Lupus Pathogenesis Research Articles

Estrogen Receptor α Deficiency Modulates TLR Ligand-Mediated PDC-TREM Expression in Plasmacytoid Dendritic Cells in Lupus-Prone Mice.

Female lupus-prone NZM2410 estrogen receptor α (ERα)-deficient mice are protected from renal disease and have prolonged survival compared with wild-type littermates; however, the mechanism of protection is unknown. Plasmacytoid dendritic cells (pDCs) and type I IFN drive lupus pathogenesis. Estrogen acting via ERα enhances both pDC development and IFN production. The objectives for this study were to determine if ERα modulates pDC function and IFN activity in predisease NZM2410 mice as a possible protective mechanism of ERα deficiency in lupus-prone mice. We measured the effect of ERα deficiency on spleen pDC frequency, number, maturation, and activation state. ERα deficiency reduced type I IFN activity and the frequency of MHC class II(+) pDCs in the spleen without altering overall pDC frequency, number, or maturation state. Additionally, ERα-deficient NZM2410 mice had a significantly decreased frequency of pDCs expressing PDC-TREM, a modulator of TLR-mediated IFN production. After in vitro TLR9 stimulation, ERα deficiency significantly reduced the expression of PDC-TREM on pDCs from both NZM2410 and C57BL/6 mice. Thus, we have identified a significant effect of ERα deficiency on pDCs in predisease NZM2410 mice, which may represent a mechanism by which ERα deficiency protects NZM2410 mice from lupuslike disease.

Extended Antiphospholipid Antibodies Screening in Systemic Lupus Erythematosus Patients

The antiphospholipid syndrome (APS) is one of the most encountered autoimmunity in systemic lupus erythematosus (SLE) patients and pathogenesis of these two seems to be intricate. To investigate the association of antiphospholipid antibodies (APLAs) titer with the presence of secondary APS diagnosis in SLE patients. 65 patients fulfilling the 2012 Systemic Lupus Collaborating International Clinics (SLICC) SLE's criteria were included. The APS diagnosis was sustained according to the 2006 Sydney APS's criteria. Three groups of patients were defined: SLE patients with secondary APS, SLE with history of positive "criteria" APLAs but without APS clinical features, respectively SLE patients without positive APLAs or clinical APS criteria. An extended APLAs panel was searched in all cases: both IgM and IgG of anticardiolipin antibodies (aCL), anti-P2 glycoprotein I antibodies (aβ2GPI), antiphosphatidylethanolamine antibodies (aPE), antiphosphatidylserine antibodies (aPS), respectively antiprothrombin antibodies (aPT). Results. Only the aβ2GPI, both IgM and IgG serotypes, had significantly higher titers in patients with SLE and secondary APS compared to no APS (with/ without positive APLAs): median (min; max) 7.0 (0.0-300.0) vs. 1.0 (0.0-28.0) vs. 1.0 (0.0-12.0), respectively 3.0 (0.0-79.0) vs. 1.0 (0.0-3.0) vs. 1.0 (0.0-12.0) (p<0.001, Kruskal-Wallis test)]. Also, in regression logistic models, only the aβ2 GPI (IgG and IgM ) were identified as risk factors for secondary APS diagnosis in the SLE patients: OR(95%CI) 5.9 (2.2-15.7), respectively 1.3 (1.1-1.5). In regard with the SLE markers, the IgG serotypes of the "non-criteria" APLAs analyzed (aPS, aPT, aPE) were correlated with the antiDNA titers while the IgM serotypes inversely associated with the complement C3 levels. IgG aβ2 GPI are accompanied by almost 6-fold increase risk of secondary APS when screening SLE patients. On the contrary, the "non-criteria" APLAs do not seem associated with the APS diagnosis in SLE patients. Some correlates of the "non-criteria" APLAs with the antiDNA and complement C3 levels were also observed.

Predominant Role of Plasmacytoid Dendritic Cells in Stimulating Systemic Autoimmunity.

Plasmacytoid dendritic cells (pDCs), which are prominent type I interferon (IFN-I)-producing immune cells, have been extensively implicated in systemic lupus erythematosus (SLE). However, whether they participate critically in lupus pathogenesis remains unknown. Recent studies using various genetic and cell type-specific ablation strategies have demonstrated that pDCs play a pivotal role in the development of autoantibodies and the progression of lupus under diverse experimental conditions. The findings of several investigations highlight a notion that pDCs operate critically at the early stage of lupus development. In particular, pDCs have a profound effect on B-cell activation and humoral autoimmunity in vivo. This deeper understanding of the vital role of pDCs in lupus pathogenesis supports the therapeutic targeting of the pDC-IFN-I pathway in SLE.

