Diversity Outbred Research Articles

Abstract 2920: A Diversity outbred (DO) mouse model reveals regulators of HER2-driven tumorigenesis

Abstract Although humans are heterogeneous, evaluation of tumorigenesis and immunotherapy has relied on the use of inbred mouse strains, which may lead to biased results. In order to investigate the effect(s) of genetic background on tumor incidence and immune response, we have taken a unique new approach to identify genes that are associated with tumor onset time & tumor growth rate using Diversity Outbred (DO) mice. Specifically, we crossed DO female mice (J:DO, Jackson Labs) with BALB/NeuT (NeuT) mice to introduce a heterogeneous background in (BALBxDO) F1 NeuT mice. NeuT express transforming rat neu, the homologue of HER2, and develop spontaneous tumors in all 10 mammary glands between 14 and 19 weeks of age (woa). DO mice are generated by non-sibling crosses of 8 founder strains: A/J, C57BL/6J, 129S1/SvlmJ, NOD/ShiLtJ, NZO/HILtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ to encompass greater than 90% of murine polymorphic alleles. The genetic makeup of each F1 mouse is defined using 143,600 chromosomal markers (mostly SNP) in the Giga Mouse Universal Genotyping Array. SNP analysis identifies the origin of quantitative trait loci (QTL), using the R/QTL package. The program reads the GigaMUGA genotyping results to perform haplotype reconstruction and determine the founder strain haplotype of each marker locus. DO F1 mice have been successfully used to identify the critical role of MHC-IB, and NK cells, in generation of HER2 Ab response following vaccination of HER2 tolerant mice (Wei et al., 2020). In (BALBxDO) F1 NeuT female mice most spontaneous mammary tumors develop over a wider range of time from 9 to 23 woa. QTL analysis of tumor onset time revealed QTLs in chromosomes (Chr) 1 and X with peak LOD scores of 7.7 (p = 0.01) and 8.6 (p < 0.05), respectively. Tumor growth rate was associated with a QTL in Chr 10. More specifically, PWK and CAST haplotypes in these QTLs were associated with early and faster growing tumors. About 10-15 genes in the 3 QTLs contain missense SNP alterations unique to PWK and CAST, and the genes have been associated with human breast cancer prognosis. As an example, Mid2 identified in ChrX QTL interacts with BRCA1, and is negatively associated with patient survival using the TCGA BRCA database. Single cell RNA sequencing revealed that Mid2 is expressed in NeuT mammary tumor tissue. Studies are ongoing to determine the functional significance of candidate genes in HER2/neu mammary tumorigenesis. DO F1 QTL combined with scRNA transcriptomic analysis opens important new opportunities to define critical genes in cancer biology and immunotherapy, with the potential for novel intervention and disease prevention. Citation Format: Jennifer Jacob, Kuang Wei, Joyce D. Reyes, Cong-Cong Yin, Elizabeth Rondini, Qing-Sheng Mi, James Granneman, Prahlad Parajuli, K Wagner, Wei-Zen Wei. A Diversity outbred (DO) mouse model reveals regulators of HER2-driven tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2920.

Genes, other than Muc5b, play a role in bleomycin-induced lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an incurable genetic disease that affects 5 million people worldwide. The gain-of-function MUC5B promoter variant rs35705950 is the dominant genetic risk factor for IPF, yet has a low penetrance. This raises the possibility that other genes and transcripts affect the penetrance of MUC5B. Previously, we have shown that the concentration of Muc5b in bronchoalveolar epithelia is directly associated with the extent and persistence of bleomycin-induced lung fibrosis in mice. In this study, we investigated whether bleomycin-induced lung injury is Muc5b dependent in genetically divergent strains of mice. Specifically, mice from the eight Diversity Outbred (DO) founders were phenotyped for Muc5b expression and lung fibrosis 3 wk after intratracheal bleomycin administration. Although we identified strains with low Muc5b expression and minimal lung fibrosis (CAST/EiJ and PWK/PhJ) and strains with high Muc5b expression and extensive lung fibrosis (NZO/H1LtJ and WSB/EiJ), there also were strains that did not demonstrate a clear relationship between Muc5b expression and lung fibrosis (129S1/SvlmJ, NOD/ShiLtJ, and C57BL/6J, A/J). Hierarchical clustering suggests that other factors may work in concert with or potentially independent of Muc5b to promote bleomycin-induced lung injury and fibrosis. This study suggests that these strains and their recombinant inbred crosses may prove helpful in identifying the genes and transcripts that interact with Muc5b and cause lung fibrosis.

