anti-dsDNA Autoantibody Production Research Articles

Loss of interleukin-1 beta is not protective in the lupus-prone NZM2328 mouse model.

Aberrant activation of the innate immune system is a known driver of lupus pathogenesis. Inhibition of the inflammasome and its downstream signaling components in murine models of lupus has been shown to reduce the severity of disease. Interleukin-1 beta (IL-1β) is a proinflammatory cytokine released from cells following inflammasome activation. Here, we examine how loss of IL-1β affects disease severity in the lupus-prone NZM2328 mouse model. We observed a sex-biased increase in immune complex deposition in the kidneys of female mice in the absence of IL-1β that corresponds to worsened proteinuria. Loss of IL-1β did not result in changes in overall survival, anti-dsDNA autoantibody production, or renal immune cell infiltration. RNA-sequencing analysis identified upregulation of TNF and IL-17 signaling pathways specifically in females lacking IL-1β. Increases in these signaling pathways were also found in female patients with lupus nephritis, suggesting clinical relevance for upregulation of these pathways. Together, these data suggest that inhibition of the inflammasome or its downstream elements that block IL-1β signaling may need to be approached with caution in SLE, especially in patients with renal involvement to prevent potential disease exacerbation.

Depletion of microRNA-183-96-182 miRNA cluster in lymphocytes suppresses anti-dsDNA autoantibody production and IgG deposition in kidney in C57BL/6-Faslpr/lpr mice

Abstract The miR-183-96-182 (miR-183C) is a highly conserved miRNA cluster among species. Our previous work found a significant upregulation of miR-183C in the splenic cells of three different murine models of systemic lupus erythematosus (SLE). Current studies revealed that miR-183C miRNAs are critically involved in immunity and autoimmunity. In this study, we found that inhibition of miR-182 alone or miR-183C in vitro with antagomirs significantly reduced lupus-related inflammatory cytokine IFN-γ and IL-6 in activated splenocytes from MRL or MRL/lpr mice. To further characterize the pathogenic role of miR-182 and miR-183C in lupus in vivo, we developed B6-lpr mice with conditional depletion of miR-182 or miR-183C in CD2+ lymphocyte. We found that depletion of either miR-182 or miR-183C in the lymphocytes of B6/lpr mice had no obvious effect on T and B cell development as similar percentage of CD4+. CD8+, CD19+, as well as Tregs, follicular helper T (TFH), germinal center B (GCB), and plasma cells were observed in the miR-182−/− and miR-183C−/− and their respective control. Importantly, we observed a significant reduction of serum anti-dsDNA autoantibodies in miR-183C−/− mice when compared to age-matched controls and the B6/lpr mice with miR-182 or miR-183C deficiency have significantly reduced IgG deposition in the kidneys. Meanwhile, there was reduced IFN production in ex vivo activated splenocytes from the knockout mice. Furthermore, we demonstrated that miR-182 and miR-183C regulated the inflammatory response in splenocytes via targeting forkhead box O1 (Foxo1). Together, our data suggest a potential therapeutic effect of targeting miR-183C in lupus.

Systemic Lupus Erythematosus and DNA Degradation and Elimination Defects.

Introduction: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by the production of autoantibodies specific for components of the cell nucleus and that causes damage to body tissues and organs. The pathogenesis of SLE remains unclear, with numerous studies pointing to a combination of genetic and environmental factors. A critical stage in SLE development is cell necrosis, in which undegraded chromatin and nucleoproteins are released into the blood, resulting in circulating cell-free DNA and serum nucleoproteins that trigger anti-dsDNA autoantibody production. This systematic literature review aimed to examine whether SLE stems from a DNA degradation and elimination defect.Materials and Methods: An advanced literature search was conducted in PubMed using the following keywords: [(“SLE” OR “Systemic Lupus Erythematosus” OR “Lupus”)] AND [(“DNA” OR “DNA Degradation”)] AND [(“Defect Elimination”)]. More articles were obtained from the references of the identified articles and basic Google searches. Twenty-five peer-reviewed articles published within the past 10 years (2007–2018) were included for review.Results: The findings of each study are summarized in Tables 1, 2.Discussion and Conclusion: The etiopathogenesis of SLE remains controversial, which limits therapeutic inventions for this disease. However, SLE is a DNA degradation and elimination disorder caused by uncleared histones and nuclear material that leak into the extracellular space and form cell-free DNA, triggering an immune response that destroys tissues and organs. Under normal conditions, apoptosis allows DNA and other nuclear material to be efficiently cleared through degradation and additional complex mechanisms such that this material does not trigger the immune system to produce nuclear autoantibodies.

