Inbred Mouse Research Articles

A TLR4 ligand-based adjuvant for promoting the immunogenicity of typhoid subunit vaccines

None of the typhoid Vi Polysaccharide (ViPS) subunit vaccines incorporate adjuvants, and the immunogenicity of ViPS vaccines (e.g. Typbar TCV® and Typhim Vi®) is in part due to associated TLR4 ligands such as endotoxin present in these vaccines. Since endotoxin content in vaccines is variable and kept very low due to inherent toxicity, it was hypothesized that incorporating a defined amount of a non-toxic TLR4-ligand such as monophosphoryl lipid A in ViPS vaccines would improve their immunogenicity. To test this hypothesis, a monophosphoryl lipid A-based adjuvant formulation named Turbo was developed. Admixing Turbo with Typbar TCV® (ViPS-conjugated to tetanus toxoid) increased the levels of anti-ViPS IgM, IgG1, IgG2b, IgG2a/c, and IgG3 in inbred and outbred mice. In infant mice, a single immunization with Turbo adjuvanted Typbar TCV® resulted in a significantly increased and durable IgG response and improved the control of bacterial burden compared to mice immunized without Turbo. Similarly, when adjuvanted with Turbo, the antibody response and control of bacteremia were also improved in mice immunized with Typhim Vi®, an unconjugated vaccine. The immunogenicity of unconjugated ViPS is inefficient in young mice and is lost in adult mice when immunostimulatory ligands in ViPS are removed. Nevertheless, when adjuvanted with Turbo, poorly immunogenic ViPS induced a robust IgG response in young and adult mice, and this was observed even under antigen-limiting conditions. These data suggest that incorporation of Turbo as an adjuvant will make typhoid vaccines more immunogenic regardless of their intrinsic immunogenicity or conjugation status and maximize the efficacy across all ages.

Molecular Regulation of Fetal Brain Development in Inbred and Congenic Mouse Strains Differing in Longevity

The objective of this study was to investigate gene regulation of the developing fetal brain from congenic or inbred mice strains that differed in longevity. Gene expression and alternative splice variants were analyzed in a genome-wide manner in the fetal brain of C57BL/6J mice (long-lived) in comparison to B6.Cg-Cav1tm1Mls/J (congenic, short-lived) and AKR/J (inbred, short-lived) mice on day(d) 12, 15, and 17 of gestation. The analysis showed a contrasting gene expression pattern during fetal brain development in these mice. Genes related to brain development, aging, and the regulation of alternative splicing were significantly differentially regulated in the fetal brain of the short-lived compared to long-lived mice during development from d15 and d17. A significantly reduced number of splice variants was observed on d15 compared to d12 or d17 in a strain-dependent manner. An epigenetic clock analysis of d15 fetal brain identified DNA methylations that were significantly associated with single-nucleotide polymorphic sites between AKR/J and C57BL/6J strains. These methylations were associated with genes that show epigenetic changes in an age-correlated manner in mice. Together, the finding of this study suggest that fetal brain development and longevity are epigenetically linked, supporting the emerging concept of the early-life origin of longevity.

Pesticide-Induced Alterations in Locomotor Activity, Anxiety, and Depression-like Behavior Are Mediated through Oxidative Stress-Related Autophagy: A Persistent Developmental Study in Mice.

Chlorpyrifos (CPF), dichlorvos (DDV), and cypermethrin (CP), as commonly used pesticides, have been implicated in inducing neuropsychiatric disorders, such as anxiety, depression-like behaviors, and locomotor activity impairment. However, the exact molecular mechanisms of these adverse effects, particularly in both sexes and their next-generation effects, remain unclear. In this study, we conducted behavioral analysis, along with cellular assays (monodansylcadaverine staining) and molecular investigations (qRT-PCR and western blotting of mTOR, P62, and Beclin-1) to clear the potential role of autophagy in pesticide-induced behavioral alterations. For this purpose, 42 adult female and 21 male inbred ICR mice (F0) were distributed into seven groups. Maternal mice (F0) and 112 F1 offspring were exposed to 0.5 and 1 ppm of CPF, DDV, and CP through drinking water. F1 male and female animals were studied to assess the sex-specific effects of pesticides on brain tissue. Our findings revealed pronounced anxiogenic effects and impaired locomotor activity in mice. F1 males exposed to CPF (1 ppm) exhibited significantly elevated depression-like behaviors compared to other groups. Moreover, pesticide exposure reduced mTOR and P62 levels, while enhancing the Beclin-1 gene and protein expression. These changes in autophagy signaling pathways, coupled with oxidative and neurogenic damage in the cerebral cortex and hippocampus, potentially contribute to heightened locomotor activity, anxiety, and depression-like behaviors following pesticide exposure. This study underscores the substantial impact of pesticides on both physiological and behavioral aspects, emphasizing the necessity for comprehensive assessments and regulatory considerations for pesticide use. Additionally, the identification of sex-specific responses presents a crucial dimension for pharmaceutical sciences, highlighting the need for tailored therapeutic interventions and further research in this field.

