Epstein–Barr virus antibodies mark systemic lupus erythematosus and scleroderma patients negative for anti‐DNA

Systemic lupus erythematosus (SLE) is an autoimmune disease that can attack many different body organs; the triggering event is unknown. SLE has been associated with more than 100 different autoantibody reactivities – anti‐dsDNA is prominent. Nevertheless, autoantibodies to dsDNA occur in only two‐thirds of SLE patients. We previously reported the use of an antigen microarray to characterize SLE serology. We now report the results of an expanded study of serology in SLE patients and scleroderma (SSc) patients compared with healthy controls. The analysis validated and extended previous findings: two‐thirds of SLE patients reacted to a large spectrum of self‐molecules that overlapped with their reactivity to dsDNA; moreover, some SLE patients manifested a deficiency of natural IgM autoantibodies. Most significant was the finding that many SLE patients who were negative for autoantibodies to dsDNA manifested abnormal antibody responses to Epstein–Barr virus (EBV): these subjects made IgG antibodies to EBV antigens to which healthy subjects did not respond or they failed to make antibodies to EBV antigens to which healthy subjects did respond. This observation suggests that SLE may be associated with a defective immune response to EBV. The SSc patients shared many of these serological abnormalities with SLE patients, but differed from them in increased IgG autoantibodies to topoisomerase and centromere B; 84% of SLE patients and 58% of SSc patients could be detected by their abnormal antibodies to EBV. Hence an aberrant immune response to a ubiquitous viral infection such as EBV might set the stage for an autoimmune disease.     

The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-double-stranded DNA antibodies from patients with systemic lupus erythematosus

IL-10 and IL-12 are cytokines which are important in regulating immune responses. Plasma levels of IL-10 and autoantibodies against double-stranded DNA (dsDNA) often mirror disease activity in patients with SLE. IL-12 secretion from SLE patients' blood mononuclear cells also correlates with disease activity, but has an inverse relationship. The aim of this study was to measure the effect of IL-10 and of IL-12 on the production of IgG autoantibodies from patients with SLE, both cross-sectionally and longitudinally. Peripheral blood mononuclear cells (PBMC) were cultured with IL-10 (at 20 ng/ml or 2 ng/ml) or IL-12 (at 2 ng/ml or 0.2 ng/ml) or without cytokine and the supernatanants tested for the production of double-stranded DNA antibodies (dsDNA abs), single-stranded DNA antibodies (ssDNA abs) and total IgG antibodies (IgG abs) by ELISA. The BILAG disease activity index was recorded at each patient visit (a global score of six or more is regarded as active disease). In general, treatment with IL-10 caused PBMCs from patients with inactive disease to increase their antissDNA and dsDNA ab production (by upto 354% and 186%, respectively) while patients with active disease decreased their antibody production (by upto 91% and 97%, respectively). Overall there was a correlation between disease activity and change in antissDNA and dsDNA ab production (r = - 0.51; P = 0.03 and r = - 0.48; P = 0.042, respectively). Treatment with IL-12 at 0.2 ng/ml inhibited antissDNA and dsDNA antibody production, having the greatest effect on patients with active disease (decreasing antissDNA and dsDNA antibody production by upto 75% and 73%, respectively). This resulted in a significant correlation between disease activity and change in antissDNA antibody production (r = - 0.76; P = 0.03), but significance was not reached with antidsDNA antibody production (P = 0.06). Together these data suggest that the effect of these cytokines on antibody production by SLE PBMCs involves several factors; one of which is disease activity.     

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 ((1)O(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 (1)O(2) generated by methylene blue plus radiant light. Female rabbits were immunized with (1)O(2)-modified human dsDNA to raise anti-dsDNA antibodies. (1)O(2)-modified anti-dsDNA rabbit immune sera and the (1)O(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 (1)O(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, (1)O(2)-modified anti-dsDNA rabbit immune sera as well as the (1)O(2)-modified anti-dsDNA rabbit purified IgG demonstrated a strong affinity towards (1)O(2)-modified human dsDNA(.)(1)O(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 (1)O(2) may also be inducing immunogenicity in native dsDNA resulting in the production of anti-dsDNA autoantibodies as seen in SLE patients.     