Apoptotic cell-mimicking gold nanocages loaded with LXR agonist for attenuating the progression of murine systemic lupus erythematosus

Systemic lupus erythematosus (SLE) constitutes an autoimmune disease characterized by the breakdown of tolerance to self-antigens, sustained production of pathogenic autoantibodies, and damage to multiple organs and tissues. Nanoparticle (NP)-based therapeutics have demonstrated efficacy in attenuating the progression of SLE. However, investigations of nano-drugs that address the crucial initiating factor in the pathogenesis of SLE; e.g., inefficient clearance of apoptotic cells by phagocytes and consequent accumulation of self-antigens, have seldom been reported. Here, an apoptotic cell-mimicking gold nanocage (AuNC)-based nano drug carrier capable of correcting the impaired clearance of apoptotic cells in SLE was rationally designed and generated by conjugating phosphatidylserine (PS) on the surface of liposome-coated AuNCs for liver X receptor (LXR) agonist T0901317 delivery. Notably, PS-lipos-AuNC@T0901317 could efficiently enhance apoptotic cell clearance by elevating the expression of Mer, one of the pivotal phagocytosis-associated receptors on macrophages, resulting in decreased production of anti-dsDNA autoantibodies, reduced inflammatory response, and alleviation of kidney damage in lupus model mice. Additionally, PS-lipos-AuNC could be tracked by photoacoustic imaging for nano drug carrier biodistribution. By addressing the crucial pathogenic factor of SLE, the NP-based delivery system in this study is envisioned to provide a promising strategy to treat this complex and challenging disease.

Administration of activated lymphocyte-derived DNA accelerates and aggravates lupus nephritis in B6/lpr mice: a new approach to modify a lupus murine model.

B6/lpr mouse strain is a well-known systemic lupus erythematosus murine model characterized by uncontrolled lymphoproliferation and autoantibody production. However, it displays a delayed and mild development of lupus nephritis (LN), which is not conducive to the research of the pathogenesis and therapeutic strategies of this condition. Our previous study demonstrated that activated lymphocyte-derived DNA (ALD-DNA) could induce high urine protein levels and severe glomerulonephritis (GN) in BALB/c mice. In the present study, we tried to remedy delayed urine protein production and mild GN in B6/lpr mice via ALD-DNA immunization. We found that urine protein levels were enhanced significantly in B6/lpr mice 4weeks after ALD-DNA immunization compared with those in unactivated lymphocyte-derived (UnALD)-DNA- and phosphate-buffered saline (PBS)-treated controls. Moreover, more serious GN and glomerular immune complex were observed in ALD-DNA-immunized B6/lpr mice. We further explored the mechanism, and found that ALD-DNA immunization promoted T helper type 17 (Th17) cell enrichment remarkably, which enhanced the proportion of autoantibody-secreting plasma cells and promoted the production of anti-dsDNA autoantibodies, leading to accelerated and aggravated LN. Our data demonstrated that ALD-DNA immunization could remedy delayed urine protein production and mild GN in B6/lpr mouse, which makes it more suitable for studies on the pathogenesis of and therapeutic strategies against LN.

Epistatic Interactions Between Mutations of Deoxyribonuclease 1-Like 3 and the Inhibitory Fc Gamma Receptor IIB Result in Very Early and Massive Autoantibodies Against Double-Stranded DNA.