Prednisone treatment inhibits the differentiation of B lymphocytes into plasma cells in MRL/MpSlac-lpr mice.

A number of evidence shows that the differentiation of B lymphocytes into plasma cells plays an important role in lupus pathogenesis. In this study we investigated how prednisone, a classical therapeutic drug for autoimmune diseases, regulated plasma cell differentiation in MRL/MpSlac-lpr mice. MRL/lpr mice were treated with prednisone (2.5 or 5 mg·kg(-1)·d(-1), ig) for 13 weeks, and the proteinuria levels and survival times were monitored. After the mice were euthanized, blood sample, spleen and thymus were collected. The serum levels of anti-dsDNA antibody, anti-nuclear antibody, IL-21, and IL-10 were detected using ELISA kits. Subsets of splenic B and T lymphocytes were quantified with flow cytometry. Transcription factor Blimp-1 and Bcl-6 expression was determined using qPCR and Western blot. Prednisone treatment dose-dependently attenuated the lupus symptoms in MRL/lpr mice with decreased proteinuria levels, prolonged survival times, decreased serum anti-nuclear antibody levels, and reduced spleen and thymus indices. Prednisone treatment also significantly decreased the elevated percentages of plasma cells and plasma cell precursors, decreased the percentages of activated T cells, and increased the frequency of CD4(+)CD62L(+) cells, demonstrated that decreased anti-nuclear antibodies and improvements in lupus symptoms were associated with decreased plasma cells. Furthermore, prednisone treatment decreased serum IL-21 and IL-10 levels and reduced the expression of splenic Blimp-1 and Bcl-6 (two key regulatory factors for plasma cell differentiation) in MRL/lpr mice. Prednisone treatment restricts B lymphocyte differentiation into plasma cells in MRL/lpr mice, which may be correlated with the inhibition of IL-21 production and the restoration of the balance between Blimp-1 and Bcl-6.

Genome-wide differential expression reveals candidate genes involved in the pathogenesis of lupus and lupus nephritis.

Systemic lupus erythematosus (lupus) is an autoimmune disease characterized by multiorgan pathology, accelerated apoptosis and hyper-autoantibody production against self-components. The root cause of lupus remains unknown, although multiple susceptibility factors have been reported in different ethnic group. We aimed to explore the genome-wide differential expression spectrum of lupus and its severe form lupus nephritis (LN) in Arab females. A total of 98 subjects: 40 lupus, 18 LN and 40 age/gender/ethnically matched healthy controls (HC) were recruited. Carefully chosen subjects (n = 11) were employed for whole human-genome expression profiling using high-density Human Exon 1.0.ST arrays (Affymetrix) and statistical analysis was carried out using appropriate software. Validation cohorts (n = 98) were investigated to quantify the expression of the nine selected candidate genes relative to GAPDH as endogenous control. Genome-wide differential analysis revealed seven candidate genes in lupus and 36 in LN, when individually compared to HC (anova Welch t-test, P ≤ 0.005, Tukey's honestly post hoc analysis). Analysis of differentially expressed genes with a fold change of 2, revealed 16 Gene Ontology terms satisfying a P ≤ 0.05. We further detected five distinct inflammatory and metabolic pathways such as TWEAK, osteopontin, endochondral ossification, fluropyrimidine activity and urea cycle and metabolism of amino groups that significantly contribute to the pathogenesis of lupus (P < 0.05). Validation of selected candidate genes (IRF9, ABCA1, APOBEC3, CEACAM3, OSCAR, TNFA1P6, MMP9, SLC4A1) revealed significant differences in expression, indicating their promissory role in the pathogenesis of lupus. Our study provides central gene regulators of therapeutic potential, indicating the future prospects of the study.

Role of altered mitochondria functions in the pathogenesis of systemic lupus erythematosus.

