Genetic and microbial determinants of azoxymethane-induced colorectal tumor susceptibility in Collaborative Cross mice and their implication in human cancer.

The insights into interactions between host genetics and gut microbiome (GM) in colorectal tumor susceptibility (CTS) remains lacking. We used Collaborative Cross mouse population model to identify genetic and microbial determinants of Azoxymethane-induced CTS. We identified 4417 CTS-associated single nucleotide polymorphisms (SNPs) containing 334 genes that were transcriptionally altered in human colorectal cancers (CRCs) and consistently clustered independent human CRC cohorts into two subgroups with different prognosis. We discovered a set of genera in early-life associated with CTS and defined a 16-genus signature that accurately predicted CTS, the majority of which were correlated with human CRCs. We identified 547 SNPs associated with abundances of these genera. Mediation analysis revealed GM as mediators partially exerting the effect of SNP UNC3869242 within Duox2 on CTS. Intestine cell-specific depletion of Duox2 altered GM composition and contribution of Duox2 depletion to CTS was significantly influenced by GM. Our findings provide potential novel targets for personalized CRC prevention and treatment.

Late-life dietary folate restriction reduces biosynthetic processes without compromising healthspan in mice.

Folate is a vitamin required for cell growth and is present in fortified foods in the form of folic acid to prevent congenital abnormalities. The impact of low folate status on life-long health is poorly understood. We found that limiting folate levels with the folate antagonist methotrexate increased the lifespan of yeast and worms. We then restricted folate intake in aged mice and measured various health metrics, metabolites, and gene expression signatures. Limiting folate intake decreased anabolic biosynthetic processes in mice and enhanced metabolic plasticity. Despite reduced serum folate levels in mice with limited folic acid intake, these animals maintained their weight and adiposity late in life, and we did not observe adverse health outcomes. These results argue that the effectiveness of folate dietary interventions may vary depending on an individual's age and sex. A higher folate intake is advantageous during the early stages of life to support cell divisions needed for proper development. However, a lower folate intake later in life may result in healthier aging.

Nutrient-sensing growth hormone secretagogue receptor in macrophage programming and meta-inflammation.

OBJECTIVE: Obesity-associated chronic inflammation, aka meta-inflammation, is a key pathogenic driver for obesity-associated comorbidity. Growth hormone secretagogue receptor (GHSR) is known to mediate the effects of nutrient-sensing hormone ghrelin in food intake and fat deposition. We previously reported that global Ghsr ablation protects against diet-induced inflammation and insulin resistance, but the site(s) of action and mechanism are unknown. Macrophages are key drivers of meta-inflammation. To unravel the role of GHSR in macrophages, we generated myeloid-specific Ghsr knockout mice (LysM-Cre;Ghsrf/f). METHODS: LysM-Cre;Ghsrf/f and control Ghsrf/f mice were subjected to 5 months of high-fat diet (HFD) feeding to induce obesity. In vivo, metabolic profiling of food intake, physical activity, and energy expenditure, as well as glucose and insulin tolerance tests (GTT and ITT) were performed. At termination, peritoneal macrophages (PMs), epididymal white adipose tissue (eWAT), and liver were analyzed by flow cytometry and histology. For ex vivo studies, bone marrow-derived macrophages (BMDMs) were generated from the mice and treated with palmitic acid (PA) or lipopolysaccharide (LPS). For in vitro studies, macrophage RAW264.7 cells with Ghsr overexpression or Insulin receptor substrate 2 (Irs2) knockdown were studied. RESULTS: We found that Ghsr expression in PMs was increased under HFD feeding. In vivo, HFD-fed LysM-Cre;Ghsrf/f mice exhibited significantly attenuated systemic inflammation and insulin resistance without affecting food intake or body weight. Tissue analysis showed that HFD-fed LysM-Cre;Ghsrf/f mice have significantly decreased monocyte/macrophage infiltration, pro-inflammatory activation, and lipid accumulation, showing elevated lipid-associated macrophages (LAMs) in eWAT and liver. Ex vivo, Ghsr-deficient macrophages protected against PA- or LPS-induced pro-inflammatory polarization, showing reduced glycolysis, increased fatty acid oxidation, and decreased NF-κB nuclear translocation. At molecular level, GHSR metabolically programs macrophage polarization through PKA-CREB-IRS2-AKT2 signaling pathway. CONCLUSIONS: These novel results demonstrate that macrophage GHSR plays a key role in the pathogenesis of meta-inflammation, and macrophage GHSR promotes macrophage infiltration and induces pro-inflammatory polarization. These exciting findings suggest that GHSR may serve as a novel immunotherapeutic target for the treatment of obesity and its associated comorbidity.

Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of Escherichia coli leads to bacterial replication within liver abscesses, while other organs such as the spleen clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Abscess susceptibility is heightened in the C57BL lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as 1 d post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4 (Toll-like receptor 4), are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Genetic architecture of the acute and persistent immune cell response after radiation exposure.

Hematologic toxicity is a common side effect of multimodal cancer therapy. Nearly all animal studies investigating the causes of radiotherapy-induced hematologic toxicity use inbred strains with limited genetic diversity and do not reflect the diverse responses observed in humans. We used the population-based Collaborative Cross (CC) mouse resource to investigate the genetic architecture of the acute and persistent immune response after radiation exposure by measuring 22 immune parameters in 1,720 CC mice representing 35 strains. We determined relative acute and persistent radiation resistance scores at the individual strain level considering contributions from all immune parameters. Genome-wide association analysis identified quantitative trait loci associated with baseline and radiation responses. A cross-species radiation resistance score predicted recurrence-free survival in medulloblastoma patients. We present a community resource of immune parameters and genome-wide association analyses before and after radiation exposure for future investigations of the contributions of host genetics on radiosensitivity.

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.

BACKGROUND: The 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. RESULTS: Genetic 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 ß-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. CONCLUSIONS: These 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. Video Abstract.

Precision pharmacological reversal of strain-specific diet-induced metabolic syndrome in mice informed by epigenetic and transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat, high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genome-wide DNA methylation analyses for each strain and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects revealed a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by epigenetic and transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the farnesoid X receptor pathway, and found that GW4064 exerts strain-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis. Furthermore, GW4064 treatment induced inflammatory-related gene expression changes in NOD, indicating a strain-specific effect in its associated toxicities as well as its therapeutic efficacy. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention and a mouse platform for guiding this approach.

Genetic and immune determinants of E. coli liver abscess formation.

Systemic infections can yield distinct outcomes in different tissues. In mice, intravenous inoculation of E. coli leads to bacterial replication within liver abscesses while other organs such as the spleen largely clear the pathogen. Abscesses are macroscopic necrotic regions that comprise the vast majority of the bacterial burden in the animal, yet little is known about the processes underlying their formation. Here, we characterize E. coli liver abscesses and identify host determinants of abscess susceptibility. Spatial transcriptomics revealed that liver abscesses are associated with heterogenous immune cell clusters comprised of macrophages, neutrophils, dendritic cells, innate lymphoid cells, and T-cells that surround necrotic regions of the liver. Susceptibility to liver abscesses is heightened in the C57BL/6 lineage, particularly in C57BL/6N females. Backcross analyses demonstrated that abscess susceptibility is a polygenic trait inherited in a sex-dependent manner without direct linkage to sex chromosomes. As early as one day post infection, the magnitude of E. coli replication in the liver distinguishes abscess-susceptible and abscess-resistant strains of mice, suggesting that the immune pathways that regulate abscess formation are induced within hours. We characterized the early hepatic response with single-cell RNA sequencing and found that mice with reduced activation of early inflammatory responses, such as those lacking the LPS receptor TLR4, are resistant to abscess formation. Experiments with barcoded E. coli revealed that TLR4 mediates a tradeoff between abscess formation and bacterial clearance. Together, our findings define hallmarks of E. coli liver abscess formation and suggest that hyperactivation of the hepatic innate immune response drives liver abscess susceptibility.

