B6 Background Research Articles

A novel mouse model of hepatocyte-specific apoptosis-induced myeloid cell-dominant sterile liver injury and repair response.

Apoptosis, inflammation, and wound healing are critical pathophysiological events associated with various liver diseases. Currently, there is a lack of in vivo approaches to study hepatocyte apoptosis-induced liver injury and repair. To address this critical knowledge gap, we developed a unique genetically modified mouse model, namely, 3-Transgene (Tg) with inducible Hepatocyte-Specific Apoptosis Phenotype (3xTg-iHAP) in this study. The 3xTg-iHAP mice possess three transgenes including Alb-Cre, Rosa26-rtTA, and tetO-Fasl on a B6 background. These mice are phenotypically normal, viable, and fertile. After subcutaneous administration of a single dose of doxycycline (5 mg/kg, Dox) to 3xTg-iHAP mice, we observed a complete histological spectrum of sterile liver wound-healing responses: asymptomatic hepatocyte apoptosis at 8 h, necrotic liver injury and sterile inflammation at 48 h, followed by hepatocyte mitosis and regeneration within 7 days. During the injury phase, the mice exhibited an increase in the biomarkers of alanine aminotransferase (ALT), chemokine (C-X-C motif) ligand 1 (CXCL1), and IL-6 in peripheral blood, as well as α-smooth muscle actin (α-SMA) protein in liver tissues. Conversely, the mice displayed a decrease in these markers in the recovery phase. Remarkably, this model shows that the sterile liver injury following elevated hepatocyte apoptosis is associated with an increase in myeloid cells in the liver. Within 7 days post-Dox administration, the liver of Dox-treated 3xTg-iHAP mice displays a normal histological structure, indicating the completion of wound healing. Together, we established a novel mouse model of injury and regeneration induced by hepatocyte apoptosis. This tool provides a robust in vivo platform for studying the pathophysiology of sterile liver inflammation, regeneration, and new therapeutic interventions for liver diseases.NEW & NOTEWORTHY Bu et al. present a triple-transgenic mouse model, namely, 3xTg-iHAP mice that are engineered to explore hepatocyte apoptosis-triggered sterile liver injury and regeneration. This model demonstrates a full spectrum of liver wound-healing responses from asymptomatic apoptosis to injury, myeloid cell-dominant sterile inflammation, and repair after induction of hepatocyte-specific apoptosis. The robust nature of this model makes it an invaluable in vivo tool for studying sterile liver inflammation, regeneration, and new therapeutic strategies.

Tachol1 QTL on mouse chromosome 1 is responsible for hypercholesterolemia and diet-induced obesity.

Hypercholesterolemia raises the risk for cardiovascular complications and overall health. Hypercholesterolemia is common, affecting 10% of the general population of the US, and heritable. Most individuals with hypercholesterolemia have a polygenic predisposition to the condition. Previously we identified a quantitative trait locus, Tachol1, linked to hypercholesterolemia on mouse chromosome 1 (Chr1) in a cross between C57BL/6J (B6) and TALLYHO/JngJ (TH) mice, a polygenic model for human obesity, type 2 diabetes and hyperlipidemia. Subsequently, using congenic mice that carry a TH-derived genomic segment of Chr1 on a B6 background, we demonstrated that the distal segment of Chr1, where Tachol1 maps, is necessary to cause hypercholesterolemia, as well as diet-induced obesity. In this study, we generated overlapping subcongenic lines to the distal segment of congenic region and characterized subcongenic mice carrying the smallest TH region of Tachol1, ~ 16.2Mb in size (B6.TH-Chr1-16.2Mb). Both male and female B6.TH-Chr1-16.2Mb mice showed a significantly increased plasma total cholesterol levels compared to B6 on both chow and high fat (HF) diet. B6.TH-Chr1-16.2Mb mice also had greater fat mass than B6 on HF diet, without increasing food intake. The gene and protein expression levels of absent in melanoma 2 (Aim2) gene were significantly upregulated in B6.TH-Chr1-16.2Mb mice compared to B6. In summary, we confirmed the effect of Tachol1 on hypercholesterolemia and diet-induced obesity using subcongenic analysis.

Abstract 6529: Multi-lineage de-differentiation (MLDD) towards the TTF1-low phenotype is a major determinant of immune response within STK11-deficient non-small cell lung cancer

