Parental B6 Research Articles

Studies of C57BL/6J Mice Deficient in Receptors for IFNα/β and IFNγ as a Model for EV-D68 Acute Flaccid Myelitis

AbstractEnterovirus D68 (EV-D68) causes severe respiratory disease and is a strong candidate as the etiologic agent of acute flaccid myelitis. Acute flaccid myelitis is a rare neurologic disorder that typically affects adolescents less than 13 y old, manifesting as muscle paralysis or weakness. No approved antiviral compounds or vaccines exist for EV-D68. An appropriate mouse model is needed to develop therapeutics. We previously showed that the AG129 mouse strain, which has a double knockout of Ifnar1 and Ifngr1 on the 129/Sv genetic background, exhibits clear clinical signs when infected intraperitoneally with EV-D68 at postnatal day 10. The AGB6 strain, which has the same double knockout, but on the C57BL/6 (B6) genetic background, is more sensitive to dengue virus infections relative to AG129. Given the widespread use of the B6 background in mouse research, we systematically assessed EV-D68 infection in AGB6, the AB6 and GB6 strains, which have single knockouts in Ifnar1 and Ifngr1, respectively, and the parental B6 strain. AGB6 was highly susceptible to EV-D68, equivalent to AG129. No differences in susceptibility were observed between AGB6 and AB6 and GB6 and B6, suggesting that the IFNγ receptor contributes little to EV-D68 protection. Because B6 is the genetic background for many genetically modified mice, AGB6 facilitates the evaluation of the role of host genes in EV-D68 pathogenesis through genetic crosses. We introduced the knockout allele of Setd3, a gene essential for enterovirus RNA replication, into AGB6. While EV-D68 lethality was completely penetrant in AGB6-Setd3+/+ and AGB6-Setd3+/− mice, all AGB6-Setd3−/− littermates survived until the experimental endpoint. We conclude that AGB6 is well suited for testing EV-D68 therapeutics. Our study further underscores the importance of SETD3 being a promising therapeutic target for controlling EV-D68 and other enterovirus infections.

Identification of Novel Genes Involved in Hyperglycemia in Mice.

Current attempts to prevent and manage type 2 diabetes have been moderately effective, and a better understanding of the molecular roots of this complex disease is important to develop more successful and precise treatment options. Recently, we initiated the collective diabetes cross, where four mouse inbred strains differing in their diabetes susceptibility were crossed with the obese and diabetes-prone NZO strain and identified the quantitative trait loci (QTL) Nidd13/NZO, a genomic region on chromosome 13 that correlates with hyperglycemia in NZO allele carriers compared to B6 controls. Subsequent analysis of the critical region, harboring 644 genes, included expression studies in pancreatic islets of congenic Nidd13/NZO mice, integration of single-cell data from parental NZO and B6 islets as well as haplotype analysis. Finally, of the five genes (Acot12, S100z, Ankrd55, Rnf180, and Iqgap2) within the polymorphic haplotype block that are differently expressed in islets of B6 compared to NZO mice, we identified the calcium-binding protein S100z gene to affect islet cell proliferation as well as apoptosis when overexpressed in MIN6 cells. In summary, we define S100z as the most striking gene to be causal for the diabetes QTL Nidd13/NZO by affecting β-cell proliferation and apoptosis. Thus, S100z is an entirely novel diabetes gene regulating islet cell function.

182-OR: Genetic Susceptibility to Diabetic Kidney Disease Is Linked to C/EBP-XOR Axis

Diabetic kidney disease (DKD) is the leading single cause of end-stage renal disease in the United States. Approximately 30% of diabetics develop DKD with comparable blood glucose levels, indicating a significant genetic contribution for susceptibility. However, the underlying mechanisms that contribute to differential susceptibility are poorly understood. The glomerulus is the primary site of injury with hypertrophy and podocyte depletion being the hallmarks for progressive DKD. We have demonstrated that mitochondrial oxidative damage accumulation in glomerular endothelial cells, leads to podocyte loss via endothelial-to-podocyte crosstalk in DKD susceptible DBA/2J (D2) mice compared to DKD resistant C57BL/6J (B6) mice. To map genetic loci associated with podocyte depletion after long-term diabetes (6mth), we used the 39 strains of BXD, and parental B6 and D2 mice. We identified a significant cis-acting variant in the promoter of xanthine oxidoreductase (XOR). XORs catalyze the oxidation of purine substrates produce uric acid and reactive oxygen species. XOR expression in the kidney and circulating activity were significantly higher in diabetic D2 compared to B6. XOR inhibition significantly reduced albuminuria, oxidative damage in glomeruli and prevented podocyte depletion in diabetic D2 mice. We used CRISPR/Cas9 to knock-in the XOR risk variant into DKD-resistant B6 mice and generated mutant B6-XORem1 mice with significantly higher XOR activity than B6. This risk variant is a transcription factor binding site to C/EBPβ, we show higher C/EBPβ expression in glomerular endothelium from diabetic B6-XORem1 mice but not in diabetic B6. B6-XORem1 mice had increased mitochondrial oxidative stress in endothelial cells, podocyte depletion, basement membrane thickening, albuminuria, glomerulosclerosis and tubular injury, and these changes were prevented with XOR inhibition. Our data suggest that the identified promoter variant is causal for DKD susceptibility via the C/EBPβ-XOR axis. Disclosure Q. Wang: None. H. Qi: None. S. Li: None. G. Casalena: None. L. Yu: None. I. S. Daehn: None. Funding National Institutes of Health

