Mouse Strains Research Articles

Establishment of reference intervals of haematology and biochemistry analytes in ICR mice of different ages.

Outbred stocks of mice are widely used in pre-clinical research as these animals possess a diversified genetic background when compared with inbred strains of mice. It is crucial to assess particular alterations in the physiological and functional profiles of laboratory animals using haematological and biochemical indicators. These values can also differ between laboratories because they are influenced by many different factors. We aimed to provide normal values and reference intervals for selected haematology and biochemistry analytes of 570 ICR mice at three different ages: 6-8 weeks, 10-14 weeks and 6-9 months. Reference values were calculated by non-parametric methods. For comparisons between sexes, the independent-sample t-test and Mann-Whitney test were employed, and analysis of variance was used for age differences. The findings of the study revealed age-related declines in haemoglobin concentration, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin concentrations. Mice aged 6-9 months had statistically higher platelet counts in their blood than mice of other ages. The white blood cell count had a significant age effect and progressively decreased with age. As mice get older, the percentage of neutrophils, monocytes and basophils increases, but the percentage of lymphocytes decreases. For the biochemical values, age-related significant differences in glucose, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and albumin concentrations were found. It was also found that creatinine concentrations were comparable across all age ranges. The values presented in the present work can be used as a reference to interpret clinical pathology data for other studies and to evaluate health status.

Novel multiphoton intravital imaging enables real-time study of Helicobacter pylori interaction with neutrophils and macrophages in the mouse stomach.

Helicobacter pylori (H. pylori) is a bacterial pathogen that exclusively colonizes the human gastric mucosa and can cause persistent infection. In this process, H. pylori employs various strategies to avoid recognition by the human immune system. These range from passive defense strategies (e.g., altered LPS or flagellin structures) that prevent recognition by pattern recognition receptors (PRRs) to more active approaches, such as inhibition of IL-2 secretion and proliferation of T cells via VacA. Despite the growing evidence that H. pylori actively manipulates the human immune system for its own benefit, the direct interaction of H. pylori with immune cells in situ is poorly studied. Here, we present a novel intravital imaging model of the murine stomach gastric mucosa and show for the first time the in situ recruitment of neutrophils during infection and a direct H. pylori-macrophage interaction. For this purpose, we applied multiphoton intravital microscopy adapted with live drift correction software (VivoFollow) on LysM-eGFP and CX3CR1-eGFP reporter mice strains in which specific subsets of leukocytes are fluorescently labeled. Multiphoton microscopy is proving to be an excellent tool for characterizing interactions between immune cells and pathogens in vivo.

BrightMice: a low-cost do-it-yourself instrument, designed for in vivo fluorescence mouse imaging.

In vivo fluorescent imaging represents a potent means for real-time probe quantification, facilitating insights into disease pathophysiology and therapeutic responses. Nonetheless, accurate signal quantification remains challenging due to inherent factors like light scattering and tissue absorption. Existing imaging systems, though sophisticated, often entail high costs and are typically restricted to well-funded laboratory settings. This study introduces BrightMice, an innovative in vivo fluorescent imaging system that harnesses 3D printing and consumer-grade digital cameras. Tailored for various fluorophores such as EYFP and E2-crimson, the system showcases both adaptability and effectiveness in detecting in vivo fluorescent signals in several reporter mouse strains. Comparative analyses against commercial instruments confirm BrightMice's sensitivity and underscore its potential to democratize in vivo fluorescence imaging. By providing a cost-effective and accessible solution, BrightMice stands to benefit diverse research environments.