Nucleic acid sensing receptors in systemic lupus erythematosus: development of novel DNA- and/or RNA-like analogues for treating lupus

Double‐stranded (ds) DNA, DNA‐ or RNA‐associated nucleoproteins are the primary autoimmune targets in SLE, yet their relative inability to trigger similar autoimmune responses in experimental animals has fascinated scientists for decades. While many cellular proteins bind non‐specifically negatively charged nucleic acids, it was discovered only recently that several intracellular proteins are involved directly in innate recognition of exogenous DNA or RNA, or cytosol‐residing DNA or RNA viruses. Thus, endosomal Toll‐like receptors (TLR) mediate responses to double‐stranded RNA (TLR‐3), single‐stranded RNA (TLR‐7/8) or unmethylated bacterial cytosine (phosphodiester) guanine (CpG)‐DNA (TLR‐9), while DNA‐dependent activator of IRFs/Z‐DNA binding protein 1 (DAI/ZBP1), haematopoietic IFN‐inducible nuclear protein‐200 (p202), absent in melanoma 2 (AIM2), RNA polymerase III, retinoic acid‐inducible gene‐I (RIG‐I) and melanoma differentiation‐associated gene 5 (MDA5) mediate responses to cytosolic dsDNA or dsRNA, respectively. TLR‐induced responses are more robust than those induced by cytosolic DNA‐ or RNA‐ sensors, the later usually being limited to interferon regulatory factor 3 (IRF3)‐dependent type I interferon (IFN) induction and nuclear factor (NF)‐κB activation. Interestingly, AIM2 is not capable of inducing type I IFN, but rather plays a role in caspase I activation. DNA‐ or RNA‐like synthetic inhibitory oligonucleotides (INH‐ODN) have been developed that antagonize TLR‐7‐ and/or TLR‐9‐induced activation in autoimmune B cells and in type I IFN‐producing dendritic cells at low nanomolar concentrations. It is not known whether these INH‐ODNs have any agonistic or antagonistic effects on cytosolic DNA or RNA sensors. While this remains to be determined in the future, in vivo studies have already shown their potential for preventing spontaneous lupus in various animal models of lupus. Several groups are exploring the possibility of translating these INH‐ODNs into human therapeutics for treating SLE and bacterial DNA‐induced sepsis.     

Blockade of TLR9 signaling in B cells impaired anti-dsDNA antibody production in mice induced by activated syngenic lymphocyte-derived DNA immunization

We previously established a systemic lupus erythematosus (SLE) animal model in non-susceptible BALB/c mice by immunizing with activated syngeneic lymphocyte-derived DNA (ALD-DNA), manifested by high level of anti-double-stranded DNA (dsDNA) antibodies (Abs), proteinuria, glomerular deposition of immune complex and glomerulonephritis. The production of anti-dsDNA Abs is closely related with the renal inflammation and damage in this model. However, recognition of ALD-DNA and its signaling pathway within antigen-presenting cells (APC) remains not fully clarified. Herein, in this study, Toll-like receptor 9 (TLR9), a well-known pattern-recognition receptor for dsDNA with CpG motif, was found to be dynamically up-regulated in B cells during the process of the SLE disease. Knockdown of TLR9 by short interfering RNA (siRNA) in B cells in vitro and in vivo reduced the production of anti-dsDNA antibody and consequently ameliorated the SLE syndrome in mice while the affinity and isotype of the antibody remained the same. Our results implied that TLR9 signaling of B cells might play an important role in the production of anti-dsDNA Abs triggered by auto dsDNA, which would extend our understanding of TLR9 immune recognition in the pathogenesis of SLE disease.     

Anti‐dsDNA antibodies as a classification criterion and a diagnostic marker for systemic lupus erythematosus: critical remarks

Antibodies to mammalian dsDNA have, for decades, been linked to systemic lupus erythematosus (SLE) and particularly to its most serious complication, lupus nephritis. This canonical view derives from studies on its strong association with disease. The dogma was particularly settled when the antibody was included in the classification criteria for SLE that developed during the 1970s, most prominently in the 1982 American College of Rheumatology (ACR), and recently in The Systemic Lupus International Collaborating Clinics (SLICC) classification criteria. There are several problems to be discussed before the anti‐dsDNA antibody can be accepted without further distinction as a criterion to classify SLE. Old and contemporary knowledge make it clear that an anti‐dsDNA antibody is not a unifying term. It embraces antibodies with a wide spectrum of fine molecular specificities, antibodies that are produced transiently in context of infections and persistently in the context of true autoimmunity, and also includes anti‐dsDNA antibodies that have the potential to bind chromatin (accessible DNA structures) and not (specificity for DNA structures that are embedded in chromatin and therefore unaccessible for the antibodies). This critical review summarizes this knowledge and questions whether or not an anti‐dsDNA antibody, as simply that, can be used to classify SLE.     

