Chicken Immune Cell Assay to Model Adaptive Immune Responses In Vitro

Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, will allow the development of targeted nutrition strategies, prevent infectious diseases and the usage of antimicrobials, and promote the health of animals. To investigate the mechanisms of action of immunomodulating compounds, controlled in vitro systems using freshly isolated immune cells from blood represent a promising alternative to animal experiments. Immune cell isolation from the blood of chickens is a complex and difficult process since the immune cell fractions are significantly contaminated with red blood cells and platelets. To our knowledge, a robust protocol for immune cell isolation from chicken blood and the subsequent cultivation of immune cells is not available. Here, we established a protocol for blood sampling and immune cell isolation and cultivation from chicken blood, which could be applied for the investigation of direct effects of immunomodulating compounds. This protocol, combining different techniques of immune cell isolation, cultivation, and differentiation of distinct immune cell populations, will serve as a potential alternative to animal testing in vivo. By gaining knowledge about the mechanisms of action of immunomodulating compounds, this in vitro model will contribute to promote health and welfare in chicken farming. Abstract Knowledge about the modes of action of immunomodulating compounds such as pathogens, drugs, or feed additives, e.g., probiotics, gained through controlled but animal-related in vitro systems using primary cultured peripheral blood mononuclear cells (PBMCs) will allow the development of targeted nutrition strategies. Moreover, it could contribute to the prevention of infectious diseases and the usage of antimicrobials, and further promote the health of the animals. However, to our knowledge, a protocol for the isolation of PBMCs with reduced thrombocyte count from chicken blood and subsequent cell culture over several days to assess the effects of immunomodulating compounds is not available. Therefore, we established an optimized protocol for blood sampling and immune cell isolation, culture, and phenotyping for chicken PBMCs. For blood sampling commercial Na–citrate tubes revealed the highest count of vital cells compared to commercial Li–heparin (p < 0.01) and K3EDTA (p < 0.05) tubes. Using combined dextran and ficoll density gradient separation, the thrombocyte count was significantly reduced (p < 0.01) compared to slow-speed centrifugation with subsequent ficoll. For cell culture, the supplementation of RPMI-1640 medium with 10% chicken serum resulted in the lowest relative cell count of thrombocytes compared to fetal calf serum (FCS) (p < 0.05). To validate the ability of the cell culture system to respond to stimuli, concanavalin A (conA) was used as a positive control. The optimized protocol allows the isolation and cultivation of vital PBMCs with reduced thrombocyte count from chicken blood for subsequent investigation of the modes of action of immunomodulating compounds.Peer Reviewe

A 5′ UTR Mutation Contributes to Down-Regulation of Bbs7 in the Berlin Fat Mouse

The Bardet–Biedl Syndrome 7 (Bbs7) gene was identified as the most likely candidate gene causing juvenile obesity in the Berlin Fat Mouse Inbred (BFMI) line. Bbs7 expression is significantly lower in the brain, adipose tissue, and liver of BFMI mice compared to lean C57BL/6NCrl (B6N) mice. A DNA sequence comparison between BFMI and B6N revealed 16 sequence variants in the Bbs7 promoter region. Here, we tested if these mutations contribute to the observed differential expression of Bbs7. In a cell-based dual-luciferase assay, we compared the effects of the BFMI and the B6N haplotypes of different regions of the Bbs7 promotor on the reporter gene expression. A single-nucleotide polymorphism (SNP) was identified causing a significant reduction in the reporter gene expression. This SNP (rs29947545) is located in the 5′ UTR of Bbs7 at Chr3:36.613.350. The SNP is not unique to BFMI mice but also occurs in several other mouse strains, where the BFMI allele is not associated with lower Bbs7 transcript amounts. Thus, we suggest a compensatory mutation in the other mouse strains that keeps Bbs7 expression at the normal level. This compensatory mechanism is missing in BFMI mice and the cell lines tested

QTL-mapping in the obese Berlin Fat Mouse identifies additional candidate genes for obesity and fatty liver disease

The Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and the metabolic syndrome. This study aimed to identify genetic variants associated with liver weight, liver triglycerides, and body weight using the obese BFMI sub-line BFMI861-S1. BFMI861-S1 mice are insulin resistant and store ectopic fat in the liver. In generation 10, 58 males and 65 females of the advanced intercross line (AIL) BFMI861-S1xB6N were phenotyped under a standard diet over 20 weeks. QTL analysis was performed after genotyping with the MiniMUGA Genotyping Array. Whole-genome sequencing and gene expression data of the parental lines was used for the prioritization of positional candidate genes. Three QTLs associated with liver weight, body weight, and subcutaneous adipose tissue (scAT) weight were identified. A highly significant QTL on chromosome (Chr) 1 (157–168 Mb) showed an association with liver weight. A QTL for body weight at 20 weeks was found on Chr 3 (34.1–40 Mb) overlapping with a QTL for scAT weight. In a multiple QTL mapping approach, an additional QTL affecting body weight at 16 weeks was identified on Chr 6 (9.5–26.1 Mb). Considering sequence variants and expression differences, Sec16b and Astn1 were prioritized as top positional candidate genes for the liver weight QTL on Chr 1; Met and Ica1 for the body weight QTL on Chr 6. Interestingly, all top candidate genes have previously been linked with metabolic traits. This study shows once more the power of an advanced intercross line for fine mapping. QTL mapping combined with a detailed prioritization approach allowed us to identify additional and plausible candidate genes linked to metabolic traits in the BFMI861-S1xB6N AIL. By reidentifying known candidate genes in a different crossing population the causal link with specific traits is underlined and additional evidence is given for further investigations

Whole-Genome Sequencing Data Reveal New Loci Affecting Milk Production in German Black Pied Cattle (DSN)

German Black Pied (DSN) is considered an ancestral population of the Holstein breed. The goal of the current study was to fine-map genomic loci for milk production traits and to provide sequence variants for selection. We studied genome-wide associations for milk-production traits in 2160 DSN cows. Using 11.7 million variants from whole-genome sequencing of 304 representative DSN cattle, we identified 1980 associated variants (−log10(p) ≥ 7.1) in 13 genomic loci on 9 chromosomes. The highest significance was found for the MGST1 region affecting milk fat content (−log10(p) = 11.93, MAF = 0.23, substitution effect of the minor allele (ßMA) = −0.151%). Different from Holstein, DGAT1 was fixed (0.97) for the alanine protein variant for high milk and protein yield. A key gene affecting protein content was CSN1S1 (−log10(p) = 8.47, MAF = 049, ßMA = −0.055%) and the GNG2 region (−log10(p) = 10.48, MAF = 0.34, ßMA = 0.054%). Additionally, we suggest the importance of FGF12 for protein and fat yield, HTR3C for milk yield, TLE4 for milk and protein yield, and TNKS for milk and fat yield. Selection for favored alleles can improve milk yield and composition. With respect to maintaining the dual-purpose type of DSN, unfavored linkage to genes affecting muscularity has to be investigated carefully, before the milk-associated variants can be applied for selection in the small population.Federal Ministry of Food and Agriculture (BMEL)Peer Reviewe

Transmission distortion and genetic incompatibilities between alleles in a multigenerational mouse advanced intercross line

While direct additive and dominance effects on complex traits have been mapped repeatedly, additional genetic factors contributing to the heterogeneity of complex traits have been scarcely investigated. To assess genetic background effects, we investigated transmission ratio distortions (TRDs) of alleles from parent to offspring using an advanced intercross line (AIL) of an initial cross between the mouse inbred strains C57BL/6NCrl (B6N) and BFMI860-12 [Berlin Fat Mouse Inbred (BFMI)]. A total of 341 males of generation 28 and their respective 61 parents and 66 grandparents were genotyped using Mega Mouse Universal Genotyping Arrays. TRDs were investigated using allele transmission asymmetry tests, and pathway overrepresentation analysis was performed. Sequencing data were used to test for overrepresentation of nonsynonymous SNPs (nsSNPs) in TRD regions. Genetic incompatibilities were tested using the Bateson–Dobzhansky–Muller two-locus model. A total of 62 TRD regions were detected, many in close proximity to the telocentric centromere. TRD regions contained 44.5% more nsSNPs than randomly selected regions (182 vs 125.9 6 17.0, P < 1 X 10-4). Testing for genetic incompatibilities between TRD regions identified 29 genome-wide significant incompatibilities between TRD regions [P(BF) < 0.05]. Pathway overrepresentation analysis of genes in TRD regions showed that DNA methylation, epigenetic regulation of RNA, and meiotic/meiosis regulation pathways were affected independent of the parental origin of the TRD. Paternal BFMI TRD regions showed overrepresentation in the small interfering RNA biogenesis and in the metabolism of lipids and lipoproteins. Maternal B6N TRD regions harbored genes involved in meiotic recombination, cell death, and apoptosis pathways. The analysis of genes in TRD regions suggests the potential distortion of protein–protein interactions influencing obesity and diabetic retinopathy as a result of disadvantageous combinations of allelic variants in Aass, Pgx6, and Nme8. Using an AIL significantly improves the resolution at which we can investigate TRD. Our analysis implicates distortion of protein–protein interactions as well as meiotic drive as the underlying mechanisms leading to the observed TRD in our AIL. Furthermore, genes with large amounts of nsSNPs located in TRD regions are more likely to be involved in pathways that are related to the phenotypic differences between the parental strains. Genes in these TRD regions provide new targets for investigating genetic adaptation, protein–protein interactions, and determinants of complex traits such as obesity

