F1 Mice Research Articles

Effects of AS2819899, a novel selective PI3Kδ inhibitor, in a NZB/W F1 mouse lupus-like nephritis model

Phosphoinositide 3-kinases generate lipid-based second messengers that control an array of intracellular signaling pathways. In particular, phosphoinositide 3-kinases delta (PI3Kδ) is expressed primarily in hematopoietic cells and plays an important role in B-cell development and function. B cells play a critical role in autoimmune diseases by producing autoantibodies. Studies have therefore increasingly focused on PI3Kδ as a therapeutic target for the treatment of inflammatory and autoimmune diseases. One such autoimmune disease is systemic lupus erythematosus (SLE). SLE is a chronic systemic autoimmune disease with repeated recurrence and remission, and autoantibodies play an important role in its pathogenesis. Here, we examined the pharmacological profile of the novel PI3Kδ selective inhibitor AS2819899 and investigated its therapeutic potential against SLE in a NZB/W F1 mouse lupus-like nephritis model, a widely-used SLE mouse model. AS2819899 prevented B and T cell activation in vitro, and inhibited antibody production in a T-cell independent de novo antibody production mouse model. In the spontaneous NZB/W F1 mouse model, AS2819899 treatment significantly reduced anti-dsDNA antibody titers and improved kidney dysfunction. Further, AS2819899 inhibited the memory recall reaction in a T-cell dependent antibody production mouse model, suggesting that AS2819899 can potentially maintain remission of SLE. Moreover, we identified a pharmacodynamics marker for AS2819899 that may be useful in clinical studies. These results indicate that AS2819899 may be an attractive therapeutic candidate for SLE, including the maintenance of remission.

Cocaine-Induced Sensitization is Linked to Distal Chromosome 6 Region in Congenic Mouse Model

ObjectivePreviously we mapped QTL Eac2 to mouse Chr6 and identified the first gene (Grm7) as accounting for alcohol consumption in a mammalian model. Despite the central role of glutamate receptors in addiction, the effects of Grm7 gene variants are not well known. Here we test the hypothesis that genetic variation of the distal mouse Chr6 Eac2 region, location of Grm7, controls cocaine-induced locomotor sensitization. MethodC57BL/6By background and B6.C6.327.54 congenic mice were subjected to whole-genome SNP genotyping. Isogeneic (C57BL/6ByXB6.C6.327.54)F2 mice homozygous for SNPs in the BALB/c-type Eac2 region were selected to create a subcongenic strain (B6By.C6.108-120). In a 2-strain x 2-sex 2-treatment factorial design (n = 6-10) C57BL/6By and B6By.C6.108-120 mice received repeated daily cocaine or saline intraperitoneal injections, and locomotor activity was recorded for 90 minutes immediately after injection. ResultsC57BL/6By females with the G/G genotype of SNP rs3723352 of Grm7 responded to cocaine with significantly higher activity and greater cocaine-induced sensitization than those with the BALB/cJ-type T/T genotype in the congenic strain. ConclusionThe results are consistent with a large body of accumulated mechanistic evidence for a role of the mGlu7 receptor in the control of neurobiological responses to cocaine, and are consistent with the hypotheses that (1) natural variants of the Grm7 gene show pleiotropy and can modulate cocaine-induced behaviors in addition to alcohol consumption, (2) interactions between mGluR7 expression, estrogen receptors, and estradiol may explain phenotypic variation in females. Heritable variation of GRM7 may affect vulnerability to substance abuse in women.