Lupus nephritis: the central role of nucleosomes revealed

Systemic lupus erythematosus (SLE) is an autoimmune syndrome characterized by autoantibodies to nuclear constituents. Some of these antibodies are diagnostically important, whereas others act as disease-modifying factors. One clinically important factor is autoantibodies against dsDNA and nucleosomes, which have overlapping diagnostic and nephritogenic impact in SLE. Although a scientific focus for 5 decades, the molecular and cellular origin of these antibodies, and why they are associated with lupus nephritis, is still not fully understood. A consensus has, however, evolved that antibodies to dsDNA and nucleosomes are central pathogenic factors in the development of lupus nephritis. In contrast, no agreement has been reached as to which glomerular structures are bound by nephritogenic anti-nucleosome antibodies in vivo. Mutually contradictory paradigms and models have evolved simply because we still lack precise and conclusive data to provide definitive insight into how autoantibodies induce lupus nephritis and which specificity is critical in the nephritic process(es). In this review, data demonstrating the central role of nucleosomes in inducing and binding potentially nephritogenic antibodies to DNA and nucleosomes are presented and discussed. These autoimmune-inducing processes are discussed in the context of Matzinger’s danger model (Matzinger P: Friendly and dangerous signals: is the tissue in control? Nat Immunol 2007, 8:11–13; Matzinger P: The danger model: a renewed sense of self. Science 2002, 296:301–305; Matzinger P: Tolerance, danger, and the extended family. Annu Rev Immunol 1994, 12:991–1045) and Medzhitov’s and Janeway’s (Medzhitov R, Janeway CA Jr: Decoding the patterns of self and nonself by the innate immune system. Science 2002, 296:298–300; Medzhitov R, Janeway CA Jr: How does the immune system distinguish self from nonself? Semin Immunol 2000, 12:185–188; Janeway CA Jr, Medzhitov R: Innate immune recognition. Annu Rev Immunol 2002, 20:197–216) distinction of noninfectious self (NIS) and infectious nonself (INS). The mechanisms leading to production of potentially nephritogenic anti-nucleosome antibodies and to overt lupus nephritis are interpreted in the context of these paradigms.     

Clinical relevance of circulating anti‐ENA and anti‐dsDNA secreting cells from SLE patients and their dependence on STAT‐3 activation

Disturbances of plasma cell homeostasis and auto‐antibody production are hallmarks of systemic lupus erythematosus. The aim of this study was to explore the presence of circulating anti‐ENA and anti‐dsDNA antibody‐secreting cells, to determine their dependence on plasma cell‐niche cytokines and to analyze their clinical value. The study was performed in SLE patients with serum anti‐ENA and/or anti‐dsDNA antibodies (n = 57). Enriched B‐cell fractions and sorted antibody‐secreting cells (CD19^lowCD38^high) were obtained from blood. dsDNA‐ and ENA‐specific antibody‐secreting cells were identified as cells capable of active auto‐antibody production in culture. The addition of a combination of IL‐6, IL‐21, BAFF, APRIL, and CXCL12 to the cultures significantly augmented auto‐antibody production and antibody‐secreting cell proliferation, whereas it diminished apoptosis. The effect on auto‐antibody production was dependent on STAT‐3 activation as it was abrogated in the presence of the JAK/STAT‐3 pathway inhibitors ruxolitinib and stattic. Among patients with serum anti‐dsDNA antibodies, the detection of circulating anti‐dsDNA‐antibody‐secreting cells was associated with higher disease activity markers. In conclusion, auto‐antibody production in response to plasma cell‐niche cytokines that are usually at high levels in SLE patients is dependent on JAK/STAT‐3 activation. Thus, patients with circulating anti‐dsDNA antibody‐secreting cells and active disease could potentially benefit from therapies targeting the JAK/STAT3 pathway.     