Abstract Background: The loss of serine/threonine kinase 11 (STK11) in non-small cell lung cancer (NSCLC) is associated with poor prognosis and resistance to immune checkpoint blockade (ICB) in 20-25% of cases. Experimental evidence from murine models suggests that STK11 loss contributes to lineage plasticity, while clinical observations indicate its impact on differentiation phenotypes in patients. However, the intricate interplay between differentiation states, the tumor microenvironment (TME), and clinical outcomes remains poorly elucidated. This study aims to comprehensively characterize these interactions to enhance our understanding of the complex dynamics underlying STK11-deficient NSCLC pathogenesis and therapeutic responses. Methods: By leveraging large multi-omics datasets, we already demonstrated the low immune infiltration in TTF1-low and neuroendocrine MLDD subsets of LKB1-deficient tumors. In this study, we used GEMM-derived two KPL (Kras, TP53, and STK11 mutations) and one KPL-restored line to inject subcutaneously in F1 hybrid mice of B6 and Sv129 backgrounds. Tumor size was monitored at regular intervals, and upon reaching a sufficient size, tumors were harvested and partitioned for the characterization of immune cells (T-cells and NK cells) by flow cytometry and identification of the MLDD phenotypes through gene expression analysis using Nanostring technology. Results: Mice injected with the STK11 WT line (2695L) exhibited smaller tumor volumes compared to those injected with STK11 mutant lines (2695X and 2381T4). Our comprehensive analysis, combining flow cytometry and Nanostring techniques, revealed that STK11 mutant lines displayed notably low infiltration of CD8+ T-cells, an elevated proportion of exhausted T-cells, and a manifestation of the TTF1-low immune phenotype characterized by high myeloid-derived suppressor cells (MDSCs) and increased KLRC1/KLRC2 expression. The presence of MDSCs in the STK11 mutant line correlated with pro-inflammatory cytokines but exhibited an inverse correlation with MHC-2 expression. MHC-2 expression was inversely correlated with CD8+ T-cell percentage and positively correlated with naïve T-cell percentage. Conclusions: The data obtained from the study indicates that the MDSC phenotype seems to be important for immune evasion in STK11-loss tumors. Follow-up flow cytometry and gene expression experiments with combined T-cells and myeloid/PMN MDSC-related gene panels, along with further experiments involving cIAP1/PD1 inhibitors, are imperative to substantiate and expand upon our current findings. Citation Format: Rahul Shivahare, No Joon Song, Bahareh Nourmohammadi, Jemal Imam, Olivia Sanabria, Joseph M. Amann, Qin Ma, Zihai Li, David P. Carbone, Jacob Kaufman. Multi-lineage de-differentiation (MLDD) towards the TTF1-low phenotype is a major determinant of immune response within STK11-deficient non-small cell lung 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 6529.

Network-based analysis predicts interacting genetic modifiers from a meta-mapping study of spike-wave discharge in mice.

Absence seizures are characterized by brief lapses in awareness accompanied by a hallmark spike-and-wave discharge (SWD) electroencephalographic pattern and are common to genetic generalized epilepsies (GGEs). While numerous genes have been associated with increased risk, including some Mendelian forms with a single causal allele, most cases of GGE are idiopathic and there are many unknown genetic modifiers of GGE influencing risk and severity. In a previous meta-mapping study, crosses between transgenic C57BL/6 and C3HeB/FeJ strains, each carrying one of three SWD-causing mutations (Gabrg2tm1Spet(R43Q) , Scn8a8j or Gria4spkw1 ), demonstrated an antagonistic epistatic interaction between loci on mouse chromosomes 2 and 7 influencing SWD. These results implicate universal modifiers in the B6 background that mitigate SWD severity through a common pathway, independent of the causal mutation. In this study, we prioritized candidate modifiers in these interacting loci. Our approach integrated human genome-wide association results with gene interaction networks and mouse brain gene expression to prioritize candidate genes and pathways driving variation in SWD outcomes. We considered candidate genes that are functionally associated with human GGE risk genes and genes with evidence for coding or non-coding allele effects between the B6 and C3H backgrounds. Our analyses output a summary ranking of gene pairs, one gene from each locus, as candidates for explaining the epistatic interaction. Our top-ranking gene pairs implicate microtubule function, cytoskeletal stability and cell cycle regulation as novel hypotheses about the source of SWD variation across strain backgrounds, which could clarify underlying mechanisms driving differences in GGE severity in humans.

Systems genetics of influenza A virus-infected mice identifies TRIM21 as a critical regulator of pulmonary innate immune response

Tripartite motif 21 (TRIM21) is a cytosolic Fc receptor that targets antibody-bound, internalized pathogens for destruction. Apart from this intrinsic defense role, TRIM21 is implicated in autoimmune diseases, inflammation, and autophagy. Whether TRIM21 participates in host interactions with influenza A virus (IAV), however, is unknown. By computational modeling of body weight and lung transcriptome data from the BXD parents (C57BL/6 J (B6) and DBA/2 J (D2)) and 41 BXD mouse strains challenged by IAV, we reveal that a Trim21-associated gene network modulates the early host responses to IAV infection. Trim21 transcripts were significantly upregulated in infected mice of both B6 and D2 backgrounds. Its expression was significantly higher in infected D2 than in infected B6 early after infection and significantly correlated with body weight loss. We identified significant trans-eQTL on chromosome 14 that regulates Trim21 expression. Nr1d2 and Il3ra were among the strongest candidate genes. Pathway analysis found Trim21 to be involved in inflammation and immunity related pathways, such as inflammation signaling pathways (TNF, IL-17, and NF-κB), viral detection signaling pathways (NOD-like and RIG-I-like), influenza, and other respiratory viral infections. Knockdown of TRIM21 in human lung epithelial A549 cells significantly augmented IAV-induced expression of IFNB1, IFNL1, CCL5, CXCL10, and IFN-stimulated genes including DDX58 and IFIH1, among others. Our data suggest that a TRIM21-associated gene network is involved in several aspects of inflammation and viral detection mechanisms during IAV infection. We identify and validate TRIM21 as a critical regulator of innate immune responses to IAV in human lung epithelial cells.