Meniscal and ligament modifications in spontaneous and post-traumatic mouse models of osteoarthritis

BackgroundOsteoarthritis (OA) is a whole joint disease that affects all joint tissues, with changes in the articular cartilage (AC), subchondral bone and synovium. Pathologies in menisci and ligaments, however, are rarely analysed, although both are known to play vital roles in the mechanical stability of the joint. The aim of our study was to describe the pathological changes in menisci and ligament during disease development in murine spontaneous and post-traumatic surgically induced OA and to quantify tissue mineralisation in the joint space using micro-computed tomography (μCT) imaging during OA progression.MethodsKnees of Str/ort mice (spontaneous OA model; 26–40 weeks) and C57CBA F1 mice following destabilisation of medial meniscus (DMM) surgery (post-traumatic OA model; 8 weeks after DMM), were used to assess histological meniscal and ligament pathologies. Joint space mineralised tissue volume was quantified by μCT.ResultsMeniscal pathological changes in Str/ort mouse knees were associated with articular cartilage lesion severity. These meniscal changes included ossification, hyperplasia, cell hypertrophy, collagen type II deposition and Sox9 expression in the fibrous region near the attachment to the knee joint capsule. Anterior cruciate ligaments exhibited extracellular matrix changes and chondrogenesis particularly at the tibial attachment site, and ossification was seen in collateral ligaments. Similar changes were confirmed in the post-traumatic DMM model. μCT analysis showed increased joint space mineralised tissue volume with OA progression in both the post-traumatic and spontaneous OA models.ConclusionsModifications in meniscal and ligament mineralisation and chondrogenesis are seen with overt AC degeneration in murine OA. Although the aetiology and the consequences of such changes remain unknown, they will influence stability and load transmission of the joint and may therefore contribute to OA progression. In addition, these changes may have important roles in movement restriction and pain, which represent major human clinical symptoms of OA. Description of such soft tissue changes, in addition to AC degradation, should be an important aspect of future studies in mouse models in order to furnish a more complete understanding of OA pathogenesis.

Allelic variation of the Tas1r3 taste receptor gene affects sweet taste responsiveness and metabolism of glucose in F1 mouse hybrids.

In mammals, inter- and intraspecies differences in consumption of sweeteners largely depend on allelic variation of the Tas1r3 gene (locus Sac) encoding the T1R3 protein, a sweet taste receptor subunit. To assess the influence of Tas1r3 polymorphisms on feeding behavior and metabolism, we examined the phenotype of F1 male hybrids obtained from crosses between the following inbred mouse strains: females from 129SvPasCrl (129S2) bearing the recessive Tas1r3 allele and males from either C57BL/6J (B6), carrying the dominant allele, or the Tas1r3-gene knockout strain C57BL/6J-Tas1r3tm1Rfm (B6-Tas1r3-/-). The hybrids 129S2B6F1 and 129S2B6-Tas1r3-/-F1 had identical background genotypes and different sets of Tas1r3 alleles. The effect of Tas1r3 hemizygosity was analyzed by comparing the parental strain B6 (Tas1r3 homozygote) and hemizygous F1 hybrids B6 × B6-Tas1r3-/-. Data showed that, in 129S2B6-Tas1r3-/-F1 hybrids, the reduction of glucose tolerance, along with lower consumption of and lower preference for sweeteners during the initial licking responses, is due to expression of the recessive Tas1r3 allele. Hemizygosity of Tas1r3 did not influence these behavioral and metabolic traits. However, the loss of the functional Tas1r3 allele was associated with a small decline in the long-term intake and preference for sweeteners and reduction of plasma insulin and body, liver, and fat mass.

Punicalagin Ameliorates Lupus Nephritis via Inhibition of PAR2.

Lupus nephritis (LN) is the most frequent phenotype in patients with systemic lupus erythematosus (SLE) and has a high rate of progression to end-stage renal disease, in spite of intensive treatment and maintenance therapies. Recent evidence suggests that protease-activated receptor-2 (PAR2) is a therapeutic target for glomerulonephritis. In this study, we performed a cell-based high-throughput screening and identified a novel potent PAR2 antagonist, punicalagin (PCG, a major polyphenol enriched in pomegranate), and evaluated the effects of PCG on LN. The effect of PCG on PAR2 inhibition was observed in the human podocyte cell line and its effect on LN was evaluated in NZB/W F1 mice. In the human podocyte cell line, PCG potently inhibited PAR2 (IC50 = 1.5 ± 0.03 µM) and significantly reduced the PAR2-mediated activation of ERK1/2 and NF-κB signaling pathway. In addition, PCG significantly decreased PAR2-induced increases in ICAM-1 and VCAM-1 as well as in IL-8, IFN-γ, and TNF-α expression. Notably, the intraperitoneal administration of PCG significantly alleviated kidney injury and splenomegaly and reduced proteinuria and renal ICAM-1 and VCAM-1 expression in NZB/W F1 mice. Our results suggest that PCG has beneficial effects on LN via inhibition of PAR2, and PCG is a potential therapeutic agent for LN.