Autoantibodies against double-stranded DNA (anti-dsDNA) are a hallmark of systemic lupus erythematosus (SLE). It is well documented that anti-dsDNA reactive B lymphocytes are normally controlled by immune self-tolerance mechanisms operating at several levels. The evolution of high levels of IgG anti-dsDNA in SLE is dependent on somatic hypermutation and clonal selection, presumably in germinal centers from non-autoreactive B cells. Twin studies as well as genetic studies in mice indicate a very strong genetic contribution for the development of anti-dsDNA as well as SLE. Only few single gene defects with a monogenic Mendelian inheritance have been described so far that are directly responsible for the development of anti-dsDNA and SLE. Recently, among other mutations, rare null-alleles for the deoxyribonuclease 1 like 3 (DNASE1L3) and the Fc gamma receptor IIB (FCGR2B) have been described in SLE patients and genetic mouse models. Here, we demonstrate that double Dnase1l3- and FcgR2b-deficient mice in the C57BL/6 background exhibit a very early and massive IgG anti-dsDNA production. Already at 10 weeks of age, autoantibody production in double-deficient mice exceeds autoantibody levels of diseased 9-month-old NZB/W mice, a long established multigenic SLE mouse model. In single gene-deficient mice, autoantibody levels were moderately elevated at early age of the mice. Premature autoantibody production was accompanied by a spontaneous hyperactivation of germinal centers, early expansions of T follicular helper cells, and elevated plasmablasts in the spleen. Anti-dsDNA hybridomas generated from double-deficient mice show significantly elevated numbers of arginines in the CDR3 regions of the heavy-chain as well as clonal expansions and diversification of B cell clones with moderate numbers of somatic mutations. Our findings show a strong epistatic interaction of two SLE-alleles which prevent early and high-level anti-dsDNA autoantibody production. Both genes apparently synergize to keep in check excessive germinal center reactions evolving into IgG anti-dsDNA antibody producing B cells.

The Systemic Activation of Programmed Death 1-PD-L1 Axis Protects Systemic Lupus Erythematosus Model from Nephritis

Background: Systemic lupus erythematosus (SLE) is characterized by abnormal activated T cells, autoreactive B cells, and massive cytokines. The CD4+ T cells determined B-cells differentiation and cytokines production. The programmed death 1 (PD-1) is the checkpoint immunoinhibitory receptor of activated T cells, and its engagement could exhaust T cells. In this study, we investigated the role of PD-1 systemic engagement with PD-L1-Ig in lupus-like nephritis in SLE mice. Methods: The murine PD-L1-Ig was injected into SLE-prone mice. The proteinuria and survival ratio were monitored. The production of anti-dsDNA autoantibodies and cytokines in serum were measured by enzyme-linked immunosorbent assay. The cytokine-producing T cells (interferon-γ, IFN-γ and IL-17α) in kidney and spleen were detected with flowcytometry. The pathological evaluation of the Ig deposition in the glomeruliand was determined with immunofluorescence. Lymphocytes in 24-h urine were detected with flowcytometry. Results: The systemic administration of PD-L1-Ig activated PD-1-PD-L1 axis of CD4+ T lymphocytes, suppressed Th17 formation in many organs, including the spleen and the kidney, demolished abnormal production of cytokines (IFN-γ, IL-17, and IL-10) and anti-dsDNA autoantibodies in serum, inhibited immunoglobulin G deposition in the glomeruli with the decrease of proteinuria, and activated T cells in urine. Accordingly, the systemic conjugation of PD-L1-PD-1 impaired renal autoimmune injure and prolonged survival time. Conclusion: Our research demonstrated that the protective function of systemic activation of PD-1-PD-L1 axis with PD-L1-Ig attenuates the nephritis in SLE-prone mice, which facilitates us to understand the suppressive function of PD-1-PD-L1 axis in the pathogenesis and progress of the lupus nephritis, and to explore a possible effective therapeutic strategy to SLE.

