FVB/NJ Research Articles

Mice with FVB-derived sequence on chromosome 17 succumb to disseminated virus infection due to aberrant NK cell and T cell responses

Zoonotic arenavirus infections can result in viral hemorrhagic disease, characterized by platelet loss, petechia, and multi-organ injury. The mechanisms governing these outcomes are likely impacted by virus strain and infection dose, as well as an individual's genetic background and immune constitution. To better understand the processes leading to severe pathogenesis, we compared two strains of inbred mice, C57BL/6J (B6) and FVB/NJ (FVB), that have diametrically opposed outcomes during disseminated lymphocytic choriomeningitis virus (LCMV) infection. Infection caused minimal pathogenesis in B6 mice, whereas FVB mice developed acute hepatitis and perished due, in part, to aberrant NK cell and Tcell responses. Susceptible mice showed an outgrowth of cytolytic CD4+ Tcells and loss of Treg cells. B6 congenic mice with the FVB allele at a 25Mb locus on chromosome 17 recapitulated FVB pathogenesis upon infection. A locus containing a limited number of variants in immune-related genes greatly impacts survival during infection.

Analysis of strain, sex, and diet-dependent modulation of gut microbiota reveals candidate keystone organisms driving microbial diversity in response to American and ketogenic diets

BackgroundThe gut microbiota is modulated by a combination of diet, host genetics, and sex effects. The magnitude of these effects and interactions among them is important to understanding inter-individual variability in gut microbiota. In a previous study, mouse strain-specific responses to American and ketogenic diets were observed along with several QTLs for metabolic traits. In the current study, we searched for genetic variants underlying differences in the gut microbiota in response to American and ketogenic diets, which are high in fat and vary in carbohydrate composition, between C57BL/6 J (B6) and FVB/NJ (FVB) mouse strains.ResultsGenetic mapping of microbial features revealed 18 loci under the QTL model (i.e., marginal effects that are not specific to diet or sex), 12 loci under the QTL by diet model, and 1 locus under the QTL by sex model. Multiple metabolic and microbial features map to the distal part of Chr 1 and Chr 16 along with eigenvectors extracted from principal coordinate analysis of measures of β-diversity. Bilophila, Ruminiclostridium 9, and Rikenella (Chr 1) were identified as sex- and diet-independent QTL candidate keystone organisms, and Parabacteroides (Chr 16) was identified as a diet-specific, candidate keystone organism in confirmatory factor analyses of traits mapping to these regions. For many microbial features, irrespective of which QTL model was used, diet or the interaction between diet and a genotype were the strongest predictors of the abundance of each microbial trait. Sex, while important to the analyses, was not as strong of a predictor for microbial abundances.ConclusionsThese results demonstrate that sex, diet, and genetic background have different magnitudes of effects on inter-individual differences in gut microbiota. Therefore, Precision Nutrition through the integration of genetic variation, microbiota, and sex affecting microbiota variation will be important to predict response to diets varying in carbohydrate composition.EsE1wzm1oyLRxL9o4MGhk7Video

Fat Mass Gained Prior to Dietary Intervention Predicts Efficacy of Personalized Dietary Guidance in Mice

ObjectivesIn previous studies into effects of carbohydrate restriction, we demonstrated differential responses to American and ketogenic diets in C57BL/6J (B6) and FVB/NJ (FVB) mice during lifetime feeding trials. These preliminary data suggested that both sex and genetic background regulated response to carbohydrate restriction. More recently, we have examined the utility of the ketogenic diet as a dietary intervention in obese B6 and FVB mice.MethodsFour-to six-week-old B6 and FVB mice were exposed to an American (35% of energy from fat, 50% from carbohydrates) diet for 3 months. After 3 months on the American diet, half of the animals were reversed to a ketogenic dietary intervention for the remainder of the 6-month feeding trial while the other half were left on the American diet.ResultsB6 females that received the ketogenic diet as a dietary intervention after 3 months of exposure to the American diet gained less fat mass than B6 females that remained on the American diet for the duration of the feeding trial (B6 female American: 15.94g, +/− 5.00g; B6 female Reversal: 9.67g, +/− 3.98g; p = 0.007, 95% CI: 1.91–10.63g). In contrast, B6 males exposed to the dietary intervention gained more fat mass than B6 males that remained on the American diet for the duration of the feeding trial (B6 male American: 19.69g, +/− 1.42g; B6 male Reversal: 23.86g, +/− 1.28g; P < 0.001, 95% CI: −5.43 – (−2.89) g). As expected, no effect was seen in either FVB males or females. The amount of fat mass gained on the American diet prior to the introduction of the dietary intervention was highly correlated to the amount of fat mass gained at the end of the feeding trial for all strain and diet combinations (r = 0.878, p < 0.001, 95% CI: 0.81- 0.92).ConclusionsThe strong relationship observed between the amount of fat mass gained prior to the introduction of the dietary intervention and the amount of fat mass gained at the end of the feeding trial suggests that there are mechanisms contributing to obesity that are not attenuated by simple dietary intervention, even within genetic backgrounds predicted to respond to carbohydrate restriction. Further studies are under way to investigate temporally controlled windows of opportunity for intervention as well as the effects of surgical removal of accumulated lipid on the plasticity of the obese phenotype.Funding SourcesNational Institutes of Health.