Systems genetics in diversity outbred mice inform BMD GWAS and identify determinants of bone strength

Genome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we use Diversity Outbred (DO) mice to directly address these limitations by performing a systems genetics analysis of 55 complex skeletal phenotypes. We apply a network approach to cortical bone RNA-seq data to discover 66 genes likely to be causal for human BMD GWAS associations, including the genes SERTAD4 and GLT8D2. We also perform GWAS in the DO for a wide-range of bone traits and identify Qsox1 as a gene influencing cortical bone accrual and bone strength. In this work, we advance our understanding of the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.

Genetic linkage analysis using Diversity Outbred mice identifies prolactin as a host-intrinsic regulator of immune checkpoint inhibitor response

Abstract The use of immune checkpoint inhibitors (ICI) targeting CTLA-4 and PD-1 have greatly improved patient survival in metastatic melanoma, but a significant proportion of patients fail to respond to therapy. Incorporating what has been learned in autoimmune conditions, we hypothesize the host genetic background plays a significant role in determining response to immunotherapy. To implement genetic diversity, Diversity Outbred (DO) mice were crossed with C57BL/6 to generate (C57BL/6xDO)F1 mice that reliably develop B16F0 tumors after subcutaneous inoculation. (C57BL/6xDO)F1 mice (n=142) treated with combined anti-PD1/anti-CTLA-4 on days 3, 6, and 10 after inoculation show wide variation in tumor latency up to a maximum of 65 days, with 19 mice never developing tumor. Genetic linkage analysis using the R/qtl2 package shows a suggestive association within the murine prolactin family on chromosome 13. Visualization of QTL effects by DO founder identifies the NZO mouse genotype as a driver of increased tumor latency. Validation was performed using Collaborative Cross (CC) mouse lines that were either the NZO or C57BL/6 genotype within this locus. (C57BL/6xCC)F1 mice containing the NZO genotype have lower tumor burden by day 15 compared to mice with the C57BL/6 genotype, validating the locus as influential on the response to ICI. Combination of systemic prolactin treatment and ICI shows evidence of an interaction effect. Mice treated with both prolactin and ICI show lower tumor burden by day 19 versus single therapy or placebo controls. These results show that the host’s genetic background significantly contributes to response to ICI, and modulation of prolactin expression may augment ICI activity.

Leveraging outbred mice to identify genetic predictors of durable response to anti-tumor antibody

Abstract Trastuzumab is one of many monoclonal antibody (mAb) therapies targeting tumor-associated antigens (TAA) that have markedly improved cancer outcomes. Trastuzumab targets epidermal growth factor 2 (HER2). While it has long been considered the standard-of-care for HER2+ breast cancer treatment, differential response between patients suggests that host genetic background may contribute to an effective response to therapy. We utilize genetically heterogeneous Diversity Outbred (DO) mice to identify genetic elements that influence host response to TAA-mAb therapy. In a cohort of (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic Neu+ (a HER2 homolog) TUBO mammary tumors, we find a divergent response to αNeu mAb therapy (clone 7.16.4). Several phenotypes were collected to compare immunological function to projected tumor doubling time (TDT). These include activation of peripheral anti-tumor T cells and IgG, natural killer cell ADCC competence, bone marrow derived macrophage ADCP/trogocytosis efficacy, and tumor immune cell infiltrate proportions. Contrary to the prospective mechanism of action of trastuzumab, we show that neither capacity for ADCC or ADCP correlate with a robust response to therapy. Following genetic linkage analysis, we identify a locus on Chr 10 that associates with TDT. To verify this locus, we use recombinant inbred collaborative cross (CC) mouse strains (CCxBALB/c) F1 mice that contain or lack this driver genotype. We find pooled survivorship between prospective responder/non-responder strains 2 months post tumor implantation differs by ~8%. This locus alone can’t fully account for a durable response to mAb therapy highlighting the multigenic nature of host immune response.