Comparison of Chikungunya Virus-Induced Disease Progression and Pathogenesis in Type-I Interferon Receptor-Deficient Mice (A129) and Two Wild-Type (129Sv/Ev and C57BL/6) Mouse Strains

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus causing a debilitating febrile illness with rheumatic disease symptoms of arthralgia and arthritis. Since its spread outside of Africa in 2005, it continues to cause outbreaks and disseminates into new territories. Intervention strategies are urgently required, including vaccination and antiviral approaches. To test efficacy, the use of small animal models is required. Two mouse strains, A129, with a deficiency in their type-I interferon (IFN) receptor, and C57BL/6 are widely used. A direct comparison of these strains alongside the wild-type parental strain of the A129 mice, 129Sv/Ev, was undertaken to assess clinical disease progression, viral loads in key tissues, histological changes and levels of sera biomarkers. Our results confirm the severe disease course in A129 mice which was not observed in the parental 129Sv/Ev strain. Of the two wild-type strains, viral loads were higher in 129Sv/Ev mice compared to C57BL/6 counterparts. Our results have established these models and parameters for the future testing of vaccines and antiviral approaches.

Insight into noncanonical small noncoding RNAs in Influenza A virus infection

Influenza A virus (IAV) induces acute respiratory infections in birds and various mammals, including humans, and presents a significant global public health concern, with considerable economic consequences. Recently, researchers have shown keen interest in noncanonical small noncoding RNAs (sncRNAs) as carriers of epigenetic information, including tRNA-derived small RNAs (tsRNAs), rRNA-derived small RNA (rsRNAs), and Y RNA-derived small RNAs (ysRNAs). Particularly, tsRNAs and rsRNAs are detected in diverse species and demonstrate evolutionary conservation. We analyzed sncRNAs sequencing data in the pulmonary tissue of two genetically distinct mouse strains, C57BL/6J and DBA/2J, to explore strain-specific variations of sncRNAs in response to IAV infection. We systematically compiled information on noncanonical sncRNAs in these two strains and investigated the tsRNAs/rsRNAs/ysRNAs profiles influenced by IAV infection. Specifically, four noncanonical sncRNA families, including rsRNA-12S, GtsRNA-Arg-CCT, GtsRNA-Arg-TCT, and GtsRNA-Lys-TTT, exhibited upregulation upon IAV infection. Notably, DBA/2J mice showed earlier systemic differential expression of noncanonical sncRNAs after IAV infection compared to C57BL/6J mice. Additionally, our study revealed a strain-specific biogenesis of MtsRNAs in response to IAV infection. Also, distinct co-expression patterns of MtsRNAs were observed between C57BL/6J and DBA/2J mice, with DBA/2J mice showing broader positive co-expression of MtsRNAs with various sncRNA families compared to C57BL/6J mice. Our study provides a novel insight into noncanonical sncRNAs and their implications in IAV pathology and mouse strain specificity.

Analysis of CNS autoimmunity in genetically diverse mice reveals unique phenotypes and mechanisms.

Multiple sclerosis (MS) is a complex disease with significant heterogeneity in disease course and progression. Genetic studies have identified numerous loci associated with MS risk, but the genetic basis of disease progression remains elusive. To address this, we leveraged the Collaborative Cross (CC), a genetically diverse mouse strain panel, and experimental autoimmune encephalomyelitis (EAE). The thirty-two CC strains studied captured a wide spectrum of EAE severity, trajectory, and presentation, including severe-progressive, monophasic, relapsing remitting, and axial rotary (AR)-EAE, accompanied by distinct immunopathology. Sex differences in EAE severity were observed in six strains. Quantitative trait locus analysis revealed distinct genetic linkage patterns for different EAE phenotypes, including EAE severity and incidence of AR-EAE. Machine learning-based approaches prioritized candidate genes for loci underlying EAE severity (Abcc4 and Gpc6) and AR-EAE (Yap1 and Dync2h1). This work expands the EAE phenotypic repertoire and identifies novel loci controlling unique EAE phenotypes, supporting the hypothesis that heterogeneity in MS disease course is driven by genetic variation.

Innate cells and STAT1-dependent signals orchestrate vaccine-induced protection against invasive Cryptococcus infection.