Identification of novel loci controlling inflammatory bowel disease susceptibility utilizing the genetic diversity of wild-derived mice.

Inflammatory bowel disease (IBD) is a complex disorder that imposes a growing health burden. Multiple genetic associations have been identified in IBD, but the mechanisms underlying many of these associations are poorly understood. Animal models are needed to bridge this gap, but conventional laboratory mouse strains lack the genetic diversity of human populations. To more accurately model human genetic diversity, we utilized a panel of chromosome (Chr) substitution strains, carrying chromosomes from the wild-derived and genetically divergent PWD/PhJ (PWD) strain on the commonly used C57BL/6J (B6) background, as well as their parental B6 and PWD strains. Two models of IBD were used, TNBS- and DSS-induced colitis. Compared with B6 mice, PWD mice were highly susceptible to TNBS-induced colitis, but resistant to DSS-induced colitis. Using consomic mice, we identified several PWD-derived loci that exhibited profound effects on IBD susceptibility. The most pronounced of these were loci on Chr1 and Chr2, which yielded high susceptibility in both IBD models, each acting at distinct phases of the disease. Leveraging transcriptomic data from B6 and PWD immune cells, together with a machine learning approach incorporating human IBD genetic associations, we identified lead candidate genes, including Itga4, Pip4k2a, Lcn10, Lgmn, and Gpr65.

Allelic variation of the Tas1r3 taste receptor gene affects sweet taste responsiveness and metabolism of glucose in F1 mouse hybrids.

In mammals, inter- and intraspecies differences in consumption of sweeteners largely depend on allelic variation of the Tas1r3 gene (locus Sac) encoding the T1R3 protein, a sweet taste receptor subunit. To assess the influence of Tas1r3 polymorphisms on feeding behavior and metabolism, we examined the phenotype of F1 male hybrids obtained from crosses between the following inbred mouse strains: females from 129SvPasCrl (129S2) bearing the recessive Tas1r3 allele and males from either C57BL/6J (B6), carrying the dominant allele, or the Tas1r3-gene knockout strain C57BL/6J-Tas1r3tm1Rfm (B6-Tas1r3-/-). The hybrids 129S2B6F1 and 129S2B6-Tas1r3-/-F1 had identical background genotypes and different sets of Tas1r3 alleles. The effect of Tas1r3 hemizygosity was analyzed by comparing the parental strain B6 (Tas1r3 homozygote) and hemizygous F1 hybrids B6 × B6-Tas1r3-/-. Data showed that, in 129S2B6-Tas1r3-/-F1 hybrids, the reduction of glucose tolerance, along with lower consumption of and lower preference for sweeteners during the initial licking responses, is due to expression of the recessive Tas1r3 allele. Hemizygosity of Tas1r3 did not influence these behavioral and metabolic traits. However, the loss of the functional Tas1r3 allele was associated with a small decline in the long-term intake and preference for sweeteners and reduction of plasma insulin and body, liver, and fat mass.