Contribution of Genetic Background to Vascular Adaptation to Exercise Training Based on Critical Speed of Different Intensities

An impaired endothelial function is a fundamental component of the pathogenesis of cardiovascular disease. In mice, exercise training is well‐known to improve cardiorespiratory fitness and endothelial function. However, the post exercise training effect on endothelial function varies among different strains of mice, suggesting that genetic background contributes to the heterogeneous effect of exercise on vascular health. NZW/LacJ (NZW) mice are known as relatively low responders to exercise training compared to high responder strains such as FVB/NJ (FVB). NZW mice also have relatively poor endothelial function in thoracic aorta when compared with other mouse strains. Therefore, we aimed to determine the contribution of genetic background to endothelial adaptation to exercise training in FVB and NZW mice. Vasoreactivity was assessed in femoral arteries from female mice from both strains (NZW and FVB) after 6 weeks of training using two different critical speed (CS) treadmill training intensities. Mice from each strain were randomly assigned to one of three groups: sedentary (SED), 80% of CS training (CS80), and 90% of CS (CS90) training. After training, endothelium‐dependent vasorelaxation to acetylcholine (ACh) and endothelium‐independent vasorelaxation to sodium nitroprusside (SNP) were measured as well as the vasocontractile responses to phenylephrine (PE) and potassium chloride (KCl). Maximal relaxation responses and IC50 for ACh and SNP were similar between NZW groups (SED, CS80, and CS90). In femoral arteries from FVB mice, there were no differences for maximal relaxation to ACh, but there was significant difference in IC50 for ACh‐induced vasorelaxation between SED vs. CS90 groups (p=0.05). Maximal responses to ACh were similar between NZW and FVB; however, IC50 was significantly lower in FVB compared with NZW, indicating that sensitivity to ACh is greater in arteries from FVB. There was a significant strain‐dependent difference for IC50 for SNP despite no strain differences for maximal responses. There was no effect of exercise or strain on contractile responses to PE and KCl, although the EC50 for KCl induced vasocontraction was higher (p=0.0329) in NZW. Treadmill exercise training at 80% or 90% CS did not contribute to improved vasomotor function in NZW while higher intensity exercise improved sensitivity to ACh in arteries from FVB. Genetic background did not significantly impact vasomotor function or endothelial responses to exercise training in FVB and NZW mice.

Response to Endurance Training Using Critical Speed: Influence of Genetic Background and Exercise Intensity

Exercise is used as a therapeutic option to improve an individual's quality of life or as a method to attenuate the severity of cardiovascular, respiratory, or metabolic‐related diseases. However, changes in cardiorespiratory fitness in response to endurance training are often heterogenous with individuals being more or less responsive to a similar exercise protocol. Heterogeneity in responses to exercise training is observed in humans and rodents. Low responses to training might be mitigated by modifying the standard exercise training protocol by altering the frequency, intensity, or duration of training. Threshold‐based exercise training prescription, e.g., critical speed, has also been proposed as a means to reduce the variability of training responses. The aim of this study was to identify the effect of different exercise training intensities on changes in exercise capacity in two inbred strains of mice, NZW/LacJ (NZW) and FVB/NJ (FVB). NZW mice were selected because they respond poorly to moderate intensity endurance exercise whereas, FVB mice respond well to endurance training. We hypothesized that increasing the intensity of the training would reduce the number of low or non‐responding NZW mice. To test this hypothesis, female mice from each strain were assigned to one of three treadmill training groups based on critical speed (CS): 80% of their critical speed (CS80%), 90% of their critical speed (CS90%), or sedentary controls (SED). Exercise groups within each strain were volume matched and thus ran the same distance over the 6‐week training period. Pre‐training exercise capacity differed between NZW (30.5 ± 3.5 min) and FVB (36.1 ± 2.8 min) mice (P &lt; 0.001). Critical speeds also differed between strains (NZW: 25.1 ± 2.4 m/min; FVB: 32.9 ± 1.6 m/min, P &lt; 0.001). After training, both FVB exercise groups, CS80% (10.0 ± 4.9 min, P = 0.004) and CS90% (14.0 ± 7.7 min, P = 0.007), improved their endurance capacity. In contrast, NZW CS80% did not significantly improve after training (0.4 ± 4.6 min), but NZW CS90% improved significantly (3.6 ± 2.7 min, P = 0.02). Collectively, these data indicate that the high‐responder FVB strain can adapt to moderate‐intensity and high‐intensity endurance training. The low‐responder NZW strain only showed significant improvement after sufficient homeostatic stress was induced through high‐intensity endurance training. These data suggest that increasing exercise intensity can improve responses to exercise training in low‐ or non‐responding mice.