A landmark-free morphometrics pipeline for high-resolution phenotyping: application to a mouse model of Down syndrome.

ABSTRACTCharacterising phenotypes often requires quantification of anatomical shape. Quantitative shape comparison (morphometrics) traditionally uses manually located landmarks and is limited by landmark number and operator accuracy. Here, we apply a landmark-free method to characterise the craniofacial skeletal phenotype of the Dp1Tyb mouse model of Down syndrome and a population of the Diversity Outbred (DO) mouse model, comparing it with a landmark-based approach. We identified cranial dysmorphologies in Dp1Tyb mice, especially smaller size and brachycephaly (front-back shortening), homologous to the human phenotype. Shape variation in the DO mice was partly attributable to allometry (size-dependent shape variation) and sexual dimorphism. The landmark-free method performed as well as, or better than, the landmark-based method but was less labour-intensive, required less user training and, uniquely, enabled fine mapping of local differences as planar expansion or shrinkage. Its higher resolution pinpointed reductions in interior mid-snout structures and occipital bones in both the models that were not otherwise apparent. We propose that this landmark-free pipeline could make morphometrics widely accessible beyond its traditional niches in zoology and palaeontology, especially in characterising developmental mutant phenotypes.

High-throughput measurement of fibroblast rhythms reveals genetic heritability of circadian phenotypes in diversity outbred mice and their founder strains

Circadian variability is driven by genetics and Diversity Outbred (DO) mice is a powerful tool for examining the genetics of complex traits because their high genetic and phenotypic diversity compared to conventional mouse crosses. The DO population combines the genetic diversity of eight founder strains including five common inbred and three wild-derived strains. In DO mice and their founders, we established a high-throughput system to measure cellular rhythms using in vitro preparations of skin fibroblasts. Among the founders, we observed strong heritability for rhythm period, robustness, phase and amplitude. We also found significant sex and strain differences for these rhythms. Extreme differences in period for molecular and behavioral rhythms were found between the inbred A/J strain and the wild-derived CAST/EiJ strain, where A/J had the longest period and CAST/EiJ had the shortest. In addition, we measured cellular rhythms in 329 DO mice, which displayed far greater phenotypic variability than the founders—80% of founders compared to only 25% of DO mice had periods of ~ 24 h. Collectively, our findings demonstrate that genetic diversity contributes to phenotypic variability in circadian rhythms, and high-throughput characterization of fibroblast rhythms in DO mice is a tractable system for examining the genetics of circadian traits.

Leveraging mouse genetic diversity to investigate the role of brain myeloid cells in Alzheimer’s disease

AbstractBackgroundMyeloid cells have been strongly implicated in Alzheimer’s Disease and related dementias (ADRDs). Our previous study showed genetic diversity significantly modified brain myeloid cell responses and AD‐relevant phenotypes in mice. Therefore, incorporating natural genetic variation into mouse models is indispensable to understand the role of myeloid cells in AD. This study aims to characterize brain myeloid cells in our genetically diverse AD panel to determine their functions in ADRDs.MethodsBrain myeloid cells were isolated from 8‐9 months‐old female mice from our genetically diverse AD mouse panel [C57BL/6J (B6), PWK/PhJ, WSB/EiJ and CAST/EiJ strains, each carrying APP/PS1] using magnetic activated cell sorting and were subjected to single‐cell RNA‐sequencing (scRNA‐seq). Data were processed using a customized pipeline allowing strain‐specific genome alignment followed by downstream analyses using Seurat and edgeR packages. Differences in myeloid cell responses between strains are being validated using functional assays in primary microglial cultures and immunofluorescence. To identify the putative genetic variations that drive and/or modify differences in myeloid cell responses, quantitative trait locus mapping of microglia number and morphology is being performed in brains from 120 Diversity Outbred (DO) mice, derived from eight mouse strains including those listed above.ResultsscRNA‐seq uncovered heterogeneous clusters of myeloid cells that vary significantly in cell abundance and gene expression between both wild‐type and APP/PS1 of B6, PWK, WSB and CAST mice. There was a significantly lower percentage of disease‐associated microglia in WSB.APP/PS1 mice and a significantly higher percentage of interferon‐responding microglia in PWK.APP/PS1 mice, compared to other AD strains. These data agreed with previously determined phenotype differences between strains. Differential gene expression analyses (all data compared to the B6 strain) revealed robust APP/PS1‐responding genes unique to strain background (strain by genotype effect) including Rpl29, Irf7, Cd36, H2‐Eb1, Lag3 and Cd34 in CAST; Ramp, Tmem176a, Tmem176b, Sepp1 and Inpp5d in PWK, and Ccl6, Slamf8, Nme1 and Pcna in WSB strain. Validation by primary culture and analyses of DO mice are underway.ConclusionAs in humans, mouse genetic diversity significantly impacts the landscape and dynamics of brain myeloid cells in AD. These studies will improve the translatability of myeloid‐based therapies for ADRDs.