Mitochondria, main producers of reactive-oxygen species (ROS), were studied to examine their role in the pathogenesis of systemic lupus erythematosus (SLE). PBMCs and mitochondria were isolated from SLE patients and healthy volunteers for various parameters. Mitochondrial ROS, swelling, hyperpolarization and levels of cytochrome c, caspase3 in the cells were assessed by flow cytometry. ROS was significantly increased in SLE patients (SLE vs controls: 1.83 ± 1.03 vs 1.10 ± 0.35; p < 0.0001). Depolarized state of mitochondria was greater in patients (SLE vs controls: 7.10 ± 5.50% vs 2.5 ± 1.8%; p < 0.05). Mitochondria swelling was found to be significantly altered in patients (SLE vs controls: 112.65 ± 36.56 vs 60.49 ± 20.69; p < 0.001). Expression of cytochrome c and caspase 3 (SLE vs controls: 1.37 ± 0.37% vs 1.01 ± 0.03%; 1.57 ± 0.46% vs 1.06 ± 0.07%; p < 0.05) respectively was found to be significantly increased in SLE. Further, the enzymatic activity of mitochondrial complex was assessed in isolated mitochondria. A significant decrease in activity of Complex I (SLE vs controls: 11.79 ± 3.18 vs 15.10 ± 6.38 nmol NADH oxidized/min/mg protein, p < 0.05); Complex IV (SLE vs control: 9.41 ± 5.16 vs 13.56 ± 5.92 nmol cytochrome c oxidized/min/mg protein, p < 0.05) and Complex V (SLE vs controls: 4.85 ± 1.39 vs 6.17 ± 2.02 nmol ATP hydrolyzed/min/mg protein, p < 0.05) was found in SLE patients in comparison to healthy controls. However, Complex II did not show significant variation in either group (SLE vs controls: 42.2 ± 28.6 vs 61.71 ± 42.3 nmol succinate oxidized/min/mg protein; ns). The decrease in enzyme activities of mitochondrial Complexes I, IV and V on one hand and ROS, hyperpolarization and apoptosis on the other points toward a possible role of mitochondria in the pathogenesis of lupus.

An Lck-cre transgene accelerates autoantibody production and lupus development in (NZB × NZW)F1 mice.

Lupus is an autoimmune disease characterized by the development of antinuclear autoantibodies and immune complex-mediated tissue damage. T cells in lupus patients appear to undergo apoptosis at an increased rate, and this enhanced T cell apoptosis has been postulated to contribute to lupus pathogenesis by increasing autoantigen load. However, there is no direct evidence to support this hypothesis. In this study, we show that an Lck-cre transgene, which increases T cell apoptosis as a result of T cell-specific expression of cre recombinase, accelerates the development of autoantibodies and nephritis in lupus-prone (NZB × NZW)F1 mice. Although the enhanced T cell apoptosis in Lck-cre transgenic mice resulted in an overall decrease in the relative abundance of splenic CD4(+) and CD8(+) T cells, the proportion of activated CD4(+) T cells was increased and no significant change was observed in the relative abundance of suppressive T cells. We postulate that the Lck-cre transgene promoted lupus by enhancing T cell apoptosis, which, in conjunction with the impaired clearance of apoptotic cells in lupus-prone mice, increased the nuclear antigen load and accelerated the development of anti-nuclear autoantibodies. Furthermore, our results also underscore the importance of including cre-only controls in studies using the cre-lox system.

The role of neutrophils in the pathogenesis of systemic lupus erythematosus.

Recent discoveries implicate neutrophils as important regulators of innate and adaptive immunity and in the development of organ damage in systemic autoimmune diseases, including systemic lupus erythematosus (SLE). Various putative SLE biomarkers are neutrophil-related, including neutrophil granular proteins and histones undergoing post-translational modifications during neutrophil extracellular trap (NET) formation. In the bone marrow, lupus neutrophils can drive B and T cell abnormalities, at least in part, by their enhanced production of type-I interferons, tumor necrosis factor-alpha (TNFα) and the B-cell stimulating factors B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Lupus neutrophils and, in particular, lupus low-density granulocytes (a distinct pathogenic subset) display epigenetic modifications and genomic alterations that may be relevant to their deleterious roles in SLE. Proteins and enzymes externalized by lupus NETs can affect vascular health by inducing endothelial apoptosis and oxidizing lipoproteins. Hampering NET formation through peptidylarginine deiminase inhibitors abrogates lupus phenotype and atherosclerosis in murine studies. Recent discoveries support the notion that neutrophils, low-density granulocytes and aberrant NET formation and clearance play important roles in lupus pathogenesis. Future studies should focus on how to selectively target these immunostimulatory pathways in this disease.

The Role of Cell Death in the Pathogenesis of SLE: Is Pyroptosis the Missing Link?