B6 Mouse Strain: The Best Fit for LPS-Induced Interstitial Cystitis Model.

Interstitial cystitis (IC) is a chronic inflammatory disease characterized by bladder pain and increased urinary frequency. Although the C57BL/6J (B6) and FVB/NJ (FVB) mouse strains are commonly used as animal models for studies involving the urinary system, few reports have compared their lower urinary tract anatomy, despite the importance of such data. Our study aimed to characterize bladder function changes in FVB and B6 mouse strains with lipopolysaccharide (LPS)-induced IC, to understand mouse model-based bladder research. The bladder function parameters were measured by cystometrogram. Histological assay was examined by hematoxylin and eosin stain, Masson’s trichrome stain, and immunofluorescence staining. Results indicated that the two strains in the control group exhibited different bladder structures and functions, with significant anatomical differences, including a larger bladder size in the FVB than in the B6 strain. Furthermore, cystometry tests revealed differences in bladder function pressure. LPS-treated B6 mice presented significant changes in peak pressure, with decreased intercontraction intervals; these results were similar to symptoms of IC in humans. Each strain displayed distinct characteristics, emphasizing the care required in choosing the appropriate strain for bladder-model studies. The results suggested that the B6 mouse strain is more suitable for IC models.

Diabetic Embryopathy Susceptibility in Mice Is Associated with Differential Dependence on Glucosamine and Modulation of High Glucose-Induced Oxidative Stress.

The high KM glucose transporter, GLUT2 (SLC2A2), is expressed by embryos and causes high rates of glucose transport during maternal hyperglycemic episodes in diabetic pregnancies and causes congenital malformations (diabetic embryopathy). GLUT2 is also a low KM transporter of the amino sugar, glucosamine (GlcN), which enters the hexosamine biosynthetic pathway (HBP) and provides substrate for glycosylation reactions. Exogenous GlcN also increases activity of the pentose phosphate pathway (PPP), which increases production of NADPH reducing equivalents. GLUT2-transported GlcN is inhibited by high glucose concentrations. Not all mouse strains are susceptible to diabetic embryopathy. The aim of this study was to test the hypothesis that susceptibility to diabetic embryopathy is related to differential dependence on exogenous GlcN for glycosylation or stimulation of the PPP. We tested this using murine embryonic stem cell (ESC) lines that were derived from embryopathy-susceptible FVB/NJ (FVB), and embryopathy-resistant C57Bl/6J (B6), embryos in the presence of low or high glucose, and in the presence or absence of GlcN. There were no significant differences in Glut2 expression, or of glucose or GlcN transport, between FVB and B6 ESC. GlcN effects on growth and incorporation into glycoproteins indicated that FVB ESC are more dependent on exogenous GlcN than are B6 ESC. GlcN stimulated PPP activity in FVB but not in B6 ESC. High glucose induced oxidative stress in FVB ESC but not in B6 ESC. These results indicate that FVB embryos are more dependent on exogenous GlcN for glycosylation, but also for stimulation of the PPP and NADPH production, than are B6 embryos, thereby rendering FVB embryos more susceptible to high glucose to induce oxidative stress.

