Non-classical ProIL-1beta activation during mammary gland infection is pathogen-dependent but caspase-1 independent

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1

The influence of different anticoagulants and sample preparation methods on measurement of mCD14 on bovine monocytes and polymorphonuclear neutrophil leukocytes

<p>Abstract</p> <p>Background</p> <p>Membrane-CD14 (mCD14) is expressed on the surface of monocytes, macrophages and polymorphonuclear neutrophil leukocytes (PMN). mCD14 acts as a co-receptor along with Toll like receptor 4 (TLR 4) and MD-2 for the detection of lipopolysaccharide (LPS). However, studies using different sample preparation methods and anticoagulants have reported different levels of mCD14 on the surface of monocytes and neutrophils. In this study, the influence of various anticoagulants and processing methods on measurement of mCD14 on monocytes and neutrophils was examined.</p> <p>Results</p> <p>Whole blood samples were collected in vacutainer tubes containing either sodium heparin (HEPARIN), ethylenediaminetetraacetic acid (EDTA) or sodium citrate (CITRATE). mCD14 on neutrophils and monocytes in whole blood samples or isolated cells was measured by the method of flow cytometry using fluorescein isothiocyanate (FITC)-labeled monoclonal antibody. There was a significant difference (<it>p </it>< 0.05) in the mean channel fluorescence intensity (MFI) of mCD14 on neutrophils in whole blood samples anticoagulated with HEPARIN (MFI = 64.77) in comparison with those in whole blood samples anticoagulated with either EDTA (MFI = 38.25) or CITRATE (MFI = 43.7). The MFI of mCD14 on monocytes in whole blood samples anticoagulted with HEPARIN (MFI = 206.90) was significantly higher than the MFI in whole blood samples anticoagulated with EDTA (MFI = 149.37) but similar to that with CITRATE (MFI = 162.55). There was no significant difference in the percentage of whole blood neutrophils or monocytes expressing mCD14 irrespective of type of anticoagulant used. However, MFI of mCD14 on monocytes was about 3.2-folds (HEPARIN), 3.9-folds (EDTA) or 3.7 folds (CITRATE) higher than those on neutrophils. Furthermore, there was no significant difference in mCD14 levels between unprocessed whole blood monocytes and monocytes in peripheral blood mononuclear cell preparation. Conversely, a highly significant difference was observed in mCD14 between unprocessed whole blood neutrophils and isolated neutrophils (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p>From these results, it is suggested that sodium heparin should be the preferred anticoagulant for use in the reliable quantification of the surface expression of mCD14. Furthermore, measurement of mCD14 is best carried out in whole blood samples, both for neutrophils and monocytes.</p

Scans for signatures of selection in Russian cattle breed genomes reveal new candidate genes for environmental adaptation and acclimation

Domestication and selective breeding has resulted in over 1000 extant cattle breeds. Many of these breeds do not excel in important traits but are adapted to local environments. These adaptations are a valuable source of genetic material for efforts to improve commercial breeds. As a step toward this goal we identified candidate regions to be under selection in genomes of nine Russian native cattle breeds adapted to survive in harsh climates. After comparing our data to other breeds of European and Asian origins we found known and novel candidate genes that could potentially be related to domestication, economically important traits and environmental adaptations in cattle. The Russian cattle breed genomes contained regions under putative selection with genes that may be related to adaptations to harsh environments (e.g., AQP5, RAD50, and RETREG1). We found genomic signatures of selective sweeps near key genes related to economically important traits, such as the milk production (e.g., DGAT1, ABCG2), growth (e.g., XKR4), and reproduction (e.g., CSF2). Our data point to candidate genes which should be included in future studies attempting to identify genes to improve the extant breeds and facilitate generation of commercial breeds that fit better into the environments of Russia and other countries with similar climates

Polymorphisms of the prion protein gene and their effects on litter size and risk evaluation for scrapie in Chinese Hu sheep