Effect of Dietary Vitamin D3 on T Helper-Related Activities of Lupus-Prompt New Zealand Black/New Zealand White F1 Mice

We investigated the effect of dietary vitamin D3 on T helper-related activities and progress of lupus in NZB/NZW F1 mice. Mice were fed with 0.4 or 2 μg vitamin D3 dissolved in 50 μL olive oil every other day. Expression of T cell-related cytokine genes including those of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IFN-γ was monitored during the disease course by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). In addition, total serum IgG1 and IgG2a and anti-dsDNA antibody were measured. We observed a prolonged survival of mice receiving 2μg of vitamin D3 as compared with control mice. However, vitamin D3 did not significantly affect the anti-DNA antibody formation, IgG subtype proportion, or cytokine expression profile.

Multi and transgenerational epigenetic effects of di-(2-ethylhexyl) phthalate (DEHP) in liver

Di-(2-ethylhexyl) phthalate (DEHP), a ubiquitous industrial pollutant, is a known endocrine disrupter implicated in metabolic diseases. Prenatal DEHP exposure promotes epigenetic multi- and transgenerational inheritance of adult onset disease in subsequent generations (F1–F3). However, the epigenetic toxicity is less understood in the liver. In this study, CD-1 mice were prenatally exposed to 20 μg/kg/day, 200 μg/kg/day, 500 mg/kg/day, or 750 mg/kg/day DEHP from gestational day (GD) 10.5 until birth of pups. Following prenatal exposure, the multigenerational and transgenerational effects of mRNA expression of epigenetic regulators were evaluated in F1, F2, and F3 generation mouse livers at postnatal days (PNDs) 8 and 60. Results showed that DEHP exposed mice livers exhibited significant changes in global DNA methylation levels in all three generations, with the effect being different in F2 after high dosage exposure. Histopathology indicated that DEHP exposure could induce mild damage in F1 livers. The expression levels of DNA methyltransferase 1 (Dnmt1) were significantly changed in both the F1 and F2 generations at PND 8, suggesting that maintenance Dnmt1 plays a major role in the multigenerational effect that occur in the early developmental stages. Additionally, DEHP exposure caused significant changes in ten-eleven translocation methylcytosine (Tet) dioxygenases encoding Tet1 expression in all three generations and Tet2 expression in F3 at PND 60, implicating their contributions in inducing both multi- and transgenerational effects after DEHP exposure in mouse liver. Overall, our results establish that prenatal and ancestral DEHP exposure are critical for epigenetic regulation of DNA methylation in female mouse livers.

Functional organization of mouse primary auditory cortex in adult C57BL/6 and F1 (CBAxC57) mice

The primary auditory cortex (A1) plays a key role for sound perception since it represents one of the first cortical processing stations for sounds. Recent studies have shown that on the cellular level the frequency organization of A1 is more heterogeneous than previously appreciated. However, many of these studies were performed in mice on the C57BL/6 background which develop high frequency hearing loss with age making them a less optimal choice for auditory research. In contrast, mice on the CBA background retain better hearing sensitivity in old age. Since potential strain differences could exist in A1 organization between strains, we performed comparative analysis of neuronal populations in A1 of adult (~ 10 weeks) C57BL/6 mice and F1 (CBAxC57) mice. We used in vivo 2-photon imaging of pyramidal neurons in cortical layers L4 and L2/3 of awake mouse primary auditory cortex (A1) to characterize the populations of neurons that were active to tonal stimuli. Pure tones recruited neurons of widely ranging frequency preference in both layers and strains with neurons in F1 (CBAxC57) mice exhibiting a wider range of frequency preference particularly to higher frequencies. Frequency selectivity was slightly higher in C57BL/6 mice while neurons in F1 (CBAxC57) mice showed a greater sound-level sensitivity. The spatial heterogeneity of frequency preference was present in both strains with F1 (CBAxC57) mice exhibiting higher tuning diversity across all measured length scales. Our results demonstrate that the tone evoked responses and frequency representation in A1 of adult C57BL/6 and F1 (CBAxC57) mice are largely similar.

