Zealand Black Mice Research Articles

Single cell RNA sequencing reveals ferritin as a key mediator of autoimmune pre-disposition in a mouse model of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a devastating autoimmune disorder characterized by failure of self-tolerance with resultant production of autoreactive antibodies. The etiology of this syndrome is complex, involving perturbations in immune cell signaling and development. The NZBWF1 mouse spontaneously develops a lupus-like syndrome and has been widely used as a model of SLE for over 60 years. The NZBWF1 model represents the F1 generation of a cross between New Zealand Black (NZB) and New Zealand White (NZW) mice. In order to better understand the factors that contribute to the development of autoimmunity, single cell RNA sequencing was conducted using the bone marrow from female NZBWF1 mice prior to the development of overt disease. The results were contrasted with single cell RNA sequencing results from the two parental strains. The expected findings of B cell abundance and upregulation, and evidence of interferon signaling were validated in this model. In addition, several novel areas of inquiry were identified. Most notably, the data showed a marked upregulation of the ferritin light chain across all cell types in the NZBWF1 mice compared to parental controls. This data can serve as a gene expression atlas of all hematopoietic cells in the NZBWF1 bone marrow prior to the development of autoimmunity.

The NZB/WF1 mouse model for Sjögren's syndrome: A historical perspective and lessons learned.

Sjögren's syndrome (SS) is a chronic rheumatic autoimmune disorder affecting multiple organ systems. The clinical findings in SS patients show considerable heterogeneity and overlap with other autoimmune diseases. In addition, the autoimmune response in SS initiates several years before the appearance of clinical symptoms. Thus, understanding the pathogenic mechanisms involved in the disease process have been a challenge. Several animal model systems of SS-like disease have been developed to overcome these issues. The New Zealand Black (NZB) x New Zealand White (NZW) F1 (NZB/WF1) mouse represents the first spontaneous mouse model of SS. In this review, we provide a historical perspective and detailed description of this mouse model focusing on exocrine gland histopathology, autoantibody populations, and glandular dysfunction. Considering that NZB/WF1 mice also develop a systemic lupus erythematosus (SLE)-like disease, this mouse model mimics the clinical presentation of polyautoimmunity seen in a sizable subset of SS patients. It is plausible that such patients will require distinct therapeutic interventions necessary to treat both SLE and SS. Therefore, the NZB/WF1 mouse is a powerful tool to decipher pathogenic mechanisms involved in SS related polyautoimmunity and develop appropriate therapeutic strategies.

Development of an IL-17A DNA Vaccine to Treat Systemic Lupus Erythematosus in Mice.

The interleukin-17 (IL-17) family, especially IL-17A, plays an important role in the pathogenesis of systemic lupus erythematosus (SLE). This study developed an IL-17A epitope vaccine to treat SLE in NZBWF1 and MRL/lpr mouse models. A plasmid vector encoding a hepatitis B core (HBc)-IL-17A epitope fusion protein was injected using electroporation into the skeletal muscle of NZBWF1(New Zealand Black mice x New Zealand White mice F1 hybrid strain) or MRL/lpr mice three times at 2-week intervals. As a result, anti-IL-17A antibodies were successfully produced in the HBc-IL-17A group. Accordingly, serum tumor necrosis factor alpha (TNF-α) concentrations were significantly reduced in the HBc-IL-17A group. According to pathological analysis, the IL-17A DNA vaccine significantly suppressed renal tissue damage and macrophage infiltration. Consequently, the survival rate was significantly improved in the HBc-IL-17A group. In addition, we evaluated the antigen reactivity of splenocytes from IL-17A-immunized mice using an enzyme-linked immune absorbent spot (ELISPot) assay for safety evaluation. Splenocytes from IL-17A-immunized mice were significantly stimulated by the HBc epitope peptide, but not by the IL-17A epitope or recombinant IL-17A. These results indicate that the IL-17A vaccine did not induce autoreactive T cells against endogenous IL-17A. This study demonstrates for the first time that an IL-17A DNA vaccine significantly reduced organ damage and extended survival time in lupus-prone mice.

