MRL/MpJ Research Articles

Soybean Agglutinin Alters the Gut Microbiota and Promotes Inflammation in Lupus-Prone MRL/lpr Mice

BackgroundCertain foods can trigger flares in patients with systemic lupus erythematosus. Lectins in edible plants have been reported to increase inflammation. ObjectiveThis study aimed to determine the effects of 1-time intake of soybean agglutinin (SBA) on the gut microbiota and immune response in lupus-prone MRL/MpJ (MRL)/lpr mice. MethodsMRL/MpJ-Faslpr/J (MRL/lpr) and MRL mice were randomly assigned into 4 groups (8 mice/group): MRL mice + phosphate-buffered saline (PBS) (CON), MRL mice + SBA (CS), MRL/lpr mice + PBS (LPR), and MRL/lpr + SBA (LS). PBS and SBA were orally administered at 16 wk of age, and all mice were killed 24 h after oral challenge. The disease phenotype, levels of proinflammatory cytokines, and composition of the intestinal microbiota were determined. ResultsInterferon-gamma (IFN-γ) in the serum was significantly higher, whereas the level of serum IL-10 was significantly lower in LS mice than in LPR mice [fold change (FC) = 1.31 and FC = 0.36, respectively]. The expression levels of IL-6 and TNF-α in the spleen of LS mice were significantly higher than those in LPR mice (FC = 1.66 and FC = 1.96, respectively). The expression levels of IL-6, TNF-α, and IL-1β in the kidney were also significantly higher in LS mice than in LPR mice (FC = 2.89, FC = 3.78, and FC = 2.02, respectively). The relative abundances of Erysipelotrichaceae and Turicibacter in LS mice were significantly higher than those in LPR mice (FC = 1.73 and FC = 1.74, respectively). The percentage of Breg cells in the mesenteric lymph nodes was significantly lower in LS mice than in LPR mice (FC = 0.53) (P < 0.05). No change was found between SBA treatment or not in the control (MRL) mice. ConclusionsOne-time intake of SBA can promote the secretion of proinflammatory cytokines, downregulate Breg cells, and alter the intestinal flora in MRL/lpr mice within 24 h of oral challenge, which may contribute to exacerbation of lupus.

Enhanced healing outcomes in MRL/MpJ mouse tissues conserved in insertion site following surgical repair.

Surgical repair of supraspinatus tendons (SSTs) has a high failure rate at the insertion site. A significant hurdle to therapeutic development is that effective intrinsic healing mechanisms are unknown. The MRL/MpJ (MRL) mouse exhibits tissue-specific enhanced healing; however, these tissues exhibit disparate properties from the complex SST. The extent of SST healing in the complex environment of the rotator cuff is unknown. We hypothesized that MRL mice would exhibit enhanced restoration of the structurally complex insertion site, resulting in functional improvements. B6 and MRL mice underwent SST detachment and immediate surgical repair. Mice were analyzed for gait assessment after either 2 or 6 weeks and were then killed humanely for immunohistologic analysis. MRL SSTs demonstrated enhanced recovery of zonal architecture and bone structure compared with B6 SSTs. MRL SSTs exhibited decreased levels of type III collagen at 2 weeks and increased levels of type I procollagen at 6 weeks compared with B6 SSTs. MRL mice experienced initial gait deficits at 2 weeks that had recovered by 6 weeks. The temporal balance of collagen in MRL mice suggests recovery toward naive composition. Initial gait deficits in MRL mice may provide a protective loading environment that is ultimately beneficial. The mechanisms of enhanced healing observed previously in MRL mice may be conserved in the complex SST, providing a platform to interrogate specific aspects of improved healing.

Ear wound healing in MRL/MpJ mice is associated with gut microbiome composition and is transferable to non-healer mice via microbiome transplantation.