Meiotic epigenetic factor PRDM9 impacts sperm quality of hybrid mice.

Reduced fertility of male mouse hybrids relative to their parents, or hybrid sterility, is governed by the hybrid sterility 1 (Hst1) locus. Rescue experiments with transgenes carrying sequences within or near Hst1 manifested that Hst1 contains the gene encoding meiosis-specific histone methyltransferase PRDM9. The Prdm9 gene is responsible for partial meiotic arrest, testicular atrophy, and low sperm count in (C57BL/6J x PWD)F1 mouse hybrids. Here we report that these male hybrids suffer an additional reproductive disadvantage, decreased sperm quality, which is (i) further exacerbated by the introduction of long transgenes carrying sequences from Hst1 with incomplete Prdm9 into their genome and (ii) controlled by the Prdm9 dosage. These transgenic male hybrids displayed the features of severe oligoasthenoteratozoospermia (OAT), a human infertility syndrome characterized by a low number of spermatozoa with poor motility and morphological abnormalities. Analysis of spermiogenesis in these mice revealed acrosome detachment, aberrant elongation and condensation of the nucleus. As a result, the transgenic sperm had acrosome malformations, abnormal chromatin packaging, and fragmented DNA with elevated base oxidation, revealed by using multiple methods. Heterozygosity for one null Prdm9 allele improved meiotic progression and sperm quality of both non- and transgenic hybrids. Our results indicate that genomic analysis of OAT patients should include consideration of allelic variants in PRDM9, and our transgenic models can serve as tools to understand the diverse molecular processes that, when perturbed, can cause this disease.

Effects of oocyte vitrification on the behaviors and physiological indexes of aged first filial generation mice

To evaluate the long-term effects of oocyte cryopreservation on the health of the first filial generation (F1), we used B6D2F1 mice for oocyte collection, in vitro fertilization, and breeding. The female F1 mice born from the offspring of fresh mature oocytes (control group) and from the offspring of vitrified oocytes with traditional vitrification medium (VM group) and new improved vitrification medium (2P10E7D group) were maintained until 14–15 months of age for behavioral tests and 16–17 months of age for physiological analyses. Behavioral indexes, including anxiety-like status, discrimination ability, learning and memory ability, were investigated. Physiological indexes including body weight, body fat, heart rate, blood pressure, and blood lipids were also analyzed. In our results, the behavioral indexes, body weight, body fat, heart rate, blood pressure, total cholesterol (TC), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) did not show significant differences among the three groups. However, the triglyceride (TG) level of the VM group was higher than that of the 2P10E7D group. Moreover, compared with the control group, both the VM group and the 2P10E7D group showed greatly increased diastolic blood pressure. This study is the first to report that oocyte vitrification might affect metabolic physiological indexes via transgenerational inheritance rather than behaviors related to anxiety-like status and cognitive ability. Furthermore, different vitrification media might have differential transgenerational effects.

Maternal exposure to Di-(2-ethylhexyl) phthalate (DEHP) activates the PI3K/Akt/mTOR signaling pathway in F1 and F2 generation adult mouse testis