Generation and screening of mouse models of ADRD with multiple genetic manipulations to model dementia with mixed brain pathologies

AbstractBackgroundAlzheimer’s disease‐related dementias (ADRD) encompass a wide range of neurodegenerative diseases including Alzheimer’s disease, vascular dementia, frontotemporal dementia (FTD), Lewy body dementia (LBD), and mixed dementia [1‐4]. ADRDs are distinguished by the presence of pathologies including amyloid, tau, TDP‐43 inclusions, alpha‐synuclein aggregates, cerebral amyloid angiopathy, and/or hippocampal sclerosis [4‐9]. Consequently, the heterogeneity of ADRDs presents challenges for animal modeling of disease mechanisms. Our laboratory has demonstrated that genetic diversity in disease modeling is critical to recapitulate the molecular and cognitive features of AD [1,9, 10]. Here, we introduce multiple genetic manipulations to develop novel translational models of ADRD.MethodPrecision models of ADRD on multiple genetic backgrounds were generated via combinatorial CRISPR editing to knock‐in human disease‐causing mutations onto the C57BL/6J (B6) strain and crossed to either B6, DBA/2J (D2), FVB, or WSB to generate a set of 20 unique ADRD models (Fig.1). The effect of homozygous mutations were evaluated on the B6 background. Each model was assessed using a high‐capacity multi‐domain go/no‐go screen to prioritize candidate strains for future ADRD modeling of drug discovery, preclinical testing, and disease pathology.ResultAssays included frailty, open field, y‐maze, rotarod, and contextual fear conditioning. Models with one or more late‐onset behavioral deficits (12 mo) were a “go”; whereas, early behavioral deficits (6 and 9 mo), aggression, high attrition, and low fecundity and additional factors were considered unsuitable (“no‐go”). We identified several promising models of ADRD that exhibited late‐onset frailty and motor deficits as function of mutation burden and genetic background (e.g., App/Psen1/Tardbp/Mapt on the D2 background). The FVB background strain exhibited early frailty deficits (6 mo), and thus considered unsuitable (“no‐go”). The WSB background strain exhibited reserve across multiple frailty and behavioral assays up to 12 mo.ConclusionOur results demonstrate that combining ADRD mutations in genetically distinct backgrounds is a promising strategy to generate new mouse models that better model the human mid and late‐life onset of deficits associated with ADRD. The resulting model(s) will be translationally relevant and provide important insight into disease mechanisms for the future of ADRD research and drug discoveries.

Identification of Genetic Loci That Modify the Scarb1 and Apoe Atherothrombotic Phenotype

Introduction: Atherothrombosis, or the formation of a thrombus on a ruptured atherosclerotic plaque, is a major health problem in western societies and can lead to myocardial infarction and ischemic stroke. Atherosclerotic plaques form on the inner walls of blood vessels and consist of macrophages, lipids, and prothrombotic molecules such as tissue factor that initiate the blood clotting process. Despite the complexity of atherothrombotic disease, with major (and evolving) environmental contributors, genetic variants play a role in modifying the disease process. In mice, deficiency of the Scavenger Receptor Class B type 1 (Scarb1) and Apolipoprotein E (ApoE) genes play important roles in the proper maintenance of lipid levels in the body. The Scarb1 protein is an HDL receptor known for its role in the process of transporting cholesterol from peripheral tissues toward the liver for excretion. The ApoE protein combines with lipids to form lipoproteins that package cholesterol and carry it throughout the circulation. In order to study mice deficient in these proteins, our lab produced Scarb1 and ApoE double knockout (dKO) mice on the C57BL/6J (B6) and 129S1/SvImJ (129S1) background and discovered major phenotypic differences in size and survival. Mice with a mixed 129S1/B6 background succumb to lethal atherothrombosis at approximately 8 weeks of age (as seen in Figure 1), whereas dKOs generated on the B6 background die at approximately 5 days old. We hypothesized that specific 129S1 genetic variants modify the severity of atherothrombosis in dKO mice. Methods: We produced F1-F3 generation dKO mice on the mixed 129S1/B6 background for experiments. Weights were taken for the 38 dKO mice and their littermates from 3 weeks old until the death of the knockout mice at around 8 weeks of age. Purified DNA from the dKO mice was subjected to whole genome genotyping for 143,000 markers using the GigaMUGA genotyping platform (Neogen). Weight data were used as a phenotype to perform quantitative trait loci analysis (QTL) to identify potential loci responsible for the phenotypic weight differences. Results: dKO mice from F1-F3 generations were born at the expected Mendelian frequencies but exhibited 3 distinct phenotypes based on their weights relative to their littermates. dKO mouse weights fell into 3 distinct categories: (1) indistinguishable from littermates, (2) 14%-39% less than littermates, (3) greater than 39% less than littermates. QTL analysis identified 6 loci above the significance threshold of 4.04 on Chromosomes (Chr) 1 (LOD 6.82), 2 (LOD 6.44) , 3 (LOD 4.65), 6 (LOD 4.30), 12 (LOD 4.22) and 14 (LOD 5.36; Figure 2). In addition, interaction analyses identified significant interactions of Chr 12 with Chrs 1, 2, 3, and 14. The Chr 12 candidate region is between 83 and 93 Mb consisting of 89 genes. Niemann Pick type C2 ( Npc2), an intracellular cholesterol transporter, is within the candidate region. Thus, 129S1 variants in this region affecting Npc2 transcription are strong candidates for modifying the dKO phenotypes. Discussion: We have analyzed 38 dKO mice for genetic variants affecting their health, as measured by weights relative to their non dKO littermates. We identified 6 significant genetic loci, with Npc2 as a candidate gene for the Chromosome 12 locus. Studies to reinforce these findings include the analysis of an additional cohort of dKO mice to narrow the candidate QTL intervals, analysis of Npc2 expression in the B6 and 129 strain, histology of the liver to visualize lipid accumulation, blood smears to examine the blood cell size difference in dKOs, and sequencing with GigaMUGA. The identification of atherothrombosis modifier genes could result in novel treatment strategies for atherothrombosis.