Identifying modifier genes for hypertrophic cardiomyopathy

BackgroundHypertrophic cardiomyopathy (HCM) severity greatly varies among patients even with the same HCM gene mutations. This variation is largely regulated by modifier gene(s), which, however, remain largely unknown. The current study is aimed to identify modifier genes using BXD strains, a large murine genetic reference population (GRP) derived from crosses between C57BL/6 J (B6) and D2 DBA/2 J (D2) mice. D2 mice natualy carrythe genetic basis and phenotypes of HCM. MethodsMyocardial hypertrophy, the major phenotype of HCM, was determined by cardiomyocyte size on cardiac sections in 30 BXD strains, and their parental B6 and D2 strains and morphometric analysis was performed. Quantitative Trait Locus (QTL) mapping for cardiomyocyte sizes was conducted with WebQTL in GeneNetwork. Correlation of cardiomyocyte size and cardiac gene expression in BXDs accessed from GeneNetwork were evaluated. QTL candidate genes associated with cardiomyocyte sizes were prioritized based on the score system. ResultsCardiomyocyte size varied significantly among BXD strains. Interval mapping on cardiomyocyte size data showed a significant QTL on chromosome (Chr) 2 at 66– 73.5 Mb and a suggestive QTL on Chr 5 at 20.9–39.7 Mb. Further score system revealed a high QTL score for Xirp2 in Chr 2. Xirp2 encodes xin actin-binding repeat containing 2, which is highly expressed in cardiac tissue and associate with cardiomyopathy and heart failure. In Chr5 QTL, Nos3, encoding nitric oxide synthase 3, received the highest score, which is significantly correlated with cardiomyocyte size. ConclusionThese results indicate that Xirp2 and Nos3 serve as novel candidate modifier genes for myocardial hypertrophy in HCM. These candidate genes will be validated in our future studies.

The TLR7 ligand R848 prevents mouse graft-versus-host disease and cooperates with anti-interleukin-27 antibody for maximal protection and regulatory T-cell upregulation.

In spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested a pharmacological TLR7 ligand, R848, in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T-cell allo-responsiveness and antigen presentation by CD11b+ and CD8α+ dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T-cell implantation and activation. However, it decreased interferon-gamma, tumor necrosis factor-alpha and interleukin-27 while upregulating active transforming growth factor-beta 1 plasma levels. In addition, donor and recipient Foxp3+ regulatory T-cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3+ regulatory T cells that could be further increased by inhibition of interleukin-27. The combination of anti-interleukin-27p28 mono -clonal antibody and R848 showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with upregulation of regulatory T cells. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by inhibition of interleukin-27.

Bim is a candidate gene in the regulation of merocytic dendritic cell proportion

Abstract In contrast to conventional dendritic cells (cDC), when merocytic DC (mcDC) present antigens (Ag) derived from apoptotic bodies, T cell anergy is reversed rather than induced. Although helpful to tumour clearance, reversing T cell anergy is detrimental in autoimmunity. Interestingly, mcDC are present in higher proportion in type 1 diabetes (T1D)-prone NOD mice than in B6 mice. Recently, we found that the Idd13 T1D locus is linked to the control of mcDC number. Indeed, NOD.B6-Idd13 congenic mice are resistant to T1D and have the same low number of mcDC as the parental B6 mice. Thus, we hypothesize that defining the genetic factor implicated in the regulation of mcDC number could help maintain self-tolerance and prevent T1D onset. Within the Idd13 locus, both B2m and Bim are likely candidate genes. By exploiting both B2m−/−and Bim−/−mice, we show that B2m does not affect mcDC number while mcDC were significantly increased in Bim−/−mice. Competitive hematopoietic chimera validate Bim as an intrinsic factor implicated in the regulation of mcDC proportion. In line with a role for Bim in regulating mcDC number, we demonstrate that the caspase activity and Bcl2 expression is not differentially expressed between the different mouse model, suggesting that Bim is implicated in mcDC proportion regulation via a caspase-independent pathway. Together, these data demonstrate that Bim, encoded within the Idd13 locus on the chromosome 2 modulates the number of mcDC. Identifying factors that facilitate apoptosis of mcDC may help prevent autoimmunity. Still, whether restoring Bim expression in NOD mice would prevent T1D remains to be addressed.

Mapping of genetic loci that modulate differential colonization by Escherichia coli O157:H7 TUV86-2 in advanced recombinant inbred BXD mice.

