MRL-lpr Mice Research Articles

In vivo depletion of microRNA-183-96-182 cluster alleviates autoimmunity in B6-lpr lupus mice

Abstract We have previously reported a significant upregulation of microRNA-183-96-182 cluster (miR-183C) in splenocytes of several murine lupus models. In this study, we found that the upregulation of miR-183C is inversely related to the expression of Foxo1 and Foxo3a, which play important roles in the regulation of T lymphocyte homeostasis and immune responses. Importantly, we found that inhibition of miR-183C with specific antagomirs significantly reduces lupus-related inflammatory cytokines expression such as IFNg, IL-6, and IL-17 in in vitro activated splenocytes from both MRL and MRL-lpr mice. We further showed that inhibition of miR-183C increases expression of Foxo1 and that inhibition of Foxo1 with siRNA increases IFNg and IL-6 in activated splenocytes from MRL mice. These data suggest that the miR-183C may regulate inflammatory response by targeting Foxo1. To further characterize the role of miR-183C in lupus pathogenesis, we developed B6-lpr mice with conditional depletion of miR-183C in T and B lymphocyte (miR-183C−/− B6-lpr) by serial crossbreeding of B6-lpr with miR-183Cfl/fl and hCD2-iCre mice. To our surprise, there was no significant difference in the production of IFNg and IL-6 in in vitro activated splenocytes from knock out (miR-183C−/− B6-lpr) mice when compared to that from control (miR-183Cfl/fl B6-lpr) mice. Impressively, we found that miR-183C−/− B6-lpr had reduced serum anti-dsDNA levels, spleen and lymphoid node weight when compared to controls. The histopathological analysis also revealed reduced inflammation in kidney in miR-183C−/− B6-lpr mice. Together, our results suggest a potential role of miR-183C in lupus pathogenesis and that miR-183C might be a new potential target for lupus therapy.

Development of a glomerular specific targeted nanoparticles for lupus nephritis

Lupus nephritis requires immunosuppressive therapy, which unfortunately, are associated with severe side effects. Thus, development of new treatment strategies which minimize the side effects while maintaining high therapeutic efficiency is essential. Perfluorocarbon (PFC) nanoparticles (NPs), with a nominal size of ~200 nm, are primarily limited to intravascular space. Collagen IV (col4) expresses in the glomerular basement membrane (GBM), where is the only place that col4 has direct contact with blood via fenestrated capillary endothelium. Accordingly, we designed a novel col4‐targeted NPs by coupling PFC NPs with amine‐carboxyl to a col4‐targeting ligand, which selectively target glomeruli by binding to col4 of GBM.The binding specificity and efficiency of the col4‐targeted NPs were evaluated on col4 surface, in vitro and in vivo. First, rhodamine labeled col4 targeted or non‐targeted PFC NPs were applied in col4 pre‐coated 96 wells plate at stepwise doses (1, 2, 5, 10 and 20 ul/ml). The binding affinity was determined by fluorescence intensity with IVIS. The wells incubated with col4 targeted PFC NPs showed a dose dependent fluorescence in radiant efficiency e10 (0.37 ± 0.03, 0.51 ± 0.05, 1.33 ± 0.07, 2.38 ± 0.19, 2.71 ± 0.28) (n=5, p<0.01 vs non‐targeted NP). Second, to evaluate the targeting properties in vitro, 1 μl/ml rhodamine labeled col4 targeted or non‐targeted NPs were applied on mouse primary glomerular endothelial cells and mouse primary mesangial cells for 2 h at 37°C. At the same confocal imaging settings, significant cellular uptake of the col4 targeted NPs was visualized, while it was not detectable in the cells incubated with non‐targeted NPs (n=3). Then, to evaluate the targeting properties in vivo, C57BL/6 mice were administered i.v. with 100 μl rhodamine labeled col4 targeted or non‐targeted NPs. At 24 hours after injections, col4 targeted NPs were selectively visualized at glomeruli in kidney sections, while non‐targeted NPs were barely detectable anywhere (n=3).Furthermore, the therapeutic effect of the col4 targeted NPs loaded with prednisone for lupus nephritis was evaluated in MRL‐lpr mice, a lupus‐prone animal model. The prednisone loaded col4 targeted NPs (1 ml/kg) were intravenously injected in MRL‐lpr mice (male, 8 weeks old) twice a week for 8 weeks. Compared with untreated animals, MRL‐lpr mice receiving therapeutic NPs exhibited less glomerular deposition of IgG (142±25 vs.193±18) and C3 (28±11 vs.41±13) evaluated by intensity of immunofluorescence (n=5, p<0.05 vs control), decreased proteinuria (54±17 vs.82±21 mg/dl) measured by with MultistixTM dipsticks (n=6, p<0.05 vs control), a better preservation of GFR (157±16 vs. 118±12 ml/min) assessed by clearance of plasma FITC‐inulin after a single intravenous bolus injection (n=6, p<0.01 vs control), and reduced glomerular pathology (1.3±0.8 vs. 2.4±0.6) evaluated by scoring 50 glomerular cross sections per kidney with periodic acid‐Schiff stain (n=5, p<0.05 vs control).In conclusion, we developed novel NPs that selectively target glomeruli by binding to col4 of GBM, which provide a novel tool to achieve precise therapeutics with minimum side effects for lupus nephritis.Support or Funding InformationNIH: DK099276 DK098582 HL137987 DK102691 AR067491 HL073646‐08ASN Fellowship Awards AHA: 18CDA34110441This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Cathepsin E gene expression by the transcription factor Kaiso in MRL/lpr mice derived CD4+ T cells