Imputation of 3D genome structure by genetic–epigenetic interaction modeling in mice

Gene expression is known to be affected by interactions between local genetic variation and DNA accessibility, with the latter organized into three-dimensional chromatin structures. Analyses of these interactions have previously been limited, obscuring their regulatory context, and the extent to which they occur throughout the genome. Here, we undertake a genome-scale analysis of these interactions in a genetically diverse population to systematically identify global genetic–epigenetic interaction, and reveal constraints imposed by chromatin structure. We establish the extent and structure of genotype-by-epigenotype interaction using embryonic stem cells derived from Diversity Outbred mice. This mouse population segregates millions of variants from eight inbred founders, enabling precision genetic mapping with extensive genotypic and phenotypic diversity. With 176 samples profiled for genotype, gene expression, and open chromatin, we used regression modeling to infer genetic–epigenetic interactions on a genome-wide scale. Our results demonstrate that statistical interactions between genetic variants and chromatin accessibility are common throughout the genome. We found that these interactions occur within the local area of the affected gene, and that this locality corresponds to topologically associated domains (TADs). The likelihood of interaction was most strongly defined by the three-dimensional (3D) domain structure rather than linear DNA sequence. We show that stable 3D genome structure is an effective tool to guide searches for regulatory elements and, conversely, that regulatory elements in genetically diverse populations provide a means to infer 3D genome structure. We confirmed this finding with CTCF ChIP-seq that revealed strain-specific binding in the inbred founder mice. In stem cells, open chromatin participating in the most significant regression models demonstrated an enrichment for developmental genes and the TAD-forming CTCF-binding complex, providing an opportunity for statistical inference of shifting TAD boundaries operating during early development. These findings provide evidence that genetic and epigenetic factors operate within the context of 3D chromatin structure.

Imputation of 3D genome structure by genetic-epigenetic interaction modeling in mice.

Gene expression is known to be affected by interactions between local genetic variation and DNA accessibility, with the latter organized into three-dimensional chromatin structures. Analyses of these interactions have previously been limited, obscuring their regulatory context, and the extent to which they occur throughout the genome. Here, we undertake a genome-scale analysis of these interactions in a genetically diverse population to systematically identify global genetic-epigenetic interaction, and reveal constraints imposed by chromatin structure. We establish the extent and structure of genotype-by-epigenotype interaction using embryonic stem cells derived from Diversity Outbred mice. This mouse population segregates millions of variants from eight inbred founders, enabling precision genetic mapping with extensive genotypic and phenotypic diversity. With 176 samples profiled for genotype, gene expression, and open chromatin, we used regression modeling to infer genetic-epigenetic interactions on a genome-wide scale. Our results demonstrate that statistical interactions between genetic variants and chromatin accessibility are common throughout the genome. We found that these interactions occur within the local area of the affected gene, and that this locality corresponds to topologically associated domains (TADs). The likelihood of interaction was most strongly defined by the three-dimensional (3D) domain structure rather than linear DNA sequence. We show that stable 3D genome structure is an effective tool to guide searches for regulatory elements and, conversely, that regulatory elements in genetically diverse populations provide a means to infer 3D genome structure. We confirmed this finding with CTCF ChIP-seq that revealed strain-specific binding in the inbred founder mice. In stem cells, open chromatin participating in the most significant regression models demonstrated an enrichment for developmental genes and the TAD-forming CTCF-binding complex, providing an opportunity for statistical inference of shifting TAD boundaries operating during early development. These findings provide evidence that genetic and epigenetic factors operate within the context of 3D chromatin structure.

Amygdalar neurotransmission alterations in the BTBR mice model of idiopathic autism.