Heterozygous SOD1 deficiency in mice with an NZW background causes male infertility and an aberrant immune phenotype

New Zealand white (NZW) mouse is a mutant strain that has a larval defect in the immune system, and a F1 hybrid between NZW and New Zealand Black mouse spontaneously develops systemic lupus erythematosus (SLE). In meantime, the ablation of superoxide dismutase 1 (Sod1) causes autoimmune haemolytic anaemia, a clinical condition of SLE, in mice with a C57BL/6 background. On the basis of our previous studies, we hypothesised that oxidative stress may trigger this aberrant autoimmunity in NZW mice without crossing with another strain. To validate this, we attempted to establish Sod1−/−/NZW mice but this attempt failed to obtain any objective mouse. The congenic Sod1+/−/NZW male mice were completely infertile because of severe oligozoospermia attributed to a defect in spermatogenesis. The levels of the SOD1 protein were about a half in the testes of the Sod1+/−/NZW mice. Sperm from the Sod1+/−/NZW mice were largely defective and showed quite low fertilising ability in in vitro fertilisation assays. Concomitant with an increase in the oxidatively modified proteins, spermatogenic cells underwent more cell death in the testes of the Sod1+/−/NZW mice compared to those of WT/NZW mice. An examination of immunocompetent cells from Sod1+/−/NZW mice indicated an abnormality in T-cell responses. These collective results suggest that the oxidative stress caused by an SOD1 haploinsufficiency exerts deleterious effects on the testis, either directly on spermatogenic cells or via the destabilisation of the autoimmune response in Sod1+/−/NZW mice.

Sodium 4-phenylbutyrate treatment protects against renal injury in NZBWF1 mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease predominantly affecting women and often leading to lupus nephritis and kidney damage. Endoplasmic reticulum (ER) stress has been implicated in several forms of kidney disease, but whether ER stress contributes to renal injury in SLE is unknown. To investigate this, a small molecule chaperone, sodium 4-phenylbutyrate (4-PBA), was administered to the New Zealand Black x New Zealand White F1 hybrid (NZBWF1) mouse model of SLE. In a prevention study, treatment with 4-PBA from 20 weeks of age (prior to the development of renal injury) delayed the onset of albuminuria and significantly reduced additional indices of renal injury compared with vehicle-treated NZBWF1 mice at 36 weeks of age, including collagen deposition, tubular casts, renal cell apoptosis, and blood urea nitrogen (BUN) concentration. To test whether ER stress contributes to the progression of renal injury once albuminuria has developed, mice were monitored for the onset of albuminuria (3+ or ≥300 mg/dl by dipstick measurement of 24-h urine sample) and once established, were either killed (onset group), or underwent 4-PBA or vehicle treatment for 4 weeks. Treatment with 4-PBA blocked the worsening of glomerular injury, reduced the number of dilated or cast-filled tubules, and reduced the number of apoptotic cells compared with vehicle-treated mice. BUN and left ventricle to bodyweight ratio (LV:BW) were also reduced by 4-PBA treatment. Renal expression of the endogenous chaperones, protein disulphide isomerase (PDI), and 78 kDa glucose-regulated protein (GRP78, also known as binding Ig protein (BiP)), were increased in 4-PBA-treated mice. Together, these results suggest a therapeutic potential for agents like 4-PBA in combating renal injury in SLE.

Compromised NLRP3 and AIM2 inflammasome function in autoimmune NZB/W F1 mouse macrophages.

Inflammasomes are protein complexes activated by infection and cellular stress that promote caspase-1 activation and subsequent inflammatory cytokine processing and cell death. It has been anticipated that inflammasome activity contributes to autoimmunity. However, we previously showed that macrophages from autoimmune New Zealand Black (NZB) mice lack NLRP3 inflammasome function, and their absent in melanoma 2 (AIM2) inflammasome responses are compromised by high expression of the AIM2 antagonist protein p202. Here we found that the point mutation leading to lack of NLRP3 expression occurred early in the NZB strain establishment, as it is shared with the related obese strain New Zealand Obese, but not with the unrelated New Zealand White (NZW) strain. The first cross progeny of NZB and NZW mice develop more severe lupus nephritis than the NZB strain. We have compared AIM2 and NLRP3 inflammasome function in macrophages from NZB, NZW, and NZB/W F1 mice. The NZW parental strain showed strong inflammasome function, whereas the NZB/W F1 have haploinsufficient expression of NLRP3 and show reduced NLRP3 and AIM2 inflammasome responses, particularly at low stimulus strength. It remains to be established whether the low inflammasome function could contribute to loss of tolerance and the onset of autoimmunity in NZB and NZB/W F1. However, with amplifying inflammatory stimuli through the course of disease, the NLRP3 response in the NZB/W F1 may be sufficient to contribute to kidney damage at later stages of disease.