Di-(2-ethylhexyl) phthalate (diethylhexyl phthalate, DEHP) can cause male reproductive damage in rodents and human. Moreover, DEHP is known to promote transgenerational inheritance of adult-onset disease in subsequent generations after maternal exposure during fetal gonadal development. The PI3K/Akt/mTOR signaling pathway has been implicated in germ cell survival following testicular damage. In this study, a F0 gestation DEHP exposure and transgenerational inheritance testis injury model was established to study the testis injury phenotype and the expression and activation of members of PI3K/Akt/mTOR signaling pathway in the testis of F1–F3 generation mice. We found that the bodyweight and the anogenital distance (AGD) are reduced only in F1 mice, the sperm motility and deformity decreased in F1–F3 mice, and the testicular histomorphology damagedin F1–F3 mice; however the sperm motility and deformity rates are increased and the histomorphological injury is repaired during the transgenerational process. We also found the activation of PI3K/Akt/mTOR signaling pathway is enhanced in F1 and F2, and the number of apoptotic cells is decreased in F3 generation mice compared to the control group. These results suggest that the PI3K/Akt/mTOR signaling pathway may be activated to promote the proliferation and differentiation and protect testicular cells from apoptosis in the F1 and F2 generation mice after direct exposure to DEHP.

DNA vaccine encoding heat shock protein 90 protects from murine lupus

BackgroundSystemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of autoantibodies to multiple self-antigens, including heat shock proteins (HSP). Because of the increased expression of HSP90 and abnormal immune responses to it in SLE, we investigated whether an HSP90 DNA vaccine could modulate the development and clinical manifestations of SLE in lupus-prone mice.Methods(NZB x NZW)F1 (NZB/W) mice were vaccinated with DNA constructs encoding HSP90 or control plasmids or vehicle. The mice were then monitored for survival, circulating anti-dsDNA autoantibodies, and immune phenotypes. Renal disease was evaluated by immunohistochemistry and by the measurement of proteinuria.ResultsVaccination with HSP90 DNA reduced lupus disease manifestations and prolonged the survival of NZB/W mice. The protective effects of the HSP90 DNA vaccine associated with the induction of tolerogenic dendritic cells (DCs) and an expansion of T regulatory cells (Tregs).ConclusionsThe beneficial effects of DNA vaccination with HSP90 in murine SLE support the possibility of HSP90-based therapeutic modalities of intervention in SLE.

A breeding strategy to identify modifiers of high genetic risk for methamphetamine intake.

Trace amine-associated receptor 1 (Taar1) impacts methamphetamine (MA) intake. A mutant allele (Taar1m1J ) derived from the DBA/2J mouse strain codes for a non-functional receptor, and Taar1m1J/m1J mice consume more MA than mice possessing the reference Taar1+ allele. To study the impact of this mutation in a genetically diverse population, heterogeneous stock-collaborative cross (HS-CC) mice, the product of an eight-way cross of standard and wild-derived strains, were tested for MA intake. HS-CC had low MA intake, so an HS-CC by DBA/2J strain F2 intercross was created to transfer the mutant allele onto the diverse background, and used for selective breeding. To study residual variation in MA intake existing in Taar1m1J/m1J mice, selective breeding for higher (MAH) vs lower (MAL) MA intake was initiated from Taar1m1J/m1J F2 individuals; a control line of Taar1+/+ individuals (MAC) was retained. The lines were also examined for MA-induced locomotor and thermal responses, and fluid and tastant consumption. Taar1m1J/m1J F2 mice consumed significantly more MA than Taar1+/+ F2 mice. Response to selection was significant by generation 2 and there were corresponding differences in fluid consumed. Fluid consumption was not different in non-MA drinking studies. Taar1m1J/m1J genotype (MAL or MAH vs MAC mice) was associated with heighted MA locomotor and reduced hypothermic responses. MAL mice exhibited greater sensitization than MAH mice, but the selected lines did not consistently differ for thermal or tastant phenotypes. Residual variation among high-risk Taar1m1J/m1J mice appears to involve mechanisms associated with neuroadaptation to MA, but not sensitivity to hypothermic effects of MA.

Heat-Killed Lactobacillus reuteri GMNL-263 Inhibits Systemic Lupus Erythematosus-Induced Cardiomyopathy in NZB/W F1 Mice.