Molecular Analyses of Anti-DNA Antibodies Induced by Polyomavirus BK in BALB/c Mice

In the present experiments, two groups of BALB/c mice (five individuals in each group) were hyperimmunized through four consecutive immunizations with either BK virus (Group 1) or BK dsDNA complexed with methylated BSA (Group 2). All immune sera taken after the fourth immunization from both groups reacted strongly with polyomavirus BK dsDNA as well as with calf thymus dsDNA, and all sera contained antibodies that bound in the Crithidia luciliae assay. This indicates that polyomavirus BK was able to induce antibodies with binding characteristics similar to SLE anti-DNA antibodies. To further characterize these induced anti-DNA responses, 10 monoclonal anti-DNA antibodies (four from Group 1, and six from Group 2) were generated and selected for reactivity with Sl-nuclease digested CT dsDNA. Their specificity for BK and CT dsDNA molecules, as well as their light and heavy chain variable region cDNA nucleotide sequences were analysed to compare them with known SLE derived anti-DNA antibodies. All of the 10 antibodies bound strongly to BK dsDNA, while seven also bound to CT dsDNA in competitive ELISA experiments. V-region analysis revealed that the induced antibodies resembled anti-DNA antibodies characteristic for murine SLE, and all but one contained arginine in the V_H CDR3 region. The arginines present in the monoclonal antibodies originated either from an RF shift from RF1RF3 of the D-genes or from N-sequence additions. Taken together, the data demonstrate that anti-DNA antibodies in response to hyperimmun-ization with polyomavirus BK have the same characteristics as of those occuring spontaneously in SLE. As virus infection/replication in vivo implies expression of immunogenic (non-self) DNA-binding proteins that may render DNA immunogenic, the present results may therefore suggest one physiological mechanism for production of SLE-related anti-DNA antibodies.     

Relationship between natural and infection‐induced antibodies in systemic autoimmune diseases (SAD): SLE,SSc and RA

Infection or vaccine‐induced T cell‐dependent immune response and the subsequent high‐affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease‐specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine‐induced and disease‐related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme‐linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti‐CS) and F4 fragment (anti‐F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti‐measles IgG and anti‐dsDNA IgG/IgM autoantibodies were measured using commercial and in‐house indirect ELISA tests. In all SAD groups the anti‐measles IgG‐seropositive cases showed significantly higher anti‐CS IgG titers (P = 0·011). In anti‐dsDNA IgG‐positive SLE patients, we detected significantly higher levels of anti‐CS and anti‐F4 IgG nAAbs (P = 0·001 and < 0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti‐CS and anti‐F4 nAAbs in anti‐dsDNA IgM‐positive SLE patients (P = 0·002 and 0·016, respectively). The association between IgG isotypes of pathogen‐ or autoimmune disease‐related antibodies and the IgG nAAbs may underscore the immune response‐inducible nature of the diseases investigated. The relationship between protective anti‐dsDNA IgM and the IgM isotype of anti‐F4 and anti‐CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.     

Pathogenic anti-DNA antibodies in SLE: idiotypic families and genetic origins

We have adopted an idiotypic approach to study the double stranded DNA (dsDNA) binding antibodies of systemic lupus erythematosus (SLE). Three anti-idiotypic reagents, 8.12, 3I, and F4, identify cross reactive idiotypes that are each expressed on anti-dsDNA antibodies in the sera of many patients with SLE. These idiotypic antibodies are implicated in the pathogenesis of SLE as they are present in immune complex deposits in the kidneys of patients with SLE glomerulonephritis. The autoantibody associated idiotypes are also expressed on antibodies that do not bind DNA. We are investigating the origin of the pathogenic anti-dsDNA antibodies of SLE by comparing the autoantibodies, the antibodies to foreign antigens, and the myeloma proteins that express each SLE associated idiotype. In conjunction with serological analysis of these idiotypic systems, molecular genetic studies indicate that both the 8.12 and the 3I autoantibody associated idiotypes may be germline encoded, while the F4 idiotype is generated by somatic mutation. The data further suggest that the antigenic specificity of the pathogenic anti-DNA antibodies of SLE is acquired through somatic mutation of germline immunoglobulin genes. By studying the regulation of genes capable of encoding pathogenic autoantibodies, in both SLE patients and non-autoimmune individuals, we may be able to elucidate the pathogenesis of autoimmune disease and begin to design more effective therapeutic interventions.     