Altered toll-like receptor responsiveness underlies a dominant heritable defect in Bcell tolerance in autoimmune New Zealand Black mice.

Systemic lupus erythematosus is a debilitating autoimmune disease in which autoantibodies and autoreactive T cells destroy kidneys and other organs. Disease is clinically and genetically heterogeneous, suggesting that underlying mechanisms vary between patients. We previously used an autoantibody transgenic mouse reporter system to examine the effect of different autoimmune backgrounds on B-cell tolerance, failure of which is a fundamental defect in lupus. We identified a defect consistent with reversible anergy induced by endotoxin stimulation of Bcells from Ig transgenic New Zealand Black (NZB) mice. Herein we report that the tolerance defect is revealed by TLR7 and TLR9 as well as TLR4 ligands, with additive effect, and is partially reversed by Mek inhibition. Gene expression analysis reveals significant differences in transcription of multiple TLR pathway genes and ptpn22 in stimulated NZB compared to B6 Bcells. Additionally, the defect is detected in Ig transgenic NZB F1 hybrid strains (NZBxNZW)F1 and (B6xNZB)F1. These results implicate an inherited defect wherein NZB anergic Bcells maintain coordinated TLR/BCR signaling that permits autoantibody production. Agents targeting these pathways may have therapeutic benefit in the subset of lupus patients that manifest similar defects in B-cell regulation.

Induction of IL-10 cytokine and the suppression of T cell proliferation by specific peptides from red cell band 3 and in vivo effects of these peptides on autoimmune hemolytic anemia in NZB mice

PurposeThe anion channel protein band 3 is the main target of the pathogenic red blood cells (RBC) autoantibodies in New Zealand black (NZB) mice. CD4 T cells from NZB mice with autoimmune hemolytic anemia respond to band 3. Previously, we have shown that IL-10 and peptides containing a dominant T cell epitope from red cell band 3 modulate autoimmune hemolytic anemia in NZB mice. Because of the immunoregulatory role of IL-10 in autoimmune diseases, we aim to identify individual band 3 peptides that induce high IL-10 production and simultaneously suppress CD4 T cell proliferation and to investigate the effect intranasal administration of IL-10 producing band 3 peptides on autoantibody responses of NZB mice.MethodsSplenic CD4 T cells of NZB mice were isolated and stimulated by co-culture of T cells with individual band 3 peptides. IL-10 production was measured by enzyme-linked immunosorbent assay and proliferative response of CD4 T cells was estimated by incorporation of [3H] thymidine assay. NZB mice were given either PBS, or peptides 25 (241–251) and 29 (282–296) or both peptides intranasally on three occasions at 2-day intervals. The mice were bled at 6, 10 and 18 weeks after peptide inhalation, and the number of RBC auto-antibodies was measured by DELAT and hematocrit values were assessed.ResultsPeptides 25 (241–251) and 29 (282–296) induced the highest IL-10 production by CD4 T cells. These peptides also inhibited the peak T cell proliferative response. 6 and 10 weeks after peptide inhalation, the total IgG, IgG1 and IgG2a in mice treated with both peptides 241–251 and 282–296 were significantly higher than control (P < 0.05). However, no significant difference in the mean hematocrit between of the peptide-treated mice and the control group was found.ConclusionsAlthough band 3 peptides 241–251 and 282–296 induced to the highest IL-10 production by CD4 T cells in vitro but fail to reverse the RBC autoantibody response in vivo. Modifications to improve solubility these peptides might help to modulate the immune response toward a T helper-2 profile and decrease the severity of anemia.