Adult elastic cartilage has limited repair capacity. MRL/MpJ (MRL) mice, by contrast, are capable of spontaneously healing ear punctures. This study was undertaken to characterize microbiome differences between healer and non-healer mice and to evaluate whether this healing phenotype can be transferred via gut microbiome transplantation. We orally transplanted C57BL/6J (B6) mice with MRL/MpJ cecal contents at weaning and as adults (n = 57) and measured ear hole closure 4 weeks after a 2.0mm punch and compared to vehicle-transplanted MRL and B6 (n = 25) and B6-transplanted MRL (n = 20) mice. Sex effects, timing of transplant relative to earpunch, and transgenerational heritability were evaluated. In a subset (n = 58), cecal microbiomes were profiled by 16S sequencing and compared to ear hole closure. Microbial metagenomes were imputed using PICRUSt. Transplantation of B6 mice with MRL microbiota, either in weanlings or adults, improved ear hole closure. B6-vehicle mice healed ear hole punches poorly (0.25±0.03mm, mm ear hole healing 4 weeks after a 2mm ear hole punch [2.0mm-final ear hole size], mean±SEM), whereas MRL-vehicle mice healed well (1.4±0.1mm). MRL-transplanted B6 mice healed roughly three times as well as B6-vehicle mice, and half as well as MRL-vehicle mice (0.74±0.05mm, P = 6.9E-10 vs. B6-vehicle, P = 5.2E-12 vs. MRL-vehicle). Transplantation of MRL mice with B6 cecal material did not reduce MRL healing (B6-transplanted MRL 1.3±0.1 vs. MRL-vehicle 1.4±0.1, p = 0.36). Transplantation prior to ear punch was associated with the greatest ear hole closure. Offspring of transplanted mice healed significantly better than non-transplanted control mice (offspring:0.63±0.03mm, mean±SEM vs. B6-vehicle control:0.25±0.03mm, n = 39 offspring, P = 4.6E-11). Several microbiome clades were correlated with healing, including Firmicutes (R = 0.84, P = 8.0E-7), Lactobacillales (R = 0.65, P = 1.1E-3), and Verrucomicrobia (R = -0.80, P = 9.2E-6). Females of all groups tended to heal better than males (B6-vehicle P = 0.059, MRL-transplanted B6 P = 0.096, offspring of MRL-transplanted B6 P = 0.0038, B6-transplanted MRL P = 1.6E-6, MRL-vehicle P = 0.0031). Many clades characteristic of female mouse cecal microbiota vs. males were the same as clades characteristic of MRL and MRL-transplanted B6 mice vs. B6 controls, including including increases in Clostridia and reductions in Verrucomicrobia in female mice. In this study, we found an association between the microbiome and tissue regeneration in MRL mice and demonstrate that this trait can be transferred to non-healer mice via microbiome transplantation. We identified several microbiome clades associated with healing.

Initial displacement of the intra-articular surface after articular fracture correlates with PTA in C57BL/6 mice but not "superhealer" MRL/MpJ mice.

Posttraumatic arthritis (PTA) occurs commonly after articular fracture and may arise, in part, from joint surface incongruity after injury. MRL/MpJ (MRL) "super-healer" mice are protected from PTA compared to C57BL/6 (B6) mice following articular fracture. However, the relationship between the initial displacement of the articular surface, biologic response, and susceptibility to PTA after fracture remains unclear. The objective of this study was to assess whether joint incongruity after articular fracture, as measured by in vivo micro-computed tomography (microCT), could predict pathomechanisms of PTA in mice. B6 and MRL mice (n = 12/strain) received a closed articular fracture (fx) of the left tibial plateau. Articular incongruity was quantified as bone surface deviations (BSD) for each in vivo microCT scan obtained from pre-fx to 8 weeks post-fx, followed by histologic assessment of arthritis. Serum concentrations of bone formation (PINP) and bone resorption (CTX-I) biomarkers were quantified longitudinally. Both strains showed increases in surface incongruity over time, as measured by increases in BSD. In B6 mice, acute surface incongruity was significantly correlated to the severity of PTA (R 2 = 0.988; p = .0006), but not in MRL mice (R 2 = 0.224; p = .220). PINP concentrations significantly decreased immediately post-fx in B6 mice (p = .023) but not in MRL mice, indicating higher bone synthesis in MRL mice. MRL/MpJ mice demonstrate a unique biologic response to articular fracture such that the observed articular bone surface displacement does not correlate with the severity of subsequent PTA. Clinical Relevance: Identifying therapies to enhance acute biologic repair following articular fracture may mitigate the risk of articular surface displacement for PTA.