Deletion of Vβ3+CD4+ T cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8+ T cells.

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vβ3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vβ3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders.

Genetic background influences the 5XFAD Alzheimer's disease mouse model brain proteome.

There is an urgent need to improve the translational validity of Alzheimer's disease (AD) mouse models. Introducing genetic background diversity in AD mouse models has been proposed as a way to increase validity and enable the discovery of previously uncharacterized genetic contributions to AD susceptibility or resilience. However, the extent to which genetic background influences the mouse brain proteome and its perturbation in AD mouse models is unknown. In this study, we crossed the 5XFAD AD mouse model on a C57BL/6J (B6) inbred background with the DBA/2J (D2) inbred background and analyzed the effects of genetic background variation on the brain proteome in F1 progeny. Both genetic background and 5XFAD transgene insertion strongly affected protein variance in the hippocampus and cortex (n = 3,368 proteins). Protein co-expression network analysis identified 16 modules of highly co-expressed proteins common across the hippocampus and cortex in 5XFAD and non-transgenic mice. Among the modules strongly influenced by genetic background were those related to small molecule metabolism and ion transport. Modules strongly influenced by the 5XFAD transgene were related to lysosome/stress responses and neuronal synapse/signaling. The modules with the strongest relationship to human disease-neuronal synapse/signaling and lysosome/stress response-were not significantly influenced by genetic background. However, other modules in 5XFAD that were related to human disease, such as GABA synaptic signaling and mitochondrial membrane modules, were influenced by genetic background. Most disease-related modules were more strongly correlated with AD genotype in the hippocampus compared with the cortex. Our findings suggest that the genetic diversity introduced by crossing B6 and D2 inbred backgrounds influences proteomic changes related to disease in the 5XFAD model, and that proteomic analysis of other genetic backgrounds in transgenic and knock-in AD mouse models is warranted to capture the full range of molecular heterogeneity in genetically diverse models of AD.

Multiple and Consecutive Genome Editing Using i-GONAD and Breeding Enrichment Facilitates the Production of Genetically Modified Mice.

Genetically modified (GM) mice are essential tools in biomedical research. Traditional methods for generating GM mice are expensive and require specialized personnel and equipment. The use of clustered regularly interspaced short palindromic repeats (CRISPR) coupled with improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) has highly increased the feasibility of producing GM mice in research laboratories. However, genetic modification in inbred mouse strains of interest such as C57BL/6 (B6) is still challenging because of their low fertility and embryo fragility. We have successfully generated multiple novel GM mouse strains in the B6 background while attempting to optimize i-GONAD. We found that i-GONAD reduced the litter size in superovulated pregnant females but did not impact pregnancy rates. Natural mating or low-hormone dose did not increase the low fertility rate observed in superovulated B6 females. However, diet enrichment had a positive effect on pregnancy success. We also optimized breeding conditions to increase the survival of small litters by co-housing i-GONAD-treated pregnant B6 females with synchronized pregnant FVB/NJ companion mothers. Thus, GM mice generation was increased by an enriched diet and shared pup rearing with highly fertile females such as FVB/NJ. In the present study, we generated 16 GM mice using a CRISPR/Cas system to target individual and multiple loci simultaneously or consecutively. We also compared homology-directed repair efficiency using different methods for LoxP insertion for conditional knockout mouse production. We found that a two-step serial LoxP insertion, in which each LoxP sequence was inserted individually in different i-GONAD procedures, was a low-risk high-efficiency method for generating floxed mice.

Nitric Oxide (NO) regulation of Dendritic cells (DC) metabolism and immune effector functions

Abstract Dendritic cell (DC) activation is characterized by a sustained commitment to glycolysis, a requirement for survival in DC subsets expressing inducible nitric oxide synthase (iNOS, encoded by Nos2 gene). Nitric oxide (NO) can mediate both host-protective and host-cytotoxic effects by interfering with pathogen replication and/or by inhibiting the host mitochondrial respiration, resulting in metabolic rewiring in DCs. These phenomena primarily have been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6), where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced post-activation survival in LPS-stimulated PWD DCs due to attenuated NO production. To genetically map PWD’s NO phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2; 11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. The 11.2 DCs show comparable iNOS expression with lower NO production levels than B6 DCs. We demonstrate that activated 11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production and suggest a useful model to investigate the contribution/regulation of NO in DC metabolism and immune function. This work was supported by the National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (P30GM118228, 1R21AI135385-01A, T32AI055402-15, and R21 AI145306), the NIH, National Institute of Neurological Disorders and Stroke (R01 NS097596), and the National Multiple Sclerosis Society (RG-1901-33309).