Global DNA hypomethylation in CD4+ cells in systemic lupus erythematosus (SLE) was suggested to play a key role in the pathogenesis. To identify new methylation-sensitive genes, we integrated genome-wide DNA methylation and mRNA profiling data in CD4+ cells of MRL/lpr (MRL) and C57BL6/J (B6) mice. We identified Cathepsin E (Ctse), in which 13 methyl-CpGs within 583 bp region of intron 1 were hypomethylated, and Ctse mRNA upregulated in MRL compared with B6 mice. One of methyl-CpGs, mCGCG was 93.3 ± 2.05% methylated in B6 mice, while 80.0 ± 6.2% methylated and mutated to CGGG in MRL mice. Kaiso is known to bind to mCGCG and we hypothesized that it represses expression of Ctse in B6 mice. The binding of Kaiso to mCGCG site in B6 mice was reduced in MRL mice revealed by ChIP-PCR. EL4 cells treated with 5-azaC and/or Trichostatin A showed the suppression of binding of Kaiso to mCGCG motif by ChIP-PCR and the overexpression of Ctse was demonstrated by qPCR. Ctse gene silencing by siRNA in EL4 cells resulted in reduction of IL-10 secretion. The hypomethylation of mCGCG motif, reduced recruitment of Kaiso, and increased expression of Ctse and Il-10 in CD4+ cells may be involved in the pathogenesis of SLE.

Procyanidin B2 prevents lupus nephritis development in mice by inhibiting NLRP3 inflammasome activation.

Lupus nephritis (LN) is a multifactorial event that contributes to the long-term mortality of systemic lupus erythematosus (SLE). Activation of NLRP3 inflammasome has been known to play a role in SLE pathogenesis. We evaluated the renal protection effects of procyanidin B2 (PCB2) and the involvement of NLRP3 in a mouse model involving MRL/lpr and MRL/MpJ mice. Kidney injury was evaluated by measuring the renal clinical and pathological features, renal immune complex deposition, and serum anti-double-stranded (anti-dsDNA) Abs. ELISA and Western blotting were used to detect NLRP3 inflammasome activation and IL-1β/IL-18 production. NLRP3 gene silencing was introduced into MRL/lpr mice by short hairpin RNA, and the renal damage was compared with the treatment of PCB2. PCB2 remarkably reduced renal damage in MRL/lpr mice, reflected by the reduced proteinuria, and serum levels of blood urea nitrogen and creatinine, as well as pathological features with less renal injury. PCB2 significantly reduced renal immune complex deposition and serum anti-dsDNA levels, notably inhibited the NLRP3 inflammasome activation, and reduced the renal and serum levels of IL-1β and IL-18 in MRL/lpr mice compared with those of NLRP3 gene-silenced MRL-lpr mice. PCB2 significantly suppressed LN in MRL-lpr mice by inhibiting the activation of NLRP3 inflammasome and subsequent IL-1β and IL-18 production. This finding explores a novel mechanism by which procyanidin exerts inflammatory suppression effects and its clinical benefits in LN prevention.

