MicroRNA expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli in vitro

Background Coliform mastitis is a symptom of postpartum dysgalactia syndrome (PDS), a multifactorial infectious disease of sows. Our previous study showed gene expression profile change after bacterial challenge of porcine mammary epithelial cells (PMECs). These mRNA expression changes may be regulated through microRNAs (miRNAs) which play critical roles in biological processes. Therefore, miRNA expression profile was investigated in PMECs. Results PMECs were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogen Escherichia coli (E. coli) for 3 h and 24 h, in vitro. At 3 h post-challenge with E. coli, target gene prediction identified a critical role of miRNAs in regulation of host immune responses and homeostasis of PMECs mediated by affecting pathways including cytokine binding (miR-202, miR-3277, miR-4903); IL-10/PPAR signaling (miR-3277, miR-4317, miR-548); and NF-ĸB/TNFR2 signaling (miR-202, miR-2262, miR-885-3p). Target genes of miRNAs in PMECs at 24 h were significantly enriched in pathways associated with interferon signaling (miR-210, miR-23a, miR-1736) and protein ubiquitination (miR-125, miR-128, miR-1280). Conclusions This study provides first large-scale miRNA expression profiles and their predicted target genes in PMECs after contact with a potential mastitis-causing E. coli strain. Both, highly conserved miRNAs known from other species as well as novel miRNAs were identified in PMECs, representing candidate predictive biomarkers for PDS. Time-dependent pathogen clearance suggests an important role of PMECs in inflammatory response of the first cellular barrier of the porcine mammary gland

Identification of expression QTL (eQTL) of genes expressed in porcine M. longissimus dorsi and associated with meat quality traits

<p>Abstract</p> <p>Background</p> <p>Genetic analysis of transcriptional profiles is a promising approach for identifying and dissecting the genetics of complex traits like meat performance. Accordingly, expression levels obtained by microarray analysis were taken as phenotypes in a linkage analysis to map eQTL. Moreover, expression levels were correlated with traits related to meat quality and principle components with high loadings of these traits. By using an up-to-date annotation and localization of the respective probe-sets, the integration of eQTL mapping data and information of trait correlated expression finally served to point to candidate genes for meat quality traits.</p> <p>Results</p> <p>Genome-wide transcriptional profiles of <it>M. longissimus dorsi </it>RNAs samples of 74 F2 animals of a pig resource population revealed 11,457 probe-sets representing genes expressed in the muscle. Linkage analysis of expression levels of these probe-sets provided 9,180 eQTL at the suggestive significance threshold of LOD > 2. We mapped 653 eQTL on the same chromosome as the corresponding gene and these were designated as 'putative <it>cis-</it>eQTL'. In order to link eQTL to the traits of interest, probe-sets were addressed with relative transcript abundances that showed correlation with meat quality traits at p ≤ 0.05. Out of the 653 'putative <it>cis-</it>eQTL', 262 transcripts were correlated with at least one meat quality trait. Furthermore, association of expression levels with composite traits with high loadings for meat quality traits generated by principle component analysis were taken into account leading to a list of 85 genes exhibiting <it>cis-</it>eQTL and trait dependent expression.</p> <p>Conclusion</p> <p>Holistic expression profiling was integrated with QTL analysis for meat quality traits. Correlations between transcript abundance and meat quality traits, combined with genetic positional information of eQTL allowed us to prioritise candidate genes for further study.</p

Microarray-based transcriptional profiling of Eimeria bovis-infected bovine endothelial host cells

Within its life cycle Eimeria bovis undergoes a long lasting intracellular development into large macromeronts in endothelial cells. Since little is known about the molecular basis of E. bovis-triggered host cell regulation we applied a microarray-based approach to define transcript variation in bovine endothelial cells early after sporozoite invasion (4 h post inoculation (p.i.)), during trophozoite establishment (4 days p.i.), during early parasite proliferation (8 days p.i.) and towards macromeront maturation (14 days p.i.). E. bovis infection led to significant changes in the abundance of many host cell gene transcripts. As infection progressed, the number of regulated genes increased such that 12, 45, 175 and 1184 sequences were modulated at 4 h, 4, 8 and 14 days p.i., respectively. These genes significantly interfered with several host cell functions, networks and canonical pathways, especially those involved in cellular development, cell cycle, cell death, immune response and metabolism. The correlation between stage of infection and the number of regulated genes involved in different aspects of metabolism suggest parasite-derived exploitation of host cell nutrients. The modulation of genes involved in cell cycle arrest and host cell apoptosis corresponds to morphological in vitro findings and underline the importance of these aspects for parasite survival. Nevertheless, the increasing numbers of modulated transcripts associated with immune responses also demonstrate the defensive capacity of the endothelial host cell. Overall, this work reveals a panel of novel candidate genes involved in E. bovis-triggered host cell modulation, providing a valuable tool for future work on this topic