Autism Spectrum Disorders (ASD) are principally diagnosed by three core behavioural symptoms, such as stereotyped repertoire, communication impairments and social dysfunctions. This complex pathology has been linked to abnormalities of corticostriatal and limbic circuits. Despite experimental efforts in elucidating the molecular mechanisms behind these abnormalities, a clear etiopathogenic hypothesis is still lacking. To this aim, preclinical studies can be really helpful to longitudinally study behavioural alterations resembling human symptoms and to investigate the underlying neurobiological correlates. In this regard, the BTBR T+ Itpr3tf/J (BTBR) mice are an inbred mouse strain that exhibits a pattern of behaviours well resembling human ASD-like behavioural features. In this study, the BTBR mice model was used to investigate neurochemical and biomolecular alterations, regarding Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF), together with GABAergic, glutamatergic, cholinergic, dopaminergic and noradrenergic neurotransmissions and their metabolites in four different brain areas, i.e. prefrontal cortex, hippocampus, amygdala and hypothalamus. In our results, BTBR strain reported decreased noradrenaline, acetylcholine and GABA levels in prefrontal cortex, while hippocampal measurements showed reduced NGF and BDNF expression levels, together with GABA levels. Concerning hypothalamus, no differences were retrieved. As regarding amygdala, we found reduced dopamine levels, accompanied by increased dopamine metabolites in BTBR mice, together with decreased acetylcholine, NGF and GABA levels and enhanced glutamate content. Taken together, our data showed that the BTBR ASD model, beyond its face validity, is a useful tool to untangle neurotransmission alterations that could be underpinned to the heterogeneous ASD-like behaviours, highlighting the crucial role played by amygdala.

CFP/Yit: An Inbred Mouse Strain with Slow Gastrointestinal Transit.

Gastrointestinal transit (GIT) is influenced by factors including diet, medications, genetics, and gut microbiota, with slow GIT potentially indicating a functional disorder linked to conditions, such as constipation. Although GIT studies have utilized various animal models, few effectively model spontaneous slow GIT. We aimed to characterize the GIT phenotype of CFP/Yit (CFP), an inbred mouse strain with suggested slow GIT. Female and male CFP mice were compared to Crl:CD1 (ICR) mice in GIT and assessed based on oral gavage of fluorescent-labeled 70-kDa dextran, feed intake, fecal amount, and fecal water content. Histopathological analysis of the colon and analysis of gut microbiota were conducted. CFP mice exhibited a shorter small intestine and a 1.4-fold longer colon compared to ICR mice. The median whole-GIT time was 6.0-fold longer in CFP mice than in ICR mice. CFP mice demonstrated slower gastric and cecal transits than ICR mice, with a median colonic transit time of 4.1h (2.9-fold longer). CFP mice exhibited lower daily feed intakes and fecal amounts. Fecal water content was lower in CFP mice, apparently attributed to the longer colon. Histopathological analysis showed no changes in CFP mice, including tumors or inflammation. Moreover, CFP mice had a higher Firmicutes/Bacteroidota ratio and a relative abundance of Erysipelotrichaceae in cecal and fecal contents. This study indicates that CFP mice exhibit slow transit in the stomach, cecum, and colon. As a novel mouse model, CFP mice can contribute to the study of gastrointestinal physiology and disease.

Into the Wild: A novel wild-derived inbred strain resource expands the genomic and phenotypic diversity of laboratory mouse models.

The laboratory mouse has served as the premier animal model system for both basic and preclinical investigations for over a century. However, laboratory mice capture only a subset of the genetic variation found in wild mouse populations, ultimately limiting the potential of classical inbred strains to uncover phenotype-associated variants and pathways. Wild mouse populations are reservoirs of genetic diversity that could facilitate the discovery of new functional and disease-associated alleles, but the scarcity of commercially available, well-characterized wild mouse strains limits their broader adoption in biomedical research. To overcome this barrier, we have recently developed, sequenced, and phenotyped a set of 11 inbred strains derived from wild-caught Mus musculus domesticus. Each of these "Nachman strains" immortalizes a unique wild haplotype sampled from one of five environmentally distinct locations across North and South America. Whole genome sequence analysis reveals that each strain carries between 4.73-6.54 million single nucleotide differences relative to the GRCm39 mouse reference, with 42.5% of variants in the Nachman strain genomes absent from current classical inbred mouse strain panels. We phenotyped the Nachman strains on a customized pipeline to assess the scope of disease-relevant neurobehavioral, biochemical, physiological, metabolic, and morphological trait variation. The Nachman strains exhibit significant inter-strain variation in >90% of 1119 surveyed traits and expand the range of phenotypic diversity captured in classical inbred strain panels. These novel wild-derived inbred mouse strain resources are set to empower new discoveries in both basic and preclinical research.