Tcell-derived IFN-γ downregulates protective group 2 innate lymphoid cells in murine lupus erythematosus.

Innate lymphoid cells (ILCs) are important regulators of the immune response and play a crucial role in the restoration of tissue homeostasis after injury. GATA-3+ IL-13- and IL-5-producing group 2 innate lymphoid cells (ILC2s) have been shown to promote tissue repair in barrier organs, but despite extensive research on ILCs in the recent years, their potential role in autoimmune diseases is still incompletely understood. In the present study, we investigate the role of ILC2s in the MRL/MpJ-Faslpr (MRL-lpr) mouse model for severe organ manifestation of systemic lupus erythematosus (SLE). We show that in these MRL-lpr mice, progression of lupus nephritis is accompanied with a reduction of ILC2 abundance in the inflamed renal tissue. Proliferation/survival and cytokine production of kidney-residing ILC2s was suppressed by IFN-γ and, to a lesser extent, by IL-27 which were produced by activated Tcells and myeloid cells in the nephritic kidney, respectively. Most importantly, restoration of ILC2 numbers by IL-33-mediated expansion ameliorated lupus nephritis and prevented mortality in MRL-lpr mice. In summary, we show here that development of SLE-like kidney inflammation leads to a downregulation of the renal ILC2 response and identify an ILC2-expanding therapy as a promising treatment approach for autoimmune diseases.

A(1-7) reduces pathologies associated with SLE in MRL-lpr mice

Abstract Patients with Systemic Lupus Erythematosus (SLE) suffer from a chronic inflammatory autoimmune disease that causes damage to various organ systems. Even with the array of current treatments, patients with lupus are still in need of effective therapies to treat this complex, multi-system disease. Recent studies of the Renin Angiotensin System (RAS), widely known for its role in blood pressure regulation, have uncovered immunomodulatory effects. One of the more recently discovered components of RAS, angiotensin (1-7) [A(1-7)], is a bioactive peptides that affects various systems. In our study, MRL-lpr mice were treated daily with saline or A(1-7) for 6 weeks starting at 8 weeks of age. Proteinuria was monitored throughout the duration of the study. MRL-lpr mice treated with saline had higher levels of proteinuria compared to the MRL/MpJ- control mice, and A(1-7) treated MRL-lpr mice. A glomerulonephritis score revealed that the MRL-lpr mice had significant glomerular pathologies compared to MRL-MpJ mice of the same age. A(1-7) treatment significantly reduced the glomerular pathologies in the MRL-lpr mice. Treatment with A(1-7) reduced the size of both inguinal and axillary lymph nodes in MRl-lpr mice, and levels of anti-dsDNA IgG compared to the saline treated MRL-lpr mice. Treatment with A(1-7) also significantly reduced the severity of cutaneous pathologies resulting in only mild inflammation and hair loss compared to the saline treated MRL-lpr mice. Daily systemic treatment with A(1-7) significantly ameliorated disease in one of the most severe mouse models of SLE, making Mas agonists a potential target for future SLE therapies.