Using the collaborative cross to identify the role of host genetics in defining the murine gut microbiome.

BACKGROUND: The human gut microbiota is a complex community comprised of trillions of bacteria and is critical for the digestion and absorption of nutrients. Bacterial communities of the intestinal microbiota influence the development of several conditions and diseases. We studied the effect of host genetics on gut microbial composition using Collaborative Cross (CC) mice. CC mice are a panel of mice that are genetically diverse across strains, but genetically identical within a given strain allowing repetition and deeper analysis than is possible with other collections of genetically diverse mice. RESULTS: 16S rRNA from the feces of 167 mice from 28 different CC strains was sequenced and analyzed using the Qiime2 pipeline. We observed a large variance in the bacterial composition across CC strains starting at the phylum level. Using bacterial composition data, we identified 17 significant Quantitative Trait Loci (QTL) linked to 14 genera on 9 different mouse chromosomes. Genes within these intervals were analyzed for significant association with pathways and the previously known human GWAS database using Enrichr analysis and Genecards database. Multiple host genes involved in obesity, glucose homeostasis, immunity, neurological diseases, and many other protein-coding genes located in these regions may play roles in determining the composition of the gut microbiota. A subset of these CC mice was infected with Salmonella Typhimurium. Using infection outcome data, an increase in abundance of genus Lachnospiraceae and decrease in genus Parasutterella correlated with positive health outcomes after infection. Machine learning classifiers accurately predicted the CC strain and the infection outcome using pre-infection bacterial composition data from the feces. CONCLUSION: Our study supports the hypothesis that multiple host genes influence the gut microbiome composition and homeostasis, and that certain organisms may influence health outcomes after S. Typhimurium infection. Video Abstract.

An unanticipated discourse of HB-EGF with VANGL2 signaling during embryo implantation.

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo-uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

Precision pharmacological reversal of genotype-specific diet-induced metabolic syndrome in mice informed by transcriptional regulation.

Diet-related metabolic syndrome is the largest contributor to adverse health in the United States. However, the study of gene-environment interactions and their epigenomic and transcriptomic integration is complicated by the lack of environmental and genetic control in humans that is possible in mouse models. Here we exposed three mouse strains, C57BL/6J (BL6), A/J, and NOD/ShiLtJ (NOD), to a high-fat high-carbohydrate diet, leading to varying degrees of metabolic syndrome. We then performed transcriptomic and genomic DNA methylation analyses and found overlapping but also highly divergent changes in gene expression and methylation upstream of the discordant metabolic phenotypes. Strain-specific pathway analysis of dietary effects reveals a dysregulation of cholesterol biosynthesis common to all three strains but distinct regulatory networks driving this dysregulation. This suggests a strategy for strain-specific targeted pharmacologic intervention of these upstream regulators informed by transcriptional regulation. As a pilot study, we administered the drug GW4064 to target one of these genotype-dependent networks, the Farnesoid X receptor pathway, and found that GW4064 exerts genotype-specific protection against dietary effects in BL6, as predicted by our transcriptomic analysis, as well as increased inflammatory-related gene expression changes in NOD. This pilot study demonstrates the potential efficacy of precision therapeutics for genotype-informed dietary metabolic intervention, and a mouse platform for guiding this approach.

Refining risk estimates for lead in drinking water based on the impact of genetics and diet on blood lead levels using the Collaborative Cross mouse population.

