NZM2410 Mouse Model Research Articles

Lupus susceptibility gene Esrrg regulates Treg development and suppressive function through mitochondrial metabolism

Abstract Estrogen-Related Receptor Gamma (Esrrg) is the candidate gene for the Sle1c2 susceptibility locus, which encodes the orphan nuclear receptor ERR-g, in the NZM2410 mouse model of Systemic Lupus Erythematosus (SLE). Congenic C57BL/6 (B6) mice expressing this locus have an increased IFN-g production and dysregulated metabolism in their CD4 T cells, but the role of ERR-g in CD4 T cells is still unclear. In muscle, heart, iPSCs, pancreatic β cells and neurons, Esrrg has been identified as a major regulator of mitochondrial metabolism. We generated Treg conditional Esrrg knockout mice Foxp3cre Esrrgf/f (Esrrg-KO) and compared CD4+ T cells between Esrrg-KO and B6 mice. Tregs from Esrrg-KO mice showed a reduced expression of Foxp3 and CD25, and other markers associated with Treg suppressive activity. Esrrg-KO mice demonstrated non-Treg global CD4 T cell activation, including the increased frequency and number of CD69 and T effector memory cells, as well as increased spontaneous T follicular helper (Tfh) cells and decreased T follicular regulatory (Tfr) cell frequencies. In the T-dependent NP-KLH immunization, Esrrg-KO mice produced increased Tfh and germinal center B cells and less Tfr, which led to high affinity anti-NP IgG. Esrrg-deficiency in Tregs significantly altered the expression of genes and metabolites, ATP, NAD+ and ROS, involved in mitochondria function. Esrrg-KO mice also exhibited a high expression of mTORC1 and Hif1-a, a pathway that is known to destabilize Foxp3 expression. Finally, Esrrg-deficiency decreased both respiration and glycolysis in Tregs. These results demonstrate that Esrrg-deficiency results in defective mitochondrial function in Tregs, which leads to defective Tregs and global CD4 T cell hyperactivation.

Contribution of B-1a cells to systemic lupus erythematosus in the NZM2410 mouse model.

Systemic lupus erythematosus (SLE) is an autoimmune disease of complex etiology in which B cells play a central role. An expanded number of B-1a cells have been consistently associated with murine lupus, and more recently with human SLE. We have identified Cdkn2c, a gene that controls cell cycle progression, as a key regulator of B-1a cell numbers and have associated Cdkn2c deficiency with autoimmune pathology, including the production of autoantibodies and the skewing of CD4(+) Tcells toward inflammatory effector functions. We review the genetic studies that have led to these findings, as well as the possible mechanisms by which B-1a cell expansion and Cdkn2c deficiency are related to SLE pathogenesis.

Activation of rheumatoid factor-specific B cells is antigen dependent and occurs preferentially outside of germinal centers in the lupus-prone NZM2410 mouse model.

AM14 rheumatoid factor (RF) B cells in the MRL/lpr mice are activated by dual BCR and TLR7/9 ligation and differentiate into plasmablasts via an extrafollicular (EF) route. It was not known whether this mechanism of activation of RF B cells applied to other lupus-prone mouse models. We investigated the mechanisms by which RF B cells break tolerance in the NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) strain in comparison with C57BL/6 (B6) controls, each expressing the AM14 H chain transgene in the presence or absence of the IgG2a(a) autoantigen. The TC, but not B6, genetic background promotes the differentiation of RF B cells into Ab-forming cells (AFCs) in the presence of the autoantigen. Activated RF B cells preferentially differentiated into plasmablasts in EF zones. Contrary to the MRL/lpr strain, TC RF B cells were also located within germinal centers, but only the formation of EF foci was positively correlated with the production of RF AFCs. Immunization of young TC.AM14 H chain transgenic mice with IgG2a(a) anti-chromatin immune complexes (ICs) activated RF B cells in a BCR- and TLR9-dependent manner. However, these IC immunizations did not result in the production of RF AFCs. These results show that RF B cells break tolerance with the same general mechanisms in the TC and the MRL/lpr lupus-prone genetic backgrounds, namely the dual activation of the BCR and TLR9 pathways. There are also distinct differences, such as the presence of RF B cells in GCs and the requirement of chronic IgG2a(a) anti-chromatin ICs for full differentiation of RF AFCs.

Dysregulated cytokine production by dendritic cells modulates B cell responses in the NZM2410 mouse model of lupus.