Sex-specific genetic architecture in response to American and ketogenic diets

Background/objectivesThere is a growing appreciation for individual responses to diet. In a previous study, mouse strain-specific responses to American and ketogenic diets were observed. In this study, we searched for genetic variants underlying differences in the responses to American and ketogenic diets between C57BL/6J (B6) and FVB/NJ (FVB) mouse strains.ResultsGenetic mapping of fat and lean mass gain revealed QTLs on Chromosome (Chr) 1 at 191.6 Mb (Fmgq1) (P < 0.001, CI = 180.2–194.4 Mb), Chr5 at 73.7 Mb (Fmgq2, Lmgq1) (P < 0.001, CI = 66.1–76.6 Mb), and Chr7 at 40.5 Mb (Fmgq3) (P < 0.01, CI = 36.6–44.5 Mb). Analysis of serum HDL cholesterol concentration identified a significant (P < 0.001, CI = 160.6–176.1 Mb) QTL on Chr1 at 168.6 Mb (Hdlq1). Causal network inference suggests that HDL cholesterol and fat mass gain are both linked to Fmgq1.ConclusionsStrong sex effects were identified at both Fmgq2 and Lmgq1, which are also diet-dependent. Interestingly, Fmgq2 and Fmgq3 affect fat gain directly, while Fmgq1 influences fat gain directly and via an intermediate change in serum cholesterol. These results demonstrate how precision nutrition will be advanced through the integration of genetic variation and sex in physiological responses to diets varied in carbohydrate composition.

Genetic and Pharmacological Manipulations of Glyoxalase 1 Mediate Ethanol Withdrawal Seizure Susceptibility in Mice.

Central nervous system (CNS) hyperexcitability is a clinically significant feature of acute ethanol withdrawal. There is evidence for a genetic contribution to withdrawal severity, but specific genetic risk factors have not been identified. The gene glyoxalase 1 (Glo1) has been previously implicated in ethanol consumption in mice, and GLO1 inhibition can attenuate drinking in mice and rats. Here, we investigated whether genetic and pharmacological manipulations of GLO1 activity can also mediate ethanol withdrawal seizure severity in mice. Mice from two transgenic lines overexpressing Glo1 on different genetic backgrounds (C57BL/6J (B6) and FVB/NJ (FVB)) were tested for handling-induced convulsions (HICs) as a measure of acute ethanol withdrawal. Following an injection of 4 g/kg alcohol, both B6 and FVB mice overexpressing Glo1 showed increases in HICs compared to wild-type littermates, though only the FVB line showed a statistically significant difference. We also administered daily ethanol injections (2 g/kg + 9 mg/kg 4-methylpyrazole) to wild-type B6 mice for 10 days and tested them for HICs on the 10th day following treatment with either a vehicle or a GLO1 inhibitor (S-bromobenzylglutathione cyclopentyl diester (pBBG)). Treatment with pBBG reduced HICs, although this effect was only statistically significant following two 10-day cycles of ethanol exposure and withdrawal. These results provide converging genetic and pharmacological evidence that GLO1 can mediate ethanol withdrawal seizure susceptibility.

Host Genetic Background and Gut Microbiota Contribute to Differential Metabolic Responses to Fructose Consumption in Mice

It is unclear how high fructose consumption induces disparate metabolic responses in genetically diverse mouse strains. We aimed to investigate whether the gut microbiota contributes to differential metabolic responses to fructose. Eight-week-old male C57BL/6J (B6), DBA/2J (DBA), and FVB/NJ (FVB) mice were given 8% fructose solution or regular water (control) for 12 wk. The gut microbiota composition in cecum and feces was analyzed using 16S ribosomal DNA sequencing, and permutational multivariate ANOVA (PERMANOVA) was used to compare community across mouse strains, treatments, and time points. Microbiota abundance was correlated with metabolic phenotypes and host gene expression in hypothalamus, liver, and adipose tissues using Biweight midcorrelation. To test the causal role of the gut microbiota in determining fructose response, we conducted fecal transplants from B6 to DBA mice and vice versa for 4 wk, as well as gavaged antibiotic-treated DBA mice with Akkermansia for 9 wk, accompanied with or without fructose treatment. Compared with B6 and FVB, DBA mice had significantly higher Firmicutes to Bacteroidetes ratio and lower baseline abundance of Akkermansia and S24-7 (P <0.05), accompanied by metabolic dysregulation after fructose consumption. Fructose altered specific microbial taxa in individual mouse strains, such as a 7.27-fold increase in Akkermansia in B6 and 0.374-fold change in Rikenellaceae in DBA (false discovery rate <5%), which demonstrated strain-specific correlations with host metabolic and transcriptomic phenotypes. Fecal transplant experiments indicated that B6 microbes conferred resistance to fructose-induced weight gain in DBA mice (F=43.1, P <0.001), and Akkermansia colonization abrogated the fructose-induced weight gain (F=17.8, P<0.001) and glycemic dysfunctions (F=11.8, P=0.004) in DBA mice. Our findings support that differential microbiota composition between mouse strains is partially responsible for host metabolic sensitivity to fructose, and that Akkermansia is a key bacterium that confers resistance to fructose-induced metabolic dysregulation.

Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.

The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.

Differential metabolic and multi-tissue transcriptomic responses to fructose consumption among genetically diverse mice

Understanding how individuals react differently to the same treatment is a major concern in precision medicine. Metabolic challenges such as the one posed by high fructose intake are important determinants of disease mechanisms. We embarked on studies to determine how fructose affects differential metabolic dysfunctions across genetically dissimilar mice, namely, C57BL/6 J (B6), DBA/2 J (DBA) and FVB/NJ (FVB), by integrating physiological and gene regulatory mechanisms. We report that fructose has strain-specific effects, involving tissue-specific gene regulatory cascades in hypothalamus, liver, and white adipose tissues. DBA mice showed the largest numbers of genes associated with adiposity, congruent with their highest susceptibility to adiposity gain and glucose intolerance across the three tissues. In contrast, B6 and FVB mainly exhibited cholesterol phenotypes, accompanying the largest number of adipose genes correlating with total cholesterol in B6, and liver genes correlating with LDL in FVB mice. Tissue-specific network modeling predicted strain-and tissue-specific regulators such as Fgf21 (DBA) and Lss (B6), which were subsequently validated in primary hepatocytes. Strain-specific fructose-responsive genes revealed susceptibility for human diseases such that genes in liver and adipose tissue in DBA showed strong enrichment for human type 2 diabetes and obesity traits. Liver and adipose genes in FVB were mostly related to lipid traits, and liver and adipose genes in B6 showed relevance to most cardiometabolic traits tested. Our results show that fructose induces gene regulatory pathways that are tissue specific and dependent on the genetic make-up, which may underlie interindividual variability in cardiometabolic responses to high fructose consumption.

Human IL12p80 and Neural Stem Cells Enhance Sciatic Nerve Regeneration

Background: We previously identified mouse interleukin-12p80 (mIL12p80, a homodimer of IL12p40) in the cell extracts of implanted poly(L-lactic acid) (PLA) nerve conduit combined with neural stem cells (NSCs) and showed that mIL12p80 enhances the functional recovery and nerve regeneration in mice. Here, we showed that human IL12p80 (hIL12p80) improves the sciatic nerve regeneration in mice. Methods: Implantation of hIL12p80 and NSCs into nerve conduit promotes repair of the sciatic nerve injury in mouse model. Findings: NSCs transplanted with hIL12p80 showed the best recovery than the other groups in sciatic functional index (SFI), compound muscle action potential (CMAP) and Rotarod analyses. In addition, the group with Conduit+NSC+hIL12p80 (CNI) outperformed the other groups in SFI and Rotarod tests beginning at the fourth-week post-surgery. Immunohistochemistry showed that the CNI group increases the diameter of newly regenerated nerve 98%. In vitro studies showed that hIL12p80 stimulates differentiation of mouse NSCs to oligodendrocyte lineages through phosphorylation of Stat3 at Y705. Further, implantation using poly(L-lactic-co-glycolic acid) (PLGA) nerve conduits (C 2.0 and C 2.1) showed better recovery in Rotarod test and CMAP than using PLA conduits (C 1.0) in FVB/NJ (FVB) mice. In C57BL/6J (B6) mice, the group with C 2.1+NSC+hIL12p80 (C 2.1NI) not only promotes sciatic functional recovery but also reduces experimental autotomy. Interpretation: These results suggest that hIL12p80 together with NSCs enhance the functional recovery and accelerate the regeneration of damaged nerves in mice. These findings could be used in devising potential strategies for nerve injury treatment. Funding Statement: This research is supported by National Health Research Institutes, Taiwan (CS-107-PP-02), and Ministry of Science and Technology, Taiwan (107-1901-01-19-03; 107-0324-01-19-03). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: In this study, all animal experimental procedures followed ethical guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Health Research Institutes (Protocol No. NHRI-IACUC-101067A).