Deletion of Vβ3 +CD4 +T-cells by endogenous mouse mammary tumor virus 3 prevents type 1 diabetes induction by autoreactive CD8 +T-cells

Abstract In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T-cells. Interactions between both helper CD4 +and cytotoxic CD8 +T-cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T-cells arise from deleterious interactions between relatively common genes which regulate aspects of T-cell activation/effector function (Ctla-4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T-cell receptor signaling (Ptpn22). In this study, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded Mtv3 provirus as a genetic element affecting CD4 +/CD8 +T-cell interactions through an additional mechanism, altering the T-cell receptor (TCR) repertoire. Mtv3 encodes a superantigen that deletes the majority of Vβ3 +thymocytes in NOD mice. Ablating Mtv3 and restoring Vβ3 +T-cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8 +T-cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifest by Vβ3 +CD4 +T-cells, which unless deleted by Mtv3, accumulate in insulitic lesions and trigger in B6 background mice the pathogenic activation of diabetogenic CD8 +T-cells. Our findings provide evidence that endogenous Mtv superantigens can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vβ repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human disorders. Supported by grants from the American Diabetes Association (1-14-BS-051) and NIH (AI130656)

The Genetic Background Is Shaping Cecal Enlargement in the Absence of Intestinal Microbiota.

Germ-free (GF) rodents have become a valuable tool for studying the role of intestinal microbes on the host physiology. The major characteristic of GF rodents is an enlarged cecum. The accumulation of mucopolysaccharides, digestion enzymes and water in the intestinal lumen drives this phenotype. Microbial colonization normalizes the cecum size in ex-GF animals. However, whether strain genetics influences the cecal enlargement is unknown. Here we investigated the impact of mouse genetic background on the cecal size in five GF strains frequently used in biomedical research. The cecal weight of GF mice on B6 background (B6J and B6N) represented up to 20% of total body weight. GF NMRI and BALBc mice showed an intermediate phenotype of 5-10%, and those on the C3H background of up to 5%. Reduced cecal size in GF C3H mice correlated with decreased water content, increased expression of water transporters, and reduced production of acidic mucins, but was independent of the level of digestive enzymes in the lumen. In contrast, GF B6J mice with greatly enlarged cecum showed increased water content and a distinct metabolic profile characterized by altered amino acid and bile acid metabolism, and increased acidic mucin production. Together, our results show that genetic background influences the cecal enlargement by regulating the water transport, production of acidic mucins, and metabolic profiles.

Absence of Apolipoprotein E Exacerbates Prion Pathology and Promote Microglial Mediated Neurodegeneration

AbstractBackgroundBoth Alzheimer’s disease and prionoses are neurogenerative disorders, which involve misfolded protein deposition and transition of homeostatic microglia to their neurodegenerative phenotype. In prionoses, accumulation of toxic PrPSc protein constitutes the culprit of pathogenesis. Neurodegenerative phenotype microglia (MGnD) appear early in the course of disease and play opposing roles in PrPSc mediated neurodegeneration. Whilst clearance of PrPSc has a disease‐limiting effect, MGnD‐driven neuroinflammation is deleterious to neurons. Apolipoprotein (apo) E is a pleiotropic protein, which controls MGnD phagocytic and inflammatory characteristics. Despite prominent role of microglia in prionoses, involvement of apoE in their pathogenesis has not been established.MethodWild type (WT) and Apoe‐/‐ mice, both on the B6 background, were inoculated with 22L mouse adapted scrapie strain or normal brain homogenate. Neuropathological and biochemical analyses were performed 15 and 23 weeks post inoculation (WPI) on asymptomatic and overly symptomatic animals, respectively. NanoStringTM analysis of microglia and astrocytic transcriptomic markers was performed at 23WPI.ResultPrion infection of WT mice upregulates total brain apoE level and effects cell‐type shift in the apoE expression, downregulating astrocytic apoE while increasing microglial apoE 24‐folds. 22L Apoe‐/‐ mice have significantly shorter disease incubation period, higher load of spongiform changes, and increased total brain PrP and PrPSc levels compared to 22LWT mice. In 22L Apoe ‐/‐ mice astro and micro gliosis appears earlier and is more robust compared to 22LWT mice. Transcriptomic analysis shows significantly higher level of PAN and A1, but not A2 astrocytic markers and MGnD markers in 22L Apoe ‐/‐ mice compared to 22LWT animals. Microglia in 22L Apoe ‐/‐ mice feature impaired neuronal phagocytosis and produce increased level of pro‐inflammatory interleukins and cytokines. 22L Apoe ‐/‐ mice show higher neuronal loss and greater tempo of neuronal degeneration than 22LWT mice.ConclusionOur results indicate the net effect of apoE in prion pathogenesis is protective. ApoE facilitates clearance of PrPSc and dying neurons by microglia, which becomes ineffective in the absence of apoE. Remnants of disintegrating neurons promote MGnD phenotype transition and inflammatory cascade propagating the vicious cycle of neuronal death and neuroinflammation. Our data also indicate homeostatic microglia transit to the MGnD even if they do not express apoE.

Calcineurin Inhibitors Inhibit Tolerance Induction By Suppressing Terminal Differentiation of Donor Exhausted T Cells after Allogeneic SCT