A comprehensive phenotyping program for the MRL-lpr mouse lupus model

Abstract MRL/MpJ-Faslpr/J (MRL-lpr) mice develop an autoimmune disease resembling systemic lupus erythematosus, a disease affecting > 1.5 million Americans. The causative mutation, Faslpr, promotes survival of self-reactive lymphocytes, leading to immune proliferation, lymphadenopathy, emergence of anti-DNA antibodies, and fatal immune complex glomerulonephrosis. Like all spontaneous autoimmunity models, MRL-lpr colonies are susceptible to inadvertent selective pressure when mice with absent or delayed phenotypes are bred. We will explain a program for promoting genetic and phenotypic integrity within MRL-lpr breeding colonies. In addition to confirmation of genetic background and Faslpr genotype, breeder phenotypes were assessed at the conclusion of the breeding period. White blood cell counts (WBC) were used to measure lymphoproliferation: at 17 weeks of age, median WBC (103 cells/μL) was 14.2 for females (range = 3.4 – 97.3; N=155) and 10.5 for males (range = 3.7 – 51.4; N=125). Urinalysis to characterize renal damage at 18 weeks of age showed a median albumin/creatinine ratio of 623 for females (range = 1 – 22806; N=163) and 95 for males (range = 1 – 6013; N=123). Median spleen weights (grams) at 18 weeks of age were 0.5464 in females (range = 0.1112 – 2.1348; N=177) and 0.4410 in males (range = 0.2078 – 1.5516; N=135). We will describe a scoring system for clinical signs present at gross necropsy, including appearance of fur/skin, lymph nodes, and organs. We will show that phenotypic integrity can be ensured over time by breeding mice from lineages showing optimal disease development criteria. This program has value beyond breeder assessment, as it can be applied to MRL-lpr mice on study and at conclusion.

CD95 and the MRL-lpr Mouse Model.

CD95 (Fas-ligand) is a key mediator of cell death in multiple setting, thus its loss within the MRL-lpr (Faslpr) homozygote mice results in spontaneous autoimmunity. This is characterized by the development of arthritis and immune complex glomerulonephrosis making this strain a useful model for studying systemic lupus erythematosus. Herein we describe a method to exploit the heterozygote offspring of this strain in a model to study the effects of a CD95L blocking peptide on lupus-like disease in vivo.

Sex-specific effects of LiCl treatment on preservation of renal function and extended life-span in murine models of SLE: perspective on insights into the potential basis for survivorship in NZB/W female mice.

Considerable research effort has been invested in attempting to understand immune dysregulation leading to autoimmunity and target organ damage. In systemic lupus erythematosus (SLE), patients can develop a systemic disease with a number of organs involved. One of the major target organs is the kidney, but patients vary in the progression of the end-organ targeting of this organ. Some patients develop glomerulonephritis only, while others develop rapidly progressive end organ failure. In murine models of SLE, renal involvement can also occur. Studies performed over the past several years have indicated that treatment with LiCl of females, but not males of the NZB/W model, at an early age during the onset of disease, can prevent development of end-stage renal disease in a significant percentage of the animals. While on Li treatment, up to 80 % of the females can exhibit long-term survival with evidence of mild glomerulonephritis which does not progress to renal failure in spite of on-going autoimmunity. Stopping the treatment led to a reactivation of the disease and renal failure. Li treatment of other murine models of SLE was less effective and decreased survivorship in male BxSB mice, exhibited little effect on male MRL-lpr mice, and only modestly improved survivorship in female MRL-lpr mice. This perspective piece discusses the findings of several related studies which support the concept that protecting target organs such as the kidney, even in the face of continued immune insults and some inflammation, can lead to prolonged survival with retention of organ function. Some possible mechanisms for the effectiveness of Li treatment in this context are also discussed. However, the detailed mechanistic basis for the sex-specific effects of LiCl treatment particularly in the NZB/W model remains to be elucidated. Elucidating such details may provide important clues for development of effective treatment for patients with SLE, ~90 % of which are females.