Coadministration of doxorubicin with vitamin D3, Lactobacillus acidophilus, and Lactobacillus casei in the 4T1 mouse model of breast cancer: anticancer and enteroprotective effects.

The use of doxorubicin (Dox) in the treatment of breast cancer negatively affects the intestines and other tissues. Many studies have proven that probiotics and vitamin D3 have antitumor and intestinal tissue-protecting properties. To achieve effectiveness and minimize side effects, the current study aims to administer Dox together with probiotics (Lactobacillus acidophilus and Lactobacillus casei) and vitamin D3. Forty-two female BALB/c inbred mice were divided into six groups: Group 1 (Control), Group 2 (Dox), Group 3 (Dox and probiotics), Group 4 (Dox and vitamin D3), Group 5 (Dox, probiotics, and vitamin D3), and Group 6 (probiotics and vitamin D3). The 4T1 mouse carcinoma cell line was injected into the mammary fat pad of each mouse. Gene expression was examined using quantitative real-time PCR. The treated groups (except group 6) showed significantly reduced tumor volume and weight compared to the control group (P < 0.05, P < 0.01). Probiotics/vitamin D3 with Dox reduced chemotherapy toxicity and a combination of supplements had a significant protective effect against Dox (P < 0.05, 0.01, 0.001). The treated groups (except 6) had significantly higher expression of Bax/Caspase 3 genes and lower expression of Bcl-2 genes than the control group (P < 0.05, 0.01). Coadministration of Dox with probiotics and vitamin D3 showed promising results in reducing tumor size, protecting intestinal tissue and influencing gene expression, suggesting a strategy to enhance the effectiveness of breast cancer treatment while reducing side effects.

Genetics and diet shape the relationship between islet function and whole body metabolism.

Despite the fact that genes and the environment are known to play a central role in islet function, our knowledge of how these parameters interact to modulate insulin secretory function remains relatively poor. Presently, we performed ex vivo glucose-stimulated insulin secretion and insulin content assays in islets of 213 mice from 13 inbred mouse strains on chow, Western diet (WD), and a high-fat, carbohydrate-free (KETO) diet. Strikingly, among these 13 strains, islets from the commonly used C57BL/6J mouse strain were the least glucose responsive. Using matched metabolic phenotyping data, we performed correlation analyses of isolated islet parameters and found a positive correlation between basal and glucose-stimulated insulin secretion, but no relationship between insulin secretion and insulin content. Using in vivo metabolic measures, we found that glucose tolerance determines the relationship between ex vivo islet insulin secretion and plasma insulin levels. Finally, we showed that islet glucose-stimulated insulin secretion decreased with KETO in almost all strains, concomitant with broader phenotypic changes, such as increased adiposity and glucose intolerance. This is an important finding as it should caution against the application of KETO diet for beta-cell health. Together these data offer key insights into the intersection of diet and genetic background on islet function and whole body glucose metabolism.NEW & NOTEWORTHY Thirteen strains of mice on chow, Western diet, and high-fat, carbohydrate-free (KETO), correlating whole body phenotypes to ex vivo pancreatic islet functional measurements, were used. The study finds a huge spectrum of functional islet responses and insulin phenotypes across all strains and diets, with the ubiquitous C57Bl/6J mouse exhibiting the lowest secretory response of all strains, highlighting the overall importance of considering genetic background when investigating islet function. Ex vivo basal and stimulated insulin secretion are correlated in the islet, and KETO imparts widescale downregulation of islet insulin secretion.

