The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial and multigenetic. The apoptosis genes, fas and fas ligand (fasL), are candidate contributory genes in human SLE, as mutations of these genes result in autoimmunity in several murine models of this disease. In humans, fas mutations result in a familial autoimmune lymphoproliferative syndrome, but defects in FasL have not yet been identified. In this study, DNA from 75 patients with SLE was screened by single-stranded conformational polymorphism analysis for potential mutations of the extracellular domain of FasL. A heterozygous single-stranded conformational polymorphism for FasL, was identified in one SLE patient, who exhibited lymphadenopathy. Molecular cloning and sequencing indicated that the genomic DNA of this patient contained an 84-bp deletion within exon 4 of the fasL gene, resulting in a predicted 28 amino acid in-frame deletion. Analysis of PBMC from this patient revealed decreased FasL activity, decreased activation-induced cell death, and increased T cell proliferation after activation. This is the first report of defective FasL-mediated apoptosis related to a mutation of the human Fasl, gene in a patient with SLE and suggests that fasL mutations are an uncommon cause of the disease.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by high autoantibody levels and multiorgan tissue damage, including kidney and skin. Cutaneous manifestations are frequent in patients with SLE, yet the etiology and pathogenesis of skin injury in SLE remains unclear. We reasoned that lupus serum containing high levels of autoreactive Ig contributes to skin injury. In this article, we report that serum from SLE patients and lupus-prone mice induces skin inflammation following intradermal injection into normal mice. Lupus serum depleted of IgG failed to cause skin inflammation. Monocytes, but not lymphocytes, were found to be crucial in the development of lupus serum-induced skin inflammation, and lupus serum IgG induced monocyte differentiation into dendritic cells (DCs). TNF-α and TNFR1, but not TNFR2, were required for the development of lupus serum-induced skin inflammation. TNFR1, not TNFR2, represented the main molecule expressed in the skin lesions caused by injected lupus serum. Our studies demonstrated that lupus serum IgG causes skin injury by involving the TNFR1 signaling pathway and monocyte differentiation to DCs. Accordingly, disruption of the TNFR1-mediated signaling pathway and blockade of DC generation may prove to be of therapeutic value in patients with cutaneous lupus erythematosus.
Purpose of review
Epigenetic mechanisms regulate gene expression, and epigenetic gene dysregulation is implicated in the pathogenesis of a growing number of disorders. Of the autoimmune diseases, epigenetic mechanisms are most clearly involved in human systemic lupus erythematosus (SLE). Herein, we summarize earlier work on epigenetic mechanisms contributing to human SLE. We first focus on the roles of DNA demethylation and DNA methyltransferase enzyme dysregulation, and we then review recent and important advances in this field.
Many advances in the past year have been made. The importance of DNA demethylation in SLE was confirmed through twin studies. New T lymphocyte immune genes that are activated by DNA demethylation, and that may participate in autoreactivity, were identified. Finally, novel mechanisms contributing to DNA demethylation in SLE were discovered.
A comprehensive understanding of the epigenetic mechanisms contributing to SLE will likely enable development of new therapeutic agents and strategies that target the dysregulated genes or correct the aberrant epigenetic modifications. Although specific agents have not yet been tested in SLE, the studies reviewed hold promise that these approaches will be useful in the treatment of human lupus.
chromatin; DNA methylation; epigenetics; histone; lupus
Continued antibody gene rearrangement, termed receptor editing, is an important mechanism of central B cell tolerance that may be defective in some autoimmune individuals. We describe a quantitative assay for recombining sequence (RS) rearrangement that we use to estimate levels of antibody light chain receptor editing in various B cell populations. RS rearrangement is a recombination of a noncoding gene segment in the κ antibody light chain locus. RS rearrangement levels are highest in the most highly edited B cells, and are inappropriately low in autoimmune mouse models of systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), including those without overt disease. Low RS rearrangement levels are also observed in human subjects with SLE or T1D.
Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex multifactorial pathogenesis. T lymphocytes play a critical role in disease pathogenesis and display abnormal gene expression and poor interleukin (IL)-2 production. We previously showed that the expression of the transcriptional repressor cyclic AMP response element modulator α (CREMα) is increased in SLE T cells and contributes to reduced IL-2 production. Although estrogen is implicated in the onset and exacerbation of SLE, the precise nature of molecular events regulated by estrogen in immune cell function is not well understood. Here, we asked whether estrogen regulates the expression of CREMα in human T lymphocytes. We show that exposure of human T cells to 17-β-estradiol leads to a dose-dependent increase in CREMα mRNA expression, and this increase appears to be mediated through the estrogen receptors α and β. We show that the increased expression of CREMα is due to increased transcriptional activity of the CREM promoter and is mediated by increased expression and binding of the Sp1 transcriptional activator. We further show that estrogen treatment leads to a dose-dependent decrease in IL-2 mRNA and cytokine production by T cells. Finally, the effect of β-estradiol on CREMα is observed more frequently in T cells from women than from men. We conclude that estrogen can modulate the expression of CREMα and lead to IL-2 suppression in human T lymphocytes, thus revealing a molecular link between hormones and the immune system in SLE.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the deposition of immune complexes due to widespread loss of immune tolerance to nuclear self-antigens. Deposition in the renal glomeruli results in the development of lupus nephritis (LN), the leading cause of morbidity and mortality in SLE. In addition to the well-recognized genetic susceptibility to SLE, disease pathogenesis is influenced by epigenetic regulators such as microRNAs (miRNAs). miRNAs are small, noncoding RNAs that bind to the 3′ untranslated region of target mRNAs resulting in posttranscriptional gene modulation. miRNAs play an important and dynamic role in the activation of innate immune cells and are critical in regulating the adaptive immune response. Immune stimulation and the resulting cytokine milieu alter miRNA expression while miRNAs themselves modify cellular responses to stimulation. Here we examine dysregulated miRNAs implicated in LN pathogenesis from human SLE patients and murine lupus models. The effects of LN-associated miRNAs in the kidney, peripheral blood mononuclear cells, macrophages, mesangial cells, dendritic cells, and splenocytes are discussed. As the role of miRNAs in immunopathogenesis becomes delineated, it is likely that specific miRNAs may serve as targets for therapeutic intervention in the treatment of LN and other pathologies.
RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.
This study is to investigate the expression of progranulin (PGRN) in systemic lupus erythematosus (SLE) patients and the effect of glucocorticoid (GC) treatment on its expression.
Thirty newly diagnosed severe SLE patients and 30 healthy subjects were enrolled in this study. The serum levels of PGRN and the inflammatory factors of SLE were detected by ELISA and the mRNA expression of these proteins were detected by real-time PCR.
The serum levels of PGRN, IL-6, PR3, TNFR, TNF-α and anti-dsDNA antibody in SLE patients were increased significantly compared with healthy controls (P < 0.05). The relative expression of PGRN mRNA was increased by 4.88-fold in pre-treatment SLE patients compared with controls (P < 0.05). After prednisone treatment, the serum levels of PGRN decreased significantly, and the relative expression of PGRN mRNA was decreased by 1.34-fold compared with the untreated controls (P < 0.01). Moreover, Serum concentration of PGRN was correlated with serum levels of IL-6, TNF-α, TNFR and anti-dsDNA antibody in both pre-treatment and post-treatment SLE patients.
PGRN is up-regulated in the SLE patients and is correlated with pro-inflammatory cytokines and anti-dsDNA antibody. Glucocorticoids can down-regulate the expression of PGRN in SLE patients.
Systemic lupus erythematosus; Progranulin; Glucocorticoid; IL-6
Systemic lupus erythematosus (SLE) highlights the dangers of dysregulated B cells and the importance of initiating and maintaining tolerance. In addition to central deletion, receptor editing, peripheral deletion, receptor revision, anergy, and indifference, we have described a new mechanism of B cell tolerance wherein dendritic cells (DCs) and macrophages (MΦs) regulate autoreactive B cells during innate immune responses. In part, DCs and MΦs repress autoreactive B cells by releasing IL-6 and soluble CD40L (sCD40L). This mechanism is selective in that IL-6 and sCD40L do not affect Ig secretion by naïve cells during innate immune responses, allowing immunity in the absence of autoimmunity. In lupus-prone mice, DCs and MΦs are defective in secretion of IL-6 and sCD40L and cannot effectively repress autoantibody secretion suggesting that defects in DC/MΦ-mediated tolerance may contribute to the autoimmune phenotype. Further, these studies suggest that reconstituting DCs and MΦs in SLE patients might restore regulation of autoreactive B cells and provide an alternative to immunosuppressive therapies.