Abstract 5146: Administration of Parabacteroides distasonis in the attenuation of colorectal tumorigenesis in a carcinogenic murine model of colorectal cancer

Abstract Colorectal cancer (CRC) is the second leading cause of related deaths in the United States. Interindividual differences in genetic background result in a complex and dynamic system between the gut microbiome and variable phenotypic response to diet as it relates to CRC risk. Previous research from our laboratory investigated CRC susceptibility in an azoxymethane (AOM) murine model using FVB/NJ (FVB) and C57Bl/6J (B6) mice fed a ketogenic (high fat, no carbohydrate) American (high fat, high carbohydrate) or standard (moderate fat, moderate carbohydrate) mouse chow. Tumor multiplicity was dependent on both strain and diet with a four-fold increase reported in FVB mice fed ketogenic diet relative to those fed standard chow. This response was not observed in FVB mice fed the American diet or in B6 mice on any of the three diets. A subsequent study that investigated the physiological response to diet in multiple genetic backgrounds revealed a decrease in the abundance of the bacterial species Parabacteroides distasonis in FVB mice fed a ketogenic diet. This established an emphasis on the interaction between variable response to diet, gut microbiome and further implications to gut health. To further investigate this relationship in FVB mice we are using an AOM-induced carcinogenic model on FVB mice fed a ketogenic diet supplemented with a biweekly gavage of P. distasonis. Upon necropsy, tumor load of mice receiving the experimental gavage will be compared to the control mice. Droplet digital PCR will be used to quantify the absolute abundance of P. distasonis in fecal samples at several time points before and after the initiation of diet and administration of AOM. Ultimately, we aim to determine the relationship between P. distasonis and CRC tumorigenesis, which will lead to a further understanding of the etiology of colorectal cancer. Citation Format: Camilo Anthony G. Gacasan. Administration of Parabacteroides distasonis in the attenuation of colorectal tumorigenesis in a carcinogenic murine model of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5146.

Differential effects of high fat diet and diet-induced obesity on skeletal acquisition in female C57BL/6J vs. FVB/NJ Mice

The effects of obesity on bone metabolism are complex, and may be mediated by consumption of a high fat diet and/or by obesity-induced metabolic dysregulation. To test the hypothesis that both high fat (HF) diet and diet-induced metabolic disease independently decrease skeletal acquisition, we compared effects of HF diet on bone mass and microarchitecture in two mouse strains: diet-induced obesity (DIO)-susceptible C57BL/6J (B6) and DIO-resistant FVB/NJ (FVB). At 3 wks of age we weaned 120 female FVB and B6 mice onto normal (N, 10% Kcal/fat) or HF diet (45% Kcal/fat) and euthanized them at 6, 12 and 20 weeks of age (N = 10/grp). Outcomes included body mass; percent fat and whole-body bone mineral density (WBBMD, g/cm2) via DXA; cortical and trabecular bone architecture at the midshaft and distal femur via μCT; and marrow adiposity via histomorphometry. In FVB HF, body mass, percent body fat, WBBMD and marrow adiposity did not differ vs. N, but trabecular bone mass was lower at 6 wks of age only (p < 0.05), cortical bone geometric properties were lower at 12 wks only, and bone strength was lower at 20 wks of age only in HF vs. N (p < 0.05). In contrast, B6 HF had higher body mass, percent body fat, and leptin vs. N. B6 HF also had higher WBBMD (p < 0.05) at 9 and 12 wks of age but lower distal femur trabecular bone mass at 12 wks of age, and lower body mass-adjusted cortical bone properties at 20 wks of age compared to N (p < 0.05). Marrow adiposity was also markedly higher in B6 HF vs. N. Overall, HF diet negatively affected bone mass in both strains, but was more deleterious to trabecular bone microarchitecture and marrow adiposity in B6 than in FVB mice. These data suggest that in addition to fat consumption itself, the metabolic response to high fat diet independently alters skeletal acquisition in obesity.

Genetic and pharmacological manipulation of glyoxalase 1 regulates voluntary ethanol consumption in mice.

Previous studies have identified an association between the gene glyoxalase 1 (Glo1) and anxiety-like behavior in mice and have shown that the substrate of GLO1, methylglyoxal, is a competitive partial agonist at GABAA receptors. Given the well-established role of GABAA receptors in the behavioral effects of ethanol (EtOH), we investigated the role of Glo1 in voluntary EtOH consumption in mice using the drinking in the dark (DID) paradigm. Transgenic mice overexpressing Glo1 on both FVB/NJ (FVB) or C57BL/6J (B6) backgrounds showed increased voluntary EtOH consumption compared to their wild-type littermates in DID. Furthermore, transgenic Glo1 knockdown mice on a B6 background showed decreased voluntary EtOH consumption in DID. These genetic manipulations of Glo1 had no effect on sucrose, saccharin or water consumption. Finally, we found that a small molecule GLO1 inhibitor (S-bromobenzylglutathione cyclopentyl diester (pBBG; 6.25, 12.5 mg/kg)) reduced EtOH consumption compared to vehicle treated B6 mice without altering saccharin or water consumption. Sucrose consumption was only reduced by the higher (12.5 mg/kg) dose of pBBG. We did not observe differences in the loss of righting reflex (LORR) or EtOH-induced foot slips on the balance beam in response to acute EtOH administration (LORR: 4 g/kg, Balance Beam: 1.25 g/kg) in B6 or FVB mice overexpressing Glo1, nor in B6 mice treated with pBBG. These data are the first to implicate Glo1 in EtOH-related behaviors and suggest that GLO1 inhibitors may have therapeutic potential for the treatment of alcohol use disorders.

