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Cross-talk between liver and mammary tissue after experimental Escherichia coli mastitis in Holstein dairy cows using RNAseq
Abstract
Abstract Text: Using RNAseq combined with bioinformatics tools, our objective was to identify cross-talk between liver and mammary tissue and key pathways altered during intramammary (IMI) challenge with Escherichia coli (E. coli). Six cows were inoculated with ~20-40 CFU of live E. coli into one mammary quarter at ~4-6 weeks in lactation. Biopsies were performed at -144 and 24 hours relative to challenge in liver and at 24h in both rear quarters (i.e. infected and non-infected) of the mammary gland. Each sample was sequenced using a 100 bp paired-end approach. Sequence reads were aligned to the Bovine genome and the number of reads that mapped to each of the 24,616 Ensembl genes was determined. A generalized linear model was fitted for the read count of each gene and differential expression was assessed using a likelihood ratio test statistic after adjustment for multiple testing (FDR). Ingenuity Pathway Analysis coupled with the Dynamic Impact Approach analysis of differentially expressed genes (overall time effect FDR≤0.05, post-hoc P≤0.05) indicated that IMI induced a large biological response in the liver and mammary tissue with a strong inhibition of metabolism, especially related to lipid, glucose, and xenobiotic metabolism, in the former and induction of inflammatory response/immune cells activation in the latter. Analysis of upstream regulators indicated a prominent role of several cytokines, growth factors, and transcription regulators in the two tissues’ transcriptomics adaptation to IMI, clearly lipid-related and inhibited in the liver and inflammatory-related and activated in mammary tissue. The analysis uncovered a substantial cross-talk between the two tissues during IMI with a communication almost unidirectional (i.e., from mammary to the liver) via the induction of the hepatic proliferation, regeneration, and inflammatory response due to a large number of cytokines with an increased expression in the mammary gland and able to interact with highly induced hepatic receptors. The analysis indicated that only 3 proteins (SPP1, EPO, and GRP) with an increased hepatic expression due to IMI could potentially interact with receptors involved in leukocytes differentiation/proliferation with an increased expression due to IMI in mammary tissue. The larger enrichment of immune cell-related functions in the data from the mammary tissue suggests increased recruitment of active immune-cells to the mammary tissue. The analysis uncovered a large communication from the mammary to the liver to coordinate the inflammatory response with very few factors potentially released by the liver to control mammary gland response.
Keywords: Dynamic Impact Approach, Liver, Mastitis, RNAseq
... Wang, Chinese Academy of Agricultural Science (CAAS), Beijing, China, personal communication). Using a similar approach, a large increase of information from mammary tissue to the liver during mastitis was uncovered, likely to control liver metabolism and inflammatory response (Bionaz et al., 2014). Bionaz and Loor (2012), assembling microarray data encompassing pregnancy through early lactation in studies of liver, mammary, and adipose tissue form Holstein dairy cows, uncovered and provided a visualization of the coordinated dynamic metabolic and signaling adaptations among the 3 tissues. ...
The advent of high-throughput technologies to study an animal's genome, proteome, and metabolome (i.e., "omics" tools) constituted a setback to the use of reductionism in livestock research. More recent development of "next-generation sequencing" tools was instrumental in allowing in-depth studies of the microbiome in the rumen and other sections of the gastrointestinal tract. Omics, along with bioinformatics, constitutes the foundation of modern systems biology, a field of study widely used in model organisms (e.g., rodents, yeast, humans) to enhance understanding of the complex biological interactions occurring within cells and tissues at the gene, protein, and metabolite level. Application of systems biology concepts is ideal for the study of interactions between nutrition and physiological state with tissue and cell metabolism and function during key life stages of livestock species, including the transition from pregnancy to lactation, in utero development, or postnatal growth. Modern bioinformatic tools capable of discerning functional outcomes and biologically meaningful networks complement the ever-increasing ability to generate large molecular, microbial, and metabolite data sets. Simultaneous visualization of the complex intertissue adaptations to physiological state and nutrition can now be discerned. Studies to understand the linkages between the microbiome and the absorptive epithelium using the integrative approach are emerging. We present examples of new knowledge generated through the application of functional analyses of transcriptomic, proteomic, and metabolomic data sets encompassing nutritional management of dairy cows, pigs, and poultry. Published work to date underscores that the integrative approach across and within tissues may prove useful for fine-tuning nutritional management of livestock. An important goal during this process is to uncover key molecular players involved in the organismal adaptations to nutrition.
... Wang, Chinese Academy of Agricultural Science (CAAS), Beijing, China, personal communication). Using a similar approach, a large increase of information from mammary tissue to the liver during mastitis was uncovered, likely to control liver metabolism and inflammatory response (Bionaz et al., 2014). , assembling microarray data encompassing pregnancy through early lactation in studies of liver, mammary, and adipose tissue form Holstein dairy cows, uncovered and provided a visualization of the coordinated dynamic metabolic and signaling adaptations among the 3 tissues. ...
The advent of high-throughput technologies to study an animal’s genome, proteome, and metabolome (i.e., ‘omics’ tools) constituted a setback to the use of reductionism in livestock research. More recent development of ‘next-generation sequencing’ tools was instrumental in allowing in-depth studies of the microbiome in the rumen and other sections of the gastrointestinal tract. Omics, along with bioinformatics, constitutes the foundation of modern systems biology, a field of study widely-used in model organisms (e.g., rodents, yeast, humans) to enhance understanding of the complex biological interactions occurring within cells and tissues at the gene, protein, and metabolite level. Application of systems biology concepts is ideal for the study of interactions between nutrition and physiological state with tissue and cell metabolism and function during key life stages of livestock species, including the transition from pregnancy to lactation, in utero development, or post-natal growth. Modern bioinformatic tools capable of discerning functional outcomes and biologically-meaningful networks complement the ever increasing ability to generate large molecular, microbial, and metabolite datasets. Simultaneous visualization of the complex inter-tissue adaptations to physiological state and nutrition can now be discerned. Studies to understand the linkages between the microbiome and the absorptive epithelium using the integrative approach are emerging. We present examples of new knowledge generated through the application of functional analyses of transcriptomic, proteomic, and metabolomic datasets encompassing nutritional management of dairy cows, pigs, and poultry. Published work to date underscores that the integrative approach across and within tissues may prove useful for fine-tuning nutritional management of livestock. An important goal during this process is to uncover key molecular players involved in the organismal adaptations to nutrition.
The metabolic response of the liver during periods of inflammation is poorly understood. The objective of this study was to characterize the effects of nutrient supply and lipopolysaccharide (LPS) challenge on hepatic intermediate metabolism of early- and mid-lactation cows by employing gas chromatography-mass spectrometry with stable isotope tracer. Twelve multiparous Holstein-Friesian cows in early (n = 6; 12 ± 4.2 d in milk) and mid (n = 6; 115 ± 13.5 d in milk) lactation were used for this study. Liver biopsies were performed on all cows. Liver slices (40-60 mg) were incubated in a 37°C water bath for 2 h with either control (phosphate buffered saline), pyruvate (PYR; 1 mM unlabeled pyruvate and 1 mM [(13)C3]pyruvate), pyruvate + propionate (PYR+PRO; 1 mM unlabeled pyruvate, 1 mM [(13)C3]pyruvate, and 2 mM sodium propionate), or pyruvate + AA (PYR+AA; 1 mM unlabeled pyruvate, 1 mM [(13)C3]pyruvate, and 2 mM AA solution), and LPS (0.0 or 0.2 μg/mL) was added to flasks per treatment. Enrichment of isotopomers in metabolic equilibrium with Krebs cycle intermediates was assessed. Pyruvate fluxes and the enzymatic activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) and phosphoenol pyruvate carboxykinase (PEPCK) were calculated. Media were analyzed for concentrations of tumor necrosis factor-α (TNF-α), glucose, and haptoglobin. Data were analyzed as randomized block (stage of lactation) design in a factorial arrangement of nutrient treatments by LPS dose. Challenge with LPS increased the mRNA abundance of TNF-α, haptoglobin, and serum amyloid A 2, and the concentration of TNF-α in media. Challenge with LPS increased mRNA abundance of PC but reduced the enrichment of (13)C1[M1] and (13)C2[M2]alanine and tended to reduce the enzymatic activity of PEPCK. Incubation with PYR+PRO and PYR+AA increased the flux of pyruvate to acetyl CoA. However, only PYR+PRO increased the enzymatic activity of PEPCK and PDH versus PC and decreased the mRNA abundance of PC. Cows in early lactation tended to receive a greater contribution of pyruvate to the oxaloacetate flux via the lower PDH versus PC activity and a higher mRNA abundance of PC than cows in mid lactation. Our results suggest that regardless of stage of lactation and nutrient supplement, hepatic gluconeogenesis was impaired during inflammation. Further research examining how various nutrients support liver function and improve the immunometabolic response of liver during inflammation is warranted.
Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Obesity is a metabolic state associated with excess of positive energy balance. While adipose tissues are considered the major contributor for complications associated with obesity, they influence a variety of tissues and inflict significant metabolic and inflammatory alterations. Unfortunately, the communication network between different cell-types responsible for such systemic alterations has been largely unexplored. Here we study the inter-tissue crosstalk during progression and cure of obesity using multi-tissue gene expression data generated through microarray analysis. We used gene expression data sets from 10 different tissues from mice fed on high-fat-high-sugar diet (HFHSD) at various stages of disease development and applied a novel analysis algorithm to deduce the tissue crosstalk. We unravel a comprehensive network of inter-tissue crosstalk that emerges during progression of obesity leading to inflammation and insulin resistance. Many of the crosstalk involved interactions between well-known modulators of obesity and associated pathology like inflammation. We then used similar datasets from mice that in addition to HFHSD were also administered with a herbal concoction known to circumvent the effects of HFHSD in the diet induced model of obesity in mice. We propose, the analysis presented here could be applied to understand systemic details of several chronic diseases.
Background
The objective of this study was to characterize the changes in various metabolic parameters in blood and milk during IMI challenge with Escherichia coli (E. coli) for dairy cows during early lactation. Thirty, healthy primiparous Holstein cows were infused (h = 0) with ~20-40 cfu of live E. coli into one front mammary quarter at ~4-6 wk in lactation. Daily feed intake and milk yield were recorded. At –12, 0, 3, 6, 12, 18, 24, 36, 48, 60, 72, 96, 108, 120, 132, 144, 156, 168, 180 and 192 h relative to challenge rectal temperatures were recorded and quarter foremilk was collected for analysis of shedding of E. coli. Composite milk samples were collected at -180, -132, -84, -36, -12, 12, 24, 36, 48, 60, 72, 84, 96, 132 and 180 h relative to challenge (h = 0) and analyzed for lactate dehydrogenase (LDH), somatic cell count, fat, protein, lactose, citrate, beta-hydroxybutyrate (BHBA), free glucose (fglu), and glucose-6-phosphate (G6P). Blood was collected at -12, 0, 3, 6, 12, 18, 24, 36, 60, 72, 84, 132 and 180 h relative to challenge and analyzed for plasma non-esterified fatty acids (NEFA), BHBA and glucose concentration. A generalized linear mixed model was used to determine the effect of IMI challenge on metabolic responses of cows during early lactation.
Results
By 12 h, E. coli was recovered from challenged quarters and shedding continued through 72 h. Rectal temperature peaked by 12 h post-challenge and returned to pre-challenge values by 36 h post-IMI challenge. Daily feed intake and milk yield decreased (P <0.05) by 1 and 2 d, respectively, after mastitis challenge. Plasma BHBA decreased (12 h; P <0.05) from 0.96 ± 1.1 at 0 h to 0.57 ± 0.64 mmol/L by 18 h whereas concentration of plasma NEFA (18 h) and glucose (24 h) were significantly greater, 11 and 27%, respectively, after challenge. In milk, fglu, lactose, citrate, fat and protein yield were lower whereas yield of BHBA and G6P were higher after challenge when compared to pre-challenge values.
Conclusions
Changes in metabolites in blood and milk were most likely associated with drops in feed intake and milk yield. However, the early rise in plasma NEFA may also signify enhanced adipose tissue lipolysis. Lower concentrations of plasma BHBA may be attributed to an increase transfer into milk after IMI. Decreases in both milk lactose yield and % after challenge may be partly attributed to reduced conversion of fglu to lactose. Rises in G6P yield and concentration in milk after challenge (24 h) may signify increased conversion of fglu to G6P. Results identify changes in various metabolic parameters in blood and milk after IMI challenge with E. coli in dairy cows that may partly explain the partitioning of nutrients and changes in milk components after IMI for cows during early lactation.
Aim:
To investigate over-expression of Osteopontin (OPN) pathway expression and mechanisms of action in human alcoholic liver disease (ALD), in vivo and in vitro acute alcohol models.
Methods:
OPN pathway was evaluated in livers from patients with progressive stages of human ALD and serum from drinkers with and without liver cirrhosis. In vitro stellate LX2 cells exposed to acute alcohol and in vivo in acute alcoholic steatosis mouse models were also investigated for OPN pathway expression and function. WT and OPN(-/-) mice were administered an acute dose of alcohol and extent of liver injury was examined by histopathology and liver biochemistry after 16-24 h. The causative role of OPN was studied in OPN knockout animals and in vitro in stellate LX2 cells, utilizing siRNA, aptamer and neutralizing antibodies to block OPN and OPN pathway. OPN pathway expression and downstream functional consequences were measured for signaling by Western blotting, plasmin activation by spectrophotometric assays and cell migration by confocal imaging and quantitation.
Results:
OPN expression positively correlated with disease severity in patients with progressive stages of ALD. In vivo, associated with alcoholic steatosis, a single dose of acute alcohol significantly increased hepatic OPN mRNA and protein, and a cleaved OPN form in a dose dependent manner. OPN mRNA and secreted OPN also increased in parallel with activation of LX2 stellate cells within 4 h of a single dose of alcohol. Expression of OPN receptors, αvβ3-integrin and CD44, increased in human ALD, and in vivo and in vitro with alcohol administration. This was accompanied by downstream phosphorylation of Akt and Erk, increased mRNA expression of several fibrogenesis, fibrinolysis and extracellular matrix pathway genes, plasmin activation and hepatic stellate cell (HSC) migration. Inhibition of OPN and OPN-receptor mediated signaling partially inhibited alcohol-induced HSC activation, plasmin activity and cell migration.
Conclusion:
OPN is a key mediator of the alcohol-induced effects on hepatic stellate cell functions and liver fibrogenesis.
The objective of this study was to investigate the relationship between activation of acute phase response and fatty liver in transition dairy cows. Fatty liver was induced in dairy cows by feeding 8 kg of cracked corn 1 mo before the expected day of parturition. Liver and blood samples were obtained at days -4, 3, 8, 12, 14, 22, 27, and 36 postpartum. Cows that developed fatty liver (n = 4) reached peak total lipids in the liver at day 12 postpartum with 11.4% (wet wt.) compared with 6.6% in control cows (n = 4). Cows with fatty liver had greater plasma tumor necrosis factor-alpha (TNF-α), nonesterified fatty acids (NEFA), and lower lactate concentrations than did control cows at day -4. During highest concentrations of total lipids in the liver, for at least one time-point, fatty-liver cows had greater concentrations of plasma serum amyloid A (SAA), haptoglobin, and NEFA and lower concentrations of calcitonin gene-related peptide (CGRP), prostaglandin E2 (PGE 2), cortisol, and TNF-α than did control cows. Concentrations of total lipids in the liver at 12 d postpartum were correlated (a) positively with plasma TNF-α, SAA, and NEFA and negatively with plasma CGRP before calving, (b) positively with plasma SAA, haptoglobin, and NEFA and negatively with plasma PGE2, CGRP, total cholesterol, and glucose at days 3, 8, and 12 postpartum, and (c) negatively with concentrations of plasma glucose, lactate, and total bilirubin after day 12 postpartum. In conclusion, this study indicates that the acute phase response occurs in cows with fatty liver as well as strong relationships between mediators of immune response and fatty liver.
Since its first description more than 20 years ago osteopontin has emerged as an active player in many physiological and pathological processes, including biomineralization, tissue remodeling and inflammation. As an extracellular matrix protein and proinflammatory cytokine osteopontin is thought to facilitate the recruitment of monocytes/macrophages and to mediate cytokine secretion in leukocytes. Modulation of immune cell response by osteopontin has been associated with various inflammatory diseases and may play a pivotal role in the development of adipose tissue inflammation and insulin resistance. Here we summarize recent findings on the role of osteopontin in metabolic disorders, particularly focusing on diabetes and obesity.
Mastitis is a serious production-limiting disease, with effects on milk yield, milk quality, and conception rate, and an increase in the risk of mortality and culling. The objective of this study was 2-fold: (1) to develop an economic optimization model that incorporates all the different types of pathogens that cause clinical mastitis (CM) categorized into 8 classes of culture results, and account for whether the CM was a first, second, or third case in the current lactation and whether the cow had a previous case or cases of CM in the preceding lactation; and (2) to develop this decision model to be versatile enough to add additional pathogens, diseases, or other cow characteristics as more information becomes available without significant alterations to the basic structure of the model. The model provides economically optimal decisions depending on the individual characteristics of the cow and the specific pathogen causing CM. The net returns for the basic herd scenario (with all CM included) were 216.11. The CM cases comprised (incidences, %) Staphylococcus spp. (1.6), Staphylococcus aureus (1.8), Streptococcus spp. (6.9), Escherichia coli (8.1), Klebsiella spp. (2.2), other treated cases (e.g., Pseudomonas; 1.1), other not treated cases (e.g., Trueperella pyogenes; 1.2), and negative culture cases (12.7). The average cost per case, even under optimal decisions, was greatest for Klebsiella spp. (361), other treated cases (280). This was followed by the gram-positive pathogens; among these, the greatest cost per case was due to Staph. aureus (174) and Staphylococcus spp. (115). The model recommended treatment for most CM cases (>85%); the range was 86.2% (Klebsiella spp.) to 98.5% (Staphylococcus spp.). In general, the optimal recommended time for replacement was up to 5 mo earlier for cows with CM compared with cows without CM. Furthermore, although the parameter estimates implemented in this model are applicable to the dairy farms in this study, the parameters may be altered to be specific to other dairy farms. Cow rankings and values based on disease status, pregnancy status, and milk production can be extracted; these provide guidance when determining which cows to keep or cull.
Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting posttranslational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed.
While infection-induced placental inflammation is a common mechanism of adverse pregnancy outcome, some pathogens can also trigger placental apoptosis, and Toll-like receptors (TLRs) mediate this response. Treatment of human first trimester trophoblast cells with bacterial peptidoglycan (PDG) reduces their constitutive secretion of IL-6 protein and induces apoptosis. This apoptotic response is dependent upon the cell's expression of TLR1, TLR2 and TLR10, and their lack of TLR6, such that ectopic expression of TLR6 prevents PDG-induced apoptosis and restores IL-6 production. In this current study we have identified three microRNAs (miRs) that regulate TLR2-mediated responses in the human trophoblast. Herein we report that miR-329 plays a pivotal role in mediating PDG-induced trophoblast apoptosis and inhibition of IL-6 mRNA expression by targeting the NF-κB subunit, p65. TLR2 activation by PDG upregulates miR-329 expression and inhibits NF-κB p65 and IL-6 mRNA, and this is reversed by the presence of TLR6. Moreover, inhibition of miR-329 prevents PDG-induced inhibition of NF-κB p65 and IL-6 mRNA expression, and restores cell survival. In addition, we have found miR-23a and let-7c to directly regulate PDG-mediated inhibition of IL-6 mRNA. TLR2 activation by PDG upregulates miR23a and let-7c expression and this is reversed by the presence of TLR6. Furthermore, inhibition of both miR23a and let-7c prevents PDG-inhibition of trophoblast IL-6 mRNA expression. Together, our findings suggest that multiple miRs are involved in the molecular regulation of TLR2-mediated responses in the trophoblast towards gram-positive bacterial components.
We describe Bioconductor infrastructure for representing and computing on annotated genomic ranges and integrating genomic data with the statistical computing features of R and its extensions. At the core of the infrastructure are three packages: IRanges, GenomicRanges, and GenomicFeatures. These packages provide scalable data structures for representing annotated ranges on the genome, with special support for transcript structures, read alignments and coverage vectors. Computational facilities include efficient algorithms for overlap and nearest neighbor detection, coverage calculation and other range operations. This infrastructure directly supports more than 80 other Bioconductor packages, including those for sequence analysis, differential expression analysis and visualization.
A minimally invasive biopsy technique was evaluated for udder tissue collection in dairy cows with Escherichia coli mastitis. Meanwhile, the effect of taking repeated liver and udder biopsies on the systemic and local acute phase response (APR) of the dairy cows was investigated during the disease. The cows were divided into a biopsy group (B) (n = 16) and a no-biopsy group (NB) (n = 16) and were sampled in the acute disease stage and in the recovery stage. The cows' pre-disease period served as a control period for establishing baseline values for the investigated parameters. A total of 32 Holstein-Friesian cows were inoculated with 20 to 40 colony-forming units (cfu) of E. coli in one front quarter at 0 hour. Liver biopsies were collected at -144, 12, 24 and 192 h, and udder biopsies were collected at 24 and 192 h post E. coli inoculation (PI) using a minimally invasive biopsy technique. Effects of combined biopsying were investigated by recording production traits, clinical response, and measuring inflammatory milk and blood parameters: E. coli, somatic cell count, milk amyloid A (MAA) levels, white blood cell count, polymorphonuclear neutrophilic leukocyte numbers and serum amyloid A levels at several time points. E. coli inoculation changed all production parameters and the clinical and inflammatory response in all cows except one that was not infected. Combined biopsying had no constant or transient effect on the daily feed intake, the clinical responsiveness or the blood parameters, but affected the daily milk yield and some milk parameters transiently, that is, the presence of blood in milk, increased E. coli counts and MAA levels during the acute disease stage. Combined biopsying had no effect on the parameters in the recovery stage apart from the presence of blood in the milk. In conclusion, although, a minimally invasive biopsy technique was used, tissue damages could not be avoided when biopsying and they transiently affected the inflammatory parameters in the mammary gland. Nevertheless, we believe combined biopsying of liver and udder is as an acceptable approach to study the systemic and local APR in dairy cows during E. coli mastitis, if the timing of biopsying and other types of sampling is planned accordingly.
Characterization and biological roles of the peroxisome proliferator-activated receptor (PPAR) isotypes are well known in monogastrics, but not in ruminants. However, a wealth of information has accumulated in little more than a decade on ruminant PPARs including isotype tissue distribution, response to synthetic and natural agonists, gene targets, and factors affecting their expression. Functional characterization demonstrated that, as in monogastrics, the PPAR isotypes control expression of genes involved in lipid metabolism, anti-inflammatory response, development, and growth. Contrary to mouse, however, the PPARγ gene network appears to controls milk fat synthesis in lactating ruminants. As in monogastrics, PPAR isotypes in ruminants are activated by long-chain fatty acids, therefore, making them ideal candidates for fine-tuning metabolism in this species via nutrients. In this regard, using information accumulated in ruminants and monogastrics, we propose a model of PPAR isotype-driven biological functions encompassing key tissues during the peripartal period in dairy cattle.
Read alignment is an ongoing challenge for the analysis of data from sequencing technologies. This article proposes an elegantly
simple multi-seed strategy, called seed-and-vote, for mapping reads to a reference genome. The new strategy chooses the mapped
genomic location for the read directly from the seeds. It uses a relatively large number of short seeds (called subreads)
extracted from each read and allows all the seeds to vote on the optimal location. When the read length is <160 bp, overlapping
subreads are used. More conventional alignment algorithms are then used to fill in detailed mismatch and indel information
between the subreads that make up the winning voting block. The strategy is fast because the overall genomic location has
already been chosen before the detailed alignment is done. It is sensitive because no individual subread is required to map
exactly, nor are individual subreads constrained to map close by other subreads. It is accurate because the final location
must be supported by several different subreads. The strategy extends easily to find exon junctions, by locating reads that
contain sets of subreads mapping to different exons of the same gene. It scales up efficiently for longer reads.
The large increase in milk yield and the structural changes in the dairy industry have caused major changes in the housing, feeding and management of the dairy cow. However, while large improvements have occurred in production and efficiency, the disease incidence, based on veterinary records, does not seem to be improved. Earlier reviews have covered critical periods such as the transition period in the cow and its influence on health and immune function, the interplay between the endocrine system and the immune system and nutrition and immune function. Knowledge on these topics is crucial for our understanding of disease risk and our effort to develop health and welfare improving strategies, including proactive management for preventing diseases and reducing the severity of diseases. To build onto this the main purpose of this review will therefore be on the effect of physiological imbalance (PI) on immune function, and to give perspectives for prevention of diseases in the dairy cow through nutrition. To a large extent, the health problems during the periparturient period relate to cows having difficulty in adapting to the nutrient needs for lactation. This may result in PI, a situation where the regulatory mechanisms are insufficient for the animals to function optimally leading to a high risk of a complex of digestive, metabolic and infectious problems. The risk of infectious diseases will be increased if the immune competence is reduced. Nutrition plays a pivotal role in the immune response and the effect of nutrition may be directly through nutrients or indirectly by metabolites, for example, in situations with PI. This review discusses the complex relationships between metabolic status and immune function and how these complex interactions increase the risk of disease during early lactation. A special focus will be placed on the major energetic fuels currently known to be used by immune cells (i.e. glucose, non-esterified fatty acids, beta-hydroxybutyrate and glutamine) and how certain metabolic states, such as degree of negative energy balance and risk of PI, contribute to immunosuppression during the periparturient period. Finally, we will address some issues on disease prevention through nutrition.
Physiological imbalance (PI) is a situation in which physiological parameters deviate from the normal, and cows consequently have an increased risk of developing production diseases and reduced production or reproduction. Our objectives were to (1) determine the effect of stage of lactation and milk yield on metabolic and production responses of cows during a nutrient restriction period to experimentally increase PI; (2) identify major metabolites that relate to degree of PI; and (3) identify potential biomarkers in milk for on-farm detection of PI throughout lactation. Forty-seven Holstein cows in early [n=14; 49±22 d in milk (DIM); parity=1.6±0.5], mid (n=15; 159±39 DIM; parity=1.5±0.5), and late (n=18; 273±3 DIM; parity=1.3±0.5) lactation were used. Prior to restriction, all cows were fed the same total mixed ration ad libitum. All cows were then nutrient restricted for 4 d by supplementing the ration with 60% wheat straw to induce PI. After restriction, cows returned to full feed. Daily milk yield was recorded and composite milk samples were analyzed for fat, protein, lactose, citrate, somatic cells, uric acid, alkaline phosphatase, β-hydroxybutyrate (BHBA), and milk urea nitrogen. Blood was collected daily and analyzed for metabolites: nonesterified fatty acids (NEFA), BHBA, glucose, plasma urea nitrogen, and insulin. The revised quantitative insulin sensitivity check index (RQUICKI) was calculated for each cow. Liver biopsies collected before and during restriction were analyzed for triglycerides, glycogen, phospholipids, glucose, and total lipid content. A generalized linear mixed model was used to determine the effect of stage of lactation on responses during restriction. Regression analyses were used to examine the effect of pre-restriction levels on changes during restriction. Similar decreases in milk yield among groups indicate that the capacity of individual responses is dependent on milk yield but the coping strategies used are dependent on stage of lactation. Milk yield was a better predictor of feed intake than DIM. Plasma glucose decreased for all cows, and cows in early lactation had increased plasma BHBA, whereas cows in later lactation had increased NEFA during restriction. Milk citrate had the greatest increase (58%) during restriction for all cows. Results reported here identified metabolites (i.e., glucose, NEFA, BHBA, cholesterol) as predictors of PI and identified milk citrate as a promising biomarker for PI on farm.