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by the production of antinuclear antibodies (ANAs) in association with systemic inflammation and organ damage. In addition to genetic factors, a contribution of infection to disease induction has been proposed. In the pathogenesis of lupus, immune complexes of ANAs with nuclear antigens can form and both deposit in the tissue and stimulate cytokine production to intensify inflammation. As such, the extracellular release of nuclear antigens to form pathogenic immune complexes is an important step in the pathway to disease. This release has been considered the consequence of cell death, with apoptotic cells the relevant source of nuclear material. While apoptosis could serve this role, other death forms may act similarly. Among these death forms, pyroptosis, which can be induced by inflammasome activation during infection, has features suggesting involvement in lupus. Thus, unlike apoptosis, pyroptosis is a pro-inflammatory process. Furthermore, pyroptosis leads to the release of intracellular contents including HMGB1 and ATP, both of which can act as DAMPs (deathassociated molecular patterns) to stimulate further inflammation. Importantly, pyroptosis can lead to the release of intact nuclei, suggesting a relationship to events in the formation of LE cells. While the role of pyroptosis in SLE is hypothetical at this time, further analysis of this death form should provide new insights into lupus pathogenesis and provide the missing link between infection and the initiation of lupus by products of dead and dying cells.

TWEAK/Fn14 Signaling Involvement in the Pathogenesis of Cutaneous Disease in the MRL/lpr Model of Spontaneous Lupus

TNF-like weak inducer of apoptosis (TWEAK, TNFSF12) and its sole receptor Fn14, belonging to the TNF ligand and receptor superfamilies respectively, are involved in cell survival and cytokine production. The role of TWEAK/Fn14 interactions in the pathogenesis of cutaneous lupus has not been explored. TWEAK treatment of murine PAM212 keratinocytes stimulated the secretion of RANTES via Fn14, and promoted apoptosis. Parthenolide, but not wortmanin or the MAPK inhibitor PD98059, significantly decreased production of RANTES, indicating that this effect of TWEAK is mediated via NF-κB signaling. Ultraviolet-B irradiation significantly upregulated the expression of Fn14 on keratinocytes in vitro and in vivo, and increased RANTES production. MRL/lpr Fn14 knockout lupus mice were compared with MRL/lpr Fn14 wild-type mice to evaluate for any possible differences in the severity of cutaneous lesions and the presence of infiltrating immune cells. MRL/lpr Fn14 knockout mice had markedly attenuated cutaneous disease as compared to their Fn14 wild-type littermates, as evidenced by the well maintained architecture of the skin and significantly decreased skin infiltration of T cells and macrophages. Our data strongly implicate TWEAK/Fn14 signaling in the pathogenesis of the cutaneous manifestations in the MRL/lpr model of spontaneous lupus, and suggest a possible target for therapeutic intervention.

A CD153+CD4+ T follicular cell population with cell-senescence features plays a crucial role in lupus pathogenesis via osteopontin production.

Immune aging results in diminished adaptive immunity and increased risk for autoimmunity. We previously reported a unique PD-1(+) CD44(high)CD4(+) T cell population that increases with age in normal mice. In this study, we indicate that the age-dependent PD-1(+) CD44(high)CD4(+) T cells develop as unique T follicular (TF) cells in a B cell-dependent manner and consist of two subpopulations, as follows: CD153(+) cells preferentially secreting abundant osteopontin on TCR stimulation and CD153(-) cells that are apparently TCR anergic. These unique TF cells with essentially similar features increase much earlier and are accumulated in the spontaneous germinal centers (GCs) in lupus-prone female BWF1 (f-BWF1) mice. These TF cells showed characteristic cell-senescence features and developed in association with extensive CD4(+) T cell proliferation in vivo, suggesting replicative senescence. Although the CD153(+) TF cells were defective in proliferation capacity, they were quite stable and specifically responded to self GC-B cells to secret abundant osteopontin, which inhibited B cell receptor-induced GC-B cell apoptosis in f-BWF1 mice. Transfer of CD153(+) PD-1(+) CD4(+) T cells promoted the growth of spontaneous GCs, whereas administration of anti-osteopontin Ab suppressed GC enlargement and anti-nuclear Ab production and ameliorated clinical lupus nephritis of f-BWF1 mice. Current results suggest that senescent CD153(+) TF cells generated as a consequence of extensive endogenous CD4(+) T cell proliferation play an essential, if not sufficient, role in lupus pathogenesis in lupus-prone genetic background and may also contribute to an increased autoimmunity risk with age.

Roles of B Cell-Intrinsic TLR Signals in Systemic Lupus Erythematosus.