Aberrant IgG isotype generation in mice with abnormal behaviors

BTBR T+tf/J (BTBR) mice were recently cited as a suitable animal model for the study of autism because of their behavioral characteristics and immunological changes similar to those reported from autistic subjects. The BTBR mouse was reported to have significantly higher levels of serum IgG, brain IgG deposits and anti-brain IgG than highly social C57BL/6 mice, suggesting involvement of aberrant immune responses in the occurrence of autism. Up-regulation of IgG production was investigated here, with a focus on the pattern of IgG isotype distribution compared with that in FVB/NJ (FVB) mice, another highly social control strain. The results indicated that levels of serum IgG1, IgG2b and IgG3 in post-natal day 21 BTBR mice was significantly higher than FVB mice, regardless of sex, resulting in higher IgG1:IgG2a ratios in BTBR mice than in FVB mice (statistical significance in males). A similar outcome regarding the IgG1:IgG2a ratio was observed in culture supernatants of bone marrow cells from these hosts. A presence of brain-reactive IgG in the sera of BTBR was higher than in FVB mice; levels of brain-reactive IgG against whole brain homogenates were higher in BTBR than in FVB mice, with significant differences seen in the striatum and substantia nigra regions. Levels of IgG1 deposited in the cerebellum, cortex, hippocampus or striatum of both BTBR male and female mice were significantly higher than in FVB counterparts. Overall, these results suggest that alterations in IgG isotype production or deposition in the brain could be implicated in the aberrant immune reactivities of BTBR mice.

Distinct Genetic Loci Are Associated with Platelet Aggregation Responses to Collagen and Collagen Synthetic Related Peptide (SRP) in Mice

There is substantial inter-individual variation in responsiveness of human platelets to different agonists in platelet aggregation studies. Much of this variation is likely heritable, and some of this variation can be attributed to genetic variants in platelet agonist receptors. However, beyond variants in known candidate receptor genes, the genetic factors underlying variation in platelet aggregation are undefined. In this study, we are taking a genome wide approach to identify genetic factors associated with platelet aggregation responses to different agonists in mice. We developed a method to quantify platelet aggregation responses to multiple agonists in single mouse donors and tested platelets from C57BL6/J (B6) and FVB/NJ (FVB) inbred mouse strains for their patterns of response to collagen (type I), collagen synthetic reactive peptide (SRP), the thromboxane A2 (TxA2) mimetic U44069, and PAR-4 activating peptide (AYPGKF) (B6 and FVB, n=9 for each strain). The platelet aggregation response was then quantified as an area under the aggregation curve (AUC) for each agonist. There was a significant difference between B6 and FVB in platelet responsiveness to collagen (9.06 x 103 ± 1.44 x 103 vs 3.7 x 103 ± 1.77 x 103. p<0.018), collagen SRP (2.04 x 103 ± 1.31 x 103 vs 1.25 x 104 ± 1.55 x 103. p<5 x 10-5), and U44069 (1.2 x 104 ± 2.19 x 103 vs 6.47 x 103 ± 1.85 x 103. p<0.05), respectively. No difference was observed in response to PAR-4 AP (7.07 x 103 ± 6.92 x 102 vs 8.24 x 103 ± 1.25 x 103. p=0.2). We then performed a B6 x FVB F2 intercross which generated 152 mice which were phenotyped for platelet aggregation responsiveness to collagen, collagen SRP, and U44069, using AYPGKF as a positive control. Thus far, we have performed genome-wide association studies in 54 mice using a 1449 SNP Illumina BeadChip platform. Quantitative trait loci (QTL) analysis was performed to identify QTLs associated with platelet responsiveness to collagen, SRP, and U44069. In this interim analysis, we have identified several significant QTL for platelet aggregation response to collagen and SRP. A single highly significant QTL for collagen SRP was identified on proximal chromosome 7 (LOD = 8.26) that spans ~23 Mb (5’ end to rs6239372), termed Plsrp1. Additionally, statistically significant but broad QTLs were identified for collagen on chromosome 14 (Plcoll1 and Plcoll2, peak LOD scores 3.78 and 3.87, respectively), with additional suggestive regions on chromosome 10 and chromosome 7. Intriguingly, the QTL with the strongest associations for collagen on chromosome 14 (Plcoll1 and Plcoll2) are necessarily distinct from the single collagen SRP QTL (Plsrp1) on chromosome 7. Interestingly, Plcoll1 and Plcoll2 would have been suspected from previous studies of collagen responsiveness in mice as chromosome 14 does not harbor the major candidate genes thought to interact with collagen, in contrast to Plsrp1 which contains Gp6. These findings indicate distinct and previously unsuspected genetic elements underlie the differences in platelet responsiveness to collagen and collagen SRP in mice. DisclosuresNo relevant conflicts of interest to declare.