Bacterial amyloid curli acts as a carrier for DNA to elicit an autoimmune response via TLR2 and TLR9

Abstract Bacterial biofilms are associated with numerous human infections. The predominant protein expressed in enteric biofilms is amyloid curli which forms immunogenic complexes with DNA. Infection with curli-expressing bacteria or systemic exposure to purified curli-DNA complexes triggers autoimmunity via the generation type I interferons (IFNs) and anti-double stranded (ds) DNA autoantibodies. DNA complexed with curli stimulates Toll-like receptor (TLR) 9 through a two-step mechanism. First, the cross beta-sheet structure of curli is bound by cell-surface TLR2, enabling internalization of the complex into endosomes. After internalization, the curli-DNA immune complex binds strongly to TLR9, inducing production of type I IFNs. Upon stimulation of macrophages with curli-DNA complexes, only 5% of TLR2−/− macrophages harbored intracellular curli, suggesting that TLR2 drives the internalization of curli-DNA complexes. Suppression of TLR2 internalization led to a significant decrease in Ifnβ expression. Structural analysis using small-angle X-ray scattering revealed that incorporation of DNA into curli fibrils results in the formation of ordered curli-DNA immune complexes resulting in optimal spacing to TLR9. Production of anti-dsDNA autoantibodies and type I IFNS in response to curli-DNA fibers was attenuated in the TLR2−/−, TLR9 mutant, and TLR2−/− - TLR9 mutant mice compared to wildtype mice, suggesting that TLR2 and TLR9 are critical for the autoimmune immune response. Overall, our results identify a pivotal series of events that leads to the severe pro-autoimmune effects of amyloid-expressing bacteria.

Genome-Wide Assessment of Differential DNA Methylation Associated with Autoantibody Production in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is characterized by the development of autoantibodies associated with specific clinical manifestations. Previous studies have shown an association between differential DNA methylation and SLE susceptibility, but have not investigated SLE-related autoantibodies. Our goal was to determine whether DNA methylation is associated with production of clinically relevant SLE-related autoantibodies, with an emphasis on the anti-dsDNA autoantibody. In this study, we characterized the methylation status of 467,314 CpG sites in 326 women with SLE. Using a discovery and replication study design, we identified and replicated significant associations between anti-dsDNA autoantibody production and the methylation status of 16 CpG sites (pdiscovery<1.07E-07 and preplication<0.0029) in 11 genes. Associations were further investigated using multivariable regression to adjust for estimated leukocyte cell proportions and population substructure. The adjusted mean DNA methylation difference between anti-dsDNA positive and negative cases ranged from 1.2% to 19%, and the adjusted odds ratio for anti-dsDNA autoantibody production comparing the lowest and highest methylation tertiles ranged from 6.8 to 18.2. Differential methylation for these CpG sites was also associated with anti-SSA, anti-Sm, and anti-RNP autoantibody production. Overall, associated CpG sites were hypomethylated in autoantibody positive compared to autoantibody negative cases. Differential methylation of CpG sites within the major histocompatibility region was not strongly associated with autoantibody production. Genes with differentially methylated CpG sites represent multiple biologic pathways, and have not been associated with autoantibody production in genetic association studies. In conclusion, hypomethylation of CpG sites within genes from different pathways is associated with anti-dsDNA, anti-SSA, anti-Sm, and anti-RNP production in SLE, and these associations are not explained by genetic variation. Thus, studies of epigenetic mechanisms such as DNA methylation represent a complementary method to genetic association studies to identify biologic pathways that may contribute to the clinical heterogeneity of autoimmune diseases.

Allogenic Mesenchymal Stem Cell Transplantation Ameliorates Nephritis in Lupus Mice via Inhibition of B-Cell Activation