Identification of Cartilage Microbial DNA Signatures and Associations With Knee and Hip Osteoarthritis.

Alterations of the gut microbiota have been implicated in many forms of arthritis, but an examination of cartilage microbial patterns has not been performed. This study was undertaken to characterize the microbial DNA profile of articular cartilage and determine changes associated with osteoarthritis (OA). We performed 16S ribosomal RNA gene deep sequencing on eroded and intact cartilage samples from knee OA patients (n = 21 eroded and 21 intact samples) and hip OA patients (n = 34 eroded and 33 intact samples) and cadaver controls (n = 10 knee samples and 10 hip samples). Microbial DNA diversity was assessed, groups were compared, and metagenomic profiles were reconstructed. Confirmation was performed in an independent cohort by clade-specific quantitative polymerase chain reaction. Findings in human cartilage were compared to those in cartilage from OA-susceptible C57BL/6 (B6) mice and OA-resistant MRL/MpJ (MRL) mice. Germ-free B6 mouse cartilage was analyzed as a methodologic control. Alpha diversity was reduced in human OA versus control samples (P < 0.0001), and in hip versus knee samples (P < 0.0001). Numerous clades were different in human OA versus control samples, and similar findings were noted in comparisons of murine B6 versus MRL mice. Hip samples were microbiologically distinct from knee samples. OA microbial DNA demonstrated increased gram-negative constituents (P = 0.02). Functional analysis demonstrated increases in lipopolysaccharide production (P = 9.9 × 10-3 ), phosphatidylinositol signaling (P = 4.2 × 10-4 ), and nitrogen metabolism (P = 8 × 10-3 ) and decreases in sphingolipid metabolism (P = 7.7 × 10-4 ) associated with OA. Our study reveals a microbial DNA signature in human and mouse cartilage. Alterations in this signature, including increases in gram-negative constituents, occur during the development and progression of human OA. Furthermore, our findings indicate that strain-specific signatures exist within mouse cartilage that mirror human patterns. Further study of the establishment and potential pathogenic role of these DNA signatures is needed.

Abstract 98: Harnessing Novel Gene Expression Analyses To Identify Drivers Of Ear Regeneration In MRL Mice

Purpose: The “super-healing” MRL/MpJ mouse is uniquely able to regenerate ear punch wounds, while dorsal wounds heal in a typical scarring fashion. Identification of the genes responsible for ear regeneration in these mice could provide insights toward novel anti-scarring therapies. However, previous methods for identifying the genetic basis of species-/strain-specific traits are imprecise and extremely resource-intensive. Recent studies have revealed that, by outbreeding a strain with a trait of interest (here, regenerative ear healing), assessing the hybrid offspring for spatial/tissue-specific differences in relative gene expression from the two parent alleles (“differential allele-specific expression”) can reveal genes underlying that phenotype. We hypothesized that differential allele-specific expression could represent a novel approach to identify genes driving regenerative ear healing in MRL mice. Methods: MRL/MpJ (MRL) and CAST/EiJ (CAST) mouse genome sequences were obtained. Variant calling was performed to identify sites where the sequences differed. MRL and CAST mice were cross-bred, F1 offspring underwent dorsal excisional and ear punch wounding (standard protocols), and wounds were harvested on POD7. Following incubation in ammonium thiocyanate, epidermis and underlying cartilage (for ear wounds) were removed under a surgical microscope. Dorsal and ear wound dermis were digested, and inflammatory/immune cells (CD45+; chosen because prior studies have implicated altered wound inflammatory response in MRL regeneration) were isolated by FACS and subjected to bulk RNA-sequencing. Reads were mapped to the MRL or CAST genome based on strain-specific variants, and genes with significantly differing expression (Benjamini-Hochberg method, FDR=5%) from the MRL vs. CAST allele and from dorsal vs. ear wounds were identified. Results: Ear wounds in MRLxCAST hybrid mice exhibited grossly apparent regeneration over time, consistent with prior reports. Roughly equal proportions of RNA-seq reads mapped to MRL vs. CAST alleles, indicating minimal mapping bias. Principal component analysis demonstrated that reads clustered by parent allele (MRL vs. CAST) and wound site (ear vs. dorsum), with good reproducibility between biological replicates (n=4). Importantly, the number of genes with significantly different expression from MRL vs. CAST allele was substantially (>10-fold) greater in the ear than the dorsum (2531 vs. 159 unique genes. The majority of allele-specific expression in the dorsum was also observed in the ear, overlapping region; 878 shared genes vs. 159 unique to dorsum). Conclusions: In MRL hybrid mice, inflammatory cells exhibit markedly greater allele-specific gene expression in ear wounds (regenerative) than dorsal wounds (scarring). This finding would be consistent with MRL-specific gene cis-regulatory activity leading to the improved healing phenotype seen in the ear. Our results suggest that probing differential allele-specific gene expression may represent a powerful computational approach for identifying the genetic basis of complex traits in mammals. We will examine inflammatory and other cell types (e.g., fibroblasts) in order to identify specific genes with differential allele-specific expression in regenerative versus scarring wounds, with the ultimate goal of identifying novel targets for preventing scarring.