Annexin II-binding immunoglobulins in patients with lupus nephritis and their correlation with disease manifestations.

Annexin II on mesangial cell surface mediates the binding of anti-dsDNA antibodies and consequent downstream inflammatory and fibrotic processes. We investigated the clinical relevance of circulating annexin II-binding immunoglobulins (Igs) in patients with severe proliferative lupus nephritis, and renal annexin II expression in relation to progression of nephritis in New Zealand Black and White F1 mice (NZBWF1/J) mice. Annexin II-binding Igs in serum were measured by ELISA. Ultrastructural localization of annexin II was determined by electron microscopy. Seropositivity rates for annexin II-binding IgG and IgM in patients with active lupus nephritis were significantly higher compared with controls (8.9%, 1.3% and 0.9% for annexin II-binding IgG and 11.1%, 4.0% and 1.9% for annexin II-binding IgM for patients with active lupus nephritis, patients with non-lupus renal disease and healthy subjects respectively). In lupus patients, annexin II-binding IgM level was higher at disease flare compared with remission. Annexin II-binding IgG and IgM levels were associated with that of anti-dsDNA and disease activity. Annexin II-binding IgG and IgM levels correlated with histological activity index in lupus nephritis biopsy samples. In NZBWF1/J mice, serum annexin II-binding IgG and IgM levels and glomerular annexin II and p11 expression increased with progression of active nephritis. Annexin II expression was present on mesangial cell surface and in the mesangial matrix, and co-localized with electron-dense deposits along the glomerular basement membrane. Our results show that circulating annexin II-binding IgG and IgM levels are associated with clinical and histological disease activity in proliferative lupus nephritis. The co-localization of annexin II and p11 expression with immune deposition in the kidney suggests pathogenic relevance.

Disruption of Type I-IFN Signaling Decreases Autoimmune Development and Kidney Damage, But Not Anxiety-Like Behaviors in Lupus NZB Mice

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder that is accompanied by neuropsychiatric manifestations such as anxiety. Despite undefined etio-pathogenesis for SLE, emerging evidence supports the importance of type I interferon (IFN) in the pathogenesis of autoimmune formation and renal damage both in SLE patients and lupus mice. Linkage mapping identified a quantitative trait locus (QTL) for elevated plus-maze (EPM) performance on the segment of chromosome 4 in lupus-prone New Zealand black (NZB) mice where the type I IFN-α genes are harbored. To determine possible roles of type I IFNs for anxiety-like behaviors in NZB mice, we evaluated the anxiety profile by EPM test in NZB mice with deficiency of type I-IFN receptor (IFNARKO). Consistent with previous observation, disruption of the type I-IFN signaling resulted in a dramatic attenuation in glomerulonephritis, splenomegaly and plasma anti-nuclear antibodies (ANA) in NZB mice. However, blockade of type I-IFN signaling had no effect on performance in the EPM by NZB/IFNARKO mice in comparison to wild type controls. The results support a pathogenic role for type I-IFN in autoimmune development and kidney inflammation. Nonetheless, type I-IFN signaling is not responsible for increased anxiety profile developed in these autoimmune mice.

The SOD1 transgene expressed in erythroid cells alleviates fatal phenotype in congenic NZB/NZW-F1 mice

Oxidative stress due to a superoxide dismutase 1 (SOD1) deficiency causes anemia and autoimmune responses, which are phenotypically similar to autoimmune hemolytic anemia (AIHA) and systemic lupus erythematosus (SLE) in C57BL/6 mice and aggravates AIHA pathogenesis in New Zealand black (NZB) mice. We report herein on an evaluation of the role of reactive oxygen species (ROS) in a model mouse with inherited SLE, that is, F1 mice of the NZB × New Zealand white (NZW) strain. The ROS levels within red blood cells (RBCs) of the F1 mice were similar to the NZW mice but lower compared to the NZB mice throughout adult period. Regarding SLE pathogenesis, we examined the effects of an SOD1 deficiency or the overexpression of human SOD1 in erythroid cells by establishing corresponding congenic F1 mice. A SOD1 deficiency caused an elevation in ROS production, methemoglobin content, and hyperoxidation of peroxiredoxin in RBC of the F1 mice, which were all consistent with elevated oxidative stress. However, while the overexpression of human SOD1 in erythroid cells extended the life span of the congenic F1 mice, the SOD1 deficiency had no effect on life span compared to wild-type F1 mice. It is generally recognized that NZW mice possess a larval defect in the immune system and that NZB mice trigger an autoimmune reaction in the F1 mice. Our results suggest that the oxidative insult originated from the NZB mouse background has a functional role in triggering an aberrant immune reaction, leading to fatal responses in F1 mice.