Effects of dietary iron deficiency or overload on bone: Dietary details matter

PurposeBone is susceptible to fluctuations in iron homeostasis, as both iron deficiency and overload are linked to poor bone strength in humans. In mice, however, inconsistent results have been reported, likely due to different diet setups or genetic backgrounds. Here, we assessed the effect of different high and low iron diets on bone in six inbred mouse strains (C57BL/6J, A/J, BALB/cJ, AKR/J, C3H/HeJ, and DBA/2J). MethodsMice received a high (20,000 ppm) or low-iron diet (~10 ppm) after weaning for 6–8 weeks. For C57BL/6J males, we used two dietary setups with similar amounts of iron, yet different nutritional compositions that were either richer (“TUD study”) or poorer (“UCLA study”) in minerals and vitamins. After sacrifice, liver, blood and bone parameters as well as bone turnover markers in the serum were analyzed. ResultsAlmost all mice on the UCLA study high iron diet had a significant decrease of cortical and trabecular bone mass accompanied by high bone resorption. Iron deficiency did not change bone microarchitecture or turnover in C57BL/6J, A/J, and DBA/2J mice, but increased trabecular bone mass in BALB/cJ, C3H/HeJ and AKR/J mice. In contrast to the UCLA study, male C57BL/6J mice in the TUD study did not display any changes in trabecular bone mass or turnover on high or low iron diet. However, cortical bone parameters were also decreased in TUD mice on the high iron diet. ConclusionThus, these data show that cortical bone is more susceptible to iron overload than trabecular bone and highlight the importance of a nutrient-rich diet to potentially mitigate the negative effects of iron overload on bone.

Abstract 6034: PSMA targeted alpha radiopharmaceutical [211At]PSAt-3 inhibits tumor growth in a preclinical model of prostate cancer

Abstract Introduction: Prostate cancer continues to be a highly prevalent cancer diagnosis among men and a leading cause of cancer related deaths worldwide. With the introduction[177Lu]Lu-PSMA-617, targeted radionuclide therapy has proven successful in increasing survival time for patients with metastasized castration resistant prostate cancer. However, despite encouraging results, such patients tend to develop progressive disease, and thus, enhanced efficacy is warranted. To this end, the use of radiopharmaceuticals harnessing alpha-emitters is a promising venue due to the potent energy deposition and short range of alpha particles in tissue. Here, we employ the PSMA targeting vector, [211At]PSAt-3, for targeted alpha radionuclide therapy in a preclinical prostate cancer model. Methods: [211At]PSAt-3, a structural derivative of PSMA-617, in which the L-2-naphtylalanine was replaced by L-3-[211At]astato-phenylalanine, was obtained by electrophilic [211At]astatodesilylation. Evaluation of [211At]PSAt-3 was conducted in male inbred nude mice of Balb/c background xenografted subcutaneously with human prostate cancer cells (LNCaP). Prior biodistribution studies were carried out with approximately 100 kBq intravenous injections, and subsequent biodistribution 2, 6 and 24h post injection (P.I.). For establishment of therapeutic effect, a single intravenous dose of [211At]PSAt-3 was administered. To asses hematological toxicity, blood cell composition was assessed weekly. Results: Favorable tumor uptake of 15.2±6 %ID/g was observed 2h after injection of [211At]PSAt-3 in LNCaP xenografted nude mice, with subsequent 9±5.1% ID/g and 6.4±2.4% ID/g 6 and 24h P.I., respectively. [211At]PSAt-3 cleared mainly by the kidneys, in which uptake was 72, 38 and 6% ID/g at 2, 6 and 24h P.I., respectively. Thyroid/throat uptake remained low at ~0.1% ID for all timepoints investigated, indicating a low degree of de-astatination. Initial efficacy studies using a single intravenous dose of 0.5 MBq (~20 MBq/kg) demonstrated significant growth delay and tumor volume reduction compared with control mice (saline). Blood platelet count was reduced after administration of [211At]PSAt-3 and subsequently recovered. No signs of severe toxicity, and no significant weight loss between control and treated mice was observed, suggesting that the treatment is well tolerated. Conclusion: 0.5 MBq [211At]PSAt-3 was well tolerated in mice and demonstrated tumor volume reduction and significant delay of tumor growth. These findings suggest [211At]PSAt-3 as a promising candidate for clinical translation. Citation Format: Lars Hvass, Marius Müller, Anne Clausen, Matthias Herth, Andreas Kjaer. PSMA targeted alpha radiopharmaceutical [211At]PSAt-3 inhibits tumor growth in a preclinical model of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6034.

Impact of ChAdOx1 or DNA Prime Vaccination on Magnitude, Breadth, and Focus of MVA-Boosted Immunogen-Specific T Cell Responses.