Automatic discovery of clinically interpretable imaging biomarkers for Mycobacterium tuberculosis supersusceptibility using deep learning.

BackgroundIdentifying which individuals will develop tuberculosis (TB) remains an unresolved problem due to few animal models and computational approaches that effectively address its heterogeneity. To meet these shortcomings, we show that Diversity Outbred (DO) mice reflect human-like genetic diversity and develop human-like lung granulomas when infected with Mycobacterium tuberculosis (M.tb) .MethodsFollowing M.tb infection, a “supersusceptible” phenotype develops in approximately one-third of DO mice characterized by rapid morbidity and mortality within 8 weeks. These supersusceptible DO mice develop lung granulomas patterns akin to humans. This led us to utilize deep learning to identify supersusceptibility from hematoxylin & eosin (H&E) lung tissue sections utilizing only clinical outcomes (supersusceptible or not-supersusceptible) as labels.FindingsThe proposed machine learning model diagnosed supersusceptibility with high accuracy (91.50 ± 4.68%) compared to two expert pathologists using H&E stained lung sections (94.95% and 94.58%). Two non-experts used the imaging biomarker to diagnose supersusceptibility with high accuracy (88.25% and 87.95%) and agreement (96.00%). A board-certified veterinary pathologist (GB) examined the imaging biomarker and determined the model was making diagnostic decisions using a form of granuloma necrosis (karyorrhectic and pyknotic nuclear debris). This was corroborated by one other board-certified veterinary pathologist. Finally, the imaging biomarker was quantified, providing a novel means to convert visual patterns within granulomas to data suitable for statistical analyses.ImplicationsOverall, our results have translatable implication to improve our understanding of TB and also to the broader field of computational pathology in which clinical outcomes alone can drive automatic identification of interpretable imaging biomarkers, knowledge discovery, and validation of existing clinical biomarkers.FundingNational Institutes of Health and American Lung Association.

Diversity Outbred Mice Reveal the Quantitative Trait Locus and Regulatory Cells of HER2 Immunity.

The genetic basis and mechanisms of disparate antitumor immune response was investigated in Diversity Outbred (DO) F1 mice that express human HER2. DO mouse stock samples nearly the entire genetic repertoire of the species. We crossed DO mice with syngeneic HER2 transgenic mice to study the genetics of an anti-self HER2 response in a healthy outbred population. Anti-HER2 IgG was induced by Ad/E2TM or naked pE2TM, both encoding HER2 extracellular and transmembrane domains. The response of DO F1 HER2 transgenic mice was remarkably variable. Still, immune sera inhibited HER2+ SKBR3 cell survival in a dose-dependent fashion. Using DO quantitative trait locus (QTL) analysis, we mapped the QTL that influences both total IgG and IgG2(a/b/c) Ab response to either Ad/E2TM or pE2TM. QTL from these four datasets identified a region in chromosome 17 that was responsible for regulating the response. A/J and NOD segments of genes in this region drove elevated HER2 Ig levels. This region is rich in MHC-IB genes, several of which interact with inhibitory receptors of NK cells. (B6xA/J)F1 and (B6xNOD)F1 HER2 transgenic mice received Ad/E2TM after NK cell depletion, and they produced less HER2 IgG, demonstrating positive regulatory function of NK cells. Depletion of regulatory T cells enhanced response. Using DO QTL analysis, we show that MHC-IB reactive NK cells exert positive influence on the immunity, countering negative regulation by regulatory T cells. This new, to our knowledge, DO F1 platform is a powerful tool for revealing novel immune regulatory mechanisms and for testing new interventional strategies.