BackgroundShiga toxin (Stx)-producing E. coli (STEC) are responsible for foodborne outbreaks that can result in severe human disease. During an outbreak, differential disease outcomes are observed after infection with the same STEC strain. One question of particular interest is why some infected people resolve infection after hemorrhagic colitis whereas others progress to the hemolytic uremic syndrome (HUS). Host age and infection dose have been implicated; however, these parameters do not appear to fully account for all of the observed variation in disease severity. Therefore, we hypothesized that additional host genetic factors may play a role in progression to HUS.Methods and ResultsTo mimic the genetic diversity in the human response to infection by STEC, we measured the capacity of an O157:H7 outbreak isolate to colonize mouse strains from the advanced recombinant inbred (ARI) BXD panel. We first infected the BXD parental strains C57BL/6 J (B6) and DBA/2 J (D2) with either 86–24 (Stx2a+) or TUV86-2, an Stx2a-negative isogenic mutant. Colonization levels were determined in an intact commensal flora (ICF) infection model. We found a significant difference in colonization levels between the parental B6 and D2 strains after infection with TUV86-2 but not with 86–24. This observation suggested that a host factor that may be masked by Stx2a affects O157:H7 colonization in some genetic backgrounds. We then determined the TUV86-2 colonization levels of 24 BXD strains in the ICF model. We identified several quantitative trait loci (QTL) associated with variation in colonization by correlation analyses. We found a highly significant QTL on proximal chromosome 9 (12.5–26.7 Mb) that strongly predicts variation in colonization levels and accounts for 15–20 % of variance. Linkage, polymorphism and co-citation analyses of the mapped region revealed 36 candidate genes within the QTL, and we identified five genes that are most likely responsible for the differential colonization.ConclusionsThe identification of the QTL on chromosome 9 supports our hypothesis that individual genetic makeup affects the level of colonization after infection with STEC O157:H7.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2127-7) contains supplementary material, which is available to authorized users.

Borrelia burgdorferi arthritis-associated locus Bbaa1 regulates Lyme arthritis and K/B×N serum transfer arthritis through intrinsic control of type I IFN production.

Localized upregulation of type I IFN was previously implicated in development of Borrelia burgdorferi-induced arthritis in C3H mice, and was remarkable due to its absence in the mildly arthritic C57BL/6 (B6) mice. Independently, forward genetics analysis identified a quantitative trait locus on Chr4, termed B. burgdorferi-associated locus 1 (Bbaa1), that regulates Lyme arthritis severity and includes the 15 type I IFN genes. Involvement of Bbaa1 in arthritis development was confirmed in B6 mice congenic for the C3H allele of Bbaa1 (B6.C3-Bbaa1), which developed more severe Lyme arthritis and K/B×N model of rheumatoid arthritis (RA) than did parental B6 mice. Administration of a type I IFN receptor blocking mAb reduced the severity of both Lyme arthritis and RA in B6.C3-Bbaa1 mice, formally linking genetic elements within Bbaa1 to pathological production of type I IFN. Bone marrow-derived macrophages from Bbaa1 congenic mice implicated this locus as a regulator of type I IFN induction and downstream target gene expression. Bbaa1-mediated regulation of IFN-inducible genes was upstream of IFN receptor-dependent amplification; however, the overall magnitude of the response was dependent on autocrine/paracrine responses to IFN-β. In addition, the Bbaa1 locus modulated the functional phenotype ascribed to bone marrow-derived macrophages: the B6 allele promoted expression of M2 markers, whereas the C3H allele promoted induction of M1 responses. This report identifies a genetic locus physically and functionally linked to type I IFN that contributes to the pathogenesis of both Lyme and RA.

Abstract 663: Identification of 2 Novel Candidate Genes of Atherosclerosis Susceptibility on Mouse Chromosome 3: Pla2g12a and Elovl6

Quantitative trait locus (QTL) mapping in an F2 intercross (n=452) of atherosclerosis-susceptible C57BL/6 (B6) and atherosclerosis-resistant FVB mice on the LDL-receptor deficient background revealed a novel atherosclerosis susceptibility locus on mouse chromosome (Chr) 3. In previous work the susceptible genetic region on Chr3 was narrowed to 80 - 160 MB and validated by congenic FVB.Chr3 B6/B6 mice. We hypothesized that underlying genetic variation in this region leads to differential expression of causal genes, thereby affecting atherosclerosis susceptibility. We performed transcriptome-wide expression analyses in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=4/4) using Illumina Ref-8 arrays followed by validation in livers of congenic FVB.Chr3 B6/B6 and FVB mice (n=8/9) as well as in livers of B6 and FVB mice (n=5/5) by quantitative real-time PCR (qRT-PCR). C is -regulation was investigated in F2 livers (n=47) by correlating the expression to the genotype. Tissue-specific expression of genes was examined by qRT-PCR in parental B6 and FVB mice. Western blot analysis and immunohistochemical staining (IHC) were performed. Mechanisms of atherogenesis were investigated by RNAi. Pla2g12a and Elovl6 were identified as candidate genes co-segregating with the atherosclerosis QTL at marker rs13464244. Pla2g12a mRNA expression was inversely correlated (r 2 =0.2, p=0.002) with atherosclerotic lesion size in F2 mice while Elovl6 expression was positively correlated (r 2 =0.18, p=0.002). qRT-PCR revealed a strong expression of Pla2g12a in muscle and fat tissues whereas Elovl6 was highly expressed in liver and fat tissues. Western blot analysis revealed significantly decreased protein expression of Pla2g12a in livers of B6 compared to FVB and an increased expression of Elovl6 in B6 mice. IHC staining of Pla2g12a and Elovl6 in aortic roots indicated high expression in macrophages and predominantly in endothelial cells. siRNA knockdown of Elovl6 was associated with reduced adhesion and increased apoptosis. In conclusion, we identified Elovl6 and Pla2g12a as promising candidate genes of atherosclerosis susceptibility on mouse Chr3. Further work is necessary to better understand the influence of these two genes on atherosclerosis development.