The breakdown in tolerance of autoreactive B cells in the lupus-prone NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) mice results in the secretion of autoantibodies. TC dendritic cells (DCs) enhance B cell proliferation and antibody secretion in a cytokine-dependent manner. However, the specific cytokine milieu by which TC DCs activate B cells was not known. In this study, we compared TC and C57BL/6 (B6) control for the distribution of DC subsets and for their production of cytokines affecting B cell responses. We show that TC DCs enhanced B cell proliferation through the production of IL-6 and IFN-γ, while antibody secretion was only dependent on IL-6. Pre-disease TC mice showed an expanded PDCA1+ cells prior to disease onset that was localized to the marginal zone and further expanded with age. The presence of PDCA1+ cells in the marginal zone correlated with a Type I Interferon (IFN) signature in marginal zone B cells, and this response was higher in TC than B6 mice. In vivo administration of anti-chromatin immune complexes upregulated IL-6 and IFN-γ production by splenic DCs from TC but not B6 mice. The production of BAFF and APRIL was decreased upon TC DC stimulation both in vitro and in vivo, indicating that these B cell survival factors do not play a role in B cell modulation by TC DCs. Finally, TC B cells were defective at downregulating IL-6 expression in response to anti-inflammatory apoptotic cell exposure. Overall, these results show that the TC autoimmune genetic background induces the production of B cell-modulating inflammatory cytokines by DCs, which are regulated by the microenvironment as well as the interplay between DC.

The SLE-associated Pbx1-d isoform acts as a dominant-negative transcriptional regulator

Pbx1 is a transcription factor involved in multiple cellular processes, including the maintenance of self-renewal of hematopoietic progenitors. We have shown that the CD4(+) T-cell expression of a novel splice isoform of Pbx1, Pbx1-d, is associated with lupus susceptibility in the NZM2410 mouse and in lupus patients. The function of Pbx1 in T cells is unknown, but the splicing out of the DNA-binding domain in Pbx1-d predicts a dominant-negative function. In support of this hypothesis, we have shown that Pbx1-d transduction accelerates differentiation of MC3T3-E1 osteoblast pregenitors and mimics the effect of short hairpin RNA silencing of Pbx1. Conversely, Pbx1-d transduction reduced the expression of Sox3, a gene strongly transactivated by Pbx1, and Pbx1-d did not bind the Sox3 promoter. These results constitute a first step towards the understanding on how Pbx1-d contributes to systemic autoimmunity in the NZM2410 mouse model as well as in lupus patients.

Regulation of T cell metabolism and autoimmunity by the orphan nuclear receptor ERRγ (123.47)

Abstract Autoreactive T cells and abnormal cytokine production are key features of human and mouse systemic lupus erythematosus (SLE). The SLE susceptibility locus Sle1c2 is associated with CD4+ T cell hyperactivation, hyperproliferation and hyperproduction of IFNγ in the NZM2410 mouse model. Sle1c2 contains two genes, only one of which, Esrrg, is expressed in mouse CD4+ T cells. qRT-PCR and Western blot analysis showed that Esrrg is expressed in CD4+ T cells, albeit at a low level. Esrrg expression is significantly decreased in B6.Sle1c2 CD4+ T cells as compared to B6, and negatively correlates with T cell activation. In vitro, Esrrg expression is dramatically decreased after CD4+ T cells are stimulated with anti-CD3/CD28. These data suggest that Esrrg is required to maintain T cells in a quiescent state. Esrrg is a key regulator of cell metabolism. Consistently, Sle1c2 is associated with increased expression of genes involved in the glycolytic pathway, such as MCT4 and Hif1a, which have been previously associated with enhanced T cell effector functions. Sle1c2 is also associated with a decreased mitochondrial mass and functions, which is consistent with Esrrg playing a key role in these processes. Furthermore, treatment of CD4+ T cells with ERRγ agonist and reverse agonist affects T cell survival, proliferation, Foxp3 expression, and IL-2 and IFNγ production. Our study suggests that decreased Esrrg expression contributes to T cell autoimmunity through regulating cell metabolism.

Mechanisms for non-antigen specific rheumatoid factor B cell activation in the NZM2410 mouse model of lupus (159.9)

Abstract Systemic lupus erythematosus is characterized by the presence of autoantibodies, including rheumatoid factor (RF), due to a breakdown in B cell tolerance. In the AM14 model, the AM14 transgenic heavy chain combined to Vκ8 produces a RF specific for IgG2aa. Contrary to the MRL/lpr lupus model, RF B cell activation required the presence of chromatin-IgG immune complexes (ICs), but could occur in the absence of the IgG2aa autoantigen in the B6.NZM2410.Sle1.Sle2.Sle3 (TC) strain, which contains the three major NZM2410-derived lupus susceptibility loci (Sle1, Sle2, Sle3). Treatment of young TC.AM14 and control B6.AM14 mice with a TLR7/9 antagonist did not decrease RF B cell activation after immunization with antigen non-specific anti-chromatin ICs. Splenic DCs from mice injected with anti-chromatin hybridomas showed elevated expression of IL-6 and IFN-γ mRNA in TC mice in comparison to B6 controls. B cell co-cultures with TC-derived BMDCs induced greater B cell proliferation than B6-derived BMDCs. Finally, B cell activation of aged AM14 mice expressing Sle1 or Sle2 showed that while Sle1 promoted antigen specific activation, Sle2 induced B-cell activation is polyclonal in nature. Therefore, our results show that while cognate-antigen independent RF B cell activation is not affected by inhibition of TLR7/9 signaling, it is enhanced by DC-derived cytokines and the expression of the Sle loci.