Fungal pathogens are underappreciated causes of significant morbidity and mortality worldwide. In previous studies, we determined that a heat-killed, Cryptococcus neoformans fbp1-deficient strain (HK-fbp1) is a potent vaccine candidate. We determined that vaccination with HK-fbp1 confers protective immunity against lethal Cryptococcosis in an interferon γ (IFNγ)-dependent manner. In this study, we set out to uncover cellular sources and relevant targets of the protective effects of IFNγ in response to the HK-fbp1 vaccine. We found that early IFNγ production peaks at day 3 and that monocytes and neutrophils are important sources of this cytokine after vaccination. Neutralization of IFNγ at day 3 results in impaired CCR2+ monocyte recruitment and reduced differentiation into monocyte-derived dendritic cells (Mo-DC). In turn, depletion of CCR2+ cells prior to immunization results in impaired activation of IFNγ-producing CD4 and CD8 T cells. Thus, monocytes are important targets of innate IFNγ and help promote further IFNγ production by lymphocytes. We employed monocyte-fate mapper and conditional STAT1 knockout mice to uncover that STAT1 activation in CD11c+ cells, including alveolar macrophages, Mo-DCs, and monocyte-derived macrophages (Mo-Mac) is essential for HK-fbp1 vaccine-induced protection. Altogether, our aggregate findings suggest critical roles for innate cells as orchestrators of vaccine-induced protection against Cryptococcus infection.IMPORTANCEThe number of patients susceptible to invasive fungal infections across the world continues to rise at an alarming pace yet current antifungal drugs are often inadequate. Immune-based interventions and novel antifungal vaccines hold the promise of significantly improving patient outcomes. In previous studies, we identified a Cryptococcus neoformans mutant strain (Fbp1-deficient) as a potent, heat-inactivated vaccine candidate capable of inducing homologous and heterologous antifungal protection. In this study, we used a combination of methods together with a cohort of conditional knockout mouse strains to interrogate the roles of innate cells in the orchestration of vaccine-induced antifungal protection. We uncovered novel roles for neutrophils and monocytes as coordinators of a STAT1-dependent cascade of responses that mediate vaccine-induced protection against invasive cryptococcosis. This new knowledge will help guide the future development of much-needed antifungal vaccines.

Proteins required for stereocilia elongation during mammalian hair cell development ensure precise and steady heights during adult life

The hair bundle, or stereocilia bundle, is the mechanosensory compartment of hair cells (HCs) in the inner ear. To date, most mechanistic studies have focused on stereocilia bundle morphogenesis, and it remains unclear how this organelle critical for hearing preserves its precise dimensions during life in mammals. The GPSM2-GNAI complex occupies the distal tip of stereocilia in the tallest row and is required for their elongation during development. Here, we ablate GPSM2-GNAI in adult mouse HCs after normal stereocilia elongation is completed. We observe a progressive height reduction of the tallest row stereocilia totaling ~600 nm after 12 wk in Gpsm2 mutant inner HCs. To measure GPSM2 longevity at tips, we generated a HaloTag-Gpsm2 mouse strain and performed pulse-chase experiments in vivo. Estimates using pulse-chase or tracking loss of GPSM2 immunolabeling following Gpsm2 inactivation suggest that GPSM2 is relatively long-lived at stereocilia tips with a half-life of 9 to 10 d. Height reduction coincides with dampened auditory brainstem responses evoked by low-frequency stimuli in particular. Finally, GPSM2 is required for normal tip enrichment of elongation complex (EC) partners MYO15A, WHRN, and EPS8, mirroring their established codependence during development. Taken together, our results show that the EC is also essential in mature HCs to ensure precise and stable stereocilia height and for sensitive detection of a full range of sound frequencies.

Variations in the germinal center response revealed by genetically diverse mouse strains.