Recent evidence indicates that bone marrow-derived mesenchymal stem cells (BM-MSCs) possess immunosuppressive properties both in vitro and in vivo. We have previously demonstrated that transplantation of human MSCs can significantly improve the autoimmune conditions in MRL/lpr mice. The current study aimed to determine the mechanisms by which murine BM-MSC transplantation (MSCT) ameliorates nephritis in MRL/lpr mice. In this study, we found that MSCT can significantly prolong the survival of MRL/lpr mice. Eight weeks after transplantation, MSCT-treated mice showed significantly smaller spleens than control animals, with fewer marginal zones (MZs), T1, T2, activated B-cells, and plasma cells. Moreover, serum levels of B-cell activating factor (BAFF) and IL-10 in MSCT-treated mice decreased significantly compared to those in the control group, while levels of serum TGF-β were increased. Notably, decreased BAFF expression in both spleen and kidney was accompanied by decreased production of anti-dsDNA autoantibodies and proteinuria in MSCT-treated mice. Since BAFF is mainly expressed by T-cells and dendritic cells, we incubated BM-MSCs and DCs together and found that the production of BAFF by DCs was suppressed by MSCs. Thus, our findings suggest that MSCT may suppress the excessive activation of B-cells via inhibition of BAFF production in MRL/lpr mice.

Soluble human CD83 ameliorates lupus in NZB/W F1 mice

In the present study we explored the immunomodulatory potential of prokaryotically expressed soluble CD83 in the treatment of murine lupus using the NZB/W F1 mouse model. Therefore female NZB/W F1 lupus mice were treated either with sCD83 or PBS for 4 weeks. sCD83 treated mice showed a significantly delayed onset of anti-dsDNA autoantibody production when compared with the control group. Importantly, during the treatment period with sCD83 none of the mice showed elevated levels of anti-dsDNA autoantibodies. In addition, NZB/W F1 mice which received sCD83 displayed lower concentrations of anti-histone IgG autoantibodies. Furthermore, there was no difference in total IgG antibodies, indicating a modulatory role for sCD83 in the production of self-reactive antibodies without decreasing total IgG. These results indicate that administration of sCD83 has profound immune-modulatory effects on the induction of autoantibodies in NZB/W F1 lupus mice and may thus be a promising approach to interfere with autoimmunity in SLE and other autoantibody-driven diseases.

17-β Estradiol reduces atherosclerosis without exacerbating lupus in ovariectomized systemic lupus erythematosus-susceptible LDLr−/− mice

ObjectiveTo test the hypothesis that estrogen treatment in a radiation chimera mouse model of systemic lupus erythematosus (SLE) and atherosclerosis will increase SLE-associated atherosclerosis by increasing autoantibody production and inflammation. MethodsWe used a radiation chimera mouse model in which bone marrow from the polygenic B6.Sle1.2.3 model of SLE was transferred to the low density lipoprotein receptor knock out (LDLr−/−) model of atherosclerosis on a C57BL/6 background (Sle/LDLr−/−). Ovariectomized chimeric mice were treated for 10 weeks with either 5.6 μg/day of 17β-estradiol or placebo; outcomes included atherosclerosis plaque size, anti-dsDNA autoantibody production and renal pathology. ResultsMean atherosclerosis plaque size was 67.4 ± 7.6% smaller in the estrogen treated group (p < 0.0001). Estrogen treated Sle/LDLr−/− mice had no significant difference in serum cholesterol concentration, lipoprotein distribution, anti-dsDNA autoantibody concentration, antibody isotype concentration and renal histopathology score compared to placebo. However, they had significantly lower mean urine protein to urine creatinine ratio (UP:UC). There was no correlation between atherosclerosis lesion size and either the renal histology score or UP:UC ratio in Sle/LDLr−/− mice. ConclusionThese results indicate that 17β-estradiol is atheroprotective within the context of murine SLE independent of changes in serum cholesterol concentration, autoantibody concentration, or renal pathology. The SLE phenotype in Sle/LDLr−/− mice is not exacerbated by exogenous 17β-estradiol administration, and the reduced UP:UC ratio suggests a protective effect against lupus nephritis.

