Browsing by Subject "Lupus Erythematosus, Systemic"
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Item Common Alleles of the SLAM/CD2 Family are Associated with Murine Lupus(2005-04-25) Limaye, Nisha; Wakeland, Edward K.The Sle1b locus on telomeric mouse chromosome 1 mediates a break in tolerance to chromatin in the NZM2410 model of the autoimmune disease Systemic Lupus Erythematosus (SLE). B6.Sle1b congenic mice produce anti-nuclear autoantibodies (ANAs), and have elevated activated B and CD4+ T cells, and mild splenomegaly. Fine mapping of Sle1b positioned it within a ~900 kb region between 171.3 and 172.2 Mb. A contig of 100 B6-derived Bacterial Artificial Chromosomes (BACs) was constructed across Sle1b, and sequencing of six BACs that form an overlapping tiling path across it revealed that the interval contains 24 genes, 19 of which are expressed in the spleen, and 14 of which are in B and CD4+ T cells. We carried out extensive candidate gene analyses on the spleen-expressed genes, including sequencing of all the exons and flanking introns in the lupus-resistant B6 and susceptible B6.Sle1b parental strains, as well as Quantitative Real-time PCR on B and CD4+ T cell cDNA to detect any potentially functional polymorphisms between them. These analyses showed that the SLAM/CD2 family of seven immunoregulatory receptors, clustered within the locus, are by far the best candidates to be the Sle1b gene(s). The members of this family play important roles in intercellular interactions, activation, and function, by engaging in homophilic interactions with themselves or with each other, on a wide variety of immune cell types. Sequence analyses of their extracellular ligand-binding immunoglobulin (Ig) domains revealed that the cluster forms two stable, linked haplotypes of alleles in 33 common inbred laboratory strains of mice. The B6-like haplotype is found only in a small set of C57-strains, while the B6.Sle1b-like haplotype is found in all of the remaining, including the autoimmune-prone MRL, NOD, and NZB, as well as non-autoimmune strains like 129, Balb/c, and C3H. Introgression of this common haplotype from 129 onto B6 also potentiates autoimmunity, causing phenotypes similar to those of B6.Sle1b mice, which derive this interval from the NZW parent of NZM2410. Autoimmunity is mediated, not by a rare mutation peculiar to the region from NZW, but instead by common polymorphic variants of this family, in combination with the downstream signaling and effector molecules and pathways present in the B6 genetic background, underlining the importance of epistasis in such complex, multigenic autoimmune phenotypes. An examination of the SLAM/CD2 Ig domains in a large group of wild-outbred and wild-derived inbred strains belonging to different species of Mus, and sub-species of Mus musculus, has shown that the "disease" alleles of this family are also very common in these populations, demonstrating that their prevalence in the lab strains is not simply an artifact of their inbreeding. The genes also show the presence of ancestral or trans-species polymorphisms, indicative of maintenance of these alleles by balancing selection, although we do not yet know what precisely drives it. The small size of the Sle1b susceptibility interval, and the presence of this linked cluster of attractive candidate genes within it, makes it hard to identify which gene or combination of genes within this family is actually responsible for the autoimmunity by any further recombinational analysis. We have instead turned to a BAC-transgenic rescue strategy by which to localize the gene to a single B6-derived BAC, by its ability to complement the ANA-production phenotype and rescue autoimmunity in B6.Sle1b mice. We believe the strategy is feasible because Sle1b has a strong allele dose effect, so that the presence of a B6 allele of the Sle1b gene causes a large drop in penetrance of ANA-production, from about 90% in nine month old B6.Sle1b females, to about 33% in (B6 X B6.Sle1b) F1s. Our data show that none of the non-SLAM/CD2 candidates within the region is able to rescue B6.Sle1b mice, despite being demonstrably expressed from their BAC-transgenes. BACs carrying certain SLItem Haplotype-Specific Effects of the Slam/Cd2 Family on the Immune Response(2007-05-22) Nguyen, Charles Minh; Wakeland, Edward K.The Sle1b susceptibility interval mediates a breach in tolerance to nuclear antigens in the NZM2410 model of systemic lupus erythematosus (SLE). Congenic B6 mice