The humoral response is complex and involves multiple cellular populations and signaling pathways. Bacterial and viral infections, as well as immunization regimens, can trigger this type of response, promoting the formation of microanatomical cellular structures called germinal centers (GCs). GCs formed in secondary lymphoid organs support the differentiation of high-affinity plasma cells and memory B cells. There is growing evidence that the quality of the humoral response is influenced by genetic variants. Using 12 genetically divergent mouse strains, we assessed the impact of genetics on GC cellular traits. At steady state, in the spleen, lymph nodes and Peyer's patches, we quantified GC B cells, plasma cells and follicular helper Tcells. These traits were also quantified in the spleen of mice following immunization with a foreign antigen, namely, sheep red blood cells, in addition to the number and size of GCs. We observed both strain- and organ-specific variations in cell type abundance, as well as for GC number and size. Moreover, we find that some of these traits are highly heritable. Importantly, the results of this study inform on the impact of genetic diversity in shaping the GC response and identify the traits that are the most impacted by genetic background.

Beneficial Impact of Inhaled 25(OH)-Vitamin D3 and 1,25(OH)2-Vitamin D3 on Pulmonary Response in the Murine Model of Hypersensitivity Pneumonitis.

Despite numerous scientific reports on the negative impact of vitamin D3 deficiency on many respiratory diseases, little is known about the influence of this phenomenon on the development and progression of hypersensitivity pneumonitis (HP). The presented study is an attempt to shed light on this occurrence. The research was performed on mouse strain C57BL/6J exposed to the antigen of Pantoea agglomerans (etiological factor of HP). To induce vitamin D3 deficiency, mice received a diet with a 10 times lower amount of cholecalciferol than the main control group. VD3-deficient mice inhaled 25(OH)-VD3 or 1,25(OH)2-VD3 used separately or with SE-PA. At the beginning of the experiment and after 14 and 28 days of inhalation, respiratory function was examined using whole-body plethysmography. Moreover, at indicated time points, mice were sacrificed and samples collected for histological examination, flow cytometry, and ELISA. The performed study revealed that inhalations with 25(OH)-VD3 and 1,25(OH)2-VD3 effectively eliminated most of the negative changes in the respiratory system caused by vitamin D3 deficiency by restoring the physiological concentration of 1,25(OH)2-VD3 in the body. VD3-deficient mice which inhaled P. agglomerans antigen and vitamin D3 metabolites also demonstrated the ability of the tested compounds to eliminate, or at least weaken, the negative effects of the HP causative factor and desired effect, including improvement of respiratory functions and attenuation of inflammation and signs of fibrosis. The obtained results suggested that the beneficial influence of inhaled vitamin D3 metabolites on HP development was associated with the restoration of the physiological concentration of 1,25(OH)2-VD3 in the pulmonary compartments in VD3-deficient mice.

The mutant mouse resource and research center (MMRRC) consortium: the US-based public mouse repository system

AbstractNow in its 25th year, the Mutant Mouse Resource and Research Center (MMRRC) consortium continues to serve the United States and international biomedical scientific community as a public repository and distribution archive of laboratory mouse models of human disease for research. Supported by the National Institutes of Health (NIH), the MMRRC consists of 4 regionally distributed and dedicated vivaria, offices, and specialized laboratory facilities and an Informatics Coordination and Service Center (ICSC). The overarching purpose of the MMRRC is to facilitate groundbreaking biomedical research by offering an extensive repertoire of mutant mice that are essential for advancing the understanding of human physiology and disease. The function of the MMRRC is to identify, acquire, evaluate, characterize, cryopreserve, and distribute mutant mouse strains to qualified biomedical investigators around the nation and the globe. Mouse strains accepted from the research community are held to the highest scientific standards to optimize reproducibility and enhance scientific rigor and transparency. All submitted strains are thoroughly reviewed, documented, and validated using extensive scientific quality control measures. In addition, the MMRRC conducts resource-related research on cryopreservation, mouse genetics, environmental conditions, and other topics that enhance operations of the MMRRC. Today, the MMRRC maintains an archive of mice, cryopreserved embryos and sperm, embryonic stem (ES) cell lines, and murine hybridomas for nearly 65,000 alleles. Since its inception, the MMRRC has fulfilled more than 20,000 orders from 13,651 scientists at 8441 institutions worldwide. The MMRRC also provides numerous services to assist researchers, including scientific consultation, technical assistance, genetic assays, microbiome analysis, analytical phenotyping, pathology, cryorecovery, husbandry, breeding and colony management, infectious disease surveillance, and disease modeling. The ICSC coordinates MMRRC operations, interacts with researchers, and manages the website (mmrrc.org) and online catalogue. Researchers benefit from an expansive list of well-defined mouse models of disease that meet the highest scientific standards while submitting investigators benefit by having their mouse strains cryopreserved, protected, and distributed in compliance with NIH policies.