Systemic lupus erythematosus; B cell tolerance; Autoimmunity; Dendritic cell; Macrophage; Smith antigen
Systemic lupus erythematosus (SLE) is a heterogeneous disease that can affect multiple organs. A hallmark of this disease, like for other autoimmune diseases, is the presence of large amounts of autoantibodies. As such SLE is considered to be a B cell disease perpetuated by the expansion of autoreactive T and B cells. The T cells involved have long been considered to be Th1 and Th17 cells as these potent pro-inflammatory cells can be found in the tissues of SLE patients. Recent advances point to a role for the Th2 environment in contributing to SLE through promotion of autoantibody production. Here we describe the recent work focusing on autoreactive IgE and the activation of basophils as promoting the production of autoantibodies in SLE. The findings, both in a mouse model of SLE and in human SLE subjects, support the concept that the activation of the basophil by autoreactive IgE-containing immune complexes serves to amplify the production of autoantibodies and contributes to the pathogenesis of disease. We propose that therapeutic targeting of this amplification loop by reducing the levels of circulating autoreactive IgE may have benefit in SLE.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by an overproduction of autoantibodies. The loss of self-tolerance in SLE is believed to be caused by the dysregulation of both innate and adaptive immune systems. Neutrophils, the most abundant effector cells of innate immunity, have long been shown to be associated with SLE. However, their role in the pathogenesis of SLE was not clear until recent studies discovered abnormal regulation of neutrophil extracellular traps (NETs) in SLE patients. NETs are web-like structures composed of chromatin backbones and granular molecules. They are released by activated neutrophils through a process called “NETosis”. Nets were first described in 2004 as a novel host defense mechanism to trap and kill foreign pathogens. Recent evidence shows that NETs also participate in the pathogenesis of a variety of inflammatory and autoimmune diseases, including SLE. An imbalance between NET formation and clearance in SLE patients may play a prominent role in the perpetuation of autoimmunity and the exacerbation of disease, as well as the induction of end-organ manifestations. This review summarizes the current findings regarding the contribution of NETs to the pathogenesis of SLE.
Neutrophils; Neutrophil extracellular traps; Lupus; Pathogenesis; Autoimmune; Interferon
Autoreactivity to histones is a pervasive feature of several human autoimmune disorders, including systemic lupus erythematosus (SLE). Specific post-translational modifications (PTMs) of histones within neutrophil extracellular traps (NETs) may potentially drive the process by which tolerance to these chromatin-associated proteins is broken. We hypothesized that NETs and their unique histone PTMs might be capable of inducing autoantibodies that target histones.
We developed a novel and efficient method for the in vitro production, visualization, and broad profiling of histone-PTMs of human and murine NETs. We also immunized Balb/c mice with murine NETs and profiled their sera on autoantigen and histone peptide microarrays for evidence of autoantibody production to their immunogen.
We confirmed specificity toward acetyl-modified histone H2B as well as to other histone PTMs in sera from patients with SLE known to have autoreactivity against histones. We observed enrichment for distinctive histone marks of transcriptionally silent DNA during NETosis triggered by diverse stimuli. However, NETs derived from human and murine sources did not harbor many of the PTMs toward which autoreactivity was observed in patients with SLE or in MRL/lpr mice. Further, while murine NETs were weak autoantigens in vivo, there was only partial overlap in the immunoglobulin G (IgG) and IgM autoantibody profiles induced by vaccination of mice with NETs and those seen in patients with SLE.
Isolated in vivo exposure to NETs is insufficient to break tolerance and may involve additional factors that have yet to be identified.
Mitochondrial transcripts in kinetoplastids undergo remarkable posttranscriptional editing by uridylate insertion and deletion. The often dramatic remodeling of pre-mRNA sequences is directed by small guide RNAs (gRNAs) to produce mature mRNAs. In vitro analyses of editing have been used to determine the mechanism of editing and show that editing occurs by a series of enzyme-catalyzed steps. They also show that chimeric gRNA/mRNA molecules are not editing intermediates as proposed but are aberrant end products of editing. The complexes and molecules that catalyze editing are now being identified and characterized. The origin of editing, its developmental regulation which helps control the switching between terminal respiratory systems during the life cycle of trypanosomes, and other areas for future study are discussed.