Synergistic Activity of Deguelin and Fludarabine in Cells from Chronic Lymphocytic Leukemia Patients and in the New Zealand Black Murine Model.

B-cell chronic lymphocytic leukemia (CLL) remains an incurable disease, and despite the improvement achieved by therapeutic regimes developed over the last years still a subset of patients face a rather poor prognosis and will eventually relapse and become refractory to therapy. The natural rotenoid deguelin has been shown to induce apoptosis in several cancer cells and cell lines, including primary human CLL cells, and to act as a chemopreventive agent in animal models of induced carcinogenesis. In this work, we show that deguelin induces apoptosis in vitro in primary human CLL cells and in CLL-like cells from the New Zealand Black (NZB) mouse strain. In both of them, deguelin dowregulates AKT, NFκB and several downstream antiapoptotic proteins (XIAP, cIAP, BCL2, BCL-XL and survivin), activating the mitochondrial pathway of apoptosis. Moreover, deguelin inhibits stromal cell-mediated c-Myc upregulation and resistance to fludarabine, increasing fludarabine induced DNA damage. We further show that deguelin has activity in vivo against NZB CLL-like cells in an experimental model of CLL in young NZB mice transplanted with spleen cells from aged NZB mice with lymphoproliferation. Moreover, the combination of deguelin and fludarabine in this model prolonged the survival of transplanted mice at doses of both compounds that were ineffective when administered individually. These results suggest deguelin could have potential for the treatment of human CLL.

Alterations in the mir-15a/16-1 Loci Impairs Its Processing and Augments B-1 Expansion in De Novo Mouse Model of Chronic Lymphocytic Leukemia (CLL).

New Zealand Black (NZB) mice, a de novo model of CLL, share multiple characteristics with CLL patients, including decreased expression of miR-15a/16-1. We previously discovered a point mutation and deletion in the 3' flanking region of mir-16-1 of NZB and a similar mutation has been found in a small number of CLL patients. However, it was unknown whether the mutation is the cause for the reduced miR-15a/16-1 expression and CLL development. Using PCR and in vitro microRNA processing assays, we found that the NZB sequence alterations in the mir-15a/16-1 loci result in deficient processing of the precursor forms of miR-15a/16-1, in particular, we observe impaired conversion of pri-miR-15a/16-1 to pre-miR-15a/16-1. The in vitro data was further supported by derivation of congenic strains with replaced mir-15a/16-1 loci at one or both alleles: NZB congenic mice (NmiR+/-) and DBA congenic mice (DmiR-/-). The level of miR-15a/16-1 reflected the configuration of the mir-15a/16-1 loci with DBA congenic mice (DmiR-/-) showing reduced miR-15a levels compared to homozygous wild-type allele, while the NZB congenic mice (NmiR+/-) showed an increase in miR-15a levels relative to homozygous mutant allele. Similar to Monoclonal B-cell Lymphocytosis (MBL), the precursor stage of the human disease, an overall expansion of the B-1 population was observed in DBA congenic mice (DmiR-/-) relative to wild-type (DmiR+/+). These studies support our hypothesis that the mutations in the mir-15a/16-1 loci are responsible for decreased expression of this regulatory microRNA leading to B-1 expansion and CLL development.

Identification of the SLAM Adapter Molecule EAT-2 as a Lupus-Susceptibility Gene That Acts through Impaired Negative Regulation of Dendritic Cell Signaling.