A deletion containing a CTCF-element in intron 8 of the Bbs7 gene is partially responsible for juvenile obesity in the Berlin Fat Mouse

The Berlin Fat Mouse Inbred (BFMI) line is a model for juvenile obesity. Previous studies on crosses between BFMI and C57Bl/6N (B6N) have identified a recessive defect causing juvenile obesity on chromosome 3 (jObes1). Bbs7 was identified as the most likely candidate gene for the observed effect. Comparative sequence analysis showed a 1578 bp deletion in intron 8 of Bbs7 in BFMI mice. A CTCF-element is located inside this deletion. To investigate the functional effect of this deletion, it was introduced into B6N mice using CRISPR/Cas9. Two mice containing the target deletion were obtained (B6N Bbs7emI8∆1 and Bbs7emI8∆2) and were subsequently mated to BFMI and B6N to generate two families suitable for complementation. Inherited alleles were determined and body composition was measured by quantitative magnetic resonance. Evidence for a partial complementation (13.1–15.1%) of the jObes1 allele by the CRISPR/Cas9 modified B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles was found. Mice carrying the complementation alleles had a 23–27% higher fat-to-lean ratio compared to animals which have a B6N allele (P(Bbs7emI8∆1) = 4.25 × 10–7; P(Bbs7emI8∆2) = 3.17 × 10–5). Consistent with previous findings, the recessive effect of the BFMI allele was also seen for the B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles. However, the effect size of the B6N Bbs7emI8∆1 and Bbs7emI8∆2 alleles was smaller than the BFMI allele, and thus showed only a partial complementation. Findings suggest additional variants near Bbs7 in addition to or interacting with the deletion in intron 8

Identification of four novel QTL linked to the metabolic syndrome in the Berlin Fat Mouse

Background: The Berlin Fat Mouse Inbred line (BFMI) is a model for obesity and the metabolic syndrome. This study aimed to identify genetic variants associated with impaired glucose metabolism using the obese lines BFMI861-S1 and BFMI861-S2, which are genetically closely related, but differ in several traits. BFMI861-S1 is insulin resistant and stores ectopic fat in the liver, whereas BFMI861-S2 is insulin sensitive. Methods: In generation 10, 397 males of an advanced intercross line (AIL) BFMI861-S1 × BFMI861-S2 were challenged with a high-fat, high-carbohydrate diet and phenotyped over 25 weeks. QTL-analysis was performed after selective genotyping of 200 mice using the GigaMUGA Genotyping Array. Additional 197 males were genotyped for 7 top SNPs in QTL regions. For the prioritization of positional candidate genes whole genome sequencing and gene expression data of the parental lines were used. Results: Overlapping QTL for gonadal adipose tissue weight and blood glucose concentration were detected on chromosome (Chr) 3 (95.8–100.1 Mb), and for gonadal adipose tissue weight, liver weight, and blood glucose concentration on Chr 17 (9.5–26.1 Mb). Causal modeling suggested for Chr 3-QTL direct effects on adipose tissue weight, but indirect effects on blood glucose concentration. Direct effects on adipose tissue weight, liver weight, and blood glucose concentration were suggested for Chr 17-QTL. Prioritized positional candidate genes for the identified QTL were Notch2 and Fmo5 (Chr 3) and Plg and Acat2 (Chr 17). Two additional QTL were detected for gonadal adipose tissue weight on Chr 15 (67.9–74.6 Mb) and for body weight on Chr 16 (3.9–21.4 Mb). Conclusions: QTL mapping together with a detailed prioritization approach allowed us to identify candidate genes associated with traits of the metabolic syndrome. In addition, we provided evidence for direct and indirect genetic effects on blood glucose concentration in the insulin-resistant mouse line BFMI861-S1

Modelling the Species Distribution of Flat-Headed Cats (Prionailurus planiceps), an Endangered South-East Asian Small Felid