Genome-Wide Linkage Analysis of Global Gene Expression in Loin Muscle Tissue Identifies Candidate Genes in Pigs

BACKGROUND: Nearly 6,000 QTL have been reported for 588 different traits in pigs, more than in any other livestock species. However, this effort has translated into only a few confirmed causative variants. A powerful strategy for revealing candidate genes involves expression QTL (eQTL) mapping, where the mRNA abundance of a set of transcripts is used as the response variable for a QTL scan. METHODOLOGY/PRINCIPAL FINDINGS: We utilized a whole genome expression microarray and an F(2) pig resource population to conduct a global eQTL analysis in loin muscle tissue, and compared results to previously inferred phenotypic QTL (pQTL) from the same experimental cross. We found 62 unique eQTL (FDR <10%) and identified 3 gene networks enriched with genes subject to genetic control involved in lipid metabolism, DNA replication, and cell cycle regulation. We observed strong evidence of local regulation (40 out of 59 eQTL with known genomic position) and compared these eQTL to pQTL to help identify potential candidate genes. Among the interesting associations, we found aldo-keto reductase 7A2 (AKR7A2) and thioredoxin domain containing 12 (TXNDC12) eQTL that are part of a network associated with lipid metabolism and in turn overlap with pQTL regions for marbling, % intramuscular fat (% fat) and loin muscle area on Sus scrofa (SSC) chromosome 6. Additionally, we report 13 genomic regions with overlapping eQTL and pQTL involving 14 local eQTL. CONCLUSIONS/SIGNIFICANCE: Results of this analysis provide novel candidate genes for important complex pig phenotypes

Segregation of Regulatory Polymorphisms with Effects on the Gluteus Medius Transcriptome in a Purebred Pig Population

Background: The main goal of the present study was to analyse the genetic architecture of mRNA expression in muscle, a tissue with an outmost economic importance for pig breeders. Previous studies have used F2 crosses to detect porcine expression QTL (eQTL), so they contributed with data that mostly represents the between-breed component of eQTL variation. Herewith, we have analysed eQTL segregation in an outbred Duroc population using two groups of animals with divergent fatness profiles. This approach is particularly suitable to analyse the within-breed component of eQTL variation, with a special emphasis on loci involved in lipid metabolism. Methodology/Principal Findings: GeneChip Porcine Genome arrays (Affymetrix) were used to determine the mRNA expression levels of gluteus medius samples from 105 Duroc barrows. A whole-genome eQTL scan was carried out with a panel of 116 microsatellites. Results allowed us to detect 613 genome-wide significant eQTL unevenly distributed across the pig genome. A clear predominance of trans- over cis-eQTL, was observed. Moreover, 11 trans-regulatory hotspots affecting the expression levels of four to 16 genes were identified. A Gene Ontology study showed that regulatory polymorphisms affected the expression of muscle development and lipid metabolism genes. A number of positional concordances between eQTL and lipid trait QTL were also found, whereas limited evidence of a linear relationship between muscle fat deposition and mRNA levels of eQTL regulated genes was obtained. Conclusions/Significance: Our data provide substantial evidence that there is a remarkable amount of within-breed genetic variation affecting muscle mRNA expression. Most of this variation acts in trans and influences biological processes related with muscle development, lipid deposition and energy balance. The identification of the underlying causal mutations and the ascertainment of their effects on phenotypes would allow gaining a fundamental perspective about how complex traits are built at the molecular level

The three-way relationship of polymorphisms of porcine genes encoding terminal complement components, their differential expression, and health-related phenotypes

<p>Abstract</p> <p>Background</p> <p>The complement system is an evolutionary ancient mechanism that plays an essential role in innate immunity and contributes to the acquired immune response. Three modes of activation, known as classical, alternative and lectin pathway, lead to the initiation of a common terminal lytic pathway. The terminal complement components (TCCs: C6, C7, C8A, C8B, and C9) are encoded by the genes <it>C6</it>, <it>C7</it>, <it>C8A</it>, <it>C8B</it>, <it>C8G</it>, and <it>C9</it>. We aimed at experimentally testing the porcine genes encoding TCCs as candidate genes for immune competence and disease resistance by addressing the three-way relationship of genotype, health related phenotype, and mRNA expression.</p> <p>Results</p> <p>Comparative sequencing of cDNAs of animals of the breeds German Landrace, Piétrain, Hampshire, Duroc, Vietnamese Potbelly Pig, and Berlin Miniature Pig (BMP) revealed 30 SNPs (21 in protein domains, 12 with AA exchange). The promoter regions (each ~1.5 kb upstream the transcription start sites) of <it>C6</it>, <it>C7</it>, <it>C8A</it>, <it>C8G</it>, and <it>C9</it> exhibited 29 SNPs. Significant effects of the TCC encoding genes on hemolytic complement activity were shown in a cross of Duroc and BMP after vaccination against Mycoplasma hyopneumoniae, Aujeszky disease virus and PRRSV by analysis of variance using repeated measures mixed models. Family based association tests (FBAT) confirmed the associations. The promoter SNPs were associated with the relative abundance of TCC transcripts obtained by real time RT-PCR of 311 liver samples of commercial slaughter pigs. Complement gene expression showed significant relationship with the prevalence of acute and chronic lung lesions.</p> <p>Conclusions</p> <p>The analyses point to considerable variation of the porcine TCC genes and promote the genes as candidate genes for disease resistance.</p