Toll-like receptors (TLRs) are a large family of pattern recognition receptors. TLR signals are involved in the pathogenesis of systemic lupus erythematosus. Mouse and human B cells constitutively express most TLRs. Many B cell subpopulations are highly responsive to certain TLR ligation, including B-1 B cells, transitional B cells, marginal zone B cells, germinal center B cell and memory B cells. The B cell-intrinsic TLR signals play critical roles during lupus process. In this review, roles of B cell-intrinsic TLR2, 4, 7, 8 and 9 signals are discussed during lupus pathogenesis in both mouse model and patients. Moreover, mechanisms underlying TLR ligation-triggered B cell activation and signaling pathways are highlighted.

OX40 Ligand Contributes to Human Lupus Pathogenesis by Promoting T Follicular Helper Response

Increased activity of T follicular helper (Tfh) cells plays a major pathogenic role in systemic lupus erythematosus (SLE). However, the mechanisms that cause aberrant Tfh cell responses in SLE remain elusive. Here we showed the OX40 ligand (OX40L)-OX40 axis contributes to the aberrant Tfh response in SLE. OX40L was expressed by myeloid antigen-presenting cells (APCs), but not B cells, in blood and in inflamed tissues in adult and pediatric SLE patients. The frequency of circulating OX40L-expressing myeloid APCs positively correlated with disease activity and the frequency of ICOS(+) blood Tfh cells in SLE. OX40 signals promoted naive and memory CD4(+) Tcells to express multiple Tfh cell molecules and were sufficient to induce them to become functional B cell helpers. Immune complexes containing RNA induced OX40L expression on myeloid APCs via TLR7 activation. Our study provides a rationale to target the OX40L-OX40 axis as a therapeutic modality for SLE.

Lupus erythematosus

Lupus erythematosus is an autoimmune disease with a broad spectrum of cutaneous manifestations. The pathogenesis of lupus is based on a loss of tolerance against self antigens and can be mediated by defects in apoptosis, defects in eliminating cellular remnants and increased activation of the innate as well as the adaptive immune system. The increased activation of the innate immune system can be mediated by sensing of endogenous or exogenous nucleic acids, genetic variants in the components of the receptor cascade or disturbances in restriction of self nucleic acids. The inflammatory milieu is characterized by type I interferon expression and autoantibody production. The main trigger factors of the disease are sun exposure and viral infections. Lupus erythematosus is effectively treated by glucocorticosteroids. Approved alternatives for long-term treatment are antimalarial agents and the B-cell inhibitor belimumab for patients with systemic lupus erythematosus. Future studies should more intensely analyse the effect of novel therapies on cutaneous manifestations to allow early detection of cutaneous lupus. Furthermore novel therapeutic strategies which specifically target the responsible pathogenetic mechanisms of the individual subtypes of lupus erythematosus are needed to improve the therapeutic success for this heterogeneous patient population.

The endothelial deprotection hypothesis for lupus pathogenesis: the dual role of C1q as a mediator of clearance and regulator of endothelial permeability.

Systemic lupus erythematosus (SLE) is a heterogeneous multifactorial systemic autoimmune disease affecting several organs. SLE can start relatively early in life and results in impaired quality of life and shortened life expectancy because of a gradual disease progression leading to cardiovascular, renal and neoplastic disease. The basic mechanisms of the pathogenesis of the disease still remain to be clarified. It is clear that complement proteins play a key and complex role in the development of SLE. Complement component C1q has been known to be a fundamental component of lupus development, but most explanations focus on its role in apoptotic debris removal. Importantly, C1q was recently found to play a key role in the maintenance of vascular endothelial integrity. We suggest that apoptotic products, endothelial cells and extracellular matrix components, which display negatively charged moieties, compete for binding to molecules of the innate humoral immune response, like C1q. Genetic or acquired factors leading to an increased load of apoptotic cell debris and decrease or absence of C1q therefore interfere with the regulation of endothelial permeability and integrity. Furthermore, we suggest that lupus is the net result of an imbalance between the two functions of immune clearance and vascular endothelial integrity maintenance, an imbalance triggered and sustained by autoimmunity, which skews C1q consumption by IgG-mediated complement classical pathway activation on autoantigens. In this triangle of innate clearance, autoimmunity and endothelial integrity, C1q plays a central role. Hence, we interpret the pathogenesis of lupus by identifying three key components, namely innate immune clearance, autoimmunity and endothelial integrity and we establish a link between these components based on the protective role that innate clearance molecules play in endothelial renewal. By including the vasoprotective role of C1q in the interpretation of SLE development we attempt to provide novel explanations for the symptoms, organ damage, diagnostic and therapeutic difficulties of the disease.