[Introduction] Calcineurin inhibitors (CNIs) reduce acute GVHD by inhibiting NFAT signaling in donor T cells but fail to prevent chronic GVHD (cGVHD). We have previously shown that donor alloreactive T cells differentiated into exhausted T cells with severely impaired alloreactivity after mouse allogeneic hematopoietic stem cell transplantation (allo-SCT) (Asakura S: JCI 2010). Given the critical role of NFAT signaling in T-cell exhaustion, we explored the role of CNIs in donor T-cell exhaustion and tolerance induction after allo-SCT using mouse SCT models with host alloantigen (allo-Ag)-specific 2C TCR-transgenic (2C) T cells and single cell RNA sequencing (scRNAseq) of donor T cells. [Methods] B6D2F1 recipients (H-2b/d) were lethally irradiated and transplanted with 1 x 106 2C T cells (CD8.1+) plus 1 x 106 wild type T cells (WT, CD45.1+) of B6 background (H-2b) together with T-cell-depleted bone marrow cells (TCD-BM) from WT B6 (CD45.2+) on day 0. In some experiments, recipients were p.o. injected with 25 mg/kg cyclosporin (CSP) daily on days 0-14. [Results] Flowcytometric analysis (FCM) showed 2C CD8+ T cells differentiated into terminally exhausted T cells (Tex) expressing multiple co-inhibitory receptors such as PD-1 and TIGIT on day 14 after allo-SCT, that led to unresponsiveness of donor T cells to PD-1 blockade. Next, we performed scRNAseq of donor T cells harvested from CSP- or vehicle-treated recipients on day 14 after allo-SCT. UMAP analysis identified 4 clusters of CD4+ T cells (C10-C13) and 9 clusters of CD8+ T cells (C1-C9), and all of them expressed Tox and Pdcd1, indicating that all donor T cells entered differentiation pathway to exhaustion. CSP remarkably increased C10 (CD4+) and C1 (CD8+) characterized by lower expression of Tox and Pdcd1 and higher expression of Ly6c2 compared to other clusters (Fig 1). FCM confirmed CSP significantly expanded PD-1+TIGIT-, and TOXlow cells in 2C and WT CD8+ and WT CD4+ donor T cells on day 14, and these cells persisted at least 2 wks after cessation of CSP. After adoptively transferred into irradiated secondary B6D2F1 recipients, Ly6C+ T cells purified from CSP-treated recipients were differentiated into Ly6C- Tex, while Ly6C- T cells remained to be Ly6C-, indicating that CSP-induced Ly6C+ donor T cells are precursors of Tex (pTex) like cells. We named these cells as pTex-CNI, based on lower TCF-1 expression compared to TCF-1high canonical pTex. We found that, among various agents known to suppress GVHD or Tex differentiation, only CSP and ibrutinib induced ToxlowPD-1+TIGIT-Ly6C+ pTex-CNI, suggesting that NFAT inhibition is critical for pTex-CNI induction. Importantly, only Ly6C+ donor T cells, but not Ly6C- cells, induced cGVHD after adoptive transfer, suggesting that pTex-CNI play a critical role in pathophysiology of cGVHD (Fig 2). pTex-CNI demonstrated marked proliferative response and differentiation toward Tex upon PD-1 blockade as canonical pTex did in tumor and viral infection models in the previous reports (Sade-Feldman M: Cell 2018, Hudson WH: Immunity 2019). Administration of PD-L1 mAbs from day 14 enhanced OXPHOS of donor T cells and eradicated host-type leukemia cells only in CSP-treated recipients. Post-transplant cyclophosphamide (PTCy) is widely used in clinical allo-SCT, and CNIs are started either before SCT or after PTCy typically on day 5. We found that pTex-CNI were induced only when CSP was added to PTCy from day -1, but not from day 5, suggesting that NFAT inhibition early after allo-SCT is critical for induction of pTex-CNI. Finally, we confirmed that T cells from the patients who underwent HLA-matched PBSCT with tacrolimus (TAC)-based GVHD prophylaxis started from day -1 expressed significantly lower levels of PD-1, TIGIT, and TOX on day 28 compared to those from patients underwent haploidentical PBSCT with PTCy-based GVHD prophylaxis combined with TAC started from day 5. [Conclusion] Our study for the first time demonstrated that CNI inhibits terminal differentiation of donor exhausted T cells after allo-SCT. In mouse models, pTex-CNI contributed to development of cGVHD and PD-1 blockade-induced anti-tumor response after allo-SCT. The timing of CNI initiation in PTCy-based allo-SCT could determine the persistence of donor T cell alloreactivity after allo-SCT, that could impact on the strategies of cessation of immunosuppressants and treatment of relapsed diseases. These should be tested in the future clinical studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mesenchymal stem cell transplantation alleviated atherosclerosis in systemic lupus erythematosus through reducing MDSCs

ObjectiveThe mechanism by which mesenchymal stem cell (MSC) transplantation alleviates atherosclerosis in systemic lupus erythematosus (SLE) remains elusive. In this study, we aim to explore the efficacy and mechanism of MSC in ameliorating atherosclerosis in SLE.MethodsApoE−/− and Fas−/− mice on the B6 background were cross-bred to generate SLE mice with atherosclerosis. Myeloid-derived suppressor cells (MDSCs) were sorted and quantified. The apoE−/−Fas−/− mice were either treated with anti-Gr antibody or injected with MDSCs. The lupus-like autoimmunity and atherosclerotic lesions were evaluated. Furthermore, the apoE−/−Fas−/− mice were transplanted with MSCs and lupus-like autoimmunity and atherosclerotic lesions were assessed.ResultsMDSCs in peripheral blood, spleen, draining lymph nodes increased in apoE−/−Fas−/− mice compared with B6 mice. Moreover, the adoptive transfer of MDSCs aggravated both atherosclerosis and SLE pathologies, whereas depleting MDSCs ameliorated those pathologies in apoE−/−Fas−/− mice. MSC transplantation in apoE−/−Fas−/− mice decreased the percentage of MDSCs, alleviated the typical atherosclerotic lesions, including atherosclerotic lesions in aortae and liver, and reduced serum cholesterol, triglyceride and low-density lipoprotein levels. MSC transplantation also reduced SLE pathologies, including splenomegaly, glomerular lesions, anti-dsDNA antibody in serum, urine protein and serum creatinine. Moreover, MSC transplantation regulated the generation and function of MDSCs through secreting prostaglandin E 2 (PGE2).ConclusionTaken together, these results indicated that the increased MDSCs contributed to atherosclerosis in SLE. MSC transplantation ameliorated the atherosclerosis and SLE through reducing MDSCs by secreting PGE2.Graphical