Interferon-�� and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells

Dendritic cells (DCs) are initiators of innate immunity and acquired immunity as cells linking these two bio‐defence systems through the production of cytokines such as interferon‐α (IFN‐α) and interleukin‐12 (IL‐12). Nucleic acids such as DNA from damaged cells or pathogens are important activators not only for anti‐microbial innate immune responses but also in the pathogenesis of IFN‐related autoimmune diseases. Plasmacytoid DCs are regarded as the main effectors for the DNA‐mediated innate immunity by possessing DNA‐sensing toll‐like receptor 9 (TLR9). We here found that double‐stranded DNA (dsDNA) complexed with lipotransfectants triggered activation of human monocyte‐derived DCs (moDCs), leading to the preferential production of IFN‐α but not IL‐12. This indicates that myeloid DCs also function as supportive effectors against the invasion of pathogenic microbes through the DNA‐mediated activation in innate immunity. The dsDNA with lipotransfectants can be taken up by moDCs without co‐localization of endosomal LAMP1 staining, and the dsDNA‐mediated IFN‐α production was not impaired by chloroquine. These findings indicate that moDC activation by dsDNA does not involve the endosomal TLR pathway. In contrast, single‐stranded RNA (ssRNA) stimulated moDCs to secrete IL‐12 but not IFN‐α. This process was inhibited by chloroquine, suggesting an involvement of the TLR pathway in ssRNA‐mediated moDC activation. As might be inferred from our findings, myeloid DCs may function as a traffic control between innate immunity via IFN‐α production and acquired immunity via IL‐12 production, depending on the type of nucleic acids. Our results provide a new insight into the biological action of myeloid DCs underlying the DNA‐mediated activation of protective or pathogenic immunity.     

The role of cell stress in systemic autoimmune disease]

Anti-DNA antibody is a characteristic feature of systemic lupus erythematosus (SLE) and plays an important role in pathogenesis of lupus nephritis. However, the mechanism of anti-DNA antibody production, which may directly link to the etiology of SLE, remains uncertain. Mammalian DNA alone is not immunogenic. However, some anti-DNA antibodies cross-react with self antigens, and immunization of mice with a certain peptide could induce anti-DNA antibodies. These facts raise a question as to whether an antigenic trigger of anti-DNA antibodies production is DNA itself. Therefore, molecular mimicry of DNA by non DNA antigen is a possibility for the initial production of anti-DNA antibodies. We found that the human monoclonal nephritogenic anti-DNA antibody, O-81, specifically bound to an endoplasmic reticulum stress response protein, Herp. This suggests that lupus nephritogenic anti-DNA antibody cross-react with Herp and an epitope on Herp mimics DNA. Each time cells receive stress (for example, viral infection), the synthesis of Herp protein is induced. If the epitope is immunogenic, repetitive cell stress can be a trigger of anti-DNA antibodies production.     

Invariant natural killer T cells in lupus patients promote IgG and IgG autoantibody production

IgG autoantibodies, including antibodies to double‐stranded DNA (dsDNA), are pathogenic in systemic lupus erythematosus (SLE), but the mechanisms controlling their production are not understood. To assess the role of invariant natural killer T (iNKT) cells in this process, we studied 44 lupus patients. We took advantage of the propensity of PBMCs from patients with active disease to spontaneously secrete IgG in vitro. Despite the rarity of iNKT cells in lupus blood (0.002–0.05% of CD3‐positive T cells), antibody blockade of the conserved iNKT TCR or its ligand, CD1d, or selective depletion of iNKT cells, inhibited spontaneous secretion of total IgG and anti‐dsDNA IgG by lupus PBMCs. Addition of anti‐iNKT or anti‐CD1d antibody to PBMC cultures also reduced the frequency of plasma cells, suggesting that lupus iNKT cells induce B‐cell maturation. Like fresh iNKT cells, expanded iNKT‐cell lines from lupus patients, but not healthy subjects, induced autologous B cells to secrete antibodies, including IgG anti‐dsDNA. This activity was inhibited by anti‐CD40L antibody, as well as anti‐CD1d antibody, confirming a role for CD40L‐CD40 and TCR‐CD1d interactions in lupus iNKT‐cell‐mediated help. These results reveal a critical role for iNKT cells in B‐cell maturation and autoantibody production in patients with lupus.     