It is well known that scrapie is a fatal, neurodegenerative disease in sheep and goat, which belongs to the group of transmissible spongiform encephalopathies (TSEs) or prion diseases. It has been confirmed that the polymorphisms of prion protein gene (PRNP) at codons 136, 154, and 171 have strong relationship with scrapie in sheep. In the present study, nine polymorphisms of PRNP at codons 136, 154, and 171 and other six loci (at codons 101, 112, 127, 137, 138, and 152) were detected in 180 Chinese Hu sheep. All the alleles at codons 136, 154, and 171 have been identified and resulted in three new genotypes. The frequencies of predominant alleles were 85% (A136), 99.40% (R154), and 37.78% (Q171), respectively. The predominant haplotype ARQ has a relatively high frequency of 57.77%. The frequencies of dominant genotypes of ARR/ARQ and ARQ/ARQ were 30 and 26.67%, respectively. Three new found genotypes named ARQ/TRK, ARQ/TRR, and TRR/TRQ had the same lower frequencies (0.56%). The relationship of PRNP genotype with scrapie risk and litter size showed that the predominant genotypes are corresponded to the risk score of R1 (1.67%), R2 (32.22%), and R3 (42.22%). Just at the first parity, the individuals with ARH/ARH genotype had significantly larger litter size than the mean value and those with ARQ/ARQ and ARR/ARQ genotypes. In short, this study provided preliminary information about alleles and genotypes of PRNP in Chinese Hu sheep. It could be concluded that Hu sheep has a low susceptibility to natural scrapie, and the predominant PRNP genotype at least has no significant effect on litter size

The climatic and genetic heritage of Italian goat breeds with genomic SNP data

Local adaptation of animals to the environment can abruptly become a burden when faced with rapid climatic changes such as those foreseen for the Italian peninsula over the next 70 years. Our study investigates the genetic structure of the Italian goat populations and links it with the environment and how genetics might evolve over the next 50 years. We used one of the largest national datasets including &gt; 1000 goats from 33 populations across the Italian peninsula collected by the Italian Goat Consortium and genotyped with over 50 k markers. Our results showed that Italian goats can be discriminated in three groups reflective of the Italian geography and its geo\u2011political situation preceding the country unification around two centuries ago. We leveraged the remarkable genetic and geographical diversity of the Italian goat populations and performed landscape genomics analysis to disentangle the relationship between genotype and environment, finding 64 SNPs intercepting genomic regions linked to growth, circadian rhythm, fertility, and inflammatory response. Lastly, we calculated the hypothetical future genotypic frequencies of the most relevant SNPs identified through landscape genomics to evaluate their long\u2011term effect on the genetic structure of the Italian goat populations. Our results provide an insight into the past and the future of the Italian local goat populations, helping the institutions in defining new conservation strategy plans that could preserve their diversity and their link to local realities challenged by climate change

LILRA2 Selectively Modulates LPS-Mediated Cytokine Production and Inhibits Phagocytosis by Monocytes

The activating immunoglobulin-like receptor, subfamily A, member 2 (LILRA2) is primarily expressed on the surface of cells of the innate immunity including monocytes, macrophages, neutrophils, basophils and eosinophils but not on lymphocytes and NK cells. LILRA2 cross-linking on monocytes induces pro-inflammatory cytokines while inhibiting dendritic cell differentiation and antigen presentation. A similar activating receptor, LILRA4, has been shown to modulate functions of TLR7/9 in dendritic cells. These suggest a selective immune regulatory role for LILRAs during innate immune responses. However, whether LILRA2 has functions distinct from other receptors of the innate immunity including Toll-like receptor (TLR) 4 and FcγRI remains unknown. Moreover, the effects of LILRA2 on TLR4 and FcγRI-mediated monocyte functions are not elucidated. Here, we show activation of monocytes via LILRA2 cross-linking selectively increased GM-CSF production but failed to induce IL-12 and MCP-1 production that were strongly up-regulated by LPS, suggesting functions distinct from TLR4. Interestingly, LILRA2 cross-linking on monocytes induced similar amounts of IL-6, IL-8, G-CSF and MIP-1α but lower levels of TNFα, IL-1β, IL-10 and IFNγ compared to those stimulated with LPS. Furthermore, cross-linking of LILRA2 on monocytes significantly decreased phagocytosis of IgG-coated micro-beads and serum opsonized Escherichia coli but had limited effect on phagocytosis of non-opsonized bacteria. Simultaneous co-stimulation of monocytes through LILRA2 and LPS or sequential activation of monocytes through LILRA2 followed by LPS led lower levels of TNFα, IL-1β and IL-12 production compared to LPS alone, but had additive effect on levels of IL-10 and IFNγ but not on IL-6. Interestingly, LILRA2 cross-linking on monocytes caused significant inhibition of TLR4 mRNA and protein, suggesting LILRA2-mediated suppression of LPS responses might be partly via regulation of this receptor. Taken together, we provide evidence that LILRA2-mediated activation of monocytes is significantly different to LPS and that LILRA2 selectively modulates LPS-mediated monocyte activation and FcγRI-dependent phagocytosis