The efficacy of anti-viral T-cell vaccines may greatly depend on their ability to generate high-magnitude responses targeting a broad range of different epitopes. Recently, we created the HIV T-cell immunogen HTI, designed to generate T-cell responses to protein fragments more frequently targeted by HIV controllers. In the present study, we aim to maximize the breadth and magnitude of the T-cell responses generated by HTI by combining different vaccine vectors expressing HTI. We evaluated the ability to induce strong and broad T-cell responses to the HTI immunogen through prime vaccination with DNA plasmid (D) or Chimpanzee Adenovirus Ox1 (ChAdOx1; C) vectors, followed by a Modified Virus Ankara (MVA; M) vaccine boost (DDD, DDDM, C, and CM). HTI-specific T-cell responses after vaccination were measured by IFN-γ-ELISpot assays in two inbred mice strains (C57BL/6 and BALB/c). CM was the schedule triggering the highest magnitude of the response in both mice strains. However, this effect was not reflected in an increase in the breadth of the response but rather in an increase in the magnitude of the response to specific immunodominant epitopes. Immunodominance profiles in the two mouse strains were different, with a clear dominance of T-cell responses to a Pol-derived peptide pool after CM vaccination in C57BL/6. Responses to CM vaccination were also maintained at higher magnitudes over time (13 weeks) compared to other vaccination regimens. Thus, while a ChAdOx1 prime combined with MVA booster vaccination generated stronger and more sustained T-cell responses compared to three DNA vaccinations, the ChAdOx1 primed responses were more narrowly targeted. In conclusion, our findings suggest that the choice of vaccine vectors and prime-boost regimens plays a crucial role in determining the strength, duration, breadth, and focus of T-cell responses, providing further guidance for selecting vaccination strategies.

Integration of genomic and transcriptomic data of inbred mouse models for polygenic obesity and leanness revealed “obese” and “lean” candidate alleles in polyadenylation signals

Most mammalian genes have multiple polyadenylation (PA) sites, and alternative polyadenylation (APA) has been linked to diseases such as obesity. Studies have shown that changes in the polyadenylation signal (PAS) can influence the efficiency of cleavage and affect disease susceptibility and phenotype. In our recent study we used inbred mouse models of polygenic obesity and leanness and identified single-nucleotide polymorphisms in PAS (PAS-SNPs) within several obesity candidate genes, including five with differential expression. Nevertheless, to date, there has been no systematic, whole-genome-level approach aiming to prioritise PAS-SNPs potentially affecting APA. Therefore, in this study, we build upon our previous work by integrating existing genomics data with transcriptomics. DEGs were identified in nine tissues by Affymetrix GeneChip. PA and PAS sites were from the PolyASite 2.0 portal. Prioritisation of candidate PAS-SNPs was performed based on whether they were located in DEG, the type of PAS changes they caused, locations of PA sites relative to PAS, and location(s) and expression(s) of Affymetrix probes within a given gene in various tissues. For the candidates, potential consequences due to the alteration in APA events were investigated using bioinformatics databases and tools. The analysis found 127 PAS-SNPs in 101 DEGs across different tissues and identified 12 high-priority and 7 moderate-priority PAS-SNP candidates in 10 and 7 DEGs, respectively. Candidate PAS-SNPs were in 3′ UTR of 12 protein-coding genes (Lean line: Edil3, Eif2s1, Fbxl3, Hlf, Hsf2bp, Knop1, Lair1, Nmrk1; Fat line: Ehd1, Rpl14, Spon1, Txndc9), introns of four protein-coding genes (Lean line: Abi3bp, Prr16; Fat line: Agmo, Itga7) and intron of one lncRNA (Lean line: 1700086O06Rik). The integration of whole-genome sequencing and transcriptome analyses in this study has identified potential genome-wide candidate SNPs that could affect APA by altering/disrupting PAS motifs and be related to obesity in mice. The data provides a foundation for further research into these PAS-SNPs, their genes, and their contribution to the obesity/leanness phenotype, and contributes a part in explaining missing heritability commonly observed in complex traits.

Mt10 Vaccine Protects Diversity Outbred Mice from CVB3 Infection by Producing Virus-Specific Neutralizing Antibodies and Diverse Antibody Isotypes.