Pre-B Cell Leukemia Homeobox 1 Is Associated with Lupus Susceptibility in Mice and Humans

Sle1a.1 is part of the Sle1 susceptibility locus, which has the strongest association with lupus nephritis in the NZM2410 mouse model. In this study, we show that Sle1a.1 results in the production of activated and autoreactive CD4(+) T cells. Additionally, Sle1a.1 expression reduces the peripheral regulatory T cell pool, as well as induces a defective response of CD4(+) T cells to the retinoic acid expansion of TGF-β-induced regulatory T cells. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d overexpression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells and to decrease their apoptotic response to retinoic acid. PBX1-d is expressed more frequently in the CD4(+) T cells from lupus patients than from healthy controls, and its presence correlates with an increased central memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance.

Murine Lupus Susceptibility Locus Sle1a Controls Regulatory T Cell Number and Function through Multiple Mechanisms

The Sle1 locus is a key determinant of lupus susceptibility in the NZM2410 mouse model. Within Sle1, we have previously shown that Sle1a expression enhances activation levels and effector functions of CD4(+) T cells and reduces the size of the CD4(+)CD25(+)Foxp3(+) regulatory T cell subset, leading to the production of autoreactive T cells that provide help to chromatin-specific B cells. In this study, we show that Sle1a CD4(+) T cells express high levels of ICOS, which is consistent with their increased ability to help autoreactive B cells. Furthermore, Sle1a CD4(+)CD25(+) T cells express low levels of Foxp3. Mixed bone marrow chimeras demonstrated that these phenotypes require Sle1a to be expressed in the affected CD4(+) T cells. Expression of other markers generally associated with regulatory T cells (Tregs) was similar regardless of Sle1a expression in Foxp3(+) cells. This result, along with in vitro and in vivo suppression studies, suggests that Sle1a controls the number of Tregs rather than their function on a per cell basis. Both in vitro and in vivo suppression assays also showed that Sle1a expression induced effector T cells to be resistant to Treg suppression, as well as dendritic cells to overproduce IL-6, which inhibits Treg suppression. Overall, these results show that Sle1a controls both Treg number and function by multiple mechanisms, directly on the Tregs themselves and indirectly through the response of effector T cells and the regulatory role of dendritic cells.

Several Genes Contribute to the Production of Autoreactive B and T Cells in the Murine Lupus Susceptibility LocusSle1c

The systemic lupus erythematosus 1 (Sle1) locus mediates the loss of tolerance to nuclear Ags in the NZM2410 mouse model of lupus through intrinsic defects in both B and T cells. Congenic analysis has shown that Sle1 corresponds to at least three genetic loci, Sle1a, Sle1b, and Sle1c. Telomeric Sle1c is associated with abnormal B cell responses to subthreshold stimulation with anti-IgM and C3d and with decreased T-dependent humoral immune responses. We have proposed that these phenotypes resulted from polymorphisms in the C3 complement receptor Cr2 gene. We have also found that Sle1c was associated with the production of histone-specific autoreactive CD4(+) T cells, which correlated with higher activation and proliferative responses, and a reduction in the CD4(+)CD25(+)CD62L(+)forkhead/winged helix transcription factor gene (Foxp3(+)) compartment. In this study we showed, using congenic recombinants, that the decreased humoral immune response and impaired GC formation map to the NZM2410 Cr2 allele. A chronic graft-vs-host disease model also showed that Sle1c produces significantly more autoreactive B cells than B6 controls, and that this phenotype maps to two regions excluding the Cr2 gene. Mixed bone marrow chimera demonstrated that the increased activation, proliferative response, and reduced regulatory T cell compartment were intrinsic to Sle1c-expressing CD4(+) T cells. These phenotypes mapped to the same two loci identified with the chronic graft-vs-host disease model, excluding the Cr2 region. Overall, these results show that Sle1c results in the production of autoreactive B and T cells through the expression of three different genes, one of which is consistent with Cr2, based on the phenotypes of the Cr2-deficient mice, and the other two corresponding to as yet unidentified genes.