Basal physiological parameters of two congenic mice strains: C5 deficient C57BL/6 and C5 sufficient A/J

To investigate the in vivo role of complement component C5 it is common to compare the inflammatory response between C5-normal and C5-deficient inbred mice strains. Nevertheless, it should be expected that differences in the genetic backgrounds between those strains may affect several physiological parameters, complicating the correct interpretation of results. The use of congenic mice, developed by backcrossing, is therefore preferred. Still, several physiological parameters may be distinctive in the normal and deficient strains and therefore require careful analysis before animals are selected for investigation. We generated two congenic mouse strains: C57BL/6 (B6) C5−, derived from the parental B6 C5+ strain and A/J C5+ mice derived from the parental A/J C5− strain. After confirmation by nucleotide sequencing, immunodiffusion and hemolytic activity analysis, several basal physiological parameters were analyzed in the congenic and parental strains before antigen exposition. Serum levels of liver alanine aminotransferase, alkaline phosphatase, albumin, triglycerides, cholesterol and uric acid were found to be different in C5-sufficient and C5-deficient mice from one or both genetic backgrounds (B6 and/or A/J). On the other hand, serum levels of liver aspartate aminotransferase, glucose and urea were not affected by the presence of C5 in either strain. Furthermore, in some cases, C5-dependent variations in these parameters were more evident in mice of the same gender. We conclude here that C5-deficient mice strains may present distinct systemic behaviors which should be taken in consideration before differences in the immune responses are attributed solely to the lack of circulating C5.

Autosomal Dominant Inheritance of Brain Cardiolipin Fatty Acid Abnormality in VM/DK Mice: Association with Hypoxic‐Induced Cognitive Insensitivity

Cardiolipin is a complex polyglycerol phospholipid found almost exclusively in the inner mitochondrial membrane and regulates numerous enzyme activities especially those related to oxidative phosphorylation and coupled respiration. Abnormalities in cardiolipin can impair mitochondrial function and bioenergetics. We recently demonstrated that the ratio of shorter chain saturated and monounsaturated fatty acids (C16:0; C18:0; C18:1) to longer chain polyunsaturated fatty acids (C18:2; C20:4; C22:6) was significantly greater in the brains of adult VM/DK (VM) inbred mice than in the brains of C57BL/6J (B6) mice. The cardiolipin fatty acid abnormalities in VM mice are also associated with alterations in the activity of mitochondrial respiratory complexes. In this study we found that the abnormal brain fatty acid ratio in the VM strain was inherited as an autosomal dominant trait in reciprocal B6×VM F1 hybrids. To evaluate the potential influence of brain cardiolipin fatty acid composition on cognitive sensitivity, we placed the parental B6 and VM mice and their reciprocal male and female B6VMF1 hybrid mice (3-month-old) in a hypoxic chamber (5% O2). Cognitive awareness (conscientiousness) under hypoxia was significantly lower in the VM parental mice and F1 hybrid mice (11.4±0.4 and 11.0±0.4min, respectively) than in the parental B6 mice (15.3±1.4min), indicating an autosomal dominant inheritance like that of the brain cardiolipin abnormalities. These findings suggest that impaired cognitive awareness under hypoxia is associated with abnormalities in neural lipid composition.