Group B coxsackieviruses (CVBs) cause a wide range of diseases in humans, but no vaccines are currently available to prevent these infections. Previously, we had demonstrated that a live attenuated CVB3 vaccine virus, Mutant 10 (Mt10), offers protection against multiple CVB serotypes as evaluated in various inbred mouse strains; however, the applicability of these findings to the outbred human population remains uncertain. To address this issue, we used Diversity Outbred (DO) mice, whose genome is derived from eight inbred mouse strains that may capture the level of genetic diversity of the outbred human population. To determine the efficacy of the Mt10 vaccine, we established the CVB3 infection model in the DO mice. We noted that CVB3 infection resulted mainly in pancreatitis, although viral RNA was detected in both the pancreas and heart. Histologically, the pancreatic lesions comprised of necrosis, post-necrotic atrophy, and lymphocyte infiltration. In evaluating the efficacy of the Mt10 vaccine, both male and female DO mice were completely protected in challenge studies with CVB3, and viral RNA was not detected in the heart or pancreas. Likewise, vaccine recipients of both sexes showed significant levels of virus-neutralizing antibodies. Furthermore, by using the CVB3 viral protein 1, virus-reactive antibodies were found to be diverse in the order of IgG2c, followed by IgG2a, IgG2b/IgG3, and IgG1. Together, the data suggest that the Mt10 vaccine virus can offer protection against CVB infections that may have translational significance.

Microglial reactivity in brainstem chemosensory nuclei in response to hypercapnia.

Microglia, the resident immune cells of the CNS, surveil, detect, and respond to various extracellular signals. Depending on the nature of these signals, an integrative microglial response can be triggered, resulting in a phenotypic transformation. Here, we evaluate whether hypercapnia modifies microglia phenotype in brainstem respiratory-related nuclei. Adult C57BL/6 inbred mice were exposed to 10% CO2 enriched air (hypercapnia), or pure air (control), for 10 or 30min and immediately processed for immunohistochemistry to detect the ubiquitous microglia marker, ionized calcium binding adaptor molecule 1 (Iba1). Hypercapnia for thirty, but not 10min reduced the Iba1 labeling percent coverage in the ventral respiratory column (VRC), raphe nucleus (RN), and nucleus tractus solitarius (NTS) and the number of primary branches in VRC. The morphological changes persisted, at least, for 60min breathing air after the hypercapnic challenge. No significant changes were observed in Iba1+ cells in the spinal trigeminal nucleus (Sp5) and the hippocampus. In CF-1 outbred mice, 10% CO2 followed by 60min of breathing air, resulted in the reduction of Iba1 labeling percent coverage and the number and length of primary branches in VRC, RN, and NTS. No morphological change was observed in Iba1+ cells in Sp5 and hippocampus. Double immunofluorescence revealed that prolonged hypercapnia increased the expression of CD86, an inflammatory marker for reactive state microglia, in Iba1+ cells in VRC, RN, and NTS, but not in Sp5 and hippocampus in CF-1 mice. By contrast, the expression of CD206, a marker of regulatory state microglia, persisted unmodified. In brainstem, but not in hippocampal microglia cultures, hypercapnia increased the level of IL1β, but not that of TGFβ measured by ELISA. Our results show that microglia from respiratory-related chemosensory nuclei, are reactive to prolonged hypercapnia acquiring an inflammatory-like phenotype.

Host Genetic Background Influences BCG-Induced Antibodies Cross-Reactive to SARS-CoV-2 Spike Protein.

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) protects against childhood tuberculosis; and unlike most vaccines, BCG broadly impacts immunity to other pathogens and even some cancers. Early in the COVID-19 pandemic, epidemiological studies identified a protective association between BCG vaccination and outcomes of SARS-CoV-2, but the associations in later studies were inconsistent. We sought possible reasons and noticed the study populations often lived in the same country. Since individuals from the same regions can share common ancestors, we hypothesized that genetic background could influence associations between BCG and SARS-CoV-2. To explore this hypothesis in a controlled environment, we performed a pilot study using Diversity Outbred mice. First, we identified amino acid sequences shared by BCG and SARS-CoV-2 spike protein. Next, we tested for IgG reactive to spike protein from BCG-vaccinated mice. Sera from some, but not all, BCG-vaccinated Diversity Outbred mice contained higher levels of IgG cross-reactive to SARS-CoV-2 spike protein than sera from BCG-vaccinated C57BL/6J inbred mice and unvaccinated mice. Although larger experimental studies are needed to obtain mechanistic insight, these findings suggest that genetic background may be an important variable contributing to different associations observed in human randomized clinical trials evaluating BCG vaccination on SARS-CoV-2 and COVID-19.