Hearing loss without overt metabolic acidosis in ATP6V1B1 deficient MRL mice, a new genetic model for non-syndromic deafness with enlarged vestibular aqueducts.

Mutations of the human ATP6V1B1 gene cause distal renal tubular acidosis (dRTA; OMIM #267300) often associated with sensorineural hearing impairment; however, mice with a knockout mutation of Atp6v1b1 were reported to exhibit a compensated acidosis and normal hearing. We discovered a new spontaneous mutation (vortex, symbol vtx) of Atp6v1b1 in an MRL/MpJ (MRL) colony of mice. In contrast to the reported phenotype of the knockout mouse, which was developed on a primarily C57BL/6 (B6) strain background, MRL-Atp6v1b1vtx/vtx mutant mice exhibit profound hearing impairment, which is associated with enlarged endolymphatic compartments of the inner ear. Mutant mice have alkaline urine but do not exhibit overt metabolic acidosis, a renal phenotype similar to that of the Atpbv1b1 knockout mouse. The abnormal inner ear phenotype of MRL- Atp6v1b1vtx/vtx mice was lost when the mutation was transferred onto the C57BL/6J (B6) background, indicating the influence of strain-specific genetic modifiers. To genetically map modifier loci in Atp6v1b1vtx/vtx mice, we analysed ABR thresholds of progeny from a backcross segregating MRL and B6 alleles. We found statistically significant linkage with a locus on Chr 13 that accounts for about 20% of the hearing threshold variation in the backcross mice. The important effect that genetic background has on the inner ear phenotype of Atp6v1b1 mutant mice provides insight into the hearing loss variability associated with dRTA caused by ATP6V1B1 mutations. Because MRL-Atp6v1b1vxt/vtx mice do not recapitulate the metabolic acidosis of dRTA patients, they provide a new genetic model for nonsyndromic deafness with enlarged vestibular aqueduct (EVA; OMIM #600791).

Characterization of mouse mediastinal fat-associated lymphoid clusters.

The association between adipose tissue and immunity has been established and fat-associated lymphoid clusters (FALCs) are considered as a source of immune cells. We discovered lymphoid clusters (LCs) in mouse mediastinal fat tissues (MFTs). In Th1-biased C57BL/6N (B6), Th2-biased DBA/2Cr (DBA) and autoimmune-prone MRL/MpJ (MRL) mice strains, LCs without a fibrous capsule and germinal center were observed in white-colored MFTs extending from the diaphragm to the heart. The number and size of the LCs were larger in 12-month-old mice than in 3-month-old mice in all of the examined strains. Moreover, B6 had an especially large number of LCs compared with DBA and MRL. The immune cells in the LCs consisted of mainly T-cells and some B-cells. The majority of T-cells were CD4+ helper T (Th) cells, rather than CD8+ cytotoxic T-cells and no obvious immune cell population difference was present among the strains. Furthermore, high endothelial venules and lymphatic vessels in the LCs were better developed in B6 mice than in the other strains. Interestingly, some CD133+ hematopoietic progenitor cells and some c-Kit+/CD127+ natural helper cells were detected in the LCs. BrdU+ proliferating cells were more abundant in the LCs of B6 mice than in the LCs of the other strains and the number of BrdU+ cells increased with age. This is the first report of LCs in mouse MFTs. We suggest that the mouse genetic background affects LC size and number. We term the LCs "mediastinal fat-associated lymphoid clusters". These clusters can be considered as niches for Th cell production.