It has been increasingly recognized that accelerated atherosclerosis is a major cause of morbidity and mortality in patients with systemic lupus erythematosus, a multisystem autoimmune disease. In this study, we investigated the anti-apoptotic effects of heat-killed Lactobacillus reuteri GMNL-263 on the cardiac tissue of NZB/W F1 mice. The myocardial architecture of the mice heart was observed and evaluated using different staining techniques such as hematoxylin and eosin, TUNEL assay, Masson's trichrome, and fluorescent immunohistochemistry. Additionally, the probiotics-related pathway proteins were analyzed via western blot analysis. Our results showed prevention of enlarged interstitial spaces and abnormal myocardial structures in the hearts of NZB/W F1 mice with L. reuteri GMNL-263 feeding. Significant reduction in TUNEL-positive cells, Fas death receptor-related components, and apoptosis was also detected in the cardiac tissues of the NZB/W F1 mice after L. reuteri GMNL-263 feeding compared with the control group. These findings are the first to reveal the protective effects of L. reuteri GMNL-263 against cardiac abnormalities in NZB/W F1 mice and suggest the potential clinical applications of L. reuteri GMNL-263 in the treatment of SLE-related cardiovascular diseases.

Poly dispersed acid-functionalized single walled carbon nanotubes target activated T and B cells to suppress acute and chronic GVHD in mouse model

Graft versus host disease (GVHD) results from hyper-activation of transplanted lymphocytes against the host antigens. Bone marrow transplantation in humans as well as some cases of blood transfusion and organ transplantation are associated with a strong GVH reaction resulting in GVHD that in many cases may be fatal. We had previously shown that poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) specifically target activated T and B lymphocytes and kill them. In the present study, efficacy of AF-SWCNTs to suppress the GVH reaction was tested in the mouse model. Acute GVHD was induced in mice by administering intravenously 30 or 60 million spleen cells from a parental strain (C57bl/6 mouse, MHC haplotype H-2b) to host (C57bl/6 x Balb/c) F1 mice (MHC haplotype H-2b/d)and waiting for 8–10 days. Chronic GVHD was similarly induced by administration of 30 million parent spleen cells to F1 mice and waiting for a period of 60 days. Our results demonstrate a marked decline in splenomegaly and recovery of spleen T (both CD4 and CD8) and B cells in GVHD mice treated with AF-SWCNTs. AF-SWCNTs treatment also limited T and B cell proliferation by restricting S-phage of cell cycle. Generation of anti-host cytotoxic T cells (CTLs) was also markedly suppressed by AF-SWCNT treatment of acute GVHD mice, and a significant reduction in the generation of anti-host antibodies could also be demonstrated. Taken together, our results suggest that the AF-SWCNTs can be considered as a potential therapeutic agent for treating GVHD.

OP0046 TARGETING IRAK1 AND 4 SIGNALING WITH R835, A NOVEL ORAL SMALL MOLECULE INHIBITOR: A POTENTIAL NEW TREATMENT FOR SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by loss of immunological tolerance, hyperactivation of immune cells, proinflammatory cytokine production and, ultimately, end organ damage due to immune complex deposition. Toll-like receptors (TLRs), which are essential to the innate immune response to microbes and other danger signals, play a key role in the pathogenesis of SLE by recognition of self-molecules (1). Interleukin receptor associated kinases (IRAK)1 and 4 are responsible for initiating MyD88-dependent signaling from most TLRs and Interleukin-1 Receptors (IL-1R) and represent attractive targets for the therapeutic treatment of SLE (2). We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recently completed phase 1 study.Objectives:The aim of our study was to investigate the effect of IRAK1/4 selective inhibition as potential therapeutic approach for SLE. We evaluated the effect of our clinical candidate R835 on TLR7 signaling and in a mouse model of lupus-like disease.Methods:Human primary dendritic cells and whole blood were stimulated with gardiquimod (TLR7 agonist) to evaluate the effect of R835 on Interferon-alpha (IFN-α) production. R835 was further evaluated for its efficacy on survival and disease progression in lupus-prone NZB/W F1 mice with early or active signs of disease.Results:R835 inhibited TLR7-induced cytokine production in human dendritic cells and whole blood. Given orally to mice, R835 dose-dependently decreased serum IFN-α in response to administration of a TLR7 agonist. Furthermore, treatment of NZB/W F1 lupus-prone mice with R835 reversed the progression of lupus-like disease and the establishment of a pro-inflammatory environment, as demonstrated by decreased levels of proteinuria, blood urea nitrogen and autoantibodies, and reversal of renal pathology.Conclusion:To our knowledge, R835 is the first dual IRAK1/4 inhibitor to enter clinical development and provides an attractive approach to treat a range of autoimmune and rheumatic diseases, including lupus.