The characterization analysis of pathogenic T cells in immune-mediated aplastic anemia mouse model

目的通过DsRed小鼠淋巴细胞输注加全身辐照（TBI）建立免疫介导的再生障碍性贫血（AA）C.B10-H2b/LilMcd (C.B10)小鼠模型，分析C.B10 AA小鼠模型中病理性T细胞在不同器官的动态归巢过程及特征。方法通过异体淋巴细胞输注加TBI建立AA小鼠模型，以TBI组为对照，分析外周血细胞和骨髓单个核细胞数，验证造模效果。在造模的第3、6、9、12天，处死小鼠并收集骨髓、脾脏、淋巴结和胸腺单个核细胞，流式细胞术分析DsRed+ T细胞的动态变化及效应记忆T细胞（TEM）、中央记忆T细胞（TCM）、初始T细胞（naïve T）和效应T细胞（TEFF）的比例。PCR Array分析AA小鼠不同组织内DsRed+病理性T细胞活化相关基因的表达谱。结果与TBI组相比，AA组小鼠在造模第9、12天出现严重骨髓衰竭，第12天时骨髓有核细胞数、外周血细胞数均显著减少（P值均<0.05）。AA组小鼠骨髓、脾脏、淋巴结内DsRed+ T细胞比例随时间增加而增加。骨髓中的DsRed+ T细胞比例在第3、6天低于脾脏与淋巴结，但在第12天时高于脾脏与淋巴结（P值均<0.05）。整个骨髓衰竭形成过程中，胸腺中DsRed+ T细胞比例均最低。在第12天时，AA小鼠骨髓、淋巴结和脾脏单个核细胞中，DsRed+CD3+CD4+ T细胞比例分别为（91.38±2.10）％、（39.78±6.98）％、（67.87±12.77）％，三者之间两两比较均有统计学意义（P值均<0.05）；DsRed+CD3+CD8+ T细胞比例分别为（98.21±1.49）％、（94.06±4.20）％、（96.29±1.23）％，差异均无统计学意义（P值均>0.05）。第9、12天，AA组小鼠骨髓中几乎所有的DsRed+CD4+ T或DsRed+CD8+ T细胞均为TEM，而淋巴结中包括TEM、TCM以及naïve T。PCR Array结果显示：骨髓中DsRed+CD4+或DsRed+CD8+ T细胞中CD38、IFN-γ、LAG3、CSF1、SPP1及TNFSF13B表达增高，但 DsRed+CD4+ T细胞中FOXP3、CTLA4表达降低。结论DsRed小鼠淋巴细胞输注可导致C.B10小鼠发生骨髓衰竭，并可以在该AA模型小鼠中追踪DsRed+病理性T细胞。在C.B10 AA模型小鼠体内，DsRed+CD8+和DsRed+CD4+ T细胞先归巢到脾脏与淋巴结，在其中扩增分化，最终转运至骨髓并转化为TEM。胸腺对异体T细胞的归巢过程更具抵抗性，提示胸腺在免疫介导AA模型的骨髓衰竭过程中可能是一个免疫豁免部位。在C.B10 AA模型小鼠的骨髓衰竭形成过程中，归巢到骨髓的DsRed+病理性T细胞较迁移到脾脏与淋巴结的细胞具有更强的免疫活性，而免疫抑制活性则减弱。

Abstract 2468: Genetic metastatic colorectal cancer model with in vivo imaging capabilities

Abstract Introduction: Colorectal cancer (CRC) is the second leading cancer related cause of death worldwide, responsible for nearly 2 million deaths annually, most commonly due to complications from metastasis. A mouse model that reproduces human CRC is critically important to support pre-clinical research in the field. Recently a novel inducible genetically engineered mouse model in an immune competent B6 background was developed that metastasizes to the lung, liver, and peritoneum recapitulating what is seen in CRC patients. This model uses the Lgr5 (L) promoter to drive tamoxifen inducible Cre recombinase with the following conditional alleles: Apc (A), mutant Kras G12D (K), Tgfbr2 (T), and p53 (P) (LAKTP). After establishing the model in our center, we found mice developing tumors in the absence of tamoxifen induction. Because the original model lacked the ability to monitor the tumor development and progression in vivo in real time we created a LAKTP based mouse model that would allow for easy monitoring of tumor growth over time, allowing us to determine the frequency and time course of tumor development in the absence of tamoxifen induction. Methods: C57BL/6J mice, with each individual mutation (L, A, K, T, and P), were purchased from Jackson Laboratory and used for breeding in-house. Following breeding, congenic C57BL/6J LAKTP mice were characterized prior to breeding in conditional Luciferase (Rosa26 LSL Luciferase) which was also purchased from Jackson Laboratory. Luciferin was provided by intraperitoneal injection prior to capturing the bioluminescence signal using an imager from Spectral Imaging Systems. Tissue samples were fixed in formalin and embedded in paraffin prior sectioning for histology and immunohistochemistry. Results: Approximately 30% of our LAKTP mice died or required sacrifice due to meeting humane endpoints around 12 weeks of life in the absence of tamoxifen induction. Upon dissection, these mice exhibited large tumors located in the small intestine that led to non-survival bowel obstructions. Ten percent of these mice had liver metastases as well. Breeding in the conditional Luciferase allele allowed for detection of developing, non-induced tumors as early as 6 weeks of life. In addition to being useful for monitoring tumor growth in vivo, immunostaining of tumor tissue with anti-luciferase antibodies allows for detection of cells derived from Lgr5 positive intestinal stem cells. Conclusion: The LAKTP mouse model is an important tool for studying CRC, but exhibits tumor development due to leaky Cre activity in the absence of tamoxifen in approximately 30% of mice by 12 weeks. Our strategy of breeding in conditional luciferase allows for in vivo imaging such that mice can be partitioned based on bioluminescence for trials that require equivalent tumor burden at the beginning of interventions. In addition, bioluminescence imaging also allows for tumor size to be monitored over time. Citation Format: Tyler Leiva, James Griffith, Shaoxuan Guo, Megan Lerner, Lacey McNally, William Berry, Katherine Morris. Genetic metastatic colorectal cancer model with in vivo imaging capabilities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2468.