Intra-articular Delivery of Purified Mesenchymal Stem Cells from C57BL/6 or MRL/MpJ Superhealer Mice Prevents Posttraumatic Arthritis

Joint injury dramatically enhances the onset of osteoarthritis (OA) and is responsible for an estimated 12% of OA. Posttraumatic arthritis (PTA) is especially common after intra-articular fracture, and no disease-modifying therapies are currently available. We hypothesized that the delivery of mesenchymal stem cells (MSCs) would prevent PTA by altering the balance of inflammation and regeneration after fracture of the mouse knee. Additionally, we examined the hypothesis that MSCs from the MRL/MpJ (MRL) "superhealer" mouse strain would show increased multilineage and therapeutic potentials as compared to those from C57BL/6 (B6) mice, as MRL mice have shown exceptional in vivo regenerative abilities. A highly purified population of MSCs was prospectively isolated from bone marrow using cell surface markers (CD45-/TER119-/PDGFRα+/Sca-1+). B6 MSCs expanded greater than 100,000-fold in 3 weeks when cultured at 2% oxygen and displayed greater adipogenic, osteogenic, and chondrogenic differentiation as compared to MRL MSCs. Mice receiving only a control saline injection after fracture demonstrated PTA after 8 weeks, but the delivery of 10,000 B6 or MRL MSCs to the joint prevented the development of PTA. Cytokine levels in serum and synovial fluid were affected by treatment with stem cells, including elevated systemic interleukin-10 at several time points. The delivery of MSCs did not reduce the degree of synovial inflammation but did show increased bone volume during repair. This study provides evidence that intra-articular stem cell therapy can prevent the development of PTA after fracture and has implications for possible clinical interventions after joint injury before evidence of significant OA.

Genomic analysis of the appearance of testicular oocytes in MRL/MpJ mice

Mammals produce sperm or oocytes depending on their sex; however, newborn MRL/MpJ (MRL) male mice produce oocytes within their testes. We previously reported that one of the genes responsible for this phenotype is present on the MRL-type Y chromosome (Y(MRL)), and that multiple genes, probably autosomal, are also required for the development of this phenotype. In this study we focused on the autosomal genes and examined their relationship with this phenotype by analyzing the progeny from crosses between MRL mice and other strains. We first observed the male F1 progeny from the crosses between female A/J, C57BL/6 (B6), BALB/c, C3H/He, or DBA/2 mice and male MRL mice, and two consomic strains, male B6-Y(MRL) and MRL-Y(B6). Testicular oocytes that were morphologically similar to those of MRL mice were detected in all mouse strains except BALBMRLF1; however, the incidence of testicular oocytes was significantly lower than that in MRL mice. The appearance of testicular oocytes in MRL-Y(B6) mice indicates that this phenotype is strongly affected by genomic factors present on autosomes, and that there is at least one other causative gene on the MRL-type autosomes (MRL testicular oocyte production, mtop) other than that on Y(MRL). Furthermore, a quantitative trait locus (QTL) analysis using N2 backcross progeny from crosses between female MRLB6F1 and male MRL mice revealed the presence of susceptibility loci for the appearance of testicular oocytes at 8-17 cM on Chr 15. These findings demonstrate that the appearance of testicular oocytes is regulated by the genetic factors on Chr 15 and on Y(MRL).