Blood lead (Pb) level (BLL) is a commonly used biomarker to evaluate associations with health effects. However, interventions to reduce the adverse effects of Pb require relating BLL to external exposure. Moreover, risk mitigation actions need to ensure protection of more susceptible individuals with a greater tendency to accumulate Pb. Because little data is available to quantify inter-individual variability in biokinetics of Pb, we investigated the influence of genetics and diet on BLL in the genetically diverse Collaborative Cross (CC) mouse population. Adult female mice from 49 CC strains received either a standard mouse chow or a chow mimicking the American diet while being provided water ad libitum with 1000 ppm Pb for 4 weeks. In both arms of the study, inter-strain variability was observed; however, in American diet-fed animals, the BLL was greater and more variable. Importantly, the degree of variation in BLL among strains on the American diet was greater (2.3) than the default variability estimate (1.6) used in setting the regulatory standards. Genetic analysis identified suggestive diet-associated haplotypes that were associated with variation in BLL, largely contributed by the PWK/PhJ strain. This study quantified the variation in BLL that is due to genetic background, diet, and their interactions, and observed that it may be greater than that assumed for current regulatory standards for Pb in drinking water. Moreover, this work highlights the need of characterizing inter-individual variation in BLL to ensure adequate public health interventions aimed at reducing human health risks from Pb.

Comparative transcriptome analysis of Peromyscus leucopus and C3H mice infected with the Lyme disease pathogen.

Lyme disease (LD), the most prevalent tick-borne disease of humans in the Northern Hemisphere, is caused by the spirochetal bacterium of Borreliella burgdorferi (Bb) sensu lato complex. In nature, Bb spirochetes are continuously transmitted between Ixodes ticks and mammalian or avian reservoir hosts. Peromyscus leucopus mice are considered the primary mammalian reservoir of Bb in the United States. Earlier studies demonstrated that experimentally infected P. leucopus mice do not develop disease. In contrast, C3H mice, a widely used laboratory strain of Mus musculus in the LD field, develop severe Lyme arthritis. To date, the exact tolerance mechanism of P. leucopus mice to Bb-induced infection remains unknown. To address this knowledge gap, the present study has compared spleen transcriptomes of P. leucopus and C3H/HeJ mice infected with Bb strain 297 with those of their respective uninfected controls. Overall, the data showed that the spleen transcriptome of Bb-infected P. leucopus mice was much more quiescent compared to that of the infected C3H mice. To date, the current investigation is one of the few that have examined the transcriptome response of natural reservoir hosts to Borreliella infection. Although the experimental design of this study significantly differed from those of two previous investigations, the collective results of the current and published studies have consistently demonstrated very limited transcriptomic responses of different reservoir hosts to the persistent infection of LD pathogens. Importance: The bacterium Borreliella burgdorferi (Bb) causes Lyme disease, which is one of the emerging and highly debilitating human diseases in countries of the Northern Hemisphere. In nature, Bb spirochetes are maintained between hard ticks of Ixodes spp. and mammals or birds. In the United States, the white-footed mouse, Peromyscus leucopus, is one of the main Bb reservoirs. In contrast to humans and laboratory mice (e.g., C3H mice), white-footed mice rarely develop clinical signs (disease) despite being (persistently) infected with Bb. How the white-footed mouse tolerates Bb infection is the question that the present study has attempted to address. Comparisons of genetic responses between Bb-infected and uninfected mice demonstrated that, during a long-term Bb infection, C3H mice reacted much stronger, whereas P. leucopus mice were relatively unresponsive.

Baseline Gait and Motor Function Predict Long-Term Severity of Neurological Outcomes of Viral Infection.