Genetic Dissection of the Murine Lupus Susceptibility LocusSle2: Contributions to Increased Peritoneal B-1a Cells and Lupus Nephritis Map to Different Loci

Lupus pathogenesis in the NZM2410 mouse model results from the expression of multiple interacting susceptibility loci. Sle2 on chromosome 4 was significantly linked to glomerulonephritis in a linkage analysis of a NZM2410 x B6 cross. Yet, Sle2 expression alone on a C57BL/6 background did not result in any clinical manifestation, but in an abnormal B cell development, including the accumulation of B-1a cells in the peritoneal cavity and spleen. Analysis of B6.Sle2 congenic recombinants showed that at least three independent loci, New Zealand White-derived Sle2a and Sle2b, and New Zealand Black-derived Sle2c, contribute to an elevated number of B-1a cells, with Sle2c contribution being the strongest of the three. To determine the contribution of these three Sle2 loci to lupus pathogenesis, we used a mapping by genetic interaction strategy, in which we bred them to B6.Sle1.Sle3 mice. We then compared the phenotypes of these triple congenic mice with that of previously characterized B6.Sle1.Sle2.Sle3, which express the entire Sle2 interval in combination with Sle1 and Sle3. Sle2a and Sle2b, but not Sle2c, contributed significantly to lupus pathogenesis in terms of survival rate, lymphocytic expansion, and kidney pathology. These results show that the Sle2 locus contains several loci affecting B cell development, with only the two NZW-derived loci having the least effect of B-1a cell accumulation significantly contributing to lupus pathogenesis.

Role of complement in the development of autoimmunity.

B cell complement receptors have been shown to be important in the generation of normal humoral immune responses, and they likely also participate in the development of autoimmunity. Complement component and receptor deficiencies have been associated with SLE in both animal models and patients with disease. Recent data suggest that Cr2 is a lupus susceptibility gene in the NZM2410 mouse model for lupus, as it generates complement receptors that are structurally and functionally altered. Complement deficiency may result in autoimmune disease because of the inability to appropriately clear immune complexes or apoptotic cells or by the impaired generation of C3-coated autoantigens for CR1/CR2. In turn, CR1/CR2 may participate in the maintenance of B cell tolerance by lowering the threshold for negative selection of autoreactive B cells, by targeting autoantigen to FDCs in secondary lymphoid organs, or by regulating autoreactive T cell function. The effect of CR2 has not been dissected from that of CR1 in the animal studies performed to date. Furthermore, the effects of CR1/CR2 dysfunction or partial deficiency, which are found in the NZM2410 mouse model and in patients with SLE respectively, have not been delineated from those of complete deficiency, which has been studied in several animal models of autoimmunity and tolerance. Although CR1/CR2 dysfunction or deficiency may confer only a modest phenotype in isolation, it is likely that when combined with other disease susceptibility genes it will result in a fully penetrant end-stage disease phenotype. Understanding the mechanisms by which these receptors participate in the maintenance of B cell tolerance will be critical in developing appropriate therapeutic interventions for patients with autoimmune diseases such as SLE.

Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.

Production of congenic mouse strains carrying genomic intervals containing SLE-susceptibility genes derived from the SLE-prone NZM2410 strain

Systemic lupus erythematosus is inherited as a complex polygenic trait. Four genomic intervals containing major SLE-susceptibility loci were previously identified by interval mapping in the NZM2410 mouse model. In this paper, we utilized a marker-assisted selection protocol to produce four congenic mouse strains, each carrying an NZM2410-derived SLE-susceptibility interval on a C57BL/6-resistant background. Each strain carries only one susceptibility allele derived from this polygenic model and consequently can be used to characterize the specific component phenotypes contributed by individual SLE-susceptibility genes. We illustrate the efficacy of this approach with phenotypic data for one of our congenic strains, B6.NZMH2(z). Our results indicate that this single genomic interval from Chromosome (Chr) 17 of NZM2410 can mediate increased levels of IgG autoantibodies specific for chromatin and that, similar to results obtained in our original genetic cross, B6.NZMH2(z/b) heterozygotes are more prone than B6.NZMH2(z) homozygotes to the development of humoral autoimmunity to nuclear antigens. These results illustrate the feasibility of using congenic strains to dissect the complex pathogenic mechanisms that mediate polygenic SLE. These congenic strains will be valuable tools in the genetic analysis of SLE susceptibility. In future studies, these congenic strains will be interbred to produce bi- and tri-congenic strains in order to assess the role of genetic interactions in the expression of specific components of SLE pathogenesis. They will also be instrumental to the positional cloning and identification of the genes responsible for SLE susceptibility, via the production of congenic recombinants.