We showed previously that C57BL/6 congenic mice with an introgressed homozygous 70 cM (125.6 Mb) to 100 cM (179.8 Mb) interval on c1 from the lupus-prone New Zealand Black (NZB) mouse develop high titers of antinuclear Abs and severe glomerulonephritis. Using subcongenic mice, we found that a genetic locus in the 88-96 cM region was associated with altered dendritic cell (DC) function and synergized with T cell functional defects to promote expansion of pathogenic proinflammatory T cell subsets. In this article, we show that the promoter region of the NZB gene encoding the SLAM signaling pathway adapter molecule EWS-activated transcript 2 (EAT-2) is polymorphic, which results in an ∼ 70% reduction in EAT-2 in DC. Silencing of the EAT-2 gene in DC that lacked this polymorphism led to increased production of IL-12 and enhanced differentiation of T cells to a Th1 phenotype in T cell-DC cocultures, reproducing the phenotype observed for DC from congenic mice with the NZB c1 70-100 cM interval. SLAM signaling was shown to inhibit production of IL-12 by CD40L-activated DCs. Consistent with a role for EAT-2 in this inhibition, knockdown of EAT-2 resulted in increased production of IL-12 by CD40-stimulated DC. Assessment of downstream signaling following CD40 cross-linking in the presence or absence of SLAM cross-linking revealed that SLAM coengagement blocked activation of p38 MAPK and JNK signaling pathways in DC, which was reversed in DC with the NZB EAT-2 allele. We conclude that EAT-2 negatively regulates cytokine production in DC downstream of SLAM engagement and that a genetic polymorphism that disturbs this process promotes the development of lupus.

Deficient NLRP3 and AIM2 Inflammasome Function in Autoimmune NZB Mice.

Inflammasomes are protein complexes that promote caspase activation, resulting in processing of IL-1β and cell death, in response to infection and cellular stresses. Inflammasomes have been anticipated to contribute to autoimmunity. The New Zealand Black (NZB) mouse develops anti-erythrocyte Abs and is a model of autoimmune hemolytic anemia. These mice also develop anti-nuclear Abs typical of lupus. In this article, we show that NZB macrophages have deficient inflammasome responses to a DNA virus and fungal infection. Absent in melanoma 2 (AIM2) inflammasome responses are compromised in NZB by high expression of the AIM 2 antagonist protein p202, and consequently NZB cells had low IL-1β output in response to both transfected DNA and mouse CMV infection. Surprisingly, we also found that a second inflammasome system, mediated by the NLR family, pyrin domain containing 3 (NLRP3) initiating protein, was completely lacking in NZB cells. This was due to a point mutation in an intron of the Nlrp3 gene in NZB mice, which generates a novel splice acceptor site. This leads to incorporation of a pseudoexon with a premature stop codon. The lack of full-length NLRP3 protein results in NZB being effectively null for Nlrp3, with no production of bioactive IL-1β in response to NLRP3 stimuli, including infection with Candida albicans. Thus, this autoimmune strain harbors two inflammasome deficiencies, mediated through quite distinct mechanisms. We hypothesize that the inflammasome deficiencies in NZB alter the interaction of the host with both microflora and pathogens, promoting prolonged production of cytokines that contribute to development of autoantibodies.

JNK Regulatory Molecule G5PR Induces IgG Autoantibody–Producing Plasmablasts from Peritoneal B1a Cells

Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black × New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody-secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells.

Relationship between urinary protein changes in lupus nephritis and renal pathology.

This study investigated the relationship between urinary protein excretion in lupus nephritis New Zealand black mice and renal pathology. A total of 328 lupus nephritis New Zealand black mice were established by a backcross hybridization method, and renal pathology was determined. The urinary protein excretion of the backcross mice over 24 h was compared and analyzed. Urinary protein excretion over 24 h differed significantly across different pathological types (1.9, 2.4, 2.9 and 4.9 g in types II, III, IV, and V, respectively) in the backcross mice (P < 0.05). Moreover, it correlated with pathology grade (r = 0.391, P = 0.0001) as well as activity index, chronic index, renal tubular interstitial activity index, and renal tubular interstitial lesions (P < 0.05) but not with vascular lesions (P = 0.683). Urinary protein excretion from lupus nephritis is closely associated with renal pathology. Urinary protein changes can be used to determine lupus nephritis pathology and have some clinical significance for treatment and prognosis.