Neurological dysfunction following viral infection varies among individuals, largely due to differences in their genetic backgrounds. Gait patterns, which can be evaluated using measures of coordination, balance, posture, muscle function, step-to-step variability, and other factors, are also influenced by genetic background. Accordingly, to some extent gait can be characteristic of an individual, even prior to changes in neurological function. Because neuromuscular aspects of gait are under a certain degree of genetic control, the hypothesis tested was that gait parameters could be predictive of neuromuscular dysfunction following viral infection. The Collaborative Cross (CC) mouse resource was utilized to model genetically diverse populations and the DigiGait treadmill system used to provide quantitative and objective measurements of 131 gait parameters in 142 mice from 23 CC and SJL/J strains. DigiGait measurements were taken prior to infection with the neurotropic virus Theiler's Murine Encephalomyelitis Virus (TMEV). Neurological phenotypes were recorded over 90 days post-infection (d.p.i.), and the cumulative frequency of the observation of these phenotypes was statistically associated with discrete baseline DigiGait measurements. These associations represented spatial and postural aspects of gait influenced by the 90 d.p.i. phenotype score. Furthermore, associations were found between these gait parameters with sex and outcomes considered to show resistance, resilience, or susceptibility to severe neurological symptoms after long-term infection. For example, higher pre-infection measurement values for the Paw Drag parameter corresponded with greater disease severity at 90 d.p.i. Quantitative trait loci significantly associated with these DigiGait parameters revealed potential relationships between 28 differentially expressed genes (DEGs) and different aspects of gait influenced by viral infection. Thus, these potential candidate genes and genetic variations may be predictive of long-term neurological dysfunction. Overall, these findings demonstrate the predictive/prognostic value of quantitative and objective pre-infection DigiGait measurements for viral-induced neuromuscular dysfunction.

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.

Background The 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 QTL 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/6J (B6) and FVB/NJ (FVB) mouse strains. Results Genetic 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 ß-diversity. Bilophila , Ruminiclostridium 9 , and Rikenella (Chr 1) were identified as sex and diet independent QTL candidate keystone organisms and Rikenelleceae RC9 Gut Group (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. Conclusions These 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.

Leveraging a natural murine meiotic drive to suppress invasive populations.

Invasive rodents are a major cause of environmental damage and biodiversity loss, particularly on islands. Unlike insects, genetic biocontrol strategies including population-suppressing gene drives with biased inheritance have not been developed in mice. Here, we demonstrate a gene drive strategy (tCRISPR) that leverages super-Mendelian transmission of the t haplotype to spread inactivating mutations in a haplosufficient female fertility gene (Prl). Using spatially explicit individual-based in silico modeling, we show that tCRISPR can eradicate island populations under a range of realistic field-based parameter values. We also engineer transgenic tCRISPR mice that, crucially, exhibit biased transmission of the modified t haplotype and Prl mutations at levels our modeling predicts would be sufficient for eradication. This is an example of a feasible gene drive system for invasive alien rodent population control.

Gut microbiota and host genetics modulate the effect of diverse diet patterns on metabolic health.

Metabolic diseases are major public health issues worldwide and are responsible for disproportionately higher healthcare costs and increased complications of many diseases including SARS-CoV-2 infection. The Western Diet (WD) specifically is believed to be a major contributor to the global metabolic disease epidemic. In contrast, the Mediterranean diet (MeD), Ketogenic diet (KD), and Japanese diet (JD) are often considered beneficial for metabolic health. Yet, there is a growing appreciation that the effect of diet on metabolic health varies depending on several factors including host genetics. Additionally, poor metabolic health has also been attributed to altered gut microbial composition and/or function. To understand the complex relationship between host genetics, gut microbiota, and dietary patterns, we treated four widely used metabolically diverse inbred mouse strains (A/J, C57BL/6J, FVB/NJ, and NOD/ShiLtJ) with four human-relevant diets (MeD, JD, KD, WD), and a control mouse chow from 6 weeks to 30 weeks of age. We found that diet-induced alteration of gut microbiota (α-diversity, ß-diversity, and abundance of several bacteria including Bifidobacterium, Ruminococcus, Turicibacter, Faecalibaculum, and Akkermansia) is significantly modified by host genetics. In addition, depending on the gut microbiota, the same diet could have different metabolic health effects. Our study also revealed that C57BL/6J mice are more susceptible to altered gut microbiota compared to other strains in this study indicating that host genetics is an important modulator of the diet-microbiota-metabolic health axis. Overall, our study demonstrated complex interactions between host genetics, gut microbiota, and diet on metabolic health; indicating the need to consider both host genetics and the gut microbiota in the development of new and more effective precision nutrition strategies to improve metabolic health.