An antibody profile of systemic lupus erythematosus detected by antigen microarray

Patients with systemic lupus erythematosus (SLE) produce antibodies to many different self‐antigens. Here, we investigated antibodies in SLE sera using an antigen microarray containing many hundreds of antigens, mostly self‐antigens. The aim was to detect sets of antibody reactivities characteristic of SLE patients in each of various clinical states – SLE patients with acute lupus nephritis, SLE patients in renal remission, and SLE patients who had never had renal involvement. The analysis produced two novel findings: (i) an SLE antibody profile persists independently of disease activity and despite long‐term clinical remission, and (ii) this SLE antibody profile includes increases in four specific immunoglobulin G (IgG) reactivities to double‐stranded DNA (dsDNA), single‐stranded DNA (ssDNA), Epstein–Barr virus (EBV) and hyaluronic acid; the profile also includes decreases in specific IgM reactivities to myeloperoxidase (MPO), CD99, collagen III, insulin‐like growth factor binding protein 1 (IGFBP1) and cardiolipin. The reactivities together showed high sensitivity (> 93%) and high specificity for SLE (> 88%). A healthy control subject who had the SLE antibody profile was later found to develop clinical SLE. The present study did not detect antibody reactivities that differentiated among the various subgroups of SLE subjects with statistical significance. Thus, SLE is characterized by an enduring antibody profile irrespective of clinical state. The association of SLE with decreased IgM natural autoantibodies suggests that these autoantibodies might enhance resistance to SLE.     

Soluble MHC class II‐driven therapy for a systemic lupus erythematosus murine experimental in vitro and in vivo model

Taking into consideration the multiparametric nature of systemic lupus erythematosus (SLE ), the severity and variability of symptoms and the lack of effective therapeutic approaches, this study took advantage of the recently described role of soluble major histocompatibility complex class II (sMHCII ) molecules in maintaining tolerance to the organism and attempted to apply sMHCII proteins as a treatment to murine SLE experimental models in vitro as well as in vivo. After breaking tolerance to DNA in vitro, which was accompanied by development of specific anti‐dsDNA antibodies, syngeneic or allogeneic sMHCII molecules, purified from healthy mouse serum, could significantly reduce the specific antibody levels and drive the system towards immunosuppression, as assessed by specific marker analysis on T cells and cytokine production by flow cytometry and ELISA , respectively. The in vivo experimental model consisted of pristane‐induced SLE symptoms to BALB /c mice, which developed maximal levels of anti‐dsDNA 2 months after pristane inoculation. Syngeneic or allogeneic sMHCII administration could alleviate pristane‐induced symptoms, significantly decrease specific anti‐dsDNA antibody production and develop immunosuppression to the host, as manifested by increase of CD 4 + CTLA ‐4 +  and CD 4 + CD 25 +  cell populations in the spleen. Thus, the results presented in this study introduced the ability of sMHCII proteins to suppress specific autoantigen response, opening new areas of research and offering novel therapeutic approaches to SLE with expanding features to other autoimmune diseases.     

Monoclonal anti-dsDNA antibody 2C10 escorts DNA to intracellular DNA sensors in normal mononuclear cells and stimulates secretion of multiple cytokines implicated in lupus pathogenesis

