Fatty Acid Amide Hydrolase regulates peripheral B cell receptor revision, polyreactivity and B1 cells in lupus
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ABSTRACT: FAAH expresson is induced in Sle2 splenic B cells. Increased peripheral B cell receptor revision, or selective peripheral expansion of BCR-revised B-cells, may lead to systemic autoimmunity, and FAAH is a lupus susceptibility gene that could regulate this process in Sle2 mice. To determine the gene expression profile in peripheral B cells from Sle2 mice (compared to B6 mice), we isolated splenic B cells from these two strains of mice, extracted the total mRNA and performed the microarray analysis Please note that Sle2 mouse is a congenic strain that harbors the Sle2 locus from Chromosome 4 of NZM2410 strain in a B6 background and Sle2 is the name of the lupus susceptibility locus.
Project description:FAAH expresson is induced in Sle2 splenic B cells. Increased peripheral B cell receptor revision, or selective peripheral expansion of BCR-revised B-cells, may lead to systemic autoimmunity, and FAAH is a lupus susceptibility gene that could regulate this process in Sle2 mice.
Project description:Sle1a.1 is part of the Sle1a lupus susceptibility locus which results in the production of activated and autoreactive CD4+ T cells as well as a reduction in the peripheral regulatory T cell (Treg) pool. Sle1a.1 CD4+ T cells showed a defective response to retinoic acid (RA) expansion of TGFβ-induced Tregs. At the molecular level, Sle1a.1 corresponds to an increased expression of a novel splice isoform of Pbx1, Pbx1-d. Pbx1-d over-expression is sufficient to induce an activated/inflammatory phenotype in Jurkat T cells, and to decrease their apoptotic response to RA. PBX1-d is expressed more frequently in lupus patients than in healthy controls, and its presence correlates with an increased memory T cell population. These findings indicate that Pbx1 is a novel lupus susceptibility gene that regulates T cell activation and tolerance. Total RNA from CD4+ T cells from C57BL/6 (B6) and B6.Sle1a.1 (Sle) mice was isolated, with 4 biological replicates each. Gene expression data from C57BL/6 mice were compared with data from B6.Sle2c1 mice.
Project description:Sle2c1 is an NZM2410-derived lupus susceptibility locus that induces an expansion of the B1a cell compartment. B1a cells have a repertoire enriched for autoreactivity, and an expansion of this B cell subset occurs in several mouse models of lupus. Here we showed that expression of Sle2c1 enhances NZB cellular phenotypes that have been associated with autoimmune pathogenesis. A combination of genetic mapping and candidate gene analysis presents Cdkn2c, a gene encoding for cyclin kinase inhibitor p18INK4c (p18), as the top candidate gene for inducing the Slec2c1 associated expansion of B1a cells. A novel SNP in the Cdkn2c promoter is associated with a significantly reduced Cdkn2c expression in the splenic B cells and B1a cells from Sle2c1-carrying mice, which leads to defective G1 cell cycle arrest in splenic B cells and increased proliferation of Pc B1a cells. As cell cycle is differentially regulated in B1a and B2 cells, these results suggest that Cdkn2c play a critical role in B1a cell self renewal, and that its impaired expression leads to an accumulation of these cells with high autoreactive potential. Total RNA from peritoneal cavity B cells (B1a) and splenic B cells (Bs) was isolated, with 4 biological replicates each. Gene expression data from C57BL/6 mice were compared with data from B6.Sle2c1 mice.
Project description:Sle1c is a sublocus of the NZM2410-derived Sle1 major susceptibility locus. We have previously shown that Sle1c contributes to lupus pathogenesis by conferring CD4+ T cell-intrinsic hyperactivation and increased susceptibility to chronic graft-versus-host disease (cGVHD) that mapped to the centromeric portion of the locus. In this study, we have refined the centromeric sublocus to a 675Kb interval, termed Sle1c2. Recombinant congenic strains expressing Sle1c2 exhibited a T cell-intrinsic CD4+ T cell hyperactivation and cGVHD susceptibility, similar to mice with the parental Sle1c. We performed a microarray analysis on CD4+ T cells to gain insights into the transcriptional programs that regulate the hyperactivation conferred by Sle1c2. CD4+ T cell cDNA was prepared from spenocytes from 5 mice from each strain and B6.Sle1c2 gene expression was compared to B6 gene expresion.
Project description:In this study, miRNA expression in splenic lymphocytes from three genetically disparate lupus-prone mouse models (MRL-lpr, B6-lpr and NZB/WF1) were profiled. 49 miRNAs were found to be differentially expressed in MRL-lpr mice compared to MRL mice; and 24 miRNAs were differentially expressed in B6-lpr mice compared to B6 mice. Among these dysregulated miRNAs, we noted that 15 miRNAs were common to both lpr strains. Interestingly, microarray analysis of NZB/W and NZW at 3 months of age, an age when overt lupus disease is not evident in NZB/W mice, revealed that only one miRNA, miR-148a was significantly upregulated in NZB/W mice.