Oxidative stress as a potential causal factor for autoimmune hemolytic anemia and systemic lupus erythematosus.

The kidneys and the blood system mutually exert influence in maintaining homeostasis in the body. Because the kidneys control erythropoiesis by producing erythropoietin and by supporting hematopoiesis, anemia is associated with kidney diseases. Anemia is the most prevalent genetic disorder, and it is caused by a deficiency of glucose 6-phosphate dehydrogenase (G6PD), for which sulfhydryl oxidation due to an insufficient supply of NADPH is a likely direct cause. Elevated reactive oxygen species (ROS) result in the sulfhydryl oxidation and hence are another potential cause for anemia. ROS are elevated in red blood cells (RBCs) under superoxide dismutase (SOD1) deficiency in C57BL/6 mice. SOD1 deficient mice exhibit characteristics similar to autoimmune hemolytic anemia (AIHA) and systemic lupus erythematosus (SLE) at the gerontic stage. An examination of AIHA-prone New Zealand Black (NZB) mice, which have normal SOD1 and G6PD genes, indicated that ROS levels in RBCs are originally high and further elevated during aging. Transgenic overexpression of human SOD1 in erythroid cells effectively suppresses ROS elevation and ameliorates AIHA symptoms such as elevated anti-RBC antibodies and premature death in NZB mice. These results support the hypothesis that names oxidative stress as a risk factor for AIHA and other autoimmune diseases such as SLE. Herein we discuss the association between oxidative stress and SLE pathogenesis based mainly on the genetic and phenotypic characteristics of NZB and New Zealand white mice and provide insight into the mechanism of SLE pathogenesis.

Notch2 Is Critical for CLL Development in the IRF4-/-Vh11 Mice

Chronic Lymphocytic Leukemia (CLL) is the most common adult leukemia in the western world. Despite considerable progress in our current understanding of CLL, the molecular events underlying the complex pathogenesis and clinical behavior of CLL have not been fully elucidated. IRF4 belongs to the IRF superfamily of transcription factors and plays critical roles at multiple stages of B cell development. Genome Wide Association Studies had linked Single Nucleotide Polymorphism (SNP) mediated IRF4 down regulation to the pathogenesis of human CLL (Di Bernardo et al. Nature Genetics, 2008). Moreover, low levels of IRF4 were found to correlate with poorer disease outcomes in human CLL (Chang et al. Blood, 2002). More recently, the studies from our group have established a causal relationship between low levels of IRF4 and development of CLL. We used Vh11 knock-in (Vh11) mice that express a prearranged immunoglobulin variable heavy chain (Vh11) leading to preferential generation of B1 cells (CLL precursors). Interestingly, we have shown that mice expressing the Vh11 transgene in the IRF4 deficient background (IRF4-/-Vh11) spontaneously develop CLL at complete penetrance (Shukla et al. Blood, 2013). Additionally, we have also shown that low levels of IRF4 dramatically accelerate CLL development in the New Zealand Black (NZB) mice (Ma et al. JBC, 2013). Although our studies have demonstrated a critical role for IRF4 in the pathogenesis of CLL, the molecular mechanism through which IRF4 controls CLL development remains unclear.In an attempt to identify the molecular networks regulated by IRF4 in CLL cells we performed gene expression profiling of CLL cells isolated from IRF4-/-Vh11 mice. Intriguingly, our analysis revealed that Notch signaling was hyperactive in the IRF4-/-Vh11 CLL cells compared to the IRF4+/+Vh11 B1 cells. This is particularly important because recent high depth sequencing studies have identified mutations in Notch1 protein leading to its stabilization and activation in ~10% of all CLL patients (Puente et al. Nature, 2011, Fabbri et al. JEM, 2011). Furthermore, the individuals carrying these mutations have an aggressive disease course and poor overall survival (Fabbri et al. JEM, 2011). Recently, studies by Simonetti et al have shown that the IRF4 deficient B cells express high levels of Notch receptors and display increased Notch activity (Simonetti et al. JEM, 2013). We also detected high levels of Notch2 but not Notch1 on the cell surface of IRF4-/-Vh11 CLL cells. To examine the contribution of Notch signaling in the pathogenesis of CLL, we crossed IRF4-/-Vh11 mice with Notch2 conditional mutant mice and with mice expressing cre recombinase in B cells (CD19cre). This approach allowed us to genetically delete Notch2 receptor specifically in B cells of IRF4-/-Vh11 mice (CD19creNotch2fl/flIRF4-/-Vh11). Surprisingly, CLL cells could still emerge from the majority of CD19creNotch2fl/flIRF4-/-Vh11 mice. However, further analysis revealed that those CLL cells expressed high levels of Notch2 on their surface and were apparently derived from IRF4-/-Vh11 B1 cells that escaped cre-mediated deletion. Importantly, our analysis also showed that the majority of B1 and B2 cells in the CD19creNotch2fl/flIRF4-/-Vh11 mice had lost the expression of Notch2, indicating that Notch2 is dispensable for their generation.We also performed a series of analysis to determine how Notch signaling affects B1 and CLL cells in vitro. We used a co-culture system to engage Notch receptors to their ligand (Delta like 1 or DL1) expressed on S17 stromal cells (S17-DL1). Co-culture of wild type B1 cells with S17-DL1 stromal cells led to enhanced proliferation and survival of B1 cells when compared with cells co-cultured on control vector transfected S17 stromal cells (S17-R1). Importantly, Notch2 receptor deficient B1 cells failed to respond to DL1 expressing S17 stromal cells. Similarly, IRF4-/-Vh11 CLL cells also showed enhanced survival on S17-DL1 stromal cells compared to S17-R1 stromal cells.In summary, our studies show that Notch2 is absolutely required for CLL development in the IRF4-/-Vh11 mice, thereby supporting a critical role for Notch signaling in the pathogenesis of CLL. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4791: The role of microRNA-15a/16 in early B1 cell development in a mouse model of chronic lymphocytic leukemia