Objective(s): Apoptosis is a tightly regulated process and plays a crucial role in autoimmune diseases. Because abnormalities in apoptosis are considered to be involved in the pathogenesis of systemic lupus erythematosus (SLE), in present study we studied the apoptosis in T lymphocytes from Iranian SLE patients at protein and gene expression levels for some molecules which are involved in apoptosis pathways.
Materials and Methods: Thirty five SLE patients (23 female, 12 male), and 20 age matched controls (10 female, 10 male) participated in this study. T lymphocytes were isolated from peripheral blood mononuclear cells (PBMCs) using MACS method. Apoptosis rate was studied at protein level by flow cytometer using Annexin V, and at gene expression level using semi-quantitative RT-PCR method for detection of Fas, FasL, Bcl-2, caspase 8, and caspase 9 genes.
Results: The percentage of apoptotic cells in SLE patients was not different in comparison with controls (20.2% ± 1.4 vs 21.1% ± 1.0), but the expression levels of FasL, caspase 8, and caspase 9 genes in all SLE patients and in female patients were significantly lower than controls; 0.45R vs 0.78R for FasL, 0.74R vs 1.0R for caspase 8, and 0.76R vs 1.26R for caspase 9 in all SLE patients and 0.37R vs 0.82R for FasL, 0.45R vs 1.6R for caspase 8, and 0.63R vs 1.56R for caspase 9 in female patients.
Conclusion: The expression levels of FasL, caspase 8 and caspase 9 molecules involved in apoptosis decreased in female, but not in male SLE patients.
Apoptosis; Autoimmune; Gene expression; Systemic lupuserythema- tosus
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (Tregs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. Treg abnormalities have been associated with several autoimmune diseases and there is interest in the role of Tregs in SLE. We previously demonstrated that transfer of expanded CD4+CD25+CD62LHI Tregs slows the development of lupus in (NZBxNZW)F1 (B/W) mice. However in the absence of Treg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3− effector T cells (Teffs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure Treg subset defined by CD4+CD25+ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4+CD25+ cells produced a population containing 70–85% CD4+Foxp3+Tregs. Expanded Tregs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4+CD25+ Tregs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of Teff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred Tregs or transferred Teffs expanded in the absence of Tregs. These studies demonstrate that adoptive transfer of expanded CD4+CD25+Foxp3+Tregs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured Teff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated Teffs from autoimmune patients may not pose a significant risk of promoting disease.
A synthetic Sendai virus-like recombinant RNA was used to develop a model system for pseudo-templated transcription of the P/C gene. The synthetic RNA molecule contains a 42-base stretch of nucleotide sequence derived from the RNA editing site of the P/C gene embedded into the chloramphenicol acetyltransferase gene. When this construct was rescued into Sendai virus, it was found that this 42-base sequence was sufficient to allow the Sendai virus polymerase to transcribe mRNAs with G-nucleotide insertions. Edited mRNA species containing a single nontemplated G insertion were found at a frequency of 6.5%, while rare messages had two G residues inserted. Edited viral RNA was not apparent, suggesting that this event is appropriately excluded during replication of the model genome. By progressively deleting from the 3' end, we found that a 24-nucleotide sequence spanning the G-insertion site was sufficient for pseudo-templated transcription in our system.
The cis-requirements for the first editing site in the atp9 mRNA from pea mitochondria were investigated in an in vitro RNA editing system. Template RNAs deleted 5′ of −20 are edited correctly, but with decreased efficiency. Deletions between −20 and the edited nucleotide abolish editing activity. Substitution of the sequences 3′ of the editing site has little effect, which suggests that the major determinants reside upstream. Stepwise mutated RNA sequences were used as templates or competitors that divide the cis-elements into several distinct regions. In the template RNAs, mutation of the sequence between −40 and −35 reduces the editing activity, while the region from −15 to −5 is essential for the editing reaction. In competition experiments the upstream region can be titrated, while the essential sequence near the editing site is largely resistant to excess competitor. This observation suggests that either one trans-factor attaches to these separate cis-regions with different affinities or two distinct trans-factors bind to these sequences, and one of which is present in limited amounts, wheras the other one is more abundant in the lysate.