Ovarian cysts in MRL / MpJ mice are derived from the extraovarian rete: a developmental study

MRL/MpJ (MRL) mice, commonly used as a model for autoimmune disease, have a high frequency of ovarian cysts originating from the rete ovarii. In the present study, to clarify how the rete ovarii, which are remnants of mesonephric tubules during embryogenesis, progress to cystic formation with aging, the morphology of MRL rete ovarii was analyzed and compared with that of normal C57BL/6N (B6) mice. In B6 mice, the rete ovarii consisted of a series of tubules, including the extraovarian rete (ER), the connecting rete (CR), and the intraovarian rete (IR), based on their location. Whereas the ER of B6 mice was composed of highly convoluted tubules lined by both ciliated and non-ciliated epithelia, the tubules in the CR and IR had only non-ciliated cells. In MRL mice, dilations of the rete ovarii initiated from the IR rather than the ER or CR. Although the histological types of cells lining the lumen of the rete ovarii were the same as those in B6 mice, the ER in MRL mice showed a variety in morphology. In particular, the connections between the ER and ovary tended to disappear with increasing age and the development of ovarian cysts. Furthermore, the epithelium lining the large ovarian cysts in MRL mice had ciliated cells forming the cluster. On the basis of these findings, it is suggested that cystic changes of the rete ovarii in MRL mice are caused by the dilations of the IR with invasion of the ER and CR into the ovarian medulla. These data provide new pathological mechanisms for ovarian cyst formation.

Quantitative trait locus analysis of ovarian cysts derived from rete ovarii in MRL/MpJ mice

MRL/MpJ (MRL) is a model mouse for autoimmune diseases such as dermatitis, vasculitis, arthritis, and glomerulonephritis. In addition to these immune-associated disorders, we found that older MRL mice develop ovarian cysts originating from the rete ovarii, which is lined by ciliated or nonciliated epithelium and considered remnants of mesonephric tubules. Ovarian cysts, which are reported to have several sources, are associated with female infertility, but information regarding the genetic etiology of ovarian cysts originating from the rete ovarii is rare. In this study, to elucidate the genetic background of development of ovarian cysts, we performed quantitative trait locus (QTL) analysis using 120 microsatellite markers, which cover the whole genome of murine chromosomes, and 213 backcross progenies between female MRL and male C57BL/6N mice. The quantitative trait measured was the circumferences of rete ovarii or ovarian cysts. As a result, suggestive linkages were detected on Chrs 3, 4, 6, and 11, but significant linkages were located on Chr 14 by interval mapping. We thereby designated the 27.5-cM region of Chr 14 "MRL Rete Ovarian Cysts (mroc)." The peak regions of Chrs 4 and 14 in particular showed a close additive interaction (p < 0.00001). From these results we concluded that multiple loci on Chrs 3, 4, 6, 11, and 14 interact to result in development of ovarian cysts in MRL mice.

Analysis of factors decreasing testis weight in MRL mice

MRL/MpJ (MRL) mouse testes have several unique characteristics, including the appearance of oocytes, the occurrence of metaphase-specific apoptosis of meiotic spermatocytes, and the presence of heat-shock-resistant spermatocytes. In the present study we used chromosomal mapping to determine the genomic background associated with small testis size in MRL mice. We prepared and analyzed C57BL/6-based congenic mice carrying MRL mouse loci. Quantitative trait loci (QTL) analysis revealed susceptibility loci for small testis size at 100 cM on chromosome (Chr) 1 and at around 80 cM on Chr 2. Analysis with B6.MRLc1 and B6.MRLc2 congenic mice and double-congenic mice confirmed the QTL data and showed that low testis weight in MRL mice was caused by germ cell apoptosis. Through histological examinations we found that B6.MRLc1 and B6.MRLc2 mice showed stage-specific apoptosis in their testes, the former at metaphase stage XII and the later at pachytene stage IV. Metaphase-specific apoptosis of spermatocytes occurs due to mutation of the exonuclease 1 (Exo1) gene located at 100 cM on Chr 1. Thus, the mutation of the Exo1 gene is also responsible for low testis weight caused by metaphase-specific apoptosis. In conclusion, testis weight is reduced in MRL mice due to apoptosis of germ cells caused by mutations in loci on Chrs 1 and 2.

Interferon-γ Prevents Death of Bystander Neurons during CD8 T Cell Responses in the Brain

T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.