Epistasis between mutator alleles contributes to germline mutation spectrum variability in laboratory mice.

Maintaining germline genome integrity is essential and enormously complex. Although many proteins are involved in DNA replication, proofreading, and repair, mutator alleles have largely eluded detection in mammals. DNA replication and repair proteins often recognize sequence motifs or excise lesions at specific nucleotides. Thus, we might expect that the spectrum of de novo mutations - the frequencies of C>T, A>G, etc. - will differ between genomes that harbor either a mutator or wild-type allele. Previously, we used quantitative trait locus mapping to discover candidate mutator alleles in the DNA repair gene Mutyh that increased the C>A germline mutation rate in a family of inbred mice known as the BXDs (Sasani et al., 2022, Ashbrook et al., 2021). In this study we developed a new method to detect alleles associated with mutation spectrum variation and applied it to mutation data from the BXDs. We discovered an additional C>A mutator locus on chromosome 6 that overlaps Ogg1, a DNA glycosylase involved in the same base-excision repair network as Mutyh (David et al., 2007). Its effect depends on the presence of a mutator allele near Mutyh, and BXDs with mutator alleles at both loci have greater numbers of C>A mutations than those with mutator alleles at either locus alone. Our new methods for analyzing mutation spectra reveal evidence of epistasis between germline mutator alleles and may be applicable to mutation data from humans and other model organisms.

Epistasis between mutator alleles contributes to germline mutation spectrum variability in laboratory mice

Maintaining germline genome integrity is essential and enormously complex. Although many proteins are involved in DNA replication, proofreading, and repair, mutator alleles have largely eluded detection in mammals. DNA replication and repair proteins often recognize sequence motifs or excise lesions at specific nucleotides. Thus, we might expect that the spectrum of de novo mutations – the frequencies of C&gt;T, A&gt;G, etc. – will differ between genomes that harbor either a mutator or wild-type allele. Previously, we used quantitative trait locus mapping to discover candidate mutator alleles in the DNA repair gene Mutyh that increased the C&gt;A germline mutation rate in a family of inbred mice known as the BXDs (Sasani et al., 2022, Ashbrook et al., 2021). In this study we developed a new method to detect alleles associated with mutation spectrum variation and applied it to mutation data from the BXDs. We discovered an additional C&gt;A mutator locus on chromosome 6 that overlaps Ogg1, a DNA glycosylase involved in the same base-excision repair network as Mutyh (David et al., 2007). Its effect depends on the presence of a mutator allele near Mutyh, and BXDs with mutator alleles at both loci have greater numbers of C&gt;A mutations than those with mutator alleles at either locus alone. Our new methods for analyzing mutation spectra reveal evidence of epistasis between germline mutator alleles and may be applicable to mutation data from humans and other model organisms.

The type 1 diabetes susceptibility locus Idd5 favours robust neonatal development of highly autoreactive regulatory T cells in the NOD mouse.

Regulatory T lymphocytes expressing the transcription factor Foxp3 (Tregs) play an important role in the prevention of autoimmune diseases and other immunopathologies. Aberrations in Treg-mediated immunosuppression are therefore thought to be involved in the development of autoimmune pathologies, but few have been documented. Recent reports indicated a central role for Tregs developing during the neonatal period in the prevention of autoimmune pathology. We therefore investigated the development of Tregs in neonatal NOD mice, an important animal model for autoimmune type 1 diabetes. Surprisingly, we found that, as compared with seven other commonly studied inbred mouse strains, in neonatal NOD mice, exceptionally large proportions of developing Tregs express high levels of GITR and PD-1. The latter phenotype was previously associated with high Treg autoreactivity in C57BL/6 mice, which we here confirm for NOD animals. The proportions of newly developing GITRhighPD-1+ Tregs rapidly drop during the first week of age. A genome-wide genetic screen indicated the involvement of several diabetes susceptibility loci in this trait. Analysis of a congenic mouse strain confirmed that Idd5 contributes to the genetic control of GITRhighPD-1+ Treg development in neonates. Our data thus demonstrate an intriguing and paradoxical correlation between an idiosyncrasy in Treg development in NOD mice and their susceptibility to type 1 diabetes.