The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk−/− mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk−/− mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk−/− mice. A transgene expressing a low level of Btk in B cells (Btklo) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btklo B cells. anti-DNA IgG was not observed in 56R.Btklo mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.
B cells; Systemic Lupus Erythematosus; Autoantibodies; Transgenic/Knockout Mice; Protein Kinases
Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by ∼20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C- terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3′-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates.
Systemic lupus erythematosus (SLE) is considered a prototype of systemic autoimmune diseases; however, despite considerable advances in recent years in the understanding of basic mechanisms in immunology, little progress has been made in elucidating the etiology and pathogenesis of this disease. This even holds for inbred mice, such as the lupus-prone New Zealand Black/New Zealand White (NZB/NZW) F1 mice, which are all genetically programmed to develop lupus at a predetermined age. This frustrating state of affairs calls for a fundamental change in our scientific thinking, and the opening of new directions in lupus research. Here, we suggest that intrinsic B cell tolerance mechanisms are not grossly impaired in lupus-prone mice, but that an unusually strong positive selection event recruits a small number of autoreactive B cells to the germinal centers. This event could be facilitated by nucleic acid–protein complexes that are created by somatic changes in the susceptible animal.
B cell tolerance; Lupus; Anti-DNA; NZB/NZW mice; Retroelements
Considerable evidence points to a role for B lymphocyte stimulator (BLyS) overproduction in murine and human systemic lupus erythematosus (SLE). Nevertheless, the correlation between circulating levels of BLyS protein and disease activity in human SLE is modest at best. This may be due to an inadequacy of the former to reflect endogenous BLyS overproduction faithfully, in that steady-state protein levels are affected not just by production rates but also by rates of peripheral utilization and excretion. Increased levels of BLyS mRNA may better reflect increased in vivo BLyS production, and therefore they may correlate better with biologic and clinical sequelae of BLyS overexpression than do circulating levels of BLyS protein. Accordingly, we assessed peripheral blood leukocyte levels of BLyS mRNA isoforms (full-length BLyS and ΔBLyS) and plasma BLyS protein levels in patients with SLE, and correlated these levels with laboratory and clinical features. BLyS protein, full-length BLyS mRNA, and ΔBLyS mRNA levels were greater in SLE patients (n = 60) than in rheumatoid arthritis patients (n = 60) or normal control individuals (n = 30). Although full-length BLyS and ΔBLyS mRNA levels correlated significantly with BLyS protein levels in the SLE cohort, BLyS mRNA levels were more closely associated with serum immunoglobulin levels and SLE Disease Activity Index scores than were BLyS protein levels. Moreover, changes in SLE Disease Activity Index scores were more closely associated with changes in BLyS mRNA levels than with changes in BLyS protein levels among the 37 SLE patients from whom repeat blood samples were obtained. Thus, full-length BLyS and ΔBLyS mRNA levels are elevated in SLE and are more closely associated with disease activity than are BLyS protein levels. BLyS mRNA levels may be a helpful biomarker in the clinical monitoring of SLE patients.
SS-A/Ro is a nucleocytoplasmic ribonucleoprotein (RNP) particle that is a common target of autoimmune response in Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Previously, SS-A/Ro has been shown to be composed of at least two polypeptide antigens of 60 and 52 kD noncovalently associated with a set of small RNAs, designated Y1-Y5. A serum from an SS patient was selected to screen a lambda gt11 cDNA library constructed from human T cell lymphoblastic leukemia (MOLT-4) mRNA. An immunoreactive clone was isolated that possessed a 1.8-kb cDNA insert. In vitro transcription and translation of the cDNA resulted in the synthesis of a 57.5-kD polypeptide which was specifically immunoprecipitated by SS-A/Ro antisera. The identity of the cDNA encoded protein as the 60-kD SS-A/Ro antigen was established by proteolytic peptide mapping of the cDNA-encoded protein and the 60-kD HeLa cell antigen. The sequence of the cDNA shows that the 60-kD SS-A/Ro protein possesses both RNA binding protein consensus sequences and a single zinc-finger motif. Recombinant SS-A/Ro antigen produced in bacteria proved to be a sensitive and specific reagent for detection of anti-SS-A/Ro antibodies in patient sera. The availability of the 60-kD SS-A/Ro cDNA will enable detailed analysis of the molecular structure and function of the SS-A/Ro RNP particle and its role in autoimmune pathology.