Dysregulation of TCR signal fine-tuning molecules due to increased ubiquitination in systemic autoimmune disease

Abstract T cell selection is primarily determined by the avidity of T cell receptor (TCR) for self-ligand-MHC. Evidence suggests that besides the TCR, there are other signal regulating molecules that play a role in establishing signal threshold in T cells during the process known as TCR tuning, which modulates the intensity of TCR signaling. Here, we determined the expression of putative TCR signal fine-tuning molecules (TFT) in mice and humans with systemic autoimmune diseases, and investigated their role in T-cell signaling defects and mechanisms of their alteration. We found the expression levels of a battery of TFTs to be reduced on thymocytes in young MRL/lpr and MRL+/+ mice compared to C3H controls. MRL Thymocytes displayed signaling defects, which were corrected by TFT agonists. In the periphery, T-cells in the diseased MRL mice exhibited an unexpectedly drastic reduction in the TFTs, which correlated with an increased responsiveness to TCR stimulation and to weak antigenic ligands. TFT expression was also reduced in T cells from patients with rheumatoid arthritis as compared to controls, particularly in elderly patients who experience more severe disease than younger patients. The reduced expression of TFTs in MRL peripheral T cells was due to their increased ubiquitination, which led to degradation of these molecules. Blocking proteasomal degradation using MG132 restored their expression. Finally, MRL-lpr mice had increased E3 ubiquitin ligases that associated with the TFTs expression. Thus, the ability of T cells to alter their signal threshold by altering the expression of TFTs via their post-translational modification mediated by E3 ligases may be a novel mechanism for T cells to escape tolerance and trigger autoimmune disease.

The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation.

Epigenetic factors such as DNA methylation and microRNAs (miRNAs) are now increasingly recognized as vital contributors to lupus etiology. In this study, we investigated the potential interaction of these two epigenetic factors in lupus-prone MRL-lpr mice. We recently reported dysregulated expression of miRNAs in splenocytes of MRL-lpr mice. Here, we report that a majority of the upregulated miRNAs in MRL-lpr mice is located at the genomic imprinted DLK1-Dio3 domain. Further, we show a differential magnitude of upregulation of DLK1-Dio3 miRNA cluster in purified splenic CD4+ T, CD19+ B, and splenic CD4-CD19- cells from MRL-lpr lupus mice when compared to control MRL mice. MRL-lpr splenocytes (especially CD19+ and CD4-CD19- subsets) were hypomethylated compared to cells from control, MRL mice. We further show that deliberate demethylation of splenocytes from control MRL mice, but not from MRL-lpr lupus mice, with specific DNA methylation inhibitor 5-Aza-2’-deoxycytidine significantly augmented DLK1-Dio3 miRNAs expression. These findings strongly indicate that the upregulation of DLK1-Dio3 miRNAs in lupus splenic cell subsets is associated with reduced global DNA methylation levels in lupus cells. There was a differential upregulation of DLK-Dio3 miRNAs among various demethylated splenic cell subsets, which implies varied sensitivity of DLK1-Dio3 miRNA cluster in these cell subsets to DNA hypomethylation. Finally, inhibition of select DLK1-Dio3 miRNA such as miR-154, miR-379 and miR-300 with specific antagomirs significantly reduced the production of lupus-relevant IFNγ, IL-1β, IL-6, and IL-10 in lipopolysaccharide (LPS) activated splenocytes from MRL-lpr mice. Our study is the first to show that DNA methylation regulates genomic imprinted DLK1-Dio3 miRNAs in autoimmune lupus, which suggests a connection of DNA methylation, miRNA and genomic imprinting in lupus pathogenesis.

Phenotypic and functional alterations of pDCs in lupus-prone mice.

Plasmacytoid dendritic cells (pDCs) were considered to be the major IFNα source in systemic lupus erythematosus (SLE) but their phenotype and function in different disease status have not been well studied. To study the function and phenotype of pDCs in lupus-prone mice we used 7 strains of lupus-prone mice including NZB/W F1, NZB, NZW, NZM2410, B6.NZMSle1/2/3, MRL/lpr and BXSB/Mp mice and C57BL/6 as control mice. Increased spleen pDC numbers were found in most lupus mice compared to C57BL/6 mice. The IFNα-producing ability of BM pDCs was similar between lupus and C57BL/6 mice, whereas pDCs from the spleens of NZB/W F1 and NZB mice produced more IFNα than pDCs from the spleens of C57BL/6 mice. Furthermore, spleen pDCs from MRL-lpr and NZM2410 mice showed increased responses to Tlr7 and Tlr9, respectively. As the disease progressed, IFN signature were evaluated in both BM and spleen pDC from lupus prone mice and the number of BM pDCs and their ability to produce IFNα gradually decreased in lupus-prone mice. In conclusion, pDC are activated alone with disease development and its phenotype and function differ among lupus-prone strains, and these differences may contribute to the development of lupus in these mice.