Attenuation of the post‐myocardial infarction stress response may promote improved survival and healing in the MRL mouse

Introduction: MRL/MpJ (MRL) mice heal better following myocardial infarction (MI) than C57BL/6J (C57) controls, may regenerate cardiac muscle, and have improved long‐term survival, though the mechanisms are unclear. We compared acute post‐MI survival and healing differences between the two strains as well as the LG/J strain, the major genetic background of the MRL. Methods: MI was induced in adult male MRL, LG/J, and C57 mice by permanent ligation of the left coronary artery. Infarct size was measured after 1, 5, or 30 days. Microarray analysis was performed on RNA collected before and 1 or 5 days post‐MI. Survival was tracked through 70 days. Results: Survival was significantly greater in MRL than C57 mice, due to decreased LV rupture in MRLs during days 2–5 post‐infarct. LG/J mice also appear more susceptible to rupture. Infarct size on day 1 was not different between the MRL and C57 strains, but was significantly larger in C57 mice after 30 days. Preliminary results suggest infarcts of all 3 strains expand to a similar degree after 5 days. Microarray analysis indicates the expression of transcripts associated with induction of apoptosis, the immune response, and inhibition of cellular proliferation may be reduced in the MRL day 1 infarct compared to the C57. The relative attenuation of these processes may contribute to the MRL's survival advantage and promote improved healing. Support: NIH

Tracking Differential Gene Expression in MRL/MpJ versus C57BL/6 Anergic B Cells: Molecular Markers of Autoimmunity

BACKGROUND: Anergy is a key mechanism controlling expression of autoreactive B cells and a major site for failed regulation in autoimmune diseases. Yet the molecular basis for this differentiated cell state remains poorly understood. The current lack of well-characterized surface or molecular markers hinders the isolation of anergic cells for further study. Global gene profiling recently identified transcripts whose expression differentiates anergic from naïve B cells in model mouse systems. The objective of the current study was to evaluate the molecular and cellular processes that differentiate anergic cells that develop in the healthy C57BL/6 (B6) milieu from those that develop in the autoimmune-prone MRL/MpJ (MRL) background. This approach takes advantage of B6 and MRL mice bearing an anti-laminin Ig transgene with a well characterized anergic B cell phenotype. RESULTS: Global gene expression was evaluated in purified transgenic B cells using Operon version 3.0 oligonucleotide microarray assaying >31,000 oligoprobes. Genes with a 2-fold expression difference in B6 as compared to MRL anergic B cells were identified. Expression of selected genes was confirmed using quantitative RT-PCR. This approach identified 43 probes corresponding to 37 characterized genes, including Ptpn22, CD74, Birc1f/Naip, and Ctla4, as differentially expressed in anergic B cells in the two strains. Gene Ontology classification identified differentiation, cell cycle, proliferation, development, apoptosis, and cell death as prominently represented ontology groups. Ingenuity Pathway Analysis identified two major networks incorporating 27 qualifying genes. Network 1 centers on beta-estradiol and TP53, and Network 2 encompasses RB1, p38 MAPK, and NFkB cell growth, proliferation, and cell cycle signaling pathways. CONCLUSION: Using microarray analysis we identified 37 characterized genes and two functional pathways engaged in maintenance of B cell anergy for which expression is distorted by underlying autoimmune genetic susceptibility. This approach identifes a new biological role for multiple genes and potential new therapeutic targets in autoimmunity.

Detecting Novel Bone Density and Bone Size Quantitative Trait Loci Using a Cross of MRL/MpJ and CAST/EiJ Inbred Mice

Most previous studies to identify loci involved in bone mineral density (BMD) regulation have used inbred strains with high and low BMD in generating F(2) mice. However, differences in BMD may not be a requirement in selecting parental strains for BMD quantitative trait loci (QTL) studies. In this study, we intended to identify novel QTL using a cross of two strains, MRL/MpJ (MRL) and CAST/EiJ (CAST), both of which exhibit relatively high BMD when compared to previously used strains. In addition, CAST was genetically distinct. We generated 328 MRL x CAST F(2) mice of both sexes and measured femur BMD and periosteal circumference (PC) using peripheral quantitative computed tomography. Whole-genome genotyping was performed with 86 microsatellite markers. A new BMD QTL on chromosome 10 and another suggestive one on chromosome 15 were identified. A significant femur PC QTL identified on chromosome 9 and a suggestive one on chromosome 2 were similar to those detected in MRL x SJL. QTL were also identified for other femur and forearm bone density and bone size phenotypes, some of which were colocalized within the same chromosomal positions as those for femur BMD and femur PC. This study demonstrates the utility of crosses involving inbred strains of mice which exhibit a similar phenotype in QTL identification.