The mammalian liver works to keep the body in a state of homeostasis and plays an important role in systemic acute phase response to infections. In this study we investigated the bovine hepatic acute phase response at the gene transcription level in dairy cows with experimentally Escherichia coli-induced mastitis. At time = 0, each of 16 periparturient dairy cows received 20-40 colony-forming units of live E. coli in one front quarter of the udder. A time series of liver biopsies was collected at -144, 12, 24, and 192 h relative to time of inoculation. Changes in transcription levels in response to E. coli inoculation were analyzed using the Bovine Genome Array and tested significant for 408 transcripts over the time series [adjusted p ≤ 0.05, abs(fold-change) > 2]. After 2-D clustering, transcripts represented three distinct transcription profiles: 1) regulation of gene transcription and apoptosis, 2) responses to cellular stress invoked by reactive metabolites, and 3) metabolism and turnover of proteins. The results showed that the liver went through a period of perturbations to its normal homeostatic condition during the first 24 h following the E. coli-induced intra-mammary inflammation. In previous studies, bacterial lipopolysaccharide, LPS, was used for intramammary stimulation to mimic E. coli infection. Comparing responses to LPS and E. coli, induced biochemical processes were similar but not identical (94 and 85% similarity between corresponding samples at early and late acute phase, respectively), but their kinetics were not. A notable difference concerned transcription of factors associated with oxidative stress in E. coli-induced liver responses.
The common approach to the multiplicity problem calls for controlling the familywise error rate (FWER). This approach, though, has faults, and we point out a few. A different approach to problems of multiple significance testing is presented. It calls for controlling the expected proportion of falsely rejected hypotheses – the false discovery rate. This error rate is equivalent to the FWER when all hypotheses are true but is smaller otherwise. Therefore, in problems where the control of the false discovery rate rather than that of the FWER is desired, there is potential for a gain in power. A simple sequential Bonferroni-type procedure is proved to control the false discovery rate for independent test statistics, and a simulation study shows that the gain in power is substantial. The use of the new procedure and the appropriateness of the criterion are illustrated with examples.
The overrepresented approach (ORA) is the most widely-accepted method for functional analysis of microarray datasets. The ORA is computationally-efficient and robust; however, it suffers from the inability of comparing results from multiple gene lists particularly with time-course experiments or those involving multiple treatments. To overcome such limitation a novel method termed Dynamic Impact Approach (DIA) is proposed. The DIA provides an estimate of the biological impact of the experimental conditions and the direction of the impact. The impact is obtained by combining the proportion of differentially expressed genes (DEG) with the log2 mean fold change and mean -log P-value of genes associated with the biological term. The direction of the impact is calculated as the difference of the impact of up-regulated DEG and down-regulated DEG associated with the biological term. The DIA was validated using microarray data from a time-course experiment of bovine mammary gland across the lactation cycle. Several annotation databases were analyzed with DIA and compared to the same analysis performed by the ORA. The DIA highlighted that during lactation both BTA6 and BTA14 were the most impacted chromosomes; among Uniprot tissues those related with lactating mammary gland were the most positively-impacted; within KEGG pathways 'Galactose metabolism' and several metabolism categories related to lipid synthesis were among the most impacted and induced; within Gene Ontology "lactose biosynthesis" among Biological processes and "Lactose synthase activity" and "Stearoyl-CoA 9-desaturase activity" among Molecular processes were the most impacted and induced. With the exception of the terms 'Milk', 'Milk protein' and 'Mammary gland' among Uniprot tissues and SP_PIR_Keyword, the use of ORA failed to capture as significantly-enriched (i.e., biologically relevant) any term known to be associated with lactating mammary gland. Results indicate the DIA is a biologically-sound approach for analysis of time-course experiments. This tool represents an alternative to ORA for functional analysis.
Cells are constantly exposed to a large variety of lipids. Traditionally, these molecules were thought to serve as simple energy storing molecules. More recently it has been realized that they can also initiate and regulate signaling events that will decisively influence development, cellular differentiation, metabolism and related functions through the regulation of gene expression. Multicellular organisms dedicate a large family of nuclear receptors to these tasks. These proteins combine the defining features of both transcription factors and receptor molecules, and therefore have the unique ability of being able to bind lipid signaling molecules and transduce the appropriate signals derived from lipid environment to the level of gene expression. Intriguingly, the members of a subfamily of the nuclear receptors, the peroxisome proliferator-activated receptors (PPARs) are able to sense and interpret fatty acid signals derived from dietary lipids, pathogenic lipoproteins or essential fatty acid metabolites. Not surprisingly, Peroxisome proliferator-activated receptors were found to be key regulators of lipid and carbohydrate metabolism. Unexpectedly, later studies revealed that Peroxisome proliferator-activated receptors are also able to modulate inflammatory responses. Here we summarize our understanding on how these transcription factors/receptors connect lipid metabolism to inflammation and some of the novel regulatory mechanisms by which they contribute to homeostasis and certain pathological conditions. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.
Bovine mastitis is one of the most costly and prevalent diseases affecting dairy cows worldwide. In order to develop new strategies to prevent Escherichia coli-induced mastitis, a detailed understanding of the molecular mechanisms underlying the host immune response to an E. coli infection is necessary. To this end, we performed a global gene-expression analysis of mammary gland tissue collected from dairy cows that had been exposed to a controlled E. coli infection. Biopsy samples of healthy and infected utter tissue were collected at T = 24 h post-infection (p.i.) and at T = 192 h p.i. to represent the acute phase response (APR) and chronic stage, respectively. Differentially expressed (DE) genes for each stage were analyzed and the DE genes detected at T = 24 h were also compared to data collected from two previous E. coli mastitis studies that were carried out on post mortem tissue.
Nine-hundred-eighty-two transcripts were found to be differentially expressed in infected tissue at T = 24 (P < 0.05). Up-regulated transcripts (699) were largely associated with immune response functions, while the down-regulated transcripts (229) were principally involved in fat metabolism. At T = 192 h, all of the up-regulated transcripts were associated with tissue healing processes. Comparison of T = 24 h DE genes detected in the three E. coli mastitis studies revealed 248 were common and mainly involved immune response functions. KEGG pathway analysis indicated that these genes were involved in 12 pathways related to the pro-inflammatory response and APR, but also identified significant representation of two unexpected pathways: natural killer cell-mediated cytotoxicity pathway (KEGG04650) and the Rig-I-like receptor signalling pathway (KEGG04622).
In E. coli-induced mastitis, infected mammary gland tissue was found to significantly up-regulate expression of genes related to the immune response and down-regulate genes related to fat metabolism. Up to 25% of the DE immune response genes common to the three E. coli mastitis studies at T = 24 h were independent of E. coli strain and dose, cow lactation stage and number, tissue collection method and gene analysis method used. Hence, these DE genes likely represent important mediators of the local APR against E. coli in the mammary gland.
Normal development of the mammary gland proceeds via interactions between the epithelium and the mesenchyme that start during embryogenesis and continue during pubertal outgrowth and differentiation. The function of specific peptide growth factors that bind members of the receptor tyrosine kinase family and the cytokine receptor family are required at each stage. In many cases the peptides are produced in one compartment and act on receptors in the other compartment. One of the striking differences between normal development and cancer is the loss of this cross-talk. Mammary tumor cells often produce a peptide and express the receptor on the same cell leading to autocrine activation of signaling pathways, a mechanism that is characteristic for cancer cells. We will discuss different peptides in the context of normal development and cancer in this review.
The neonatal bovine mammary fat pad (MFP) surrounding the mammary parenchyma (PAR) is thought to exert proliferative effects on the PAR through secretion of local modulators of growth induced by systemic hormones. We used bioinformatics to characterize transcriptomics differences between PAR and MFP from approximately 65 d old Holstein heifers. Data were mined to uncover potential crosstalk through the analyses of signaling molecules preferentially expressed in one tissue relative to the other.