Genetic variation regulates opioid-induced respiratory depression in mice

In the U.S., opioid prescription for treatment of pain nearly quadrupled from 1999 to 2014. The diversion and misuse of prescription opioids along with increased use of drugs like heroin and fentanyl, has led to an epidemic in addiction and overdose deaths. The most common cause of opioid overdose and death is opioid-induced respiratory depression (OIRD), a life-threatening depression in respiratory rate thought to be caused by stimulation of opioid receptors in the inspiratory-generating regions of the brain. Studies in mice have revealed that variation in opiate lethality is associated with strain differences, suggesting that sensitivity to OIRD is genetically determined. We first tested the hypothesis that genetic variation in inbred strains of mice influences the innate variability in opioid-induced responses in respiratory depression, recovery time and survival time. Using the founders of the advanced, high-diversity mouse population, the Diversity Outbred (DO), we found substantial sex and genetic effects on respiratory sensitivity and opiate lethality. We used DO mice treated with morphine to map quantitative trait loci for respiratory depression, recovery time and survival time. Trait mapping and integrative functional genomic analysis in GeneWeaver has allowed us to implicate Galnt11, an N-acetylgalactosaminyltransferase, as a gene that regulates OIRD.

Mapping the Effects of Genetic Variation on Chromatin State and Gene Expression Reveals Loci That Control Ground State Pluripotency.

Mouse embryonic stem cells (mESCs) cultured in the presence of LIF occupy a ground state with highly active pluripotency-associated transcriptional and epigenetic circuitry. However, ground state pluripotency in some inbred strain backgrounds is unstable in the absence of ERK1/2 and GSK3 inhibition. Using an unbiased genetic approach, we dissect the basis of this divergent response to extracellular cues by profiling gene expression and chromatin accessibility in 170 genetically heterogeneous mESCs. We map thousands of loci affecting chromatin accessibility and/or transcript abundance, including 10 QTL hotspots where genetic variation at a single locus coordinates the regulation of genes throughout the genome. For one hotspot, we identify a single enhancer variant ∼10 kb upstream of Lifr associated with chromatin accessibility and mediating a cascade of molecular events affecting pluripotency. We validate causation through reciprocal allele swaps, demonstrating the functional consequences of noncoding variation in gene regulatory networks that stabilize pluripotent states invitro.

Abstract A45: Diet-induced obesity and caloric restriction weight loss in Diversity Outbred (DO) mice: An experimental preclinical translational model for the investigation of pathways for prevention of obesity and cancer

Abstract Obesity has reached epidemic levels worldwide. It is associated with major chronic diseases, including cancer and cardiovascular disease. The etiology of obesity-associated cancer is poorly understood. New population-based research mouse models are required to better understand prevention and to increase the translational potential of mouse models to humans. The Collaborative Cross (CC) recombinant inbred lines of mice are a population-based model created from an 8-way multiparental advanced generation intercross with a 12% minor allele frequency (MAF) and more than 45 million SNP and CNV, including a significant number of private (unique) alleles. The Diversity Outbred (DO) mice were created from early CC incipient lines (generations G2:F2-5) by random selection and 175 randomly selected breeding pairs. The genetic diversity of the DO and CC RIL mice is ideal for forward and reverse genetics approaches, respectively, to identify potential causally related risk variants and identify etiology based upon causal relationships. Here, we present data from two cohorts (from different breeding generations) of 50 female and 50 male DO mice each (total 100 each sex). After 12-wks high-fat (60% kcal fat) diet we observed significant differences in weight gain during diet-induced obesity DIO. After switching diets for 8-wks of 30% caloric restriction of a low-fat (10% kcal fat) diet, we observed significant interindividual differences in weight loss. Body weight, lean and fat mass differences), increases in 4-hr fasted blood glucose and insulin levels, insulin resistance (HOMA-IR and increased blood branched chain amino acids levels (BCAA) were observed. Four interacting quantitative trait loci/gene (Chst10, Bmiq5, Ath8, and Tcq14) related to metabolism, body mass index, cardiovascular disease, and total cholesterol levels, respectively, have been identified. Regression analysis suggests significant interactions between fasted blood glucose and fat mass, HOMA-IR and fat mass, and BCAA and fat mass in DIO and CR outcomes. Results to date show significant amounts of interindividual variability of each of the quantified phenotypes in response to both diet interventions were demonstrated in female and male DO mice. These data suggest potential avenues of research on obesity and cancer relationships and pathways for intervention. Citation Format: Melissa VerHague, Jody Albright, Salvador Favela, Kerri Barron, Michael F. Coleman, Katie Meyer, John E. French, Stephen D. Hursting. Diet-induced obesity and caloric restriction weight loss in Diversity Outbred (DO) mice: An experimental preclinical translational model for the investigation of pathways for prevention of obesity and cancer [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A45.