Lupus prone DBA/2 (DBA) CD4 T cells exhibit greater B cell help, poorer help for CD8 cytotoxic T lymphocytes (CTL) and induce more severe renal disease compared to B10.D2 CD4 T cells in parent-into F1 (P→F1) mice. (123.27)

Abstract Transfer of parental B6 T cells into B6D2F1 (B6→F1) mice results in acute graft-vs- host disease (GVHD) due to donor CD8 CTL that eliminate host cells. Donor CD8 CTL fail to mature in the opposite parental transfer (DBA→F1) resulting in chronic lupus-like GVHD and severe renal disease. To determine whether CTL failure and subsequent lupus like disease in DBA→F1 mice is an intrinsic CD8 defect or extrinsic due to poor CD4 help, female CD4 and CD8 T cells were purified from DBA or C57/Bl background, MHC identical (H-2d) B10.D2 donor T cells and transferred in all 4 possible combinations into female F1 mice. Transfer of B10.D2 CD4 +B10.D2 CD8 (D2+D2→F1) mice and DBA+DBA→F1 mice (positive controls) exhibited the expected acute and chronic GVHD phenotype respectively at 2 weeks. For mixed subsets, D2+ DBA→F1 exhibited a 2 week phenotype not significantly different from control acute GVHD indicating that normal (D2) CD4 help could correct a DBA CD8 CTL defect seen as absent KRLG-1 upregulation. By contrast, DBA+D2→F1 mice exhibited an intermediate phenotype significantly different from either control group indicating that normal D2 CD8 CTL maturation was significantly impaired with DBA CD4 help. Long term, only DBA+DBA→F1 and DBA+D2→F1 mice exhibited lupus autoantibodies or renal disease. Thus, DBA CD4 T cells are the primary determinant of clinical lupus in this model by virtue of greater help for autoreactive B cells and poorer help for down regulatory CD8 CTL.

The effect of chromosome substitution on endurance exercise capacity in inbred and F2 generation mice

Previous research identified a region on chromosome 14 as an important regulator of endurance exercise capacity. The aim of this study was to identify the effect of chromosome substitution on endurance exercise capacity in inbred and hybrid mice. Mice from a chromosome substitution strain (CSS) derived from A/J (A) and C57Bl/6J (B6), denoted as B6.A14 were used to assess the contribution of chromosome 14 to endurance exercise capacity. To further investigate the heritability of the endurance exercise phenotype, 155 B6 x B6.A14 F2 mice were generated from a cross between B6 and B6.A14 CSS mice. All mice performed a graded exercise test to exhaustion to determine endurance exercise capacity. Endurance exercise capacity was expressed as time (min). B6 x B6.A14 F2 mice had an exercise capacity of 30.8 ± 0.1 minutes, which is significantly higher than parental B6.A14 CSS mice (28.5 ± 0.2 min) and similar to parental B6 mice (31.0 ± 0.1 min). Although the cohort mean is similar between F2 and B6 mice, the range of responses in the B6 x B6.A14 F2 mice (27.3 – 34.5 min) exceeded the range of the parental inbred strains. The range of responses in the B6 x B6.A14 F2 mice suggests that this population of mice is diverse in both phenotype and genotype and would be amenable to a chromosome‐wide scan for endurance exercise‐related genes.Supported by NIH grant HL085918 to MPM

Increased physical activity cosegregates with higher intake of carbohydrate and total calories in a subcongenic mouse strain

C57BL/6 J (B6) and CAST/EiJ (CAST), the inbred strain derived from M. musculus castaneus, differ in nutrient intake behaviors, including dietary fat and carbohydrate consumption in a two-diet-choice paradigm. Significant quantitative trait loci (QTLs) for carbohydrate (Mnic1) and total energy intake (Kcal2) are present between these strains on chromosome (Chr) 17. Here we report the refinement of the Chr 17 QTL in a subcongenic strain of the B6.CAST-( D17Mit19-D17Mit91 ) congenic mice described previously. This new subcongenic strain possesses CAST Chr 17 donor alleles from 4.8 to 45.4 Mb on a B6 background. Similar to CAST, the subcongenic mice exhibit increased carbohydrate and total calorie intake per body weight, while fat intake remains equivalent. Unexpectedly, this CAST genomic segment also confers two new physical activity phenotypes: 22% higher spontaneous physical activity levels and significantly increased voluntary wheel-running activity compared with the parental B6 strain. Overall, these data suggest that gene(s) involved in carbohydrate preference and increased physical activity are contained within the proximal region of Chr 17. Interval-specific microarray analysis in hypothalamus and skeletal muscle revealed differentially expressed genes within the subcongenic region, including neuropeptide W (Npw); glyoxalase I (Glo1); cytochrome P450, family 4, subfamily f, polypeptide 1 (Cyp4f15); phospholipase A2, group VII (Pla2g7); and phosphodiesterase 9a (Pde9a). This subcongenic strain offers a unique model for dissecting the contributions and possible interactions among genes controlling food intake and physical activity, key components of energy balance.