carrying the Sle1b locus produce anti-nuclear autoantibodies (ANAs) but do not develop lupus nephritis as seen in the parental NZM2410 strain. Fine mapping of the Sle1b locus placed it within a 900kb interval between 171.3 and 172.2Mb on chromosome 1. A Bacterial Artificial Chromosome (BACs) contig that spanned the interval was constructed, and a tiling pathway comprised of six BACs was sequenced. Sequence analysis revealed a dense region of 24 expressed genes. Expression studies determined numerous polymorphisms between B6 and the B6.Sle1b congenic and identified a cluster of genes known as the Slam/CD2 family as the primary candidates for Sle1b. These immunoregulatory receptors play a role in intercellular interactions and regulate function in several immune cell lineages. Of the seven family members within the locus, Ly108 appears to be the strongest candidate as B6.Sle1b shows a differential expression in isoforms. Ly108-1 is highly expressed, while Ly108-2 is expressed at much lower level in the congenic when compared to B6. When lymphocytes are stimulated, Ly108-2 is strongly up-regulated in B6, but not B6.Sle1b. Sequence analysis of the extra-cellular immunoglobulin domains of the Slam/CD2 family revealed two stable haplotypes in a panel of 33 common inbred strains of mice. The first haplotype is only found in B6 and other C57- related strains. The more common second haplotype is found in Sle1b and other autoimmune strains such as MRL, NOD, and NZB, as well as non-autoimmune strains such as 129Sv and Balb/c. The presence of this haplotype on B6 mediates autoimmunity as B6 congenics carrying the Sle1b locus from 129Sv also produce ANAs. Signaling studies on both B6.Sle1b and B6.129 reveal an altered pattern of calcium mobilization upon stimulation in T cells. In addition, CD4 T cells from B6.Sle1b demonstrate a reduction in IL-4 expression and secretion upon activation, suggesting that haplotype 2 of the Slam/CD2 family alters the immune response in T cells. Studies to understand the mechanisms by which this haplotype mediates autoimmunity are in progress.Item In Vivo Identification of SLE1B: LY108 Mediates Autoantibody Production(2008-05-12) Chan, Alice; Wakeland, Edward K.In the NZM2410 model of murine lupus, Sle1b mediates anti-nuclear autoantibody (ANA) production. Our goal is to determine the causative gene in the Sle1b locus. Seven members of the SLAM/CD2 family are located within the Sle1b interval, and previous work has shown that structural and expression polymorphisms in lymphocytes distinguish two major SLAM/CD2 haplotypes. To further narrow the interval, we utilized a BAC transgenic rescue approach whereby BACs carrying the lupus-resistant B6 alleles were bred to B6.Sle1b mice to identify the region mediating ANA suppression. One BAC carrying Cd84 and Ly108 suppressed autoantibody production. We then generated BAC transgenic mice carrying the lupus-susceptible (129) and lupus-resistant (B6) alleles of Ly108 on the B6 and B6.Sle1b genetic background, respectively. The B6 allele of Ly108 suppresses ANA production on the lupus-susceptible B6.Sle1b background while the 129 allele induces ANA on the lupus-resistant B6 genome. Taken together, these data identify Ly108 as a causative gene in Sle1b. While Ly108 is needed to mediate the breach in tolerance, we have also identified other SLAM family members as genetic modifiers necessary to recapitulate fully penetrant, high titer ANA production as seen in Sle1b. We found that in vitro stimulation of B6.Sle1b CD4 T cells led to altered cytokine production, such as decreased IL4 production. Interestingly, these phenotypes have also been reported in knockouts of SLAM/CD2 family members as well as in the absence of the SLAM family adaptor, SAP. Our data indicates that the presence of the Sle1b haplotype, derived from either NZM2410 or 129, recapitulates these phenotypes, independent of the absence of these molecules. While recent reports have suggested a role for SAP in ANA development, we find that the breach in tolerance in Sle1b mice is SAP-independent. However, SAP is necessary to potentiate the autoantibody production. ANAs is an important biomarker for autoimmune diseases including, Systemic Lupus Erythematosus (SLE), and potentially identifies an autoimmune-prone state. We have identified genes which contribute to the production of ANAs. Elucidating the pathways these genes dysregulate will provide critical insight into our understanding of tolerance and how tolerance can