Constitutive expression of the deubiquitinating enzyme CYLD does not affect microglia phenotype or function in homeostasis and neuroinflammation

Abstract The deubiquitinating enzyme CYLD negatively regulates NF-κB signaling by removing activating ubiquitin chains from several members of the NF-κB pathway. Thereby, CYLD is critical for the maintenance and differentiation of various immune cells. Despite the importance of the NF-κB pathway in microglia regulation, the role of CYLD in microglia has not been investigated so far. In this study, we investigated whether CYLD in microglia can protect against neuroinflammation using a newly generated conditional mouse strain (Rosa26-Cyld-tdTomato) that allows cell type-specific CYLD overexpression. Here, we show that overexpression of CYLD in microglia did not alter microglia numbers or microglia morphology in different brain regions. Additionally, CYLD overexpression did not modify the microglial response to LPS-induced neuroinflammation or the disease severity in experimental autoimmune encephalomyelitis (EAE). Finally, also immune cell infiltration into the CNS during EAE and under steady state conditions remained unaffected by microglial CYLD overexpression. Our findings suggest that CYLD overexpression does not alter microglial function, and thus does not represent a viable therapeutic strategy in neuroinflammatory conditions. This study highlights the complexity of ubiquitin-mediated signaling in neuroinflammation and the need for cell-type-specific investigations. The Rosa26-Cyld-tdTomato mouse model offers a valuable tool for studying CYLD’s role across various tissues and cell types. Key messages Novel mouse strain for cell type-specific overexpression of the deubiquitinating enzyme CYLD. CYLD overexpression in microglia did not alter microglia numbers or morphology in the steady state. CYLD overexpression in microglia did not protect mice from LPS-induced neuroinflammation or EAE. CYLD overexpression in microglia did not influence their gene expression during neuroinflammation.

Enhanced complement activation and MAC formation accelerates severe COVID-19

Emerging evidence indicates that activation of complement system leading to the formation of the membrane attack complex (MAC) plays a detrimental role in COVID-19. However, their pathogenic roles have never been experimentally investigated before. We used three knock out mice strains (1. C3−/−; 2. C7−/−; and 3. Cd59ab−/−) to evaluate the role of complement in severe COVID-19 pathogenesis. C3 deficient mice lack a key common component of all three complement activation pathways and are unable to generate C3 and C5 convertases. C7 deficient mice lack a complement protein needed for MAC formation. Cd59ab deficient mice lack an important inhibitor of MAC formation. We also used anti-C5 antibody to block and evaluate the therapeutic potential of inhibiting MAC formation. We demonstrate that inhibition of complement activation (in C3−/−) and MAC formation (in C3−/−. C7−/−, and anti-C5 antibody) attenuates severe COVID-19; whereas enhancement of MAC formation (Cd59ab−/−) accelerates severe COVID-19. The degree of MAC but not C3 deposits in the lungs of C3−/−, C7−/− mice, and Cd59ab−/− mice as compared to their control mice is associated with the attenuation or acceleration of SARS-CoV-2-induced disease. Further, the lack of terminal complement activation for the formation of MAC in C7 deficient mice protects endothelial dysfunction, which is associated with the attenuation of diseases and pathologic changes. Our results demonstrated the causative effect of MAC in severe COVID-19 and indicate a potential avenue for modulating the complement system and MAC formation in the treatment of severe COVID-19.