Global assessment of genomic variation in cattle by genome resequencing and high-throughput genotyping

<p>Abstract</p> <p>Background</p> <p>Integration of genomic variation with phenotypic information is an effective approach for uncovering genotype-phenotype associations. This requires an accurate identification of the different types of variation in individual genomes.</p> <p>Results</p> <p>We report the integration of the whole genome sequence of a single Holstein Friesian bull with data from single nucleotide polymorphism (SNP) and comparative genomic hybridization (CGH) array technologies to determine a comprehensive spectrum of genomic variation. The performance of resequencing SNP detection was assessed by combining SNPs that were identified to be either in identity by descent (IBD) or in copy number variation (CNV) with results from SNP array genotyping. Coding insertions and deletions (indels) were found to be enriched for size in multiples of 3 and were located near the N- and C-termini of proteins. For larger indels, a combination of split-read and read-pair approaches proved to be complementary in finding different signatures. CNVs were identified on the basis of the depth of sequenced reads, and by using SNP and CGH arrays.</p> <p>Conclusions</p> <p>Our results provide high resolution mapping of diverse classes of genomic variation in an individual bovine genome and demonstrate that structural variation surpasses sequence variation as the main component of genomic variability. Better accuracy of SNP detection was achieved with little loss of sensitivity when algorithms that implemented mapping quality were used. IBD regions were found to be instrumental for calculating resequencing SNP accuracy, while SNP detection within CNVs tended to be less reliable. CNV discovery was affected dramatically by platform resolution and coverage biases. The combined data for this study showed that at a moderate level of sequencing coverage, an ensemble of platforms and tools can be applied together to maximize the accurate detection of sequence and structural variants.</p

Strengthening insights into host responses to mastitis infection in ruminants by combining heterogeneous microarray data sources

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling studies of mastitis in ruminants have provided key but fragmented knowledge for the understanding of the disease. A systematic combination of different expression profiling studies via meta-analysis techniques has the potential to test the extensibility of conclusions based on single studies. Using the program Pointillist, we performed meta-analysis of transcription-profiling data from six independent studies of infections with mammary gland pathogens, including samples from cattle challenged <it>in vivo </it>with <it>S. aureus</it>, <it>E. coli</it>, and <it>S. uberis</it>, samples from goats challenged <it>in vivo </it>with <it>S. aureus</it>, as well as cattle macrophages and ovine dendritic cells infected <it>in vitro </it>with <it>S. aureus</it>. We combined different time points from those studies, testing different responses to mastitis infection: overall (common signature), early stage, late stage, and cattle-specific.</p> <p>Results</p> <p>Ingenuity Pathway Analysis of affected genes showed that the four meta-analysis combinations share biological functions and pathways (e.g. protein ubiquitination and polyamine regulation) which are intrinsic to the general disease response. In the overall response, pathways related to immune response and inflammation, as well as biological functions related to lipid metabolism were altered. This latter observation is consistent with the milk fat content depression commonly observed during mastitis infection. Complementarities between early and late stage responses were found, with a prominence of metabolic and stress signals in the early stage and of the immune response related to the lipid metabolism in the late stage; both mechanisms apparently modulated by few genes, including <it>XBP1 </it>and <it>SREBF1</it>.</p> <p>The cattle-specific response was characterized by alteration of the immune response and by modification of lipid metabolism. Comparison of <it>E. coli </it>and <it>S. aureus </it>infections in cattle <it>in vivo </it>revealed that affected genes showing opposite regulation had the same altered biological functions and provided evidence that <it>E. coli </it>caused a stronger host response.</p> <p>Conclusions</p> <p>This meta-analysis approach reinforces previous findings but also reveals several novel themes, including the involvement of genes, biological functions, and pathways that were not identified in individual studies. As such, it provides an interesting proof of principle for future studies combining information from diverse heterogeneous sources.</p