Over 9,000 differentially expressed genes (DEG; False discovery rate <or= 0.05) were found of which 1,478 had a >or=1.5-fold difference between PAR and MFP. Within the DEG highly-expressed in PAR vs. MFP (n = 736) we noted significant enrichment of functions related to cell cycle, structural organization, signaling, and DNA/RNA metabolism. Only actin cytoskeletal signaling was significant among canonical pathways. DEG more highly-expressed in MFP vs. PAR (n = 742) belong to lipid metabolism, signaling, cell movement, and immune-related functions. Canonical pathways associated with metabolism and signaling, particularly immune- and metabolism-related were significantly-enriched. Network analysis uncovered a central role of MYC, TP53, and CTNNB1 in controlling expression of DEG highly-expressed in PAR vs. MFP. Similar analysis suggested a central role for PPARG, KLF2, EGR2, and EPAS1 in regulating expression of more highly-expressed DEG in MFP vs. PAR. Gene network analyses revealed putative inter-tissue crosstalk between cytokines and growth factors preferentially expressed in one tissue (e.g., ANGPTL1, SPP1, IL1B in PAR vs. MFP; ADIPOQ, IL13, FGF2, LEP in MFP vs. PAR) with DEG preferentially expressed in the other tissue, particularly transcription factors or pathways (e.g., MYC, TP53, and actin cytoskeletal signaling in PAR vs. MFP; PPARG and LXR/RXR Signaling in MFP vs. PAR).
Functional analyses underscored a reciprocal influence in determining the biological features of MFP and PAR during neonatal development. This was exemplified by the potential effect that the signaling molecules (cytokines, growth factors) released preferentially (i.e., more highly-expressed) by PAR or MFP could have on molecular functions or signaling pathways enriched in the MFP or PAR. These bidirectional interactions might be required to coordinate mammary tissue development under normal circumstances or in response to nutrition.
The fine detail provided by sequencing-based transcriptome surveys suggests that RNA-seq is likely to become the platform of choice for interrogating steady state RNA. In order to discover biologically important changes in expression, we show that normalization continues to be an essential step in the analysis. We outline a simple and effective method for performing normalization and show dramatically improved results for inferring differential expression in simulated and publicly available data sets.
Information generated via microarrays might uncover interactions between the mammary gland and Streptococcus uberis (S. uberis) that could help identify control measures for the prevention and spread of S. uberis mastitis, as well as improve overall animal health and welfare, and decrease economic losses to dairy farmers. The main objective of this study was to determine the most affected gene networks and pathways in mammary tissue in response to an intramammary infection (IMI) with S. uberis and relate these with other physiological measurements associated with immune and/or metabolic responses to mastitis challenge with S. uberis O140J.
Streptococcus uberis IMI resulted in 2,102 (1,939 annotated) differentially expressed genes (DEG). Within this set of DEG, we uncovered 20 significantly enriched canonical pathways (with 20 to 61 genes each), the majority of which were signaling pathways. Among the most inhibited were LXR/RXR Signaling and PPARalpha/RXRalpha Signaling. Pathways activated by IMI were IL-10 Signaling and IL-6 Signaling which likely reflected counter mechanisms of mammary tissue to respond to infection. Of the 2,102 DEG, 1,082 were up-regulated during IMI and were primarily involved with the immune response, e.g., IL6, TNF, IL8, IL10, SELL, LYZ, and SAA3. Genes down-regulated (1,020) included those associated with milk fat synthesis, e.g., LPIN1, LPL, CD36, and BTN1A1. Network analysis of DEG indicated that TNF had positive relationships with genes involved with immune system function (e.g., CD14, IL8, IL1B, and TLR2) and negative relationships with genes involved with lipid metabolism (e.g., GPAM, SCD, FABP4, CD36, and LPL) and antioxidant activity (SOD1).
Results provided novel information into the early signaling and metabolic pathways in mammary tissue that are 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 potent activation of pro-inflammatory pathways that were associated with a marked inhibition of lipid synthesis, stress-activated kinase signaling cascades, and PPAR signaling (most likely PPARgamma). This latter effect may provide a mechanistic explanation for the inverse relationship between immune response and milk fat synthesis.
To determine whether inflammation can induce bovine fatty liver, we administered recombinant bovine tumor necrosis factor-alpha (rbTNF) to late-lactation Holstein cows. Cows (n = 5/treatment) were blocked by feed intake and parity and randomly assigned within block to control (CON; saline), rbTNF at 2 microg/(kg.d), or pair-fed control (saline, intake matched) treatments. Treatments were administered once daily by subcutaneous injection for 7 d. Plasma samples were collected daily for analysis of glucose and FFA and a liver biopsy was collected on d 7 for triglyceride (TG) and quantitative RT-PCR analyses. Data were analyzed using treatment contrasts to assess effects of tumor necrosis factor-alpha (TNFalpha) and decreased feed intake. By d 7, feed intake of both rbTNF and pair-fed cows was approximately 15% less than CON (P < 0.01). Administration of rbTNF resulted in greater hepatic TNFalpha mRNA and protein abundance and 103% higher liver TG content (P < 0.05) without affecting the plasma FFA concentration. Hepatic carnitine palmitoyltransferase 1 transcript abundance tended to be lower (P = 0.09) and transcript abundance of fatty acid translocase and 1-acyl-glycerol-3-phosphate acyltransferase was higher (both P < 0.05) after rbTNF treatment, consistent with increased FFA uptake and storage as TG. Transcript abundance of glucose-6-phosphatase (P < 0.05) and phosphoenolpyruvate carboxykinase 1 (P = 0.09), genes important for gluconeogenesis, was lower for rbTNF-treated cows. These findings indicate that TNFalpha promotes liver TG accumulation and suggest that inflammatory pathways may also be responsible for decreased glucose production in cows with fatty liver.
bioDBnet is an online web resource that provides interconnected access to many types of biological databases. It has integrated many of the most commonly used biological databases and in its current state has 153 database identifiers (nodes) covering all aspects of biology including genes, proteins, pathways and other biological concepts. bioDBnet offers various ways to work with these databases including conversions, extensive database reports, custom navigation and has various tools to enhance the quality of the results. Importantly, the access to bioDBnet is updated regularly, providing access to the most recent releases of each individual database.
Availability:
http://biodbnet.abcc.ncifcrf.gov.
Bacterial endotoxin/lipopolysaccharide elicits inflammatory responses and also elevates circulating levels of free fatty acids (FFAs) and impairs insulin sensitivity. Serum FFA elevation in acute endotoxemia has long been thought to be due to endotoxin dysregulating lipid disposal and counterregulatory hormones and cytokines. Here, we investigated the direct lipolysis effect of endotoxin in rodents and in isolated primary adipocytes. Endotoxin increases lipolysis in vivo in adipose tissues, elevates circulating FFA level, induces insulin resistance in rats, and directly stimulates chronic lipolysis in vitro in adipocytes. The lipolytic action of endotoxin is mediated via its lipid A moiety and is blocked by anti-endotoxin peptides. Neither adipocytokine secretion nor nuclear factor-kappaB activation is involved in endotoxin-induced lipolysis. Different from catecholamine, endotoxin stimulates lipolysis without elevating cAMP production and activating protein kinase A and protein kinase C. Instead, endotoxin induces phosphorylation of Raf-1, MEK1/2, and ERK1/2. Upon inhibition of ERK1/2 but not JNK and p38 MAPK, endotoxin-stimulated lipolysis ceases. Endotoxin causes perilipin down-regulation and phosphorylation and increases the activity and protein levels of hormone-sensitive lipase and adipose triglyceride lipase but does not induce hormone-sensitive lipase translocation to intracellular lipid droplets. In TLR4 (Toll-like receptor 4)-deficient mice and adipocytes, endotoxin fails to increase in vivo and in vitro lipolysis. These findings suggest that endotoxin stimulates lipolysis via TLR4 and ERK1/2 signaling in adipocytes. The lipolytic action of endotoxin liberates FFA efflux from adipocytes to the bloodstream, which is a possible basis for systemic FFA elevation and insulin resistance in endotoxemia or Gram-negative bacterial infection.
Liver plays a profound role in the acute phase response (APR) observed in the early phase of acute bovine mastitis caused by Escherichia coli (E. coli). To gain an insight into the genes and pathways involved in hepatic APR of dairy cows we performed a global gene expression analysis of liver tissue sampled at different time points before and after intra-mammary (IM) exposure to E. coli lipopolysaccharide (LPS) treatment.
Approximately 20% target transcripts were differentially expressed and eight co-expression clusters were identified. Each cluster had a unique time-dependent expression profile and consisted of genes involved in different biological processes. Our findings suggest that APR in the liver is triggered by the activation of signaling pathways that are involved with common and hepatic-specific transcription factors and pro-inflammatory cytokines. These mediators in turn stimulated or repressed the expression of genes encoding acute phase proteins (APP), collectins, complement components, chemokines, cell adhesion molecules and key metabolic enzymes during the APR. Hormones, anti-inflammatory and other hypothalamus-pituitary-adrenal axis (HPAA) linked mediators also seemed to participate in APR.