The expression of the new Ly108 isoform H1 weakens lupus-like disease of C57BL/6.Sle1b mice.
Studies of human systemic lupus erythematosus patients and of murine congenic mouse strains associate genes in a DNA segment on chromosome 1 with a genetic predisposition for this disease. The systematic analysis of lupus-prone congenic mouse strains suggests a role for two isoforms of the Ly108 receptor in the pathogenesis of the disease. In this study, we demonstrate that Ly108 is involved in the pathogenesis of lupus-related autoimmunity in mice. More importantly, we identified a third protein isoform, Ly108-H1, which is absent in two lupus-prone congenic animals. Introduction of an Ly108-H1–expressing transgene markedly diminishes T cell–dependent autoimmunity in congenic B6.Sle1b mice. Thus, an immune response–suppressing isoform of Ly108 can regulate the pathogenesis of lupus.
Systemic lupus erythematosus (SLE) is associated with the presence of complement proteins and immune complexes in affected organs. Since complement proteins are synthesized in hepatic and extrahepatic sites, we studied a murine model of SLE to ascertain the relative importance of local and humoral (liver) synthesis of complement. C3, C4, and C2 mRNA increase in kidney coincident with the development of nephritis in the MRL lpr/lpr mouse, a strain that spontaneously develops SLE. Two factor B messenger RNA transcripts are expressed in kidney and intestine; SLE nephritis is associated with decrease in the long factor B mRNA and increase in the short form. Increased local synthesis of C3 and B protein and a concomitant glomerular and renal interstitial macrophage infiltrate paralleled the increase in mRNA content in the (lpr/lpr) mice. In addition to kidney, an increase in C3, C4, C2 and factor B mRNA was noted in the lung, heart and intestine and to a lesser extent in liver of (lpr/lpr) in comparison to the MRL (+/+) animals. These results suggest that in SLE local expression of complement genes plays a role in the pathogenesis of chronic glomerulonephritis and in the autoimmune arteritis of other organs.
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the presence of pathogenic autoantibodies. Recent studies suggest that microRNAs (miRNAs) play an essential role in immunoregulation and may be involved in the pathogenesis of SLE. Therefore, it was of interest to investigate the potential therapeutic application of miRNAs in SLE, a concept that has not been thoroughly investigated thus far. Virus-like particles (VLPs) are a type of recombinant nanoparticle enveloped by certain proteins derived from the outer coat of a virus. Herein, we describe a novel miRNA-delivery approach via bacteriophage MS2 VLPs and investigate the therapeutic effects of miR-146a, a well-studied and SLE-related miRNA, in BXSB lupus-prone mice.
VLPs containing miR-146a, and the control VLPs, were prepared using an Escherichia coli expression system and then administered to lupus-prone mice over a 12-day period. We performed an enzyme-linked immunosorbent assay to evaluate the anti-dsDNA antibody, autoantibody to nuclear antigen (ANA), total IgG and total IgM levels in serum. The expression of miR-146a was analyzed by qRT-PCR. SLE-related cytokines as well as some toll-like receptor signaling pathway molecules were also measured.
Treatment with MS2-miR146a VLP showed profound effects on lupus-prone BXSB mice, including an increased level of mature miR-146a, which led to a significant reduction in the expression of autoantibodies and total IgG. Remarkably, these mice also exhibited reduced levels of proinflammatorycytokines, including IFN-Interferon-α (IFN-α), Interleukin-1β (Il-1β) and Interleukin-6 (Il-6). Moreover, we showed that the toll-like receptor pathway was involved in this regulation.
Restoring the loss of miR-146a was effective in eliminating the production of autoantibodies and ameliorating SLE progression in lupus-prone mice. Thus, the induction of dysregulated miRNAs by an MS2 VLP-based delivery system may lead to novel therapies.
systemic lupus erythematosus; anti-dsDNA antibody; autoantibody to nuclear antigen; Toll-like receptor; BXSB mice; gene therapy