Efficacy of the Combination of Metformin and CTLA4Ig in the (NZB × NZW)F1 Mouse Model of Lupus Nephritis.

CTLA4Ig, a reagent that inhibits CD28 signaling, has shown therapeutic efficacy in mouse models of lupus nephritis (LN) when combined with several other biologics or standard of care drugs. Unfortunately, clinical trials treating LN patients with CTLA4Ig (abatacept) have not met endpoints. Metformin, a drug used to control hyperglycemia that inhibits mitochondrial metabolism, lowered the effective dose of glucocorticoids and prevented major flares when added on to the standard of care treatment of lupus patients with low disease activity. Metformin combined with inhibition of glycolysis by 2-deoxyglucose showed therapeutic efficacy in multiple mouse models of LN. Because CD28 signaling triggers glucose metabolism in T cells, we hypothesized that combining CTLA4Ig treatment with metformin would have the same effect. In this study, we showed that the combination of metformin and CTLA4Ig decreased the development of LN in (NZB × NZW)F1 mice treated at the early stage of disease. This preventive effect was associated with a decreased expansion of CD4+ T cell effector subsets. However, contrary to the combination with 2-deoxyglucose, metformin combined with CTLA4Ig did not alter autoantibody production, suggesting different mechanisms of symptom mitigation. Overall, this study shows therapeutic efficacy of the combination of metformin and CTLA4Ig, two drugs with established safety records, in a preclinical mouse model of LN.

Cas12a Base Editors Induce Efficient and Specific Editing with Low DNA Damage Response

The advent of base editors (BEs) holds great potential for correcting pathogenic-related point mutations to treat relevant diseases. However, Cas9 nickase (nCas9)-derived BEs lead to DNA double-strand breaks, which can trigger unwanted DNA damage response (DDR). Here, we show that the original version of catalytically dead Cas12a (dCas12a)-conjugated BEs induce a basal level of DNA breaks and minimally activate DDR proteins, including H2AX, ATM, ATR, and p53. By fusing dCas12a with engineered human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A (APOBEC3A), we further develop the BEACON (base editing induced by human APOBEC3A and Cas12a without DNA break) system to achieve enhanced deamination efficiency and editing specificity. Efficient C-to-T editing is achieved by BEACON in mammalian cells at levels comparable to AncBE4max, with only low levels of DDR and minimal RNA off-target mutations. Importantly, BEACON induces invivo base editing in mouse embryos, and targeted C-to-T conversions are detected in F0 mice.

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

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

Genetic analysis and epistatic interaction association of lipid traits in a C57xBalb/c F2 mice

The quantitative trait loci (QTL) controlling lipid traits were studied using microsatellite markers in mice at two different stages of life i.e. at 12 and 28 weeks. Additionally, the epistatic interactions between QTLs were also evaluated. One significant QTL for cholesterol (Tcholq1, logarithm of odds ratio, LOD: 3.49) on chromosome 2 and two QTL for low density lipoprotein, Ldlq1 &Ldlq2 were identified on chromosome 2 and 10 with LOD value of 5.43 and 3.39 respectively. However, suggestive QTL for cholesterol was identified on chromosome 1, 2, 9 & 13, for high density lipoprotein (HDL) on chromosome 2 & 7 and for triglycerides on chromosome 11 & 14. The QTL identified for LDL on chromosome 2 near marker D2Mit75 showed the pleiotropic effect with cholesterol and HDL trait in mice. The epistatic analysis among all interacting loci for all lipid traits revealed 51 significant digenetic interactions (LOD>5.0) of which the maximum was recorded for LDL at 12 weeks, however 63 interactions were identified at 28 weeks with a maximum for cholesterol. The study suggests that these lipid traits are governed through a complex genomic interactions and these interactions are different at different stage of life.