be breached.Item Narrowing of the SLES1 Internal Reveals Complex Epistatic Interactions in the Suppression of Autoimmunity(2010-05-14) Belobrajdic, Katherine Ann; Walkeland, Edward K.Sle1 is a potent susceptibility locus for spontaneous systemic autoimmunity derived from the NZM2410 mouse strain. The NZW-derived suppressive modifier locus, Sles1, specifically prevents the spontaneous loss in tolerance mediated by the B6.Sle1 congenic. Sles1 had previously been fine-mapped to a remarkably gene-rich region on murine chromosome 17 containing nearly 70 genes. A series of mouse strains were constructed with a variety of suppressive and non-suppressive variants of Sles1 on the B6.Sle1 genomic background which have revealed multiple layers of epistatic gene interactions within the Sles1 interval. Phenotyping of a truncated recombinant interval mapped the Sles1 phenotype to an approximately 638 KB segment, which combined with genomic and expression analysis, suggested Btnl2 and the H2 genes are strong candidates for Sles1. Finally, further characterization of the Sles1 interval has revealed an allele-specific and tissue-specific reduction of major histocompatibility complex (MHC) Class II molecules on the surface of B cells, as well as a possible role for follicular helper T cells in the development of Sle1-mediated autoimmunity. Understanding how Sles1 and other modifiers suppress systemic autoimmunity will reveal important insights for developing therapeutic strategies for systemic lupus eythematosus (SLE).Item A Retrospective Cohort Study of Subacute Cutaneous Lupus Patients with and Without Systemic Lupus(2012-08-15) Taylor, April; Chong, Benjamin F.Objective: To compare subacute cutaneous lupus erythematosus (SCLE) patients with systemic lupus erythematosus (SLE) (+SCLE/+SLE) versus SCLE patients without SLE (+SCLE/-SLE) over a period of five years. Design: Retrospective cohort study. Setting: Outpatient dermatology and rheumatology clinics at an academic medical center. Patients: Forty-seven SCLE patients presenting between February 1989 and January 2012 were screened with nineteen meeting inclusion/exclusion criteria. Predictive variable: Anti-nuclear antibodies (ANA). Results: Of the nineteen patients included, thirteen (68.4%) had SCLE only (+SCLE/-SLE) for the majority of the study period and six (31.6%) had both SCLE and SLE (+SCLE/+SLE). At baseline, +SCLE/+SLE patients were more likely to have a history of discoid lesions, oral ulcers, lupus non-specific findings, and require multiple medications. Over the five year study period, +SCLE/+SLE patients were also more likely to have ANA, immunologic disease (including anti-double-stranded DNA), renal disease, proteinuria, decreased complement, and to complain of arthralgias. Anti-Ro antibodies alone were more common in +SCLE/-SLE patients. Conclusions: Various cutaneous manifestations of lupus are present early in the course of SCLE, but laboratory values in +SCLE/+SLE become more distinct over time. Thus, the aforementioned variables should be tested for evidence of disease involvement and to ensure adequate treatment among +SCLE/+SLE patients. In contrast, +SCLE/-SLE patients may be tested for the development of anti-Ro antibodies but need not be monitored for the other laboratory abnormalities, given their rarity among patients with SCLE only. Larger prospective studies comparing disease course in SCLE patients with and without SLE are needed to verify these findings.Item Role of Bruton’s Tyrosine Kinase and Interleukin-6 in Plasma Cell Accumulation and Autoantibody Production in Lyn-Deficient Mice, A Model of Lupus(2011-08-26T17:34:42Z) Gutierrez, Maria Antonietta; Satterthwaite, AnneSystemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as DNA and chromatin, resulting in autoantibody production, immune complex deposition, inflammation, and end organ damage such as glomerulonephritis (GN). Currently, only non-specific, immunosuppressive therapies are approved for use in lupus patients. These have undesirable side effects and risks. The development of more targeted therapies is necessary and requires a better understanding of the mechanisms that contribute to the production of autoantibodies. Mice deficient in Lyn, a gene associated with human lupus, develop several features characteristic of SLE, including peripheral plasma cell accumulation, anti-dsDNA antibodies, and GN. Lyn is a Src-family tyrosine kinase that, in general, inhibits B cell and myeloid cell activity. Loss