In Vivo Evaluation of the Cidec R46S Polymorphism for an Obesity QTL, tabw2a

ObjectivesA quantitative trait locus (QTL), tabw2a, was linked to obesity in TALLYHO/Jng (TH) mice, a polygenic model of obesity and type 2 diabetes. In combination with fine mapping information, we used whole genome sequencing data of TH mice to search positional candidate genes for tabw2a. From this search, the cell death-inducing DFFA-like effector c (Cidec) gene, containing a SNP identified as deleterious (likely to affect protein function) by both SIFT and PROVEAN, was raised as a potential candidate for tabw2a. CIDEC is a lipid droplet protein involved in the regulation of cellular lipid droplet size and lipid storage in adipocytes. There was one nucleotide substitution in the coding sequence of Cidec in TH mice, 136 C > A, compared to C57BL/6J (B6) reference sequence, that results in an amino acid substitution R46S (Arginine to Serine). The aim of this study was to evaluate the Cidec R46S polymorphism in vivo for the tabw2a QTL.MethodsWe generated knock-in mice where the TH allele (S46) is exchanged for the B6 allele (R46) at the Cidec gene on a B6 background. Targeting, ES clone selection, blastocyst injection, and breeding of ES cell chimeras were performed by Transgenics Core facility at Pennington Biomedical Research Center (Baton Rouge, LA). We bred the chimeras to B6 mice for 10 generation and collected heterozygous mice with germline transmission of the knock-in allele of Cidec S46. We then interbred the heterozygous mice and obtained homozygous mice for the knock-in allele of Cidec S46. Male and female Cidec R46S knock-in and B6 mice were weaned onto chow and high fat (HF) diets (D12266B, Research Diets) at 4 weeks of age and maintained on the same diets. At 20 weeks of age, body composition including fat and lean mass was measured using EchoMRI-100 whole body composition analyzer (Houston, TX).ResultsOn chow, no genotype effects were shown on adiposity between knock-in and B6 mice for both sexes. However, on HF diets, for both males and females, knock-in homozygous mice had significantly larger body fat mass than B6, although their body weights were comparable.ConclusionsWe conclude that the Cidec gene is a likely candidate for the obesity effect of tabw2a, and that Cidec S46 variant contributes to the obesity susceptibility.Funding SourcesAmerican Heart Association and National Institutes of Health.

Fibrosis of Chronic Lung Allograft Dysfunction After Orthotopic Lung Transplantation is Prevented in the Absence of Secondary Lymphoid Organs in a Mouse Model

<h3>Purpose</h3> It was reported that acute lung allograft rejection occurred without secondary lymphoid organs (SLOs) in a mouse allogenic lung transplantation model. The objective of this study was to investigate the role of SLOs in chronic lung allograft dysfunction (CLAD) after allogenic mouse lung transplantation using splenectomized alymphoplastic (aly/aly) mice as SLOs-deficient recipient. <h3>Methods</h3> Left lung of BALB/c mouse was transplanted into C57BL/6J (B6) or aly/aly mouse with B6 background. Low-dose cyclosporine (10 mg/kg, 3 times/week) and methylprednisolone (1.6 mg/kg, 3 times/week) were given in all the recipient mice until sacrifice. The recipient mice were sacrificed at day 15 (D15, n=4, each group) or day 30 (D30, n=5, each group) to evaluate histology. <h3>Results</h3> Allograft in B6 mice showed A2-3 rejection on D15 and A2-3 rejection with severe peri-airway and peri-vascular fibrosis on D30. On the contrary, allograft in aly/aly mouse showed A1-2 rejection on D15 and D30 without fibrosis (Figure 1). Quantification of peri-airway fibrosis thickness demonstrated significantly less fibrotic change in aly/aly mice than B6 mice on D30 (p = 0.012) (Figure 2). The A grade rejection score was significantly low on D30 in aly/aly mice (median A1, range A1-A2) than B6 mice (median A3, range A2-A3) (p = 0.015). <h3>Conclusion</h3> Fibrotic change with cellular rejection did not occur in the absence of SLOs in a mouse model of CLAD induced by inadequate immunosuppression, which may provide a platform for novel strategy to prevent CLAD.