Genetic Mapping of Social Interaction Behavior in B6/MSM Consomic Mouse Strains

Genetic studies are indispensable for understanding the mechanisms by which individuals develop differences in social behavior. We report genetic mapping of social interaction behavior using inter-subspecific consomic strains established from MSM/Ms (MSM) and C57BL/6J (B6) mice. Two animals of the same strain and sex, aged 10 weeks, were introduced into a novel open-field for 10 min. Social contact was detected by an automated system when the distance between the centers of the two animals became less than ~12 cm. In addition, detailed behavioral observations were made of the males. The wild-derived mouse strain MSM showed significantly longer social contact as compared to B6. Analysis of the consomic panel identified two chromosomes (Chr 6 and Chr 17) with quantitative trait loci (QTL) responsible for lengthened social contact in MSM mice and two chromosomes (Chr 9 and Chr X) with QTL that inhibited social contact. Detailed behavioral analysis of males identified four additional chromosomes associated with social interaction behavior. B6 mice that contained Chr 13 from MSM showed more genital grooming and following than the parental B6 strain, whereas the presence of Chr 8 and Chr 12 from MSM resulted in a reduction of those behaviors. Longer social sniffing was observed in Chr 4 consomic strain than in B6 mice. Although the frequency was low, aggressive behavior was observed in a few pairs from consomic strains for Chrs 4, 13, 15 and 17, as well as from MSM. The social interaction test has been used as a model to measure anxiety, but genetic correlation analysis suggested that social interaction involves different aspects of anxiety than are measured by open-field test.Electronic supplementary materialThe online version of this article (doi:10.1007/s10519-009-9312-x) contains supplementary material, which is available to authorized users.

Peripheral Alloantigen Drives Early Dysfunction and Eventual Exhaustion of CTL Following Delayed Donor Leukocyte Infusions.

T cell immunotherapies for cancer should ideally generate high levels of anti-tumor activity, with minimal host injury and permit the prolonged survival of functional memory/effector cells to prevent tumor recurrence. Following allogeneic stem cell transplantation, delayed donor leukocyte infusion (DLI) is one strategy employed to induce graft-versus-leukemia (GVL) responses while limiting the risk of host injury in terms of graft-versus-host disease. However, patients remain at significant risk of relapse following DLI and murine models of delayed DLI indicate that this results from the eventual loss of functional, alloreactive cytotoxic T lymphocytes (CTL) [Mapara et al. Transplantation 2003]. We hypothesised that the loss of functional CTL is driven by persistent stimulation of donor CD8 cells by alloantigen expressed by peripheral tissues. In order to follow and characterise an alloreactive CD8 response under conditions in which alloantigen was present or absent in peripheral tissues, we employed a model in which either parental B6 (H2b) or B6 x DBA-2 F1 (BDF1, H2dxb) mice were lethally irradiated and reconstituted with a mixture of B6 and BDF1 T cell depleted bone marrow. 8-10 weeks later congenic CD45.1 B6 splenocytes were transferred into the established mixed chimeras. This allowed us to test the importance of peripheral antigen in the loss of alloreactive CTL responses, since alloantigen was either restricted to the hematopoietic system (B6 +BDF1 → B6) or was ubiquitously expressed (B6 +BDF1 → BDF1). Following transfer of CD45.1 B6 splenocytes, the ensuing alloantigenspecific T cell response in both groups led to the elimination of alloantigen-positive (BDF1-derived) hematopoietic elements. Thereafter, alloreactive CD8 cells resided in an environment in which peripheral alloantigen was present (PA+) or absent (PA-). We observed similar kinetics of initial CD45.1+ CD8 cell proliferation and expansion and similar acquisition of a CD44highCD62Llow phenotype. However, by day 60, there were striking differences in the phenotype and function of transferred CD8 cells. In PA- hosts, CD45.1+ CD8 cells killed allogeneic target cells effectively both in vitro and in vivo, underwent rapid proliferation in a mixed leukocyte reaction and produced the effector cytokine, IFN-γ. In contrast CD45.1+ CD8 cells from PA+ hosts had little or no cytotoxic activity, did not proliferate to alloantigen and were IFN-γlow. Moreover, CD45.1+ CD8 cells from PA+ hosts displayed high levels of the co-inhibitory receptor PD-1, low levels of the IL-7Rα chain and responded poorly to IL-7 and IL-15 in vitro, a phenotype typical of the ‘exhaustion’ signature observed in CTL following chronic antigen exposure. In comparison, CD45.1+ CD8 cells from PA- hosts expressed significantly lower levels of PD-1, higher levels of IL-7Rα and demonstrated better responsiveness to IL-7 and IL-15 in vitro. In vitro PD-1 or PD-L1 blockade restored IFN-γ generation to CD45.1+ CD8 cells from PA+ hosts, suggesting that the PD-1 pathway may play a functional role in driving exhaustion of these cells. Importantly we observed no loss of long-term alloreactive CD4 responses in either PA+ or PA- hosts. This finding is consistent with a model in which peripheral alloantigen drives exhaustion since the majority of cells expressing Class II alloantigens in PA+ and PA- hosts would be restricted to the hematopoietic system and thus, would have been cleared in the initial alloresponse. The full exhausted phenotype of alloreactive CD8 cells described above was not seen until at least 30 days after transfer to PA+ hosts. However, as early as day 14, CTL primed in PA+ hosts produced less IFN-γ in comparison to those primed in PA-hosts, even though they were still equivalent in terms of their cytotoxicity. Furthermore, when CD8 cells primed in PA+ hosts were transferred to secondary antigen-free hosts, they still displayed reduced ‘fitness’ compared to CTL originally primed in PA- hosts. These data show that peripheral alloantigen qualitatively affects donor CTL function during priming and drives their eventual exhaustion. Additionally they suggest that blockade of co-inhibitory signals may have potential in restoring function to such cells as has been demonstrated in models of chronic infection.