Abstract C059: Regulatory Elements of Pancreas Development License the Initiation of Pancreatic Ductal Adenocarcinoma

Abstract Mutations in DNA are a known cause of cancer. However, they become oncogenic only under specific cellular conditions and within certain tissue contexts. Tissue injury often leads to the aberrant activation of developmental pathways, eroding markers of cellular differentiation and giving rise to transient cellular states. These states resemble developmental progenitors, characterized by higher transcriptional plasticity. Oncogenes, particularly KRAS, co-opt cellular plasticity by acting on tissue-specific enhancers, preventing the resolution of acinar plasticity and maintaining the pancreas in a pro-inflammatory phase that progresses to cancer1,2. However, the specific role of tissue-specific enhancers in pancreas regeneration and cancer initiation remains unexplored, and strategies to target these regenerative processes are yet to be developed. Our laboratory investigates non-mutational mechanisms of cancer, with a special interest in the intersection of developmental biology and cancer, aiming to elucidate the processes of cellular plasticity and cancer initiation. Recent studies have shown that acinar cells possess remarkable cellular plasticity, giving rise to a diverse population of cellular states in pancreas regeneration and cancer contexts3-5. We have discovered that the transcription factor Sox4 is necessary for the specification of tuft cells and gastric metaplasia found in chronic pancreatitis and in pancreatic intraepithelial lesions (PanINs). Moreover, we have shown that cellular plasticity has a non-cell autonomous protective effect; losing Sox4 and cellular heterogeneity in PanINs results in a fibrotic and immunosuppressive tumor microenvironment and disease progression. Our results demonstrated that targeting cellular plasticity accelerates or prevents cancer initiation through autonomous and non-autonomous cellular processes6. In this study, we leverage a comprehensive resource of ncRNAs transcribed from enhancer elements of pancreas development, together with their inferred target genes and correlations with clinical outcomes. We focused on an ncRNA associated with the risk of pancreatic cancer. To study its function, we developed two novel mouse strains for tissue-specific deletion, enabling precise manipulation and study of its role in the pancreas and other tissues. Our phenotypic analysis demonstrates that the ncRNA is activated following tissue injury and is essential for developing PanINs. Moreover, we show that it regulates cell-cell communication after tissue injury. This mechanism operates independently of cellular plasticity but significantly creates a pro-inflammatory environment that favors cancer development. Our results demonstrate that enhancer elements are actionable targets to regulate cell fate decisions in regeneration and cancer. Additionally, KRAS-induced cancer initiation depends on developmental regulatory elements, opening the window to develop prophylactic interventions to delay tumor onset. Citation Format: Luis Arnes. Regulatory Elements of Pancreas Development License the Initiation of Pancreatic Ductal Adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C059.

Cellular and Immunological Analysis of 2D2/Th Hybrid Mice Prone to Experimental Autoimmune Encephalomyelitis in Comparison with 2D2 and Th Lines.

Previously, we described the mechanisms of development of autoimmune encephalomyelitis (EAE) in 3-month-old C57BL/6, Th, and 2D2 mice. The faster and more profound spontaneous development of EAE with the achievement of deeper pathology occurs in hybrid 2D2/Th mice. Here, the cellular and immunological analysis of EAE development in 2D2/Th mice was carried out. In Th, 2D2, and 2D2/Th mice, the development of EAE is associated with a change in the differentiation profile of hemopoietic bone marrow stem cells, which, in 2D2/Th, differs significantly from 2D2 and Th mice. Hybrid 2D2/Th mice demonstrate a significant difference in these changes in all strains of mice, leading to the production of antibodies with catalytic activities, known as abzymes, against self-antigens: myelin oligodendrocyte glycoprotein (MOG), DNA, myelin basic protein (MBP), and five histones (H1-H4) hydrolyze these antigens. There is also the proliferation of B and T lymphocytes in different organs (blood, bone marrow, thymus, spleen, lymph nodes). The patterns of changes in the concentration of antibodies and the relative activity of abzymes during the spontaneous development of EAE in the hydrolysis of these immunogens are significantly or radically different for the three lines of mice: Th, 2D2, and 2D2/Th. Several factors may play an essential role in the acceleration of EAE in 2D2/Th mice. The treatment of mice with MOG accelerates the development of EAE pathology. In the initial period of EAE development, the concentration of anti-MOG antibodies in 2D2/Th is significantly higher than in Th (29.1-fold) and 2D2 (11.7-fold). As shown earlier, antibodies with DNase activity penetrate cellular and nuclear membranes and activate cell apoptosis, stimulating autoimmune processes. In the initial period of EAE development, the concentration of anti-DNA antibodies in 2D2/Th hybrids is higher than in Th (4.6-fold) and 2D2 (25.7-fold); only 2D2/Th mice exhibited a very strong 10.6-fold increase in the DNase activity of IgGs during the development of EAE. Free histones in the blood are cytotoxic and stimulate the development of autoimmune diseases. Only in 2D2/Th mice, during different periods of EAE development, was a sharp increase in the anti-antibody activity in the hydrolysis of some histones observed.

