Association of endogenous anti–interferon‐α autoantibodies with decreased interferon‐pathway and disease activity in patients with systemic lupus erythematosus

Objective

Numerous observations implicate interferon‐α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti‐IFNα autoantibodies (AIAAs) on IFN‐pathway and disease activity is unclear. The aim of this study was to characterize IFN‐pathway activity and the serologic and clinical profiles of AIAA‐positive patients with SLE.

Methods

Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA‐positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN‐regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN‐inducible chemokines, and other autoantibodies were measured using immunoassays.

Results

AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN^low and IFN^high, that differed in the levels of serum type I IFN bioactivity, IFN‐regulated gene expression, BAFF, anti–ribosomal P, and anti‐chromatin autoantibodies, and in AIAA status. The majority of AIAA‐positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN‐pathway activity, and lower disease activity compared with the IFN^high patients. AIAA‐positive sera were able to effectively neutralize type I IFN activity in vitro.

Conclusion

Patients with SLE commonly harbor AIAAs. AIAA‐positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN‐pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

     

Coordinate overexpression of interferon‐α–induced genes in systemic lupus erythematosus

Objective

To study the contribution of interferon‐α (IFNα) and IFNγ to the IFN gene expression signature that has been observed in microarray screens of peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE).

Methods

Quantitative real‐time polymerase chain reaction analysis of healthy control PBMCs was used to determine the relative induction of a panel of IFN‐inducible genes (IFIGs) by IFNα and IFNγ. PBMCs from 77 SLE patients were compared with those from 22 disease controls and 28 healthy donors for expression of IFIGs.

Results

Expression of IFNα‐inducible genes was significantly higher in SLE PBMCs than in those from disease controls or healthy donors. The level of expression of all IFIGs in PBMCs from SLE patients with IFNα pathway activation correlated highly with the inherent responsiveness of those genes to IFNα, suggesting coordinate activation of that cytokine pathway. Expression of genes preferentially induced by IFNγ was not significantly increased in SLE PBMCs compared with control PBMCs. IFNα‐regulated gene‐inducing activity was detected in some SLE plasma samples.

Conclusion

The coordinate activation of IFNα‐induced genes is a characteristic of PBMCs from many SLE patients, supporting the hypothesis that IFNα is the predominant stimulus for IFIG expression in lupus.

     

Activation of the interferon‐γ signaling pathway in systemic lupus erythematosus peripheral blood mononuclear cells

Objective

To investigate interferon‐γ (IFNγ) signaling in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) by analyzing IFNγ receptor (IFNγR) expression, STAT‐1 expression and phosphorylation, and the regulation of IFNγ‐inducible genes.

Methods

Fluorocytometry was used to investigate expression of STAT‐1, pSTAT‐1, CD95, HLA–DR, class I major histocompatibility complex (MHC), IFNγ‐inducible 10‐kd protein (IP‐10), monokine induced by IFNγ (Mig), and IFNγR in PBMCs from SLE patients and healthy individuals. STAT‐1 phosphorylation was determined by fluorocytometry and Western blotting after stimulation with IFNα or IFNγ. Quantitative polymerase chain reaction was used to assess messenger RNA (mRNA) expression of the IFNγ‐inducible genes IP‐10 and Mig shortly after preparation or after stimulation with IFNγ in monocytes.

Results

STAT‐1 expression was increased in PBMCs from SLE patients and correlated significantly with disease activity and with the IFN‐inducible expression of CD95 and HLA–DR. STAT‐1 expression also showed a trend toward association with class I MHC expression. In addition, the expression of other IFNγ‐inducible genes, such as IP‐10 or Mig, was increased in SLE monocytes. While STAT‐1 phosphorylation in SLE PBMCs and PBMCs from healthy individuals was similar after IFNα stimulation, incubation with IFNγ induced STAT‐1 phosphorylation only in SLE lymphocytes. Moreover, SLE monocytes showed a considerably higher increase in pSTAT‐1 expression upon IFNγ stimulation than monocytes from healthy individuals. Increased responsiveness of SLE monocytes to IFNγ was also confirmed on the mRNA level, where expression of the IFN‐inducible, STAT‐1–dependent genes IP‐10 and Mig was more efficiently increased in SLE cells. However, IFNγR was similarly expressed on SLE lymphocytes and monocytes and those from healthy individuals.

Conclusion

In addition to supporting the role of IFNs in SLE immunopathogenesis in general, the findings of the present study support a role of IFNγ in this disease.