Factor H uptake regulates intracellular C3 activation during apoptosis and decreases the inflammatory potential of nucleosomes.

Factor H (FH) binds apoptotic cells to limit the inflammatory potential of complement. Here we report that FH is actively internalized by apoptotic cells to enhance cathepsin L-mediated cleavage of endogenously expressed C3, which results in increased surface opsonization with iC3b. In addition, internalized FH forms complexes with nucleosomes, facilitates their phagocytosis by monocytes and induces an anti-inflammatory biased cytokine profile. A similar cytokine response was noted for apoptotic cells coated with FH, confirming that FH diminishes the immunogenic and inflammatory potential of autoantigens. These findings were supported by in vivo observations from CFH(-/-) MRL-lpr mice, which exhibited higher levels of circulating nucleosomes and necrotic cells than their CFH(+/+) littermates. This unconventional function of FH broadens the established view of apoptotic cell clearance and appears particularly important considering the strong associations with genetic FH alterations and diseases such as systemic lupus erythematosus and age-related macular degeneration.

Therapeutic targeting of BET protein BRD4 delays murine lupus

BRD4 is a member of the BET (bromodomain and extraterminal domain) family proteins that can bind acetylated histones and influence transcription, which are considered as potential therapeutic targets in many distinct diseases. And the BET inhibitor JQ1 has been proven to be effective in suppressing multiple inflammatory and autoimmune diseases. This study aimed to examine the therapeutic potential of JQ1 on a lupus model, MRL-lpr mice. Ten-week-old MRL-lpr mice were treated with JQ1 (oral administration of 200mg/kg) or vehicle for 8weeks. The proteinuria, nephritic damage, serum biochemistry, autoantibodies and cytokines were examined. Splenocytes of MRL-lpr mice were isolated for in vitro experiments. Treatment with JQ1 significantly attenuated the progression of proteinuria and nephritis. The serum concentrations of anti-dsDNA antibody as well as B-cell activating factor (BAFF), interleukin (IL)-1β, IL-6, IL-17 and INF-γ were inhibited, and IL-10 augmented by JQ1. Importantly, JQ1 improved the survival of lupus mice. In vitro, BAFF, IL-1β, IL-6, IL-17 and INF-γ were inhibited, and IL-10 augmented by JQ1 (500nM) in the cultures of splenocytes from diseased MRL-lpr mice, which was further supported by a significant reduction in immune complex-mediated activation of human monocytes in vitro by JQ1. Taken together, JQ1 effectively alleviates lupus in MRL-lpr mice by suppressing BAFF, pro-inflammatory cytokines and autoimmunity, supporting the therapeutic value of JQ1 in lupus disease.

Deletion of IL-18 Expression Ameliorates Spontaneous Kidney Failure in MRLlpr Mice.

The role of IL-18 in the pathogenesis of systemic lupus erythematosus is still not definitively solved. In this study, we generated MRLlpr mice, which develop a disease resembling systemic lupus erythematosus, genetically devoid of IL-18 expression. These mice in comparison to IL-18-competent MRLlpr mice show reduced signs of renal pathogenesis, while other parameters such as mean survival time, lymphadenopathy, constitutive interferon-γ production, and frequency of CD3+B220+ abnormal T cells were without differences. We conclude that in the systemic lupus erythematosus syndrom IL-18 is involved specifically in the renal pathogenesis.

Langerhans Cells Maintain Local Tissue Tolerance in a Model of Systemic Autoimmune Disease.

