenge model enables investigation of cellular mechanisms involved in pulmonary inflammatory processes as well as pharmacodynamic analysis of investigational drugs for the treatment of respiratory diseases. Aim of this work was to describe the transcriptomic profile of the human segmental LPS challenge model with contextualization to major respiratory diseases. Pre-challenge bronchoalveolar lavage fluid (BAL) and biopsies were sampled from twenty-eight smoking, healthy subjects, followed by segmental LPS challenge and saline control challenge. Twenty-four hours post instillation, BAL and biopsies were collected from the challenged lung segments. Total RNA of cells from BAL and biopsy samples were sequenced for subsequent data analysis. Differential gene expression analysis resulted in 6316 upregulated differentially expressed genes (DEGs) and 241 downregulated DEG in BAL, but only one downregulated DEG in biopsy samples after LPS challenge compared to saline challenge. Upregulated DEG in BAL were related to molecular functions such as “Inflammatory response” or “antimicrobial humoral immune response mediated by antimicrobial peptide”, enriched biological processes such as “chemokine receptor activity”, and upregulated pro-inflammatory pathways such as “Wnt signaling pathway”, “Ras signaling pathway” or “JAK-STAT signaling pathway”. Furthermore, the segmental LPS challenge model resembled aspects of the five most prevalent respiratory diseases chronic obstructive pulmonary disease (COPD), asthma, pneumonia, tuberculosis and lung cancer and featured similarities with acute exacerbations in COPD (AECOPD) and community-acquired pneumonia (CAP). Overall, our study provides extensive information about the transcriptomic profile from BAL cells and mucosal biopsies following LPS challenge in healthy smokers. It expands the knowledge about the LPS challenge model providing potential overlap with respiratory diseases in general and infection-triggered respiratory insults such as AECOPD in particular.

Project description:Background. Steer progeny suckled by cows fed a dried distillers grains and solubles (DDGS) diet the first three months of lactation were heavier during feedlot finishing and had significantly lower marbling and larger longissumus muscles than steer suckled by cows fed a control diet (CON). These differences were profound in that progeny were managed and fed identically from weaning until finishing, and suggested that the developmental program of muscle composition was established during the suckling period. Purpose. Transcriptomes of longissimus muscle were measured using next generation sequencing to investigate whether there were any developmental clues to the differences in marbling scores and muscle content between steers suckled by DDGS (n=5) versus control (CON; n=5) diet fed cows during lactation. Methods. Multiparous Angus × Simmental cows with male progeny were fed one of two diets supplemented with either DDGS or soybean meal (CON), from calving until day 129 postpartum (PP). After conclusion of the treatments at 129 days postpartum, cow–calf pairs were comingled and managed as one group until weaning at 219 days postpartum. Steers were then transitioned to a common diet. Longissimus muscle (LM) biopsies were obtained from steers suckled by cows fed DDGS (n=5) and CON (n=5) 10 d prior to slaughter. RNA was extracted from LM. Total RNA isolated and Library Prep Kit was used to generate library for measuring messenger RNA molecules. Library was sequenced on an Illumina HiSeq2500 platform. FastQC (v 0.10.1) was used to assess sequence quality, and quality trimming was done using FASTX toolkit (v 0.0.13). Bases with less than Phred33 score of 30 were removed and resulted in reads of at least 50 bases. Reads were mapped against the bowtie2-indexed Bos taurus genome using Tophat (v 2.0.9) with default parameters, and the genome annotation file (.gtf) from Ensembl was used to associate the genome with annotated features. Raw read counts of each sample for each gene feature were generated with HTSeq (v 0.5.3p7) using Tophat output and Bos taurus annotations. Counts were merged using custom perl scripts and used for downstream differential gene expression (DE) analysis. DE pairwise analysis between samples CON and DDGS was done with ‘R’ (Version 2.15.2). Counts matrix, library sizes, and experimental design were combined to create an object using edgeR (v 3.0.3) package. Results. Number of reads per sample ranged from 79,530,352 to 58,600,824. After trimming there were 77,569,706 to 57,307,614 per sample. The percent of reads that mapped to bovine genome per sample ranged from 77.3 to 92.3%, and averaged 86.3% across all samples. Number of reads per gene ranged from one to over two-hundred ninety thousand counts per million (CPM) reads. There were 809 genes differentially expressed (P-adj<0.1) between CON and DDGS muscle. Of these 637 were upregulated and 172 downregulated in DDGS relative to CON. Overall the DDGS versus CON muscle transcriptomic signature was pro-myogenic and anti-adipogenic. In particular, myokines/satellite cell maintenance factors were found among upregulated (LIF, CNTF, FGFB1, EPHB1) genes. The anti-adipogenic signature was typified by the upregulation of anti-inflammatory cytokines and receptors (IL1RAP, IL1RL2, IL13RA2, IL1F10,), and downregulation of expression of inflammation/inflammatory cytokines and receptor (TNF, IL6R CXCL9), which suggests a selection of differentiation pathways away from adipogenic line. While the upregulation of TGFB, SPP1 and INHBA supports selection of fibroblast lineage of cells. Conclusion. The lactation phase of production can effect meat quality by affecting transcriptional signatures that favor myogenesis and depress inflammation.

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Mammary Gland transcriptome modifications by nutrient restriction in early lactation Holstein cows challenged with intramammary lipopolysaccharide

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