Performing global gene expression analysis on liver tissue from IM LPS treated cows verified that the liver plays a major role in the APR of E. coli mastitis, and that the bovine hepatic APR follows the same pattern as other mammals when they are challenged with LPS. Our work presents the first insight into the dynamic changes in gene expression in the liver that influences the induction, kinetics and clinical outcome of the APR in dairy cows.
There has been great interest in dry matter intake regulation in lactating dairy cattle to enhance performance and improve animal health and welfare. Predicting voluntary dry matter intake (VDMI) is complex and influenced by numerous factors relating to the diet, management, housing, environment and the animal. The objective of this review is to identify and discuss important metabolic factors involved in the regulation of VDMI and their integration with metabolism. We have described the adaptations of intake and metabolism and discussed mechanisms of intake regulation. Furthermore we have reviewed selected metabolic signals involved in intake regulation. A substantial dip in VDMI is initiated in late pregnancy and continues into early lactation. This dip has traditionally been interpreted as caused by physical constraints, but this role is most likely overemphasized. The dip in intake coincides with changes in reproductive status, fat mass, and metabolic changes in support of lactation, and we have described metabolic signals that may play an equally important role in intake regulation. These signals include nutrients, metabolites, reproductive hormones, stress hormones, leptin, insulin, gut peptides, cytokines, and neuropeptides such as neuropeptide Y, galanin, and corticotrophin-releasing factor. The involvement of these signals in the periparturient dip in intake is discussed, and evidence supporting the integration of the regulation of intake and metabolism is presented. Still, much research is needed to clarify the complex regulation of VDMI in lactating dairy cows, particularly in the periparturient animal.
Retinoid X Receptors (RXRs) consist of a family of nuclear receptors that target and regulate multiple signalling pathways. The early evolutionary emergence of RXRs in comparison to other nuclear receptors may have allowed for the development of unique properties as transcriptional regulators. Indeed, the complexity of these receptors is derived from their ability to activate transcription as homodimers or as obligate heterodimeric partners of a multitude of other nuclear receptors. In addition, RXRs can regulate gene expression in a ligand-dependent (forming permissive heterodimeric complexes) or - independent (forming non-permissive heterodimeric complexes) manner. Given that ligand binding is a critical component of RXR function, this review will focus on the ligand dependent functions of RXR. The remarkably conserved ligand binding domain of RXR is a multi-functional structure that in addition to ligand binding, serves as a homo- and heterodimeric interface, and a region to bind coactivactor and corepressor molecules. RXRs have a small ligand binding pocket and therefore bind their ligands (such as 9-cis RA) with both high affinity and specificity. In the presence of ligand, permissive RXR heterodimers bind coactivators, but nonpermissive complexes can bind coactivators or corepressors depending on the activation of the RXR's heterodimeric partner. Physiologically, the temporal and tissue specific pattern of RXRs as well as the presence of phenotypic abnormalities in receptor knockout studies (most severe in RXRa -/- animals) demonstrate the important role for these receptors both during development (morphogenesis) and in adult differentiated tissues (cell proliferation, cell differentiation, cell death). These receptors also play an important regulatory role metabolic signaling pathways (glucose, fatty acid and cholesterol metabolism), including metabolic disorders such as type 2 diabetes, hyperlipidemia and atherosclerosis. RXRs function as master regulators producing diverse physiological effects through the activation of multiple nuclear receptor complexes. RXRs represent important targets for pharmacologic interventions and therapeutic applications.
Migration of polymorphonuclear neutrophil leukocytes (PMN) into the mammary gland provide the first line of defense against invading mastitis pathogens. Bacteria release potent toxins that activate white blood cells and epithelial cells in the mammary gland to secrete cytokines that recruit PMN that function as phagocytes at the site of infection. While freshly migrated PMN are active phagocytes, continued exposure of PMN to inhibitory factors in milk such as fat globules and casein, leads to altered PMN morphology and reduced phagocytosis. In the course of phagocytosing and destroying invading pathogens, PMN release chemicals that not only kill the pathogens but that also cause injury to the delicate lining of the mammary gland. This will result in permanent scarring and reduced numbers of milk secretory cells. The life span of PMN is limited by the onset of apoptosis. To minimize damage to mammary tissue, PMN undergo a specialized process of programmed cell death known as apoptosis. Macrophages quickly engulf and phagocytose apoptotic PMN, thereby minimizing the release of PMN granular contents that are damaging to tissue. The PMN possess an array of cell surface receptors that allow them to adhere and migrate through endothelium and to recognize and phagocytose bacteria. One receptor found on phagocytes that is receiving considerable attention in the control of infections by Gram-negative bacteria is CD14. Binding of lipopolysaccharide (LPS) to membrane bound CD14 causes release of tumor necrosis factor-alpha and sepsis. Binding of LPS to soluble CD14 shed from CD14-bearing cells results in neutralization of LPS and rapid recruitment of PMN to the site of infection. Recent advances in the fields of genomics and proteomics should greatly enhance our understanding of the PMN role in controlling intramammary infections in ruminants. Further, manipulation of PMN, through either recombinant proteins such as soluble CD14 that enhance PMN response or agents that mediate PMN apoptosis, may serve as novel therapeutics for the treatment of mastitis.
The body's early defence in response to trauma, inflammation or infection, the acute phase response, is a complex set of systemic reactions seen shortly after exposure to a triggering event. One of the many components is an acute phase protein response in which increased hepatic synthesis leads to increased serum concentration of positive acute phase proteins. The serum concentration of these acute phase proteins returns to base levels when the triggering factor is no longer present. This paper provides a review of the acute phase proteins haptoglobin, C-reactive protein and serum amyloid A and their possible use as non-specific indicators of health in large animal veterinary medicine such as in the health status surveillance of pigs at the herd level, for the detection of mastitis in dairy cattle and for the prognosis of respiratory diseases in horses.
The deleterious consequences of inbreeding, especially in the form of inbreeding depression, are well known. However, little is known about how inbreeding affects genome-wide gene expression. Here, we show that inbreeding changes transcription levels for a number of genes. Gene expression profiles of Drosophila melanogaster lines inbred to F approximately = 0.67 at different rates changed relative to those of noninbred lines, but the rate of inbreeding did not significantly affect gene expression patterns. Genes being differentially expressed with inbreeding are disproportionately involved in metabolism and stress responses, suggesting that inbreeding acts like an environmental stress factor.
Since its initial identification as one of the genes most highly upregulated upon T-cell activation, osteopontin (or Eta-1, as it was designated then) has been demonstrated to have many roles in the regulation of the immune response on multiple levels. It contributes to the development of immune-mediated and inflammatory diseases, and it regulates the host response to infection. In some cases, the mechanisms of these effects have been elucidated, while other mechanistic functions of the protein remain obscure. The protein itself makes these analyses complex, since it binds to a series of different integrins, and in addition to its classically secreted form, an intracellular form of osteopontin has been identified, which participates in several aspects of immune regulation. In this review, we focus on the role of osteopontin in a series of immune-related diseases, particularly those where significant advances have been made in recent years: multiple sclerosis, rheumatoid arthritis, lupus and related diseases, Sjögren's disease, colitis, and 1 area of inflammatory pathology, alcoholic and nonalcoholic liver diseases. A recurring theme in these diseases is a link between osteopontin and pathogenic T cells, particularly T helper 17 cells, where osteopontin produced by dendritic cells supports IL-17 expression, contributing to pathology. In addition, a role for osteopontin in B-cell differentiation is becoming clear. In general, osteopontin contributes to pathology in these diseases, but there are examples where it has a protective role; deciphering the mechanisms underlying these differences and the specific receptors for osteopontin will be a research challenge for the future. Aside from its newly discovered role in the development of Sjögren's disease, the role of osteopontin in inflammatory conditions in the oral cavity is still poorly understood. Elucidation of this role will be of interest.
© International & American Associations for Dental Research 2015.
The study investigated the effect of an intramammary lipopolysaccharide (LPS) challenge on the bovine mammary and liver transcriptome and its consequences on metabolic biomarkers and liver tissue composition. At 7 days of lactation, 7 cows served as controls (CTR) and 7 cows (LPS) received an intramammary Escherichia coli LPS challenge. The mammary and liver tissues for transcriptomic profiling were biopsied at 2.5 h from challenge. Liver composition was evaluated at 2.5 h and 7 days after challenge, and blood biomarkers were analyzed at 2, 3, 7 and 14 days from challenge. In mammary tissue, the LPS challenge resulted in 189 differentially expressed genes (DEG), with 20 down-regulated and 169 up-regulated. In liver tissue, there were 107 DEG in LPS compared with CTR with 42 down-regulated and 65 up-regulated. In mammary, bioinformatics analysis highlighted that LPS led to activation of NOD-like receptor signaling, Toll-like receptor signaling, RIG-I-like receptor signaling and apoptosis pathways. In liver, LPS resulted in an overall inhibition of fatty acid elongation in mitochondria and activation of the p53 signaling pathway. The LPS challenge induced changes in liver lipid composition, a systemic inflammation (rise of blood ceruloplasmin and bilirubin), and an increase in body fat mobilization. The data suggest that cells within the inflamed mammary gland respond by activating mechanisms of pathogen recognition. However, in the liver the response likely depends on mediators originating from the udder that affect liver functionality and specifically fatty acid metabolism (β-oxidation, ketogenesis, and lipoprotein synthesis).