Abstract 477: HDL Inhibition of Murine Atherosclerosis Lesion Development is Dependent on Strain Background

Inbred mouse strains vary greatly in their relative atherosensitivity, with C57BL/6 (C57) being the most sensitive and hence the preferred strain for most atherosclerosis studies. Among the resistant strains, FVBN/J (FVB) has been relatively well studied. On the ApoE knockout (E°) background, both mouse strains develop atherosclerotic lesions which are exacerbated by feeding a high fat/high cholesterol western-type diet (WTD). However, the FVB-E° mouse exhibits nearly 10-fold smaller lesions than the C57-E°. Additionally, FVB mice have plasma HDL cholesterol levels that are twice as high as C57. Wild type FVB mice have been shown to have lower inflammatory cell recruitment to the peritoneum upon thioglycollate treatment than C57. Our lab has generated ApoE/ApoA-I double knockout mice (EA°, lacking HDL) on both C57 and FVB background to compare the effect of HDL deficiency on lesion development. We hypothesize that the loss of HDL will have a greater effect on the more lesion susceptible C57 than the resistant FVB. 8 week C57 and FVB E° and EA° mice were kept on a WTD for 6 or 10 weeks and lesions were quantified in the innominate artery, ascending aorta, and aortic root. In all cases mice in the C57 background displayed significantly greater lesion size than mice in the FVB background, except in the ascending aorta of the E° mice at the earlier time point, despite higher plasma cholesterol levels in the FVB mice. The presence of ApoA-I was protective (E° vs EA°) in the C57 strain in the ascending aorta at both time points and its protection became significant in the innominate artery at the 10 week time point. No additional protection was afforded by ApoA-I in the FVB strain. ApoA-I deficiency in FVB mice did not affect inflammatory cell recruitment to the peritoneal cavity or plasma markers of LDL oxidation as greatly as in C57 mice. Plasma monocyte counts both before and after 6 weeks on WTD were higher in C57 mice with no effect of ApoA-I in either strain. In conclusion, the FVB strain is less sensitive than C57 to lesion development at 3 major arterial sites with no protection afforded by HDL/ApoA-I, whereas HDL/ApoA-I is significantly protective in the C57 strain. This protection may result from the anti-inflammatory/anti-oxidative nature of C57 HDL that is not observed in FVB mice.

Cocaine locomotor activation, sensitization and place preference in six inbred strains of mice

BackgroundThe expanding set of genomics tools available for inbred mouse strains has renewed interest in phenotyping larger sets of strains. The present study aims to explore phenotypic variability among six commonly-used inbred mouse strains to both the rewarding and locomotor stimulating effects of cocaine in a place conditioning task, including several strains or substrains that have not yet been characterized for some or all of these behaviors.MethodsC57BL/6J (B6), BALB/cJ (BALB), C3H/HeJ (C3H), DBA/2J (D2), FVB/NJ (FVB) and 129S1/SvImJ (129) mice were tested for conditioned place preference to 20 mg/kg cocaine.ResultsPlace preference was observed in most strains with the exception of D2 and 129. All strains showed a marked increase in locomotor activity in response to cocaine. In BALB mice, however, locomotor activation was context-dependent. Locomotor sensitization to repeated exposure to cocaine was most significant in 129 and D2 mice but was absent in FVB mice.ConclusionsGenetic correlations suggest that no significant correlation between conditioned place preference, acute locomotor activation, and locomotor sensitization exists among these strains indicating that separate mechanisms underlie the psychomotor and rewarding effects of cocaine.