Dissecting the Genetic Architecture of Cystatin C in Diversity Outbred Mice.

Plasma concentration of Cystatin C (CysC) level is a biomarker of glomerular filtration rate in the kidney. We use a Systems Genetics approach to investigate the genetic determinants of plasma CysC concentration. To do so we perform Quantitative Trait Loci (QTL) and expression QTL (eQTL) analysis of 120 Diversity Outbred (DO) female mice, 56 weeks of age. We performed network analysis of kidney gene expression to determine if the gene modules with common functions are associated with kidney biomarkers of chronic kidney diseases. Our data demonstrates that plasma concentrations and kidney mRNA levels of CysC are associated with genetic variation and are transcriptionally coregulated by immune genes. Specifically, Type-I interferon signaling genes are coexpressed with Cst3 mRNA levels and associated with CysC concentrations in plasma. Our findings demonstrate the complex control of CysC by genetic polymorphisms and inflammatory pathways.

Facial shape and allometry quantitative trait locus intervals in the Diversity Outbred mouse are enriched for known skeletal and facial development genes.

The biology of how faces are built and come to differ from one another is complex. Discovering normal variants that contribute to differences in facial morphology is one key to untangling this complexity, with important implications for medicine and evolutionary biology. This study maps quantitative trait loci (QTL) for skeletal facial shape using Diversity Outbred (DO) mice. The DO is a randomly outcrossed population with high heterozygosity that captures the allelic diversity of eight inbred mouse lines from three subspecies. The study uses a sample of 1147 DO animals (the largest sample yet employed for a shape QTL study in mouse), each characterized by 22 three-dimensional landmarks, 56,885 autosomal and X-chromosome markers, and sex and age classifiers. We identified 37 facial shape QTL across 20 shape principal components (PCs) using a mixed effects regression that accounts for kinship among observations. The QTL include some previously identified intervals as well as new regions that expand the list of potential targets for future experimental study. Three QTL characterized shape associations with size (allometry). Median support interval size was 3.5 Mb. Narrowing additional analysis to QTL for the five largest magnitude shape PCs, we found significant overrepresentation of genes with known roles in growth, skeletal and facial development, and sensory organ development. For most intervals, one or more of these genes lies within 0.25 Mb of the QTL’s peak. QTL effect sizes were small, with none explaining more than 0.5% of facial shape variation. Thus, our results are consistent with a model of facial diversity that is influenced by key genes in skeletal and facial development and, simultaneously, is highly polygenic.

Sexual Dimorphism of Atherosclerosis by Gut Microbiome in a Hyperlipidemic Diversity Outbred F1 Mouse Population

Abstract Objectives Atherosclerosis is a complex multifactorial disease that develops through the interaction of various genetic and environmental factors. The environmental factors affecting atherosclerosis susceptibility include gender differences and the gut microbiota. However, the association between sex, microbiota, and atherosclerosis remains unclear. We hypothesized that the association between gut microbial profiles and atherosclerosis demonstrates sexual dimorphism. In order to test this hypothesis, we examined atherosclerosis and microbiota in a population of hyperlipidemic outbred mice. Methods We collected offspring (262 female and 269 male mice) from a cross between hyperlipidemic male C57BL/6J mice, transgenic for both human apolipoprotein E-Leiden and cholesterol ester transfer protein genes, and ∼200 female Diversity Outbred (DO) mice, a population derived from 8 inbred strains. We fed the offspring a high fat/cholesterol diet for 12 weeks. We then examined over 20 cardio-metabolic traits and their fecal microbiota compositions using 16S rRNA sequencing. Results Female mice were significantly more susceptible to atherosclerosis with higher lesion area (P < 0.0001) and plasma cholesterol (P < 0.0001) than males. For gut microbiota analysis, females had higher microbial α-diversity (Shannon diversity, P < 0.0001) than males and their β-diversity (Unweighted UniFrac, R2 = 0.01, PERMANOVA < 0.001) showed that the overall community is significantly different. We also examined the association between sexually-dimorphic gut phylotypes and cardiometabolic phenotypes and identified a negative correlation between atherosclerosis and the abundance of the genus Bifidobacterium, while abundances of genus Lactococcus were positively associated with atherosclerosis. Upcoming studies will be designed to identify host genes affecting microbiota abundance and functional characterization of specific microbiota-atherosclerosis traits. Conclusions To the best of our knowledge, this study suggests the first example of complex interactions between sexually dimorphic traits and gut microbiota associated with atherosclerosis using DO-F1 mice. Funding Sources This work was supported by the National Heart, Lung, and Blood Institute.