Systemic autoimmune diseases such as lupus affect multiple organs, usually in a diverse fashion where only certain organs are affected in individual patients. It is unclear whether the "local" immune cells play a role in regulating tissue specificity in relation to disease heterogeneity in systemic autoimmune diseases. In this study, we used skin as a model to determine the role of tissue-resident dendritic cells (DCs) in local and systemic involvement within a systemic lupus disease model. Skin-resident DCs, namely, Langerhans cells (LCs), have been implicated in regulating tolerance or autoimmunity using elegant transgenic models, however, their role in local versus systemic immune regulation is unknown. We demonstrate that although lymphocytes from skin-draining lymph nodes of autoimmune-prone MRL/MpJ-Fas(lpr/lp) (r) (MRL-lpr) mice react spontaneously to a physiological skin self-Ag desmoglein-3, epicutaneous applications of desmoglein-3 induced tolerance that is dependent on LCs. Inducible ablation of LCs in adult preclinical MRL-lpr and MRL/MpJ-Fas(+/+) mice resulted in increased autoantibodies against skin Ags and markedly accelerated lupus dermatitis with increased local macrophage infiltration, but had no effect on systemic autoantibodies such as anti-dsDNA Abs or disease in other organs such as kidneys, lung, and liver. Furthermore, skin-draining lymph nodes of LC-ablated MRL-lpr mice had significantly fewer CD4(+) T cells producing anti-inflammatory cytokine IL-10 than LC-intact controls. These results indicate that a skin-resident DC population regulates local tolerance in systemic lupus and emphasize the importance of the local immune milieu in preventing tissue-specific autoimmunity, yet have no effect on systemic autoimmunity.

The upregulation of genomic imprinting DLK1-Dio3 miRNAs in lupus-prone mice is associated with DNA hypomethylation (BA4P.133)

Abstract Epigenetic factors such as DNA methylation and microRNAs (miRNAs) regulation are now increasingly recognized as vital contributors to lupus pathogenesis. Our recent studies revealed a large cluster of miRNAs, which is located at the genomic imprinted DLK1-Dio3 domain was dramatically increased in splenocytes from MRL-lpr mice when compared to control MRL mice. In this study, we found that MRL-lpr mice have reduced DNA methylation levels in whole splenocytes and also in purified splenic CD4+ T and CD19+ B cells when compared to MRL control, which was associated with the upregulation of DLK1-Dio3 miRNAs in lpr lupus cells. DNA demethylation treatment with 5-Aza-2’-deoxycytidine significantly increased DLK1-Dio3 miRNAs in splenic cells (especially Con A-activated cells) from MRL mice, but not MRL-lpr mice. This data further confirmed the contribution of DNA hypomethylation to the augmented expression of DLK1-Dio3 miRNAs in lupus splenic cells. Although the contribution of DLK1-Dio3 miRNAs to autoimmune disease remains unknown, our preliminary data revealed that inhibition of selected DLK1-Dio3 miRNA such as miR-379 reduced LPS-induced IFNγ in MRL-lpr splenocytes, suggesting a role of miR-379 in the regulation of inflammation. Together, our data is the first to show DNA methylation regulation of genomic imprinting DLK1-Dio3 miRNAs in autoimmune lupus, which may provide novel insight into epigenetic mechanisms in lupus pathogenesis.

Tissue-resident specialized T cells promote the migration of local tissue dendritic cells: immune regulation at the local tissue level (CAM4P.155)