Transcriptional responses of PBMC in psychosocially stressed animals indicate an alerting of the immune system in female but not castrated male pigs

Background[br/] Brain and immune system are linked in a bi-directional manner. To date, it remained largely unknown why immune components become suppressed, enhanced, or remain unaffected in relation to psychosocial stress. Therefore, we mixed unfamiliar pigs with different levels of aggressiveness. We separated castrated male and female pigs into psychosocially high- and low- stressed animals by skin lesions, plasma cortisol level, and creatine kinase activity obtained from agonistic behaviour associated with regrouping. Peripheral blood mononuclear cells (PBMC) were collected post-mortem and differential gene expression was assessed using the Affymetrix platform (n = 16).[br/] [br/] Results[br/] Relevant stress-dependent alterations were found only between female samples, but not between castrated male samples. Molecular routes related to TREM 1 signalling, dendritic cell maturation, IL-6 signalling, Toll-like receptor signalling, and IL-8 signalling were increased in high stressed females compared to low stressed females. This indicates a launch of immune effector molecules as a direct response. According to the shifts of transcripts encoding cell surface receptors (e.g. CD14, TLR2, TLR4, TREM1) the study highlights processes acting on pattern recognition, inflammation, and cell-cell communication.[br/] [br/] Conclusions[br/] The transcriptional response partly affected the degree of ‘stress responsiveness’, indicating that the high stressed females altered their signal transduction due to potential infections and injuries while fighting

Expression Analysis of the NLRP Gene Family Suggests a Role in Human Preimplantation Development

Background: The NLRP (Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing) family, also referred to as NALP family, is well known for its roles in apoptosis and inflammation. Several NLRPs have been indicated as being involved in reproduction as well. Methodology: We studied, using the unique human gametes and embryo materials, the expression of the NLRP family in human gametes and preimplantation embryos at different developmental stages, and compared the expression levels between normal and abnormal embryos using real-time PCR. Principal Findings: Among 14 members of the NLRP family, twelve were detected in human oocytes and preimplantation embryos, whereas seven were detected in spermatozoa. Eight NLRPs (NLRP4, 5, 8, 9, 11, 12, 13, and 14) showed a similar expression pattern: their expression levels were high in oocytes and then decreased progressively in embryos, resulting in a very low level in day 5 embryos. However, NLRP2 and NLRP7 showed a different expression pattern: their expression decreased from oocytes to the lowest level by day 3, but increased again by day 5. The expression levels of NLRP5, 9, and 12 were lower in day 1 abnormal embryos but higher in day3 and day5 arrested embryos, when compared with normal embryos at the same stages. NLRP7 was down-regulated in day 1 and day 5 abnormal embryos but over-expressed in day3 arrested embryos

Recent evolution of the NF-κB and inflammasome regulating protein POP2 in primates

<p>Abstract</p> <p>Background</p> <p>Pyrin-only protein 2 (POP2) is a small human protein comprised solely of a pyrin domain that inhibits NF-κB p65/RelA and blocks the formation of functional IL-1β processing inflammasomes. Pyrin proteins are abundant in mammals and several, like POP2, have been linked to activation or regulation of inflammatory processes. Because <it>POP2 </it>knockout mice would help probe the biological role of inflammatory regulation, we thus considered whether <it>POP2 </it>is common in the mammalian lineage.</p> <p>Results</p> <p>BLAST searches revealed that <it>POP2 </it>is absent from the available genomes of not only mice and rats, but those of other domestic mammals and New World monkeys as well. <it>POP2 </it>is however present in the genome of the primate species most closely related to humans including <it>Pan troglodytes </it>(chimpanzees), <it>Macaca mulatta </it>(rhesus macaques) and others. Interestingly, chimpanzee POP2 is identical to human POP2 (huPOP2) at both the DNA and protein level. Macaque POP2 (mqPOP2), although highly conserved is not identical to the human sequence; however, both functions of the human protein are retained. Further, <it>POP2 </it>appears to have arisen in the mammalian genome relatively recently (~25 mya) and likely derived from retrogene insertion of <it>NLRP2</it>.</p> <p>Conclusion</p> <p>Our findings support the hypothesis that the NLR loci of mammals, encoding proteins involved in innate and adaptive immunity as well as mammalian development, have been subject to recent and strong selective pressures. Since POP2 is capable of regulating signaling events and processes linked to innate immunity and inflammation, its presence in the genomes of hominids and Old World primates further suggests that additional regulation of these signals is important in these species.</p