© 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.
Abstract Text:
In early lactation, susceptibility to disease is greatest, impacting cow health, productivity and leading to economic losses. Mastitis is the most economically costly disease to the dairy industry and is most frequent at this time. Glucose and amino acids (especially glutamine) are the primary fuels used by leukocytes in other species and are essential substrates for optimal leukocyte function but has not been elucidated in bovine leukocytes. Yet, because these substrates are in high demand to support milk synthesis in early lactation, their supply to leukocytes may be compromised. Production-related metabolic diseases during early lactation, such as ketosis and hepatic lipidosis, can also adversely affect health and productivity. Risk of subsequent disease for cows during mastitis is unknown. During an inflammatory response, increases in circulating non-esterified fatty acids and glucose during an IMI in dairy cows have been reported. Previous work indicates that hepatic expression of key genes associated with gluconeogenesis (e.g. PCK1 and G6PC), ketogenesis (e.g. HMGCS2) and fatty acid metabolism (e.g. SREBF1 and PPARA) are down-regulated after intramammary infection (IMI). These results suggest a potential link between mastitis and the risk of subsequent metabolic disease for dairy cows during lactation. This presentation will discuss the complex relationships between metabolism and immune function, and how these immunometabolic interactions relate to susceptibility to mastitis and increase the risk of subsequent disease during early lactation. New strategies to prevent or control mastitis development and reduce the risk of subsequent disease during early lactation will also be discussed.
Keywords:
cow, mastitis, metabolism
Metabolic changes associated with inflammatory processes and infectious diseases can modify protein and amino acid requirements. In such circumstances, depressed food intake and growth and muscle wasting are frequently observed. This is thought to be the consequence of an increase in the production of cytokines such as interleukin 1 (IL-1), interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) acting to alter protein metabolism. During immunological stress, amino acids are redistributed away from protein production (growth, lactation, etc.) towards tissues involved in inflammation and immune response. They are used for the synthesis of inflammatory and immune proteins, to support immune cell proliferation, and for the synthesis of other compounds important for body defense functions. As a consequence, stimulation of the immune system disturbs normal body processes and in turn is able to induce specific amino acid requirements. Some experimental data obtained in humans, laboratory or farm animals have clearly shown that the metabolism of some amino acids is modified by immune response. In this context, we question whether an adequate supply of exogenous amino acids provided by the diet, drinking water or an intravenous infusion could prevent or at least limit the consequences of inflammation and immune system activation on muscle protein metabolism and production of farm animals. The second question concerns the effects of an exogenous amino acid supply on the immune system. While the benefit of specific amino acid supplementation for immune response has been established for certain amino acids like glutamine, cysteine and arginine, it needs to be considered for other amino acids like tryptophan.
The consequences of mastitis in terms of dairy cow behavior are relatively unknown. Future assessment of dairy cow welfare during mastitis will be facilitated by knowledge about the potential of mastitis to induce sickness behavior. Our aim was to examine behavior of dairy cows in the period from 2 d before (d -2 and -1) to 3 d (d 0, 1, and 2) after experimental intramammary challenge with Escherichia coli. Effects of experimentally induced mastitis on behavior were examined in 20 primiparous Danish Holstein-Friesian cows, all 3 to 6 wk after calving and kept in tie stalls. After evening milking on d 0, each cow received an intramammary infusion with 20 to 40 cfu of E. coli in 1 healthy front quarter. Paraclinical and bacteriological examinations were conducted to confirm infection. Half of the cows were subjected to liver and udder biopsies twice during the trial. Behavior was video-recorded on 5 consecutive days, d -2 to +2 after challenge when the cows were not disturbed by humans. The behavior of the animals was compared among all days. Infection with E. coli altered the behavior of the dairy cows. Time spent feeding was lower in the initial 24 h after infection compared with that on the other days (16.6±1.1, 16.5±1.0, 13.2±1.2, 18.1±1.1, and 16.0±0.8% of time for d -2, -1, 0, 1, and 2, respectively). The duration of standing idle increased on d 0 compared with that on the control days and d 1 and 2 (29.4±2.6, 28.0±2.3, 39.1±2.6, 31.4±3.8, and 25.9±2.6% of time for d -2, -1, 0, 1 and 2, respectively). The frequency of self-grooming behavior per hour decreased in the initial 24h compared with that on d -2, -1, and 2 (4.1±0.8, 5.4±1.9, 3.2±0.6, 3.6±0.6, and 4.8±1.0 for d -2, -1, 0, 1, and 2, respectively). Likewise, duration of rumination and frequency of turning the head against the udder decreased in the first days after infection (rumination: 32.2±1.6, 34.8±1.8, 27.9±1.7, 30.0±2.6, and 34.8±1.7% of time; and frequency of turning head: 0.6±0.1, 0.6±0.1, 0.3±0.1, 0.3±0.1, and 0.6±0.1 per hour for d -2, -1, 0, 1 and 2, respectively). The cows subjected to biopsies showed an overall decreased lying time during the entire observation period (36.3±1.5 vs. 46.1±2.2% of time) but not directly related to the period after the biopsies. Dairy cows show classic signs of illness behavior in the hours after intramammary challenge with E. coli. This knowledge can be useful for the development of welfare assessment protocols, early disease detection, and for future work aimed at understanding the behavioral needs of dairy cows suffering from mastitis.
Interleukin 10 (IL-10) is a cytokine with potent anti-inflammatory properties that plays a central role in limiting host immune response to pathogens, thereby preventing damage to the host and maintaining normal tissue homeostasis. Dysregulation of IL-10 is associated with enhanced immunopathology in response to infection as well as increased risk for development of many autoimmune diseases. Thus a fundamental understanding of IL-10 gene expression is critical for our comprehension of disease progression and resolution of host inflammatory response. In this review, we discuss modes of regulation of IL-10 gene expression in immune effector cell types, including signal transduction, epigenetics, promoter architecture, and post-transcriptional regulation, and how aberrant regulation contributes to immunopathology and disease progression.
The maintenance of membrane lipid composition within strict limits is critical to maintain optimum cellular function. The biophysical properties of the membrane can be influenced among other factors by the saturation/unsaturation of the phospholipid fatty acyl chain. The rate-limiting enzyme in unsaturated fatty acid biosynthesis is the desaturase enzyme which in turn is regulated by the lipid transcription factor sterol regulatory element binding protein (SREBP1). In this review, we collect some evidence suggesting SREBP1 network as an important allostatic regulator necessary to maintain the pool of unsaturated fatty acid lipid species that can be incorporated into biological membranes.
Dairy cows undergo tremendous metabolic and physiological adaptations around parturition to support lactation. The liver is central to many of these processes, including gluconeogenesis and metabolism of fatty acids mobilized from adipose tis- sue. Fat accumulation may impair normal functions of the liver and increase ketogenesis, which in turn may predispose cows to other metabolic abnormalities. Several aspects of dietary management and body condition may alter these adaptations, affect dry matter intake, and increase or decrease susceptibility to periparturient health problems. Overfeeding energy dur- ing the dry period is a prominent risk factor. Considerable progress has been made in recent years in describing the adap- tive changes in the liver and other organs in normal and abnormal states, but this knowledge has not yet identified unequiv- ocally the key steps that might be compromised during development of metabolic disorders. The potential role of signaling compounds, such as the inflammatory cytokines released in response to environmental stressors, infectious challenge, and oxidative stress, in the pathogenesis of periparturient disease is under investigation. New techniques such as functional genomics, using cDNA or oligonucleotide microarrays, as well as proteomics and metabolomics, provide additional high- throughput tools to determine the effects of nutrition, management, or stressors on tissue function in development of dis- ease. Integrative approaches should be fruitful in unraveling the complex interactions of metabolism, immune activation, stress physiology, and endocrinology that likely underlie development of periparturient disease.
Ten multiparous Holstein cows were used to determine the effects of negative energy balance (NEB) on the immune response to a Streptococcus uberis (strain O140J) mastitis challenge during midlactation. Before the study, milk from all quarters of each cow was bacteriologically negative, with a composite somatic cell count of <200,000 cells/mL. Cows were paired based on parity, days in milk, and milk yield. At approximately 77 d in milk, half the cows (n = 5) were feed-restricted to 60% of calculated net energy for lactation requirements to induce NEB. Feed restriction lasted 7 d. Control cows (n = 5) were fed the same diet ad libitum (i.e., positive energy balance; PEB). After 5 d, one rear quarter in all cows was inoculated with 5,000 cfu of Strep. uberis. Jugular blood and aseptic quarter milk samples were collected daily until inoculation and every 6 h postinoculation for 36 h. Blood was analyzed for nonesterified fatty acids, beta-hydroxybutyrate, insulin, cortisol, albumin, serum amyloid A (SAA), and haptoglobin (Hp). Periodically throughout the trial period, blood neutrophils were isolated for determination of cell morphology, chemotaxis, and phagocytosis capability in vitro. Quarter milk samples were analyzed for concentrations of SAA, Hp, cytokines (tumor necrosis factor-alpha, IL-10 and IL-1beta), and activity of respiratory burst enzymes (superoxide dismutase and glutathione peroxidase). All cows developed local and systemic signs of mastitis and calculated NEB was similar to that of cows experiencing postpartal NEB. Serum glucose and insulin concentrations increased in both groups after challenge, most likely because of enhanced glycogenolysis and gluconeogenesis; results indicate that immune cell function may be glucose dependent. Serum cortisol concentration was higher in NEB than PEB cows during feed restriction only (before inoculation), and serum albumin concentration was higher in NEB than PEB cows during the infection period. Compared with PEB, cows in NEB had lower SAA concentrations in serum after 5 d of feed restriction but higher SAA concentrations in milk after Strep. uberis challenge. Serum Hp concentration was higher by 36 h postchallenge in NEB than in PEB cows. Phagocytic capability of neutrophils was lower in NEB than in PEB cows at 0 h of infection but decreased in both PEB and NEB cows through 36 h postinfection. Our results indicate that cows subjected to dietary-induced NEB during midlactation had relatively minimal alterations in immune function.