Abstract Dendritic cells (DC) that primarily reside in tissues carry antigens to local lymphoid organs to induce immunity or tolerance. We posit that a defect in the migration of tissue-DCs may predispose to autoimmunity in a tissue-specific manner; and an improved migration of tissue-DC may ameliorate disease in the respective organ. Here, we demonstrate that treatment of lupus dermatitis-prone MRL-lpr and MRL+/+ mice that exhibit a profound defect in the migration of skin-DC with a glycolipid αGalCer reduced the severity of dermatitis and enhanced the migration of skin-DCs, more so in Langerhans cells (LC; p=0.004) than in Lang+dDC (p=0.03). This effect of αGalCer was independent of its effect on iNKT cells, but required the presence of CD1d. Furthermore, αGalCer treatment enhanced the numbers of epidermis-resident γδ T cells in MRL-lpr mice that had reduced numbers of γδ T cells as compared to MHC-matched control mice. Finally, gd T cell-deficient mice had reduced skin-DC migration, and isolated skin-gd T cells directly promoted the migration of LC via CD40L-CD40 interaction. These data elucidate a new mechanism of regulation of skin-DC homeostasis whereby skin-gd T cells normally facilitate LC migration from skin to cutaneous lymph node. Such ‘local’ control of migratory behavior of tissue-DC can regulate immune response in a tissue-specific manner. This mechanism of skin-DC homeostasis is disrupted in lupus dermatitis, but can be repaired by treatment with a glycolipid.

IgM autoantibodies: Roquin and Bob1ng to a different tune

Pathogenic autoantibodies directed against ubiquitous self-antigens such as immunoglobulin-G (IgG) anti-double-stranded DNA (anti-dsDNA) antibodies in systemic lupus erythematosus are produced by plasma cells that have undergone immunoglobulin isotype switching and affinity maturation in the germinal centre (GC).1 These autoantibodies form circulating immune complexes that are often deposited in the skin, kidneys and joints where they activate complement to cause tissue damage. In contrast, IgM (and IgA) natural autoantibodies are produced in the steady-state from birth by plasma cells derived from B1 cells, and are ‘hardwired’ to be polyreactive, low-affinity antibodies with relatively low pathogenic potential.2, 3 Nevertheless, IgM autoantibodies bound to antigen are still capable of activating the classical complement pathway and causing diseases such as autoimmune haemolytic anaemias due to cold agglutinins. In this issue, Corcoran and coworkers4 remind us of this by showing that sanroque mice harbouring mutations in Rc3h1/Roquin1 develop a GC-independent IgM-mediated autoimmune disease when crossed with mice deficient for the transcription factor Obf1/Ocab/Bob1 (Figure 1). These IgM anti-dsDNA antibodies are deposited in the kidneys where they cause glomerulonephritis. This remarkable observation adds to the growing list of insights into the regulation of autoantibody responses in the sanroque ENU-mutant mouse model, which is characterized by excessive follicular T helper (Tfh) cell activity and spontaneous GC development.5 Previous studies using this model have focussed on the role of specific genes and cellular interactions involved in the production of GC-dependent autoantibodies, such as the Bcl6, Sh2d1a, Cd28 and Icos.6, 7 However, the current study neatly turns this on its head by showing that sanroque mice develop an even more severe fatal autoimmunity when Obf1 is also deficient and they are unable to form GCs and secrete IgG autoantibodies. This unexpected finding is all the more intriguing given that Obf1 deficiency by itself does not cause autoimmunity, and can also prevent the GC-dependent development of lupus-like autoimmune diseases in aged Ailos−/− 8 and MRL-lpr mice.9

Toll-Like Receptor 2 or Toll-Like Receptor 4 Deficiency Does Not Modify Lupus in MRLlpr Mice

Systemic lupus erythematosus is an autoimmune disease with a high morbidity and nephritis is a common manifestation. Previous studies in murine lupus models have suggest a role for Toll-like receptor 2 and 4. We examined the role of these molecules in MRL lpr mice which is one of the most established and robust murine models. We compared disease parameters in Toll-like receptor 2 or Toll-like receptor 4 deficient mice with their littermate controls. We found no difference in the severity of glomerulonephritis as assessed by histology, serum creatinine and albuminuria when Toll-like receptor 2 or Toll-like receptor 4 deficient MRLlpr mice were compared with Toll-like receptor sufficient controls. We also found similar levels of anti-dsDNA and anti-ssDNA antibodies. These results show that Toll-like receptor 2 and Toll-like receptor 4 do not play a significant role in MRLlpr mice, and therefore they may not be important in human lupus.