Utilization of genetically diverse mouse model identifies genetic loci that correlate to response to immune checkpoint inhibitors to B16F0 melanoma

Abstract Historically metastatic melanoma has been associated with poor prognosis. The advent of immune checkpoint inhibitors targeting CTLA-4 and PD-1 have greatly improved patient survival in metastatic melanoma, but a significant proportion of patients fail to respond to therapy. To explore the effect of genetic background on immune checkpoint inhibitor efficacy, we have developed a tumor immunotherapy model using genetically heterogeneous Diversity Outbred (DO) mice and the C57BL/6 syngeneic B16F0 melanoma model. To implement genetic diversity, DO mice were crossed with C57BL/6 to generate (C57BL/6xDO)F1 mice. Untreated (C57BL/6xDO)F1 mice (n=34) reliably develop B16F0 tumors after subcutaneous inoculation, with some variation in tumor latency. To test the role of genetics in response to ICI, (C57BL/6xDO)F1 mice (n=142) were treated with combined anti-PD1/anti-CTLA-4 on days 3, 6, and 10 after inoculation with 2×105 B16F0 cells. Mice receiving therapy show wide variation in tumor development of up to 65 days (mean 20.86 +/− 11.04, CV 52.94%), with 19 never developing tumor by day 88. Re-challenge of tumor free mice confirmed adaptive response to tumor. Preliminary Genome Wide Association shows suggestive associations on chromosomes 8, 12, and 13. The locus on chromosome 13 contains a family of related genes with known function in autoimmune conditions and expression in several immune cell types. QTL effects show the NZO genotype is a positive driver at this locus and C57BL/6 is a negative driver, reflecting known ICI resistance in the C57BL/6 strain. These results demonstrate host genetic background significantly contributes to response to ICI and identifies genomic loci associating with primary resistance. NIH R37 CA220482.

Identifying host-intrinsic resistance mechanisms to therapeutic anti-tumor antibody

Abstract Monoclonal antibody (mAb) therapies have greatly improved outcomes for many cancer subtypes, many of which target tumor-associated antigens (TAA). Trastuzumab targets the TAA human epidermal growth factor 2 (HER2), inducing antibody dependent cellular cytotoxicity and phagocytosis (ADCC/ADCP) as well as interfering with receptor signaling. Despite its reputation as the gold standard treatment for HER2-overexpressing cancers, response rates remain low in metastatic breast cancer (≤30% as a monotherapy, ≤50% with chemotherapy). Multiple studies suggest induction of endogenous anti-tumor immunity is a determinant of therapeutic success. Utilizing the genetically heterogeneous Diversity Outbred (DO) mouse model, we seek to identify host-intrinsic mechanisms of resistance to TAA-mediated immunotherapy. We find a divergent response to αNeu (HER2 homolog) mAb clone 7.16.4 in (DOxBALB/c)F1 mice bearing established BALB/c-syngeneic TUBO mammary carcinoma. We find host genetic background regulates anti-tumor immune activity. ~25% showed complete response (CR) to therapy, ~60% a partial response (PR), and ~15% failed to respond (NR). CR mice were protected from a contralateral rechallenge, suggesting adaptive anti-tumor immunity was promoted by mAb therapy. Peripheral anti-tumor IgG, measured by flow cytometry, and IFNγ producing T cells, measured via ELISpot, also trend toward CR>PR>NR. Genetic linkage analysis using R/qtl2 identified loci associated with robust response to mAb therapy. Genes within these loci may represent potential routes of ingress toward addressing innate and acquired resistance during anti-TAA mAb therapy, particularly as regulators of immunogenicity of ADCC and ADCP. NIH R37 CA220482.