Genetic determinants of emotionality and stress response in AcB/BcA recombinant congenic mice and in silico evidence of convergence with cardiovascular candidate genes

Genomic loci bearing stress-related phenotypes were dissected in recombinant congenic strains (RCS) of mice with C57BL/6J (B6) and A/J progenitors. Adult male mice from 14 A/J and 22 B6 background lines were evaluated for emotional reactivity in open-field (OF) and elevated plus-maze tests. Core temperature was monitored by radio telemetry during immobilization and on standard as well as salt-enriched diets. In addition, urinary electrolytes were measured. Genome-wide linkage analysis of the parameters revealed over 20 significant quantitative trait loci (QTL). The highest logarithm of odds (LOD) scores were within the previously-reported OF emotionality locus on Chr 1 (LOD = 4.6), in the dopa decarboxylase region on Chr 11 for the plus-maze (LOD = 4.7), and within a novel region of calmodulin 1 on Chr 12 for Ca++ excretion after a 24-h salt load (LOD = 4.6). RCS stress QTL overlapped with several candidate loci for cardiovascular (CV) disease. In silico evidence of functional polymorphisms by comparative sequence analysis of progenitor strains assisted to ascertain this convergence. The anxious BcA70 strain showed down regulation of Atp1a2 gene expression in the heart (P < 0.001) and brain (P < 0.05) compared with its parental B6 strain, compatible with the enhanced emotionality described in knock out animals for this gene, also involved in the salt-sensitive component of hypertension. Functional polymorphisms in regulatory elements of candidate genes of the CV/inflammatory/immune systems support the hypothesis of genetically-altered environmental susceptibility in CV disease development.

Genome scan for exercise training responses

The genetic factors determining the magnitude of the response to exercise training are poorly understood. The aim of this study was to identify quantitative trait loci (QTL) associated with adaptation to exercise training in mice. Based on the marked differences in training responses in inbred B6 and FVB mice and intermediate phenotype of F1 offspring, an intercross breeding scheme was used to generate 188 F2 mice. Mice completed an exercise performance test before and after a 4-week treadmill running program. Male and female mice (n=84, 44.5%) were selected from the phenotypic extremes based on changes in distance run. Mean values for high (HIGH F2) and low (LOW F2) responders were 1195.5±7.9 m and 19.8±3.2 m, respectively. Changes in distance run were greater in HIGH F2 compared with all other groups, including mice from the parental FVB strain. Mice from the LOW F2 group showed responses similar to mice from the parental B6 strain and sedentary F2 mice indicating that the range of phenotypic responses in F2 mice were similar to or greater than the differences between FVB and B6 parental strains. Mice were genotyped at ~20 cM intervals using a panel of 103 SNPs. Using R/qtl software, significant QTL for post-training distance run were identified on chromosome 2 (peak at 74 cM, 5.72 LOD), chromosome 12 (41 cM, 3.49 LOD), and chromosome 14 (54 cM, 6.25 LOD). Significant or suggestive QTL for changes in distance run, post-training work, and changes in work were also identified at these same loci. Allele effects for the peak marker were plotted for the two most significant QTL (Chr. 2 and 14). At both loci, the allele for high post-training distance run came from the FVB and showed additive inheritance. These data suggest that one or more QTL determine exercise-training responses in mice. Supported by AHA SDG (M. P. M.) and NHLBI Grant HL-62826 (B. C. B).