Identification of quantitative trait loci that regulate obesity and serum lipid levels in MRL/MpJ × SJL/J inbred mice

The total body fat mass and serum concentration of total cholesterol, HDL cholesterol, and triglyceride (TG) differ between standard diet-fed female inbred mouse strains MRL/MpJ (MRL) and SJL/J (SJL) by 38-120% (P < 0.01). To investigate genetic regulation of obesity and serum lipid levels, we performed a genome-wide linkage analysis in 621 MRLx SJL F2 female mice. Fat mass was affected by two significant loci, D11Mit36 [43.7 cM, logarithm of the odds ratio (LOD) 11.2] and D16Mit51 (50.3 cM, LOD 3.9), and one suggestive locus at D7Mit44 (50 cM, LOD 2.4). TG levels were affected by two novel loci at D1Mit43 (76 cM, LOD 3.8) and D12Mit201 (26 cM, LOD 4.1), and two suggestive loci on chromosomes 5 and 17. HDL and cholesterol concentrations were influenced by significant loci on chromosomes 1, 3, 5, 7, and 17 that were in the regions identified earlier for other strains of mice, except for a suggestive locus on chromosome 14 that was specific to the MRL x SJL cross. In summary, linkage analysis in MRL x SJL F2 mice disclosed novel loci affecting TG, HDL, and fat mass, a measure of obesity. Knowledge of the genes in these quantitative trait loci will enhance our understanding of obesity and lipid metabolism.

Accelerated Wound Healing of Alkali-Burned Corneas in MRL Mice Is Associated with a Reduced Inflammatory Signature

The present study was conducted to investigate healing of alkali-burned corneas in MRL/MpJ (MRL) mice. Gross, clinical, and histologic criteria were used to compare healing of alkali-burned corneas in MRL and control C57BL/6J (B6) mice. Effects of neutrophil depletion of B6 mice and allogeneic reconstitution of B6 mice with MRL bone marrow on wound healing were evaluated. Gene expression patterns in normal and wounded corneas were surveyed with array-based quantitative real-time RT-PCR (AQPCR). MRL mice showed accelerated reepithelialization and decreased corneal opacity compared with B6 mice after alkali wounding. Marked inflammatory cell infiltration and fibrosis were evident in the corneas and anterior chambers of B6 mice. MRL mice showed less severe lesions, except for stromal edema. Rapid reepithelialization and reduced keratitis/iritis were also observed in neutrophil-depleted B6 mice, but not in B6 mice reconstituted with MRL bone marrow. AQPCR showed transcriptional changes of fewer genes associated with inflammation and corneal tissue homeostasis in alkali-burned corneas from MRL mice. Increased expression of an anti-inflammatory gene, Socs1, and a gene associated with healing, Mmp1a, were evident in MRL corneas. Alkali-burned corneas heal faster and more completely in MRL mice than in B6 mice, by means of rapid reepithelialization, reduced inflammation, and reduced fibrosis. Reduced inflammation, including decreased neutrophil infiltrates and the lack of a robust proinflammatory gene expression signature correlates with the rapid healing. However, the rapid-healing phenotype is not intrinsic to MRL hematopoietic progenitor cells.

Isolation of wound healing/regeneration genes using restrictive fragment differential display-PCR in MRL/MPJ and C57BL/6 mice

Wound healing in mammals can take several weeks to months and the process is always accompanied by scar formation. Wound healing mechanisms that mimic regeneration are not found in most mature mammalian tissues. However, the MRL/MPJ (MRL) mouse has the unique capacity to regenerate ear hole wound completely in less than a month. To identify genes involved in wound healing without a scar, we chose to use restriction fragment differential display-PCR to isolate genes differentially expressed in the MRL (good healer) mouse and the C57BL/6 (poor healer) mouse at different stages of wound healing. We identified 36 genes that were differentially expressed in the regenerating tissue of good and poor healer strains of which several genes are also genetically linked to wound healing and thus are potential candidate genes for scarless wound healing.