Viral Clearance and Neuroinflammation in Acute TMEV Infection Vary by Host Genetic Background.

A wide range of viruses cause neurological manifestations in their hosts. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the brain, depending in part on host genetic background. The interaction between host genetic background, neurological response to viral infection, and subsequent clinical manifestations remains poorly understood. In the present study, we used the genetically diverse Collaborative Cross (CC) mouse resource to better understand how differences in genetic background drive clinical signs and neuropathological manifestations of acute Theiler's murine encephalomyelitis virus (TMEV) infection. For the first time, we characterized variations of TMEV viral tropism and load based on host genetic background, and correlated viral load with microglial/macrophage activation. For five CC strains (CC002, CC023, CC027, CC057, and CC078) infected with TMEV, we compared clinical signs, lesion distribution, microglial/macrophage response, expression, and distribution of TMEV mRNA, and identified genetic loci relevant to the early acute (4 days post-infection [dpi]) and late acute (14 dpi) timepoints. We examined brain pathology to determine possible causes of strain-specific differences in clinical signs, and found that fields CA1 and CA2 of the hippocampal formation were especially targeted by TMEV across all strains. Using Iba-1 immunolabeling, we identified and characterized strain- and timepoint-specific variation in microglial/macrophage reactivity in the hippocampal formation. Because viral clearance can influence disease outcome, we used RNA in situ hybridization to quantify viral load and TMEV mRNA distribution at both timepoints. TMEV mRNA expression was broadly distributed in the hippocampal formation at 4 dpi in all strains but varied between radiating and clustered distribution depending on the CC strain. We found a positive correlation between microglial/macrophage reactivity and TMEV mRNA expression at 4 dpi. At 14 dpi, we observed a dramatic reduction in TMEV mRNA expression, and localization to the medial portion of field CA1 and field CA2. To better understand how host genetic background can influence pathological outcomes, we identified quantitative trait loci associated with frequency of lesions in a particular brain region and with microglial/macrophage reactivity. These QTL were located near several loci of interest: lysosomal trafficking regulator (Lyst) and nidogen 1 (Nid1), and transmembrane protein 106 B (Tmem106b). Together, these results provide a novel understanding about the influences of genetic variation on the acute neuropathological and immunopathological environment and viral load, which collectively lead to variable disease outcomes. Our findings reveal possible avenues for future investigation which may lead to more effective intervention strategies and treatment regimens.

Serum Cytokines Predict Neurological Damage in Genetically Diverse Mouse Models.

Viral infections contribute to neurological and immunological dysfunction driven by complex genetic networks. Theiler's murine encephalomyelitis virus (TMEV) causes neurological dysfunction in mice and can model human outcomes to viral infections. Here, we used genetically distinct mice from five Collaborative Cross mouse strains and C57BL/6J to demonstrate how TMEV-induced immune responses in serum may predict neurological outcomes in acute infection. To test the hypothesis that serum cytokine levels can provide biomarkers for phenotypic outcomes of acute disease, we compared cytokine levels at pre-injection, 4 days post-injection (d.p.i.), and 14 d.p.i. Each strain produced unique baseline cytokine levels and had distinct immune responses to the injection procedure itself. Thus, we eliminated the baseline responses to the injection procedure itself and identified cytokines and chemokines induced specifically by TMEV infection. Then, we identified strain-specific longitudinal cytokine profiles in serum during acute disease. Using stepwise regression analysis, we identified serum immune markers predictive for TMEV-induced neurological phenotypes of the acute phase, e.g., IL-9 for limb paralysis; and TNF-α, IL-1ß, and MIP-1ß for limb weakness. These findings indicate how temporal differences in immune responses are influenced by host genetic background and demonstrate the potential of serum biomarkers to track the neurological effects of viral infection.