Differential DNA methylation associated with anti-dsDNA autoantibody production in systemic lupus erythematosus

Differential DNA methylation associated with anti-dsDNA autoantibody production in systemic lupus erythematosus

Administration of fasudil, a ROCK inhibitor, attenuates disease in lupus-prone NZB/W F1 female mice

Accumulating evidence from murine studies suggests that the RhoA/ROCK pathway plays an important role in the development of autoimmune disorders. We previously demonstrated that ROCK inhibition ameliorates disease in MRL/lpr mice, a spontaneous model of lupus. This study aimed to explore the protective effects of the ROCK inhibitor fasudil in a distinct model of lupus, NZB/W F1 female mice, to assess the broad applicability of ROCK inhibition for the treatment of lupus. NZB/W F1 female mice were administered fasudil continuously in their drinking water starting at 18 or 24 weeks of age up until 44 weeks of age, or remained untreated. Fasudil treatment significantly improved survival and decreased proteinuria, particularly when treatment was started at 18 weeks. There was also a significant decrease in serum anti-dsDNA autoantibody production, glomerular IgG and C3 deposition, and glomerulonephritis. Analysis of the splenic lymphocyte compartment revealed reduced effector/memory CD4(+) T cell and plasma cell numbers in fasudil treated mice while the frequency of other B cell and T cell subsets was unchanged. These results thus indicate that fasudil can ameliorate disease in NZB/W F1 female mice, suggesting that ROCK inhibition might be broadly effective for the treatment of lupus.

Amelioration of Lupus Nephritis by Serum Amyloid P Component Gene Therapy with Distinct Mechanisms Varied from Different Stage of the Disease

BackgroundOur previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.Methodology/Principal FindingsThe ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.Conclusion/SignificanceThese results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.

Differential Genetic Associations for Systemic Lupus Erythematosus Based on Anti–dsDNA Autoantibody Production

Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE.

Immunogenicity of singlet oxygen modified human DNA: Implications for anti-DNA antibodies in systemic lupus erythematosus

Reactive oxygen species (ROS)-modified DNA has been shown to be a better and more discriminating immunogen than native DNA (nDNA) for the production of anti-DNA autoantibodies in SLE (systemic lupus erythematosus). Among ROS, the role of hydroxyl radical (·OH) in the induction of damage and modification of nDNA has been extensively studied while such documentation implicating singlet oxygen ( 1O 2) in inducing immunogenicity in nDNA leading to the production of anti-double-stranded (ds) DNA autoantibodies in SLE is not yet available. This prospective study was undertaken to evaluate the immunogenicity of healthy human dsDNA modified with 1O 2 generated by methylene blue plus radiant light. Female rabbits were immunized with 1O 2-modified human dsDNA to raise anti-dsDNA antibodies. 1O 2-modified anti-dsDNA rabbit immune sera and the 1O 2-modified anti-dsDNA rabbit purified immunoglobulin G (IgG) were tested against a variety of dsDNA antigenic substrates through direct enzyme-linked immunosorbent assay (ELISA). The immunogenicity of 1O 2-modified human dsDNA was further evaluated by studying its immunoreactivity with SLE patients’ sera and SLE patients’ purified anti-dsDNA IgG. As compared to healthy human sera, 1O 2-modified anti-dsDNA rabbit immune sera as well as the 1O 2-modified anti-dsDNA rabbit purified IgG demonstrated a strong affinity towards 1O 2-modified human dsDNA . 1O 2-modified human dsDNA proved to be potentially more immunogenic against SLE patients’ whole sera and SLE patients’ purified IgG as compared to healthy human sera. Our findings suggest that 1O 2 may also be inducing immunogenicity in native dsDNA resulting in the production of anti-dsDNA autoantibodies as seen in SLE patients.

Origin and anti-tumor effects of anti-dsDNA autoantibodies in cancer patients and tumor-bearing mice