The Diversity Outbred Mouse Population Is an Improved Animal Model of Vaccination against Tuberculosis That Reflects Heterogeneity of Protection.

Many studies of Mycobacterium tuberculosis infection and immunity have used mouse models. However, outcomes of vaccination and challenge with M. tuberculosis in inbred mouse strains do not reflect the full range of outcomes seen in people. Previous studies indicated that the novel Diversity Outbred (DO) mouse population exhibited a spectrum of outcomes after primary aerosol infection with M. tuberculosis Here, we demonstrate the value of this novel mouse population for studies of vaccination against M. tuberculosis aerosol challenge. Using the only currently licensed tuberculosis vaccine, we found that the DO population readily controlled systemic Mycobacterium bovis BCG bacterial burdens and that BCG vaccination significantly improved survival across the DO population upon challenge with M. tuberculosis Many individual DO mice that were vaccinated with BCG and then challenged with M. tuberculosis exhibited low bacterial burdens, low or even no systemic dissemination, little weight loss, and only minor lung pathology. In contrast, some BCG-vaccinated DO mice progressed quickly to fulminant disease upon M. tuberculosis challenge. Across the population, most of these disease parameters were at most modestly correlated with each other and were often discordant. This result suggests the need for a multiparameter metric to better characterize "disease" and "protection," with closer similarity to the complex case definitions used in people. Taken together, these results demonstrate that DO mice provide a novel small-animal model of vaccination against tuberculosis that better reflects the wide spectrum of outcomes seen in people.IMPORTANCE We vaccinated the Diversity Outbred (DO) population of mice with BCG, the only vaccine currently used to protect against tuberculosis, and then challenged them with M. tuberculosis by aerosol. We found that the BCG-vaccinated DO mouse population exhibited a wide range of outcomes, in which outcomes in individual mice ranged from minimal respiratory or systemic disease to fulminant disease and death. The breadth of these outcomes appears similar to the range seen in people, indicating that DO mice may serve as an improved small-animal model to study tuberculosis infection and immunity. Moreover, sophisticated tools are available for the use of these mice to map genes contributing to control of vaccination. Thus, the present studies provided an important new tool in the fight against tuberculosis.

Nitrosative Stress and Lipid Homeostasis as a Mechanism for Zileuton Hepatotoxicity and Resistance in Genetically Sensitive Mice.

Zileuton is an orally active inhibitor of leukotriene synthesis for maintenance treatment of asthma, for which clinical usage has been associated with idiosyncratic liver injury. Mechanistic understanding of zileuton toxicity is hampered by the rarity of the cases and lack of an animal model. A promising model for mechanistic study of rare liver injury is the Diversity Outbred (J:DO) mouse population, with genetic variation similar to that found in humans. In this study, female DO mice were administered zileuton or vehicle daily for 7 days (i.g.). Serum liver enzymes were elevated in the zileuton group, with marked interindividual variability in response. Zileuton exposure-induced findings in susceptible DO mice included microvesicular fatty change, hepatocellular mitosis, and hepatocellular necrosis. Inducible nitric oxide synthase and nitrotyrosine abundance were increased in livers of animals with necrosis and those with fatty change, implicating nitrosative stress as a possible injury mechanism. Conversely, DO mice lacking adverse liver pathology following zileuton exposure experienced decreased hepatic concentrations of resistin and increased concentrations of insulin and leptin, providing potential clues into mechanisms of toxicity resistance. Transcriptome pathway analysis highlighted mitochondrial dysfunction and altered fatty acid oxidation as key molecular perturbations associated with zileuton exposure, and suggested that interindividual differences in cytochrome P450 metabolism, glutathione-mediated detoxification, and farnesoid X receptor signaling may contribute to zileuton-induced liver injury (ZILI). Taken together, DO mice provided a platform for investigating mechanisms of toxicity and resistance in context of ZILI which may lead to targeted therapeutic interventions.