Cardiac lymphatics undergo distinct remodeling during hypertrophic and non-hypertrophic pregnancy.

Lymphatic vessels of the heart undergo dynamic remodeling in response to physiological and pathological cardiovascular events such as development, adult cardiac maintenance and injury repair. During pregnancy, the cardiovascular system undergoes physiological changes to meet the increased demand of blood supply to the fetus. These extensive physiologic changes make pregnancy and delivery a high-risk period in a woman's life. However, whether and how cardiac lymphatics change during pregnancy is largely undefined. Therefore, we used whole mount immunofluorescent labeling and quantitative morphometric analysis to characterize the changes in cardiac lymphatic vasculature during pregnancy using two genetically distinct inbred mouse strains, C57BL/6J and BALB/cJ. Compared to age-matched, non-pregnant C57BL/6J control mice, the hearts of C57BL/6J dams in late-pregnancy (gestation day 17.5 (G17.5)) undergo physiologic hypertrophy. However, there were no significant changes in the cardiac lymphatic vasculature. In contrast, BALB/cJ mice do not exhibit pregnancy-induced cardiac hypertrophy at G17.5 compared to age-matched, non-pregnant mice. Yet interestingly, the cardiac lymphatic vasculature of pregnant BALB/cJ dams undergoes extensive morphological changes, including decreased lymphatic length, number of endpoints, and vessel branchpoint junctions on the ventral side of the heart. These findings underscore the complexity of genetic and physiologic factors that contribute to the heterotypic remodeling of cardiac lymphatics during late pregnancy.

Inflammation perturbs hematopoiesis by remodeling specific compartments of the bone marrow niche.

Hematopoietic stem and progenitor cells (HSPC) are regulated by interactions with stromal cells in the bone marrow (BM) cavity, which can be segregated into two spatially defined central marrow (CM) and endosteal (Endo) compartments. However, the importance of this spatial compartmentalization for BM responses to inflammation and neoplasia remains largely unknown. Here, we extensively validate a combination of scRNA-seq profiling and matching flow cytometry isolation that reproducibly identifies 7 key CM and Endo populations across mouse strains and accurately surveys both niche locations. We demonstrate that different perturbations exert specific effects on different compartments, with type I interferon responses causing CM mesenchymal stromal cells to adopt an inflammatory phenotype associated with overproduction of chemokines modulating local monocyte dynamics in the surrounding microenvironment. Our results provide a comprehensive method for molecular and functional stromal characterization and highlight the importance of altered stomal cell activity in regulating hematopoietic responses to inflammatory challenges.

Novel Role of Endothelial CD45 in Regulating Endothelial-to-Mesenchymal Transition in Atherosclerosis.