STAT3 signaling is essential for the germinal center maintenance and pathogenesis of lupus (BA4P.122)

Abstract Antibody maturation as well as memory B and plasma cell differentiation occurs primarily in the germinal center (GC). Lupus may develop as a result of enhanced GC activity. Many factors involved in GC reaction, including T follicular helper (Tfh) cells, IL-21, IL-6, play critical roles in lupus pathogenesis. These inflammatory cytokines predominantly activate the STAT3 signaling pathway. Here, we demonstrate that STAT3 signaling in B cells is essential for antibody response as well as GC formation and maintenance. Increased cell apoptosis and down-regulated Bcl-xL expression are found in the STAT3 deficient GC B cells. The Tfh cell response is significantly decreased in B cell STAT3-deficient immunized mice. Further, STAT3 deficiency in autoreactive B cells results in decreased autoantibody production upon immunization with apoptotic cells. Results obtained using B cell STAT3 deficient MRL.Fas(lpr) mice suggest that STAT3 signaling contributes to the pathogenesis of lupus by regulation of autoantibody production. Our findings thus provide new insights into the role of B cell STAT3 signaling in the GC maintenance and pathogenesis of lupus.

Venetoclax (ABT-199), a potent and selective BCL-2 inhibitor, is efficacious in NZB/WF1 mouse model of lupus nephritis and reduces human lymphocyte lifespan in vitro (BA4P.127)

Abstract Proteins in the BCL-2 family are key regulators of apoptosis, or programmed cell death. We have conducted studies to evaluate a highly potent and orally available BCL-2 selective inhibitor, venetoclax (ABT-199), for efficacy and mechanism of action in the lupus prone NZB/W F1 mouse model and in human cells in vitro. Venetoclax treatment dose-dependently reduced the incidence of severe proteinuria and prolonged survival compared to vehicle controls and attenuated glomerulonephritis, tubular dilatation, immune cell infiltrates and IgG deposition in the kidney. Venetoclax mediated a significant reduction in the numbers of splenic T cells but conferred a preferential reduction in select B cell subsets. Interestingly, venetoclax did not impair the number of CD138+ long-lived plasma cells in the bone marrow, which was consistent with unaltered circulating anti-dsDNA titers in these animals. Venetoclax efficacy also correlated with a dose -dependent reduction of lymphocytes in peripheral blood of NZB/WF1 mice. Consistent with these findings, human lymphocytes from both healthy donors and systemic lupus erythematosus (SLE) patients treated with venetoclax in vitro have reduced lifespan. Taken together, these data underscore the essential role of BCL-2 in the pathogenesis of lupus and support further exploration of selective BCL-2 inhibition in autoimmune diseases such as SLE/Lupus Nephritis. The design, study conduct, and financial support for this research were provided by AbbVie.

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.

Editorial introductions

Editorial introductions

RGC-32 enhances suboptimal CD8 cytotoxic T cell effector function in acute graft versus host disease and promotes lupus-like disease in chronic graft versus host disease (THER2P.965)

Abstract Response Gene to Complement (RGC)-32 is an intracytoplasmatic protein expressed in a variety of cells in response to sublytic complement activation, TGFb and other cytokines. It plays a role in cell growth, fibroblast activation and renal fibrosis, Th17 and plasma cell differentiation. To assess whether RGC-32 expression promotes lupus-like disease, we used WT and RGC-32 KO mice to investigate whether lack of RGC-32 in host cells affects parameters of chronic (c)GVHD in the Bm12→B6 model and whether lack of RGC-32 in donor T cells affects parameters of acute (a)GVHD in the P→F1 model. In F1 mice injected with RGC-32 KO splenocytes, an attenuated phenotype of aGVHD was observed at suboptimal (30 x106) but not optimal (50 x106) donor cell doses as indicated by a significant decrease in donor CD8 engraftment, proliferation and host B cell elimination. Bm12→B6 cGVHD induced in RGC-32 KO hosts displayed an attenuated phenotype as indicated by decreased number of germinal center B cells and plasmablasts, mRNA expression of Prdm1, IRF-4 and Aicda and anti-dsDNA Ab production. These results suggest that RGC-32 expression in host B cells promotes autoimmune parameters in cGVHD while its expression in T cells enhances suboptimal CD8 cytotoxic T cell effector function thus preventing humoral autoimmunity in aGVHD. These data support the idea that RGC-32 blockade has the potential to reverse abnormalities in the CD8 T cell and B cell pathways that contribute to lupus pathogenesis.