Project description:In this study, miRNA expression in splenic lymphocytes from three genetically disparate lupus-prone mouse models (MRL-lpr, B6-lpr and NZB/WF1) were profiled. 49 miRNAs were found to be differentially expressed in MRL-lpr mice compared to MRL mice; and 24 miRNAs were differentially expressed in B6-lpr mice compared to B6 mice. Among these dysregulated miRNAs, we noted that 15 miRNAs were common to both lpr strains. Interestingly, microarray analysis of NZB/W and NZW at 3 months of age, an age when overt lupus disease is not evident in NZB/W mice, revealed that only one miRNA, miR-148a was significantly upregulated in NZB/W mice. The aim of this porject is to determine the common miRNA expression changes in splenocytes from different strains of murine lupus models. The splenocytes were prepared from genetically lupus-prone female mice including MRL/MpJ-Faslpr/J (MRL-lpr), NZBWF1/J (NZB/W), B6.MRL-Faslpr/J (B6-lpr) and their control mice MRL/MpJ (MRL), NZW/LacJ (NZW) and C57BL/6J (B6) mice (The Jackson laboratory, ME). Total RNAs, containing miRNAs were isolated from whole splenocytes using mirVana miRNA isolation kits (Ambion) following manufactory’s instructions and sent to LC Sciences (http://www.lcsciences.com/) for the microarray assay. The mouse miRNA array chips (Chip ID miRMouse 12.0 version), which included 617 unique, mature, mouse miRNA, based on the Sanger miRBase Release 12.0, were used in the assay.
Project description:Our previous study demonstrated a significant upregulation of a large set of miRNAs at the genomic imprinted Dlk1-Dio3 locus in lymphocytes of diverse murine lupus-prone strains. The upregulation of Dlk1-Dio3 miRNAs in lupus-prone mice is correlated with the global DNA hypomethylation. In this study, by performing genome-wide DNA methylation analysis, we reported that Dlk1-Dio3 genomic region in CD4+ T cells of MRL/lpr mice was hypomethylated, further linking hypomethylation to the increased expression of Dlk1-Dio3 miRNAs in lupus. Then, we assessed the gene expression levels of enzymes that either write (DNA methyltransferases, DNMTs) or erase DNA methylation (Ten-eleven translation proteins, TETs) to understand the molecular contributor to the DNA hypomethylation in MRL/lpr CD4+ T cells. The expression levels of Dnmt1, Dnmt3b, Tet1, and Tet2 were significantly increased in CD4+ T cells of MRL/lpr mice, as well as in B6/lpr and B6.sle123 mice, compared to their respective control mice. These data indicate the significant involvement of the TETs-mediated active demethylation pathway rather than reduced DNMTs-mediated passive demethylation pathway in the hypomethylation of murine lupus CD4+ T cells. The transcription factor, early growth response 2 (EGR2) is critically involved in regulating T cell functions and autoimmunity. In this research, we found that Egr2 deletion in B6/lpr mice notably reduced methylation-sensitive Dlk1-Dio3 cluster miRNAs expression in CD4+ T cells. Surprisingly, even though EGR2 has been shown to induce DNA demethylation by recruiting TET2, we found that deleting Egr2 in B6/lpr mice induced a higher number of hypomethylated DMRs than hypermethylated DMRs at either whole genome or the Dlk1-Dio3 locus in CD4+ T cells of B6/lpr mice. These data are the first finding on the positive role of EGR2 on the expression of Dlk1-Dio3 cluster miRNAs in lupus mice. Given that Dlk1-Dio3 miRNAs target the major signaling pathways in autoimmunity, these data provide a new perspective in understanding the potential pathogenic role of upregulated EGR2 in lupus.
Project description:Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive follicular helper T (Tfh) cells license high-affinity autoantibody production. Strikingly, the frequency of circulating Tfh is correlated with disease activity in SLE patients. As such, understanding the molecular mechanisms responsible for the aberrant Tfh cell generation and activation in lupus is of fundamental significance. We previously demonstrated that expanded Tfh cells in the B6.Sle1.Sle2.Sle3 (TC for triple congenic) lupus model exhibit high glycolysis and oxidative metabolism, which can be constrained by inhibiting glycolysis with 2-deoxyglucose (2DG). We performed RNA-seq analyses of splenic Tfh and naïve CD4+ T cells (Tn) comparing between TC and B6 mice. First, data revealed a large number of shared gene signatures in Tfh and Tn comparing between TC and B6 group, implicating that the aberrant development of Tfh initiates as early as Tn state. Further alignment of the Tfh transcriptome obtained from RNA-seq and earlier microarray assays demonstrated concerted alterations in numerous gene signatures of overactivation of T cells including dysregulated tyrosine kinase signaling and MAPK signaling pathways. Gene set enrichment analyses (GSEA) further revealed altered metabolic pathways (e.g., oxidative phosphorylation and pyruvate metabolism) among splenic TC Tfh cells.
Project description:Gene expression analysis of splenic follicular B cells and marginal zone B cells from B6 and CD19:KLF3 transgenic mice Comparing KLF3-transgenic and non-transgenic follicular B cells by RNA-microarray revealed that KLF3 regulates a subset of genes that was similarly up-/downregulated upon normal MZ B cell differentiation. Indeed, KLF3 expression overcame the lack of MZ B cells caused by different genetic alterations, such as CD19-deficiency or blockade of B-cell activating factor (BAFF)-receptor signaling, indicating that KLF3 may complement alternative NF-κB signaling. Thus, KLF3 is a driving force towards MZ B cell maturation. RNA of splenic follicular B cells and marginal zone B cells were obtained from 4 different mice per group (B6 and CD19:KLF3 mice). 16 samples = 8 individual mice x 2 B cell subsets.
Project description:Bone marrow derived macrophages from C57BL/6 or C57BL/6-Sst1S strains of mice were pretreated ± 100U/ml interferon gamma then infected for 0, 1, 2, 4, or 6h with Listeria monocytogenes. C57BL/6-Sst1S mice carry the Sst1 chromosomal locus of C3H mice. The Sst1 locus controls susceptibility to intracellular pathogens - these mice are therefore more susceptible than wild type B6.