Background: The flat-headed cat (Prionailurus planiceps) is one of the world’s least known, highly threatened felids with a distribution restricted to tropical lowland rainforests in Peninsular Thailand/Malaysia, Borneo and Sumatra. Throughout its geographic range large-scale anthropogenic transformation processes, including the pollution of fresh-water river systems and landscape fragmentation, raise concerns regarding its conservation status. Despite an increasing number of cameratrapping field surveys for carnivores in South-East Asia during the past two decades, few of these studies recorded the flatheaded cat. Methodology/Principal Findings: In this study, we designed a predictive species distribution model using the Maximum Entropy (MaxEnt) algorithm to reassess the potential current distribution and conservation status of the flat-headed cat. Eighty-eight independent species occurrence records were gathered from field surveys, literature records, and museum collections. These current and historical records were analysed in relation to bioclimatic variables (WorldClim), altitude (SRTM) and minimum distance to larger water resources (Digital Chart of the World). Distance to water was identified as the key predictor for the occurrence of flat-headed cats (.50% explanation). In addition, we used different land cover maps (GLC2000, GlobCover and SarVision LLC for Borneo), information on protected areas and regional human population density data to extract suitable habitats from the potential distribution predicted by the MaxEnt model. Between 54% and 68% of suitable habitat has already been converted to unsuitable land cover types (e.g. croplands, plantations), and only between 10% and 20% of suitable land cover is categorised as fully protected according to the IUCN criteria. The remaining habitats are highly fragmented and only a few larger forest patches remain. Conclusion/Significance: Based on our findings, we recommend that future conservation efforts for the flat-headed cat should focus on the identified remaining key localities and be implemented through a continuous dialogue between local stakeholders, conservationists and scientists to ensure its long-term survival. The flat-headed cat can serve as a flagship species for the protection of several other endangered species associated with the threatened tropical lowland forests and surface fresh-water sources in this region

The GTPase ARFRP1 controls the lipidation of chylomicrons in the Golgi of the intestinal epithelium

The uptake and processing of dietary lipids by the small intestine is a multistep process that involves several steps including vesicular and protein transport. The GTPase ADP-ribosylation factor-related protein 1 (ARFRP1) controls the ARF-like 1 (ARL1)-mediated Golgi recruitment of GRIP domain proteins which in turn bind several Rab-GTPases. Here, we describe the essential role of ARFRP1 and its interaction with Rab2 in the assembly and lipidation of chylomicrons in the intestinal epithelium. Mice lacking Arfrp1 specifically in the intestine (Arfrp1vil−/−) exhibit an early post-natal growth retardation with reduced plasma triacylglycerol and free fatty acid concentrations. Arfrp1vil−/− enterocytes as well as Arfrp1 mRNA depleted Caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triacylglycerol content. In addition, the release of apolipoprotein A-I (ApoA-I) was dramatically decreased, and ApoA-I accumulated in the Arfrp1vil−/− epithelium, where it predominantly co-localized with Rab2. The release of chylomicrons from Caco-2 was markedly reduced after the suppression of Rab2, ARL1 and Golgin-245. Thus, the GTPase ARFRP1 and its downstream proteins are required for the lipidation of chylo­microns and the assembly of ApoA-I to these particles in the Golgi of intestinal epithelial cells

Non-Invasive Quantification of White and Brown Adipose Tissues and Liver Fat Content by Computed Tomography in Mice

OBJECTIVES: Obesity and its distribution pattern are important factors for the prediction of the onset of diabetes in humans. Since several mouse models are suitable to study the pathophysiology of type 2 diabetes the aim was to validate a novel computed tomograph model (Aloka-Hitachi LCT-200) for the quantification of visceral, subcutaneous, brown and intrahepatic fat depots in mice. METHODS: Different lean and obese mouse models (C57BL/6, B6.V-Lep(ob), NZO) were used to determine the most adequate scanning parameters for the detection of the different fat depots. The data were compared with those obtained after preparation and weighing the fat depots. Liver fat content was determined by biochemical analysis. RESULTS: The correlations between weights of fat tissues on scale and weights determined by CT were significant for subcutaneous (r(2) = 0.995), visceral (r(2) = 0.990) and total white adipose tissue (r(2) = 0.992). Moreover, scans in the abdominal region, between lumbar vertebrae L4 to L5 correlated with whole-body fat distribution allowing experimenters to reduce scanning time and animal exposure to radiation and anesthesia. Test-retest reliability and measurements conducted by different experimenters showed a high reproducibility in the obtained results. Intrahepatic fat content estimated by CT was linearly related to biochemical analysis (r(2) = 0.915). Furthermore, brown fat mass correlated well with weighted brown fat depots (r(2) = 0.952). In addition, short-term cold-expose (4 °C, 4 hours) led to alterations in brown adipose tissue attributed to a reduction in triglyceride content that can be visualized as an increase in Hounsfield units by CT imaging. CONCLUSION: The 3D imaging of fat by CT provides reliable results in the quantification of total, visceral, subcutaneous, brown and intrahepatic fat in mice. This non-invasive method allows the conduction of longitudinal studies of obesity in mice and therefore enables experimenters to investigate the onset of complex diseases such as diabetes and obesity