of Lyn results in cellular hyperactivity associated with autoantibody production. Bruton’s tyrosine kinase (Btk), which is critical to B cell receptor (BCR) signaling, mediates BCR hypersensitivity and autoantibody production in lyn-/- mice. B cell hyperresponsiveness is not, however, sufficient for the autoimmune phenotype; additional Btk-dependent events are required. Btk also contributes to myeloid cell function, and generally opposes Lyn action in these cells as in B cells. However, the relative contribution of myeloid hyperactivity to autoantibody production in lyn-/- mice is unknown. Lyn-deficient mice expressing reduced Btk dosage in B cells and no Btk in myeloid cells were utilized to better define how Lyn and Btk regulate and mediate, respectively, the progression from tolerance to autoimmunity. Two major checkpoints regulating autoantibody production were identified and found to be breached in lyn-/- mice. The first checkpoint regulates Btk-mediated accumulation of long-lived plasma cells co-incident with polyclonal IgM autoreactivity. This is due in part to impaired migration of lyn-/- plasma cells towards SDF-1 and involves a B cell intrinsic effect of Lyn-deficiency. The second checkpoint regulates the class-switching of B cells with lupus-associated autoantigen specificities and the production of pathogenic autoantibodies. This step requires IL-6, which is produced in excess by lyn-/- myeloid cells in a Btk-dependent manner. These results suggest that both B and myeloid defects contribute to autoimmunity in lyn-/- mice and identify Btk and IL-6 as potential therapeutic targets for SLE.Item The Roles of Ly108, the Genetic Susceptibility Loci Sle3, and CXCR4/CXCL12 in Systemic Lupus Erythematosus(2011-08-26T17:35:26Z) Wang, Andrew; Wakeland, EdwardLy108, in the NZM2410-derived Sle1b locus, was identified to play a key role in thymic selection. B6.Sle1b thymocytes displayed aberrant cell-surface Ly108 expression and decreased sensitivity to CD3-induced cell-death. Significant V-ß usage was found in B6.Sle1b versus B6 thymocytes. Simultaneous administration of OVA and anti-Ly108 antibody led to complete protection of OVA-induced deletion in B6.Sle1b.OTII mice but not in B6.OTII controls. Significant differences between B6 and B6.Sle1b were found in the amount of Ly108 phosphorylation and subsequent SAP-binding. Calm2 was found to be differentially expressed in B6.Sle1b thymocytes following Ly108 cross-linking. B6.Sle1b thymocytes were shown to flux less calcium, as result of modulated intracellular stocks of calcium, and, be more arrested in G1-phase following Ly108 engagement compared to B6, leading to an overall reduction in thymic apoptosis. These data suggest that the autoimmune form of Ly108 impairs thymic tolerance by dampening CD3-signaling and disrupting a G1-S cell-cycle checkpoint. Sle3, an NZM2410-derived susceptibility locus, mediates transition from benign to fatal autoimmunity. Sle3 was mapped to two main sub-loci, Sle3a and Sle3b. Sle3b was mapped to a 3.4 Mb interval containing Klf13, which has a known role in regulating RANTES. We found that Klf13 mRNA expression was significantly increased and that B6.Sle3 macrophages secreted roughly 2-fold more RANTES compared to B6. Co- culture of B6.Sle3 macrophages with blocking antibody to RANTES reversed the hyperactivation phenotype to B6 levels, indicating that increased RANTES secretion due to a genetic lesion in Klf13 could be responsible for the hyperactivation of macrophages seen in B6.Sle3. Polymorphisms in Klf13 were shown to be associated with human SLE. A significant dysregulation of the CXCR4/CXCL12 axis was observed in multiple murine models of spontaneous lupus. Increased CXCR4 expression in lupus mice led to functional differences, including increased migration to positive CXCL12 gradients. Simultaneously, the ligand for CXCR4, CXCL12, was significantly upregulated in the nephritic kidneys. To assess the contribution of CXCR4/CXCL12 upregulation on lupus pathogenesis, mice were treated with a peptide antagonist of CXCR4. Both preventive and therapeutic administration of CXCR4 blockade resulted in reduced renal infiltration by inflammatory myeloid cells and prolonged survival. Finally, increased renal CXCL12 expression and increased immune-cell CXCR4 expression was also observed in human SLE. These findings underscore the pathogenic role of CXCR4/CXCL12 in lupus nephritis and highlight this axis as a new and promising therapeutic target in this disease.