There have been many studies on the mechanisms of internalization of DNA–anti-DNA immune complexes by cells, including the one used for rheumatoid factor-expressing mouse B cells. In parallel, studies on the role of intracellular DNA sensors in the pathogenesis of systemic lupus erythematosus (SLE) have been conducted, including the one using a mouse model lacking one of the sensors. These and other data have established a framework for understanding the pathogenic role of anti-DNA antibodies, but studies on normal cells are limited. Here, we used the monoclonal anti-dsDNA antibody 2C10, 2-kbp dsDNA and healthy human peripheral blood mononuclear cells (PBMCs) to test whether and how 2C10 and/or DNA cause pathology in normal cells. We found that on culture with PBMCs, 2C10 preferentially entered monocytes and that DNA enhanced this internalization. In contrast, DNA alone was not significantly internalized by monocytes, but 2C10 facilitated its internalization. This was suppressed by cytochalasin D, but not by methyl-β-cyclodextrin, chloroquine or an Fc blocker, suggesting the involvement of macropinocytosis in this process. Internalization of 2C10 and DNA together resulted in production of interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, IL-6, IL-10 and IL-33 by PBMCs. Cytokine production was suppressed by chloroquine and shikonin, but not by RU.521, suggesting dependence on activation of the Toll-like receptor (TLR)-9 and absent in melanoma 2 (AIM-2) pathways. These results established a simple model to demonstrate that anti-DNA antibodies can cause dysregulation of cytokine network mimicking systemic lupus erythematosus in culture of normal PBMCs, and emphasize again the importance of maintaining anti-DNA antibodies at low levels by treatment.     

Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes

Type I interferon (IFN) is a critical mediator of autoimmune diseases such as systemic lupus erythematosus (SLE) and Aicardi–Goutières Syndrome (AGS). The recently discovered cyclic‐GMP‐AMP (cGAMP) synthase (cGAS) induces the production of type I IFN in response to cytosolic DNA and is potentially linked to SLE and AGS. Suppressive oligodeoxynucleotides (ODN) containing repetitive TTAGGG motifs present in mammalian telomeres have proven useful in the treatment of autoimmune diseases including SLE. In this study, we demonstrate that the suppressive ODN A151 effectively inhibits activation of cGAS in response to cytosolic DNA, thereby inhibiting type I IFN production by human monocytes. In addition, A151 abrogated cGAS activation in response to endogenous accumulation of DNA using TREX1‐deficient monocytes. We demonstrate that A151 prevents cGAS activation in a manner that is competitive with DNA. This suppressive activity of A151 was dependent on both telomeric sequence and phosphorothioate backbone. To our knowledge this report presents the first cGAS inhibitor capable of blocking self‐DNA. Collectively, these findings might lead to the development of new therapeutics against IFN‐driven pathologies due to cGAS activation.     

Heterogeneity of anti-idiotypic antibodies to anti-DNA antibodies in humans.

Serum antibodies in some patients with systemic lupus erythematosus (SLE) were found to have specificity to idiotypes (Id) of 0-81 (human monoclonal anti-single-stranded DNA (ssDNA) antibody) but not to Id of NE-1 (human monoclonal anti-double-stranded DNA (dsDNA) antibody) or pooled human IgM. The interaction of the antibodies and 0-81 was blocked by the co-existence of free ssDNA. Some of SLE sera also showed preferential binding to Id determinants of NE-1, which included the antigen-binding sites of the dsDNA antibody. Some other SLE sera reacted with both Id of 0-81 and NE-13. Thus, there was heterogeneous population among human anti-Id autoantibodies to anti-DNA antibodies. The anti-Id activity was commonly detected in inactive SLE sera, and less frequently in normal controls, suggesting some regulatory role for anti-Id antibodies in the production of autoantibodies.     

Endosomal Toll-like receptors in clinically overt and silent autoimmunity

Toll-like receptors (TLRs), first identified as pattern recognition receptors, are now recognized to serve as a key interface between innate and adaptive immunity. Systemic lupus erythematosus (SLE) is characterized by both continuous and cyclic stimulation of the innate and adaptive immune system by endogenous nucleic acids released from apoptotic or necrotic cells. TLR7 and TLR9 function as innate sensors of viral infection as their ligands are ssRNA and dsDNA, respectively. Recognition of self nucleic acids by endosomal TLRs in B cells and pDCs is thought to be an important step in the pathogenesis of SLE, generating anti-nuclear antibodies and producing type I IFN. In this review, we take a specific look at how TLR7, non-coding RNA, and SSA/Ro60 can contribute to clinical autoimmunity and organ damage in the context of neonatal lupus (NL). Although 15 times less common than SLE, NL provides a unique opportunity to study two different aspects of autoimmunity: passively acquired tissue injury in a developing fetus and clinical progression of disease in an asymptomatic mother found to have anti-Ro60 autoantibodies only after identification of heart block/rash in a child. Finally, we discuss hydroxychloroquine (HCQ) use by asymptomatic subjects which may forestall the clinical expression of autoimmunity.