A minimally invasive liver biopsy technique was tested for its applicability to study the hepatic acute phase response (APR) in dairy cows with Escherichia coli lipopolysaccharide (LPS)-induced mastitis. The hepatic mRNA expression profiles of the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-6, and IL-10, and the acute phase proteins serum amyloid A isoform 3 (SAA3), haptoglobin (Hp), and alpha(1)-acid glycoprotein (AGP) were determined by real-time reverse transcription-PCR. Fourteen primiparous cows in mid lactation were challenged with 200 microg of LPS (n = 8) or NaCl solution (n = 6) in 1 front quarter. Six repeated liver biopsies were collected at -22, 3, 6, 9, 12, and 48 h relative to LPS challenge in 4 LPS-infused cows and 3 NaCl-infused cows. The remaining cows had 3 liver biopsies taken at -22, 9, and 48 h. Production data and clinical signs were recorded and white blood cell counts and somatic cell counts (SCC) were analyzed to investigate the effect of repeated liver biopsies and verify the LPS model. Plasma concentrations of TNF-alpha, SAA3, Hp, and AGP were determined for comparison with the liver expression data. Repeated liver biopsies had no effects on the production data, clinical signs, or APR of dairy cows. Compared with the NaCl-infused cows the LPS-infused cows responded to the LPS treatment by increased body temperature (38.6 +/- 0.1 vs. 39.4 +/- 0.1 degrees C), short-term leukopenia followed by leukocytosis (6.44 +/- 0.4 vs. 5.69 +/- 0.3 x 10(6) cells/mL), an increased SCC (log(10) 2.1 +/- 0.1 vs. log(10) 2.8 +/- 0.1 x 10(3) cells/mL), heart rate (76 +/- 1 vs. 93 +/- 1 beats/min), and respiratory rate (32 +/- 2 vs. 36 +/- 1 breaths/min) in the acute phase of the disease. The LPS treatment upregulated the hepatic expression of TNF-alpha (103 +/- 24 vs. 255 +/- 18 units), IL-1beta (37 +/- 23 vs. 296 +/- 18 units), IL-6 (8 +/- 17 vs. 122 +/- 12 units), and IL-10 (130 +/- 66 vs. 541 +/- 50 units), and SAA3 (64 +/- 36 vs. 128 +/- 28 units) and Hp (9 +/- 82 vs. 762 +/- 65 units) reaching maximum levels at 3 to 6 h and 9 to 12 h postinfusion, respectively. Plasma concentrations of TNF-alpha (nondetectable vs. 1.9 +/- 0.3 ng/mL), SAA (19.8 +/- 19.4 vs. 149.7 +/- 15.5 microg/mL) and Hp (71.4 +/- 143.7 vs. 1,013.8 +/- 111.5 microg/mL) were elevated in the LPS-infused cows at 4 to 12 h, 8 to 120 h, and 24 to 120 h postinfusion, respectively. The hepatic expression of AGP and the AGP plasma concentration remained unaltered in LPS-induced cows. In conclusion, a minimally invasive liver biopsy technique can be used for studying the hepatic APR in diseased cattle. Lipopolysaccharide-induced mastitis resulted in a time-dependent production of inflammatory cytokines and SAA and Hp in the liver of dairy cows.
Inflammation is proposed to impair reverse cholesterol transport (RCT), a major atheroprotective function of high-density lipoprotein (HDL). The present study presents the first integrated functional evidence that inflammation retards numerous components of RCT.
We used subacute endotoxemia in the rodent macrophage-to-feces RCT model to assess the effects of inflammation on RCT in vivo and performed proof of concept experimental endotoxemia studies in humans. Endotoxemia (3 mg/kg SC) reduced (3)H-cholesterol movement from macrophage to plasma and (3)H-cholesterol associated with HDL fractions. At 48 hours, bile and fecal counts were markedly reduced consistent with downregulation of hepatic expression of ABCG5, ABCG8, and ABCB11 biliary transporters. Low-dose lipopolysaccharide (0.3 mg/kg SC) also reduced bile and fecal counts, as well as expression of biliary transporters, but in the absence of effects on plasma or liver counts. In vitro, lipopolysaccharide impaired (3)H-cholesterol efflux from human macrophages to apolipoprotein A-I and serum coincident with reduced expression of the cholesterol transporter ABCA1. During human (3 ng/kg; n=20) and murine endotoxemia (3 mg/kg SC), ex vivo macrophage cholesterol efflux to acute phase HDL was attenuated.
We provide the first in vivo evidence that inflammation impairs RCT at multiple steps in the RCT pathway, particularly cholesterol flux through liver to bile and feces. Attenuation of RCT and HDL efflux function, independent of HDL cholesterol levels, may contribute to atherosclerosis in chronic inflammatory states including obesity, metabolic syndrome, and type 2 diabetes.
Porcine intestinal gastrin-releasing peptide (GRP) has been demonstrated to be structurally identical to the previously characterized gastric GRP. Ion-exchange and high-performance liquid chromatography of porcine intestinal extracts have identified two variant GRP forms. Studies on one of these variant forms suggest that a beta-aspartyl shift has occurred in the Asn-His structure of GRP; such a modification in an Asn-His structure occurring in a natural peptide or protein has not been previously reported. This variant GRP, although retaining bioactivity, appears to have reduced potency in elevating canine plasma gastrin levels.
Glucocorticoids are the most widely used anti-inflammatory and immunomodulatory agents, whose mechanism of action is based mainly on interference with the activity of transcription factors, such as nuclear factor kappaB (NF-kappaB) and activator protein-1 (AP-1). The precise molecular mechanisms of gene repression by glucocorticoids are a controversial matter, due to the existence of many conflicting hypotheses. We discuss the three main paradigms reported in the literature, namely the inhibitor kappaB-alpha (IkappaB-alpha) upregulatory model, the protein-protein interaction model and the competition model.
The numerous functions of the liver are controlled primarily at the transcriptional level by the concerted actions of a limited number of hepatocyte-enriched transcription factors (hepatocyte nuclear factor 1α [HNF1α], -1β, -3α, -3β, -3γ, -4α, and -6 and members of the c/ebp family). Of these, only HNF4α (nuclear receptor 2A1) and HNF1α appear to be correlated with the differentiated phenotype of cultured hepatoma cells. HNF1α-null mice are viable, indicating that this factor is not an absolute requirement for the formation of an active hepatic parenchyma. In contrast, HNF4α-null mice die during embryogenesis. Moreover, recent in vitro experiments using tetraploid aggregation suggest that HNF4α is indispensable for hepatocyte differentiation. However, the function of HNF4α in the maintenance of hepatocyte differentiation and function is less well understood. To address the function of HNF4α in the mature hepatocyte, a conditional gene knockout was produced using the Cre-loxP system. Mice lacking hepatic HNF4α expression accumulated lipid in the liver and exhibited greatly reduced serum cholesterol and triglyceride levels and increased serum bile acid concentrations. The observed phenotypes may be explained by (i) a selective disruption of very-low-density lipoprotein secretion due to decreased expression of genes encoding apolipoprotein B and microsomal triglyceride transfer protein, (ii) an increase in hepatic cholesterol uptake due to increased expression of the major high-density lipoprotein receptor, scavenger receptor BI, and (iii) a decrease in bile acid uptake to the liver due to down-regulation of the major basolateral bile acid transporters sodium taurocholate cotransporter protein and organic anion transporter protein 1. These data indicate that HNF4α is central to the maintenance of hepatocyte differentiation and is a major in vivo regulator of genes involved in the control of lipid homeostasis.
Lipids and lipid metabolism have well-documented regulatory effects on inflammatory processes. Recent work has highlighted the role of the peroxisome proliferator-activated receptors (PPARs)--a subset of the nuclear-hormone-receptor superfamily that are activated by various lipid species--in regulating inflammatory responses. Here, we describe how the PPARs, through their interactions with transcription factors and other cell-signalling systems, have important regulatory roles in innate and adaptive immunity.