Abstract New Zealand Black (NZB) mouse is a de-novo model of chronic lymphocytic leukemia (CLL) that has been studied as a model of B-cell lymphoproliferative disorder. In most human CLL and NZB there is decreased expression of the microRNA miR15a/16. In NZB (and some CLL patients) this is due a point mutation six bases downstream from pre-miR-16 region. In both NZB mouse model and human CLL, the disease is characterized by the presence of a malignant clone of B-1 cells expressing dull levels of CD5 (T cell marker), CD11b (myeloid marker) and B220 (B cell marker). In this report we investigated the molecular pathways that result in increased B-1 lineage development in the mouse model of CLL. Initially we observed that in NZB, the B-1 lineage cells had reduced expression of Pu.1 when compared to NZB B-2 cells. PU.1 is a transcription factor required for B lineage differentiation and PU.1 knockout mice were previously found by others to have only B-1 lineage cell differentiation. To study early B1/B2 divergent cell development pathways in the context of CLL and shed light on the potential malignant cell origin of this disease, we generated induced pluripotent stem cells (iPS) from NZB spleen stromal fibroblasts. Our in vivo and in vitro studies on NZB iPS and hematopoietic progenitors (LSK) differentiation towards B-cell lineage cells followed by multicolor flow based cell sorting revealed a substantial block in the maturation of NZB iPS cells compared to wild type counterparts. Preliminary data using congenic NZB mice with corrected mir-15a/16-1 locus demonstrated that normal levels of miR-15a/16 regulate B-cell differentiation towards a higher expression of PU.1 and B220 (CD45R) and lower levels of CD11b suggestive of an enhanced B2 cell maturation pathway. Our results support the hypothesis that decreased miR-15a plays a role in B1 lymphocyte development bias in the course of B cell maturation by regulating the expression of a number of genes such as B220, PU.1 and CD11b. This work will help further uncover early molecular events which determine the increased B1 versus B2 lineage commitment associated with low levels of miR-15a/16 as an initial lesion in the development of CLL. Citation Format: Chingiz Underbayev, Siddha Kasar, Heba Degheidy, Gerald Marti, Marilyn Lightfoote, Elizabeth Raveche. The role of microRNA-15a/16 in early B1 cell development in a mouse model of chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2014-4791