In the present investigation, we detected anti-dsDNA autoantibodies in cancer patients and modeled the production of anti-dsDNA autoantibodies by inoculating tumors in BALB/c mice. Moreover, induction of anti-dsDNA autoantibodies by immunization with inactivated tumor cells and their DNA indicated that DNA of tumor cells was probably the primary antigen, which was supported by the significantly increasing levels of sera free DNA in cancer patients and tumor-bearing mice. cELISA and indirect immunofluorescence assay showed that the anti-dsDNA autoantibodies could bind to the surface components of tumor cells. In vitro assay showed that immunosera at week 6 from immunized mice displayed significant cytotoxicity to tumor cells compared to that of negative control, but no cytotoxicity mediated by immunosera at week 22 was observed. In addition, by flow cytometry and electrophoresis of fragmented DNA, the cytotoxicity might probably be mediated by apoptosis. Our data also showed that the ability of the anti-dsDNA autoantibodies to induce apoptosis of SP2/0 and Wehi 164 cells was significantly correlated ( r = 0.990, p < 0.01 and r = 0.901, p < 0.05) with their functional affinity. In vivo, the growth of solid tumors was significantly inhibited in the immunized mice inoculated directly with SP2/0 and Wehi 164 cells, or in the naïve mice which were inoculated with SP2/0 cells preincubated with immunosera containing anti-dsDNA autoantibodies. In conclusion, we demonstrated the origin of anti-dsDNA autoantibodies in cancer patients and tumor-bearing mice. And our data also showed that these autoantibodies revealed anti-tumor effect by inducing apoptosis.

CD137-mediated T cell co-stimulation terminates existing autoimmune disease in SLE-prone NZB/NZW F1 mice.

T cell receptor recognition of antigen and major histocompatibility complex (signal 1) and T cell co-stimulation (signal 2) are essential for full T cell activation, differentiation, and survival of naïve and activated T cells. The proto-typical T cell co-stimulatory receptor, CD28, is a constitutively expressed type I integral transmembrane protein and member of the Ig superfamily. Since its discovery, additional T cell co-stimulatory receptors have been identified, a number of which belong to the tumor necrosis factor receptor superfamily. Included within this group is CD137 (4-1BB), an activation-inducible, type I transmembrane protein. Co-stimulation of T cells through CD137 effectively up-regulates CD8 T cell activation and survival. Although CD4(+) T cells are efficiently activated through the T cell receptor and CD137 receptor, it provokes CD4(+) T cell anergy and blockade of T-dependent humoral immune responses. Therefore, we tested whether agonistic anti-CD137 monoclonal antibodies (mAbs) would be effective in blocking the induction or progression of B cell dependent autoimmune disease. Herein, we demonstrate the protective effect of agonistic anti-CD137 mAbs in blocking systemic lupus erythematosus (SLE) disease progression in NZB/W F1 mice. Protection from SLE following anti-CD137 mAb treatment is not confined to rescuing mice from disease progression; rather, it fully protects young mice from developing any symptoms of disease. We further found that treatment of proteinuric mice with anti-CD137 blocks ongoing anti-dsDNA autoantibody production.

IL-10 regulates murine lupus.

MRL/MpJ-Tnfrsf6(lpr) (MRL/MpJ-Fas(lpr); MRL-Fas(lpr)) mice develop a spontaneous lupus syndrome closely resembling human systemic lupus erythematosus. To define the role of IL-10 in the regulation of murine lupus, IL-10 gene-deficient (IL-10(-/-)) MRL-Fas(lpr) (MRL-Fas(lpr) IL-10(-/-)) mice were generated and their disease phenotype was compared with littermates with one or two copies of an intact IL-10 locus (MRL-Fas(lpr) IL-10(+/-) and MRL-Fas(lpr) IL-10(+/+) mice, respectively). MRL-Fas(lpr) IL-10(-/-) mice developed severe lupus, with earlier appearance of skin lesions, increased lymphadenopathy, more severe glomerulonephritis, and higher mortality than their IL-10-intact littermate controls. The increased severity of lupus in MRL-Fas(lpr) IL-10(-/-) mice was closely associated with enhanced IFN-gamma production by both CD4(+) and CD8(+) cells and increased serum concentration of IgG2a anti-dsDNA autoantibodies. The protective effect of IL-10 in this lupus model was further supported by the observation that administration of rIL-10 reduced IgG2a anti-dsDNA autoantibody production in wild-type MRL-Fas(lpr) animals. In summary, our results provide evidence that IL-10 can down-modulate murine lupus through inhibition of pathogenic Th1 cytokine responses. Modulation of the level of IL-10 may be of potential therapeutic benefit for human lupus.