Protein-tyrosine-phosphatase CD45 is exclusively expressed in all nucleated cells of the hematopoietic system but is rarely expressed in endothelial cells. Interestingly, our recent study indicated that activation of the endogenous CD45 promoter in human endothelial colony forming cells (ECFCs) induced expression of multiple EndoMT marker genes. However, the detailed molecular mechanisms underlying CD45 that drive EndoMT and the therapeutic potential of manipulation of CD45 expression in atherosclerosis are entirely unknown. We generated a tamoxifen-inducible EC-specific CD45 deficient mouse strain (EC-iCD45KO) in an ApoE-deficient (ApoE-/-) background and fed with a Western diet (C57BL/6) for atherosclerosis and molecular analyses. We isolated and enriched mouse aortic endothelial cells with CD31 beads to perform single-cell RNA sequencing. Biomedical, cellular, and molecular approaches were utilized to investigate the role of endothelial CD45-specific deletion in the prevention of EndoMT in ApoE-/- model of atherosclerosis. Single-cell RNA sequencing revealed that loss of endothelial CD45 inhibits EndoMT marker expression and transforming growth factor-β signaling in atherosclerotic mice. which is associated with the reductions of lesions in the ApoE-/- mouse model. Mechanistically, the loss of endothelial cell CD45 results in increased KLF2 expression, which inhibits transforming growth factor-β signaling and EndoMT. Consistently, endothelial CD45 deficient mice showed reduced lesion development, plaque macrophages, and expression of cell adhesion molecules when compared to ApoE-/- controls. These findings demonstrate that the loss of endothelial CD45 protects against EndoMT-driven atherosclerosis, promoting KLF2 expression while inhibiting TGFβ signaling and EndoMT markers. Thus, targeting endothelial CD45 may be a novel therapeutic strategy for EndoMT and atherosclerosis.

The impact of social partners: investigating mixed-strain housing effects on aging in female mice.

Aging is a multifaceted process characterized by the gradual decline of physiological functions and can be modulated by various internal and external factors. While social interactions have been shown to affect behaviors and physiology in different species, the impact of social partners on aging-related phenotypes and lifespan in mice remains understudied. To address this question, we investigated various aging-related traits and lifespan in two mouse strains, C57BL/6J and BALB/c, under two different housing conditions: mixed-strain and same-strain housing. Analyses using a Generalized Linear Model revealed significant differences between the two strains in several phenotypes, including metabolic, anxiety-like, and electrocardiographic traits. However, surprisingly, housing conditions did not significantly affect most of the examined parameters, including overall lifespan. Only 3 out of 25 traits-body weight change in a metabolic cage, running wheel activity, and survival days of a quartiles of mice with middle lifespans-were influenced by housing conditions in a strain-dependent manner. Together, our study suggested a minimal influence of co-housing with social partners from different genetic backgrounds on aging-related phenotypes. This result demonstrates the feasibility of mixed housing for mouse husbandry and, more importantly, provides valuable insights for future research on the social influences on the aging process in mice.

Current status of immunodeficient mouse models as substitutes to reduce cat and dog use in heartworm preclinical research

Chemoprophylactic prevention of veterinary heartworm disease in companion animals, caused by the vector-borne nematode parasite Dirofilaria immitis, is a multi-billion-dollar global market. Experimental use of cats and dogs in preclinical heartworm drug testing is increasing due to evolving drug-resistance to frontline macrocyclic lactones and renewed investment in alternative preventative drug research. We and others recently published data demonstrating proof-of-concept of utilising lymphopenic severe-combined immunodeficient (SCID) or Recombination Activating Gene (RAG)2 deficient mice with additional knockout of the IL-2/7 receptor gamma chain (γc) as alternative preventative drug screening research models of dirofilariasis. Here we summarise the current knowledge of candidate immunodeficient mouse models tested, including a comparison of susceptibility using different background strains of mice, different D. immitis isolates, following use of anti-inflammatory treatments to further suppress residual innate immunity, and efficacies achieved against different reference anthelmintics. We supplement this precis with new data on treatment response to the veterinary anthelmintic, oxfendazole, and initial evaluation of D. immitis susceptibility in CB.17 SCID and C57BL/6 RAG2-/-γc-/- mice. We conclude that in addition to NSG and NXG mice, RAG2-/-γc-/- mice on either a BALB/c or C57BL/6 background offer an alternative screening model option, widening access to academic and commercial laboratories wishing to pursue initial rapid in vivo drug screening whilst avoiding potentially unnecessary cat or dog testing.