     

Interferon‐α priming promotes lipid uptake and macrophage‐derived foam cell formation: A novel link between interferon‐α and atherosclerosis in lupus

Objective

An increased risk of premature atherosclerosis has been associated with systemic lupus erythematosus (SLE), and type I interferon (IFN) has been shown to play a pathogenic role in human SLE. The aim of this study was to determine whether IFNα is involved in the development of atherosclerosis in patients with SLE by promoting lipid uptake and macrophage‐derived foam cell formation, which is a crucial step in early atherosclerosis.

Methods

The effects of IFNα on lipid uptake and foam cell formation were determined by flow cytometry and oil red O staining. Messenger RNA and protein expression of scavenger receptors (SRs) was examined. Promoter activity was detected by luciferase reporter assay. Expression of macrophage SR class A (SR‐A) and IFN‐inducible genes (IFIGs) was measured in peripheral blood mononuclear cells obtained from 42 patients with SLE and 42 healthy donors.

Results

IFNα priming increased the uptake of oxidized low‐density lipoprotein and hence enhanced foam cell formation by up‐regulating SR‐A expression. IFNα increased SR‐A expression via enhancing its promoter activities. Examination using signaling inhibitors revealed that a phosphatidylinositol 3‐kinase/Akt signaling pathway appeared to be involved in this process. Notably, SR‐A messenger RNA was significantly increased in patients with SLE compared to normal subjects and positively correlated with IFIG expression.

Conclusion

Our data suggest that IFNα priming up‐regulated the expression of SR‐A in human monocyte/macrophages, leading to increased lipid uptake and foam cell formation. Activation of the IFN signaling pathway may be linked to the risk of atherosclerosis by affecting plaque formation in patients with SLE. These findings provide novel insights into the mechanisms of and potential therapeutic approaches to premature atherosclerosis in patients with SLE.

     

Network analysis of associations between serum interferon‐α activity,autoantibodies, and clinical features in systemic lupus erythematosus

Objective

Interferon‐α (IFNα) is a primary pathogenic factor in systemic lupus erythematosus (SLE), and high IFNα levels may be associated with particular clinical manifestations. The prevalence of individual clinical and serologic features differs significantly by ancestry. This study was undertaken to detect associations between clinical and serologic disease manifestations and serum IFNα activity in a large diverse SLE cohort, using multivariate and network analyses.

Methods

We studied 1,089 SLE patients (387 African American, 186 Hispanic American, and 516 European American patients). The presence or absence of individual American College of Rheumatology (ACR) clinical criteria for SLE, autoantibodies, and serum IFNα activity data were analyzed in univariate and multivariate models. Iterative multivariate logistic regression was performed in each ancestral background group separately to establish the network of associations between variables that were independently significant following Bonferroni correction.

Results

In all ancestral backgrounds, high IFNα activity was associated with anti‐Ro and anti–double‐stranded DNA antibodies (P = 4.6 × 10⁻¹⁸ and P = 2.9 × 10⁻¹⁶, respectively). Younger age, non‐European ancestry, and anti‐RNP were also independently associated with increased serum IFNα activity (P ≤ 6.7 × 10⁻⁴). We found 14 unique associations between variables in network analysis, and only 7 of these associations were shared among >1 ancestral background. Associations between clinical criteria were different for different ancestral backgrounds, while autoantibody–IFNα relationships were similar across backgrounds. IFNα activity and autoantibodies were not associated with ACR clinical features in multivariate models.

Conclusion

Our findings indicate that serum IFNα activity is strongly and consistently associated with autoantibodies, and not independently associated with clinical features in SLE. IFNα may be more relevant to humoral tolerance and initial pathogenesis than later clinical disease manifestations.

     

Functional assay of type I interferon in systemic lupus erythematosus plasma and association with anti–RNA binding protein autoantibodies

Objective

Peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) have increased expression of genes typically induced by type I interferon (IFN). However, it has been difficult to identify and quantify the factors responsible for activation of the IFN pathway in SLE. To characterize these mediators, we developed an assay that measures the functional effects of plasma or serum components on the gene expression of cultured target cells.

Methods

WISH epithelial cell line cells were cultured with medium, with recombinant IFNα, IFNβ, or IFNγ, or with 50% plasma from SLE patients (n = 73), rheumatoid arthritis (RA) patients (n = 19), or healthy donors (n = 30). Real‐time quantitative polymerase chain reaction was used to determine WISH cell expression of IFN target genes, including PRKR, IFIT1, IFI44, MX1, and C1orf29 (preferentially induced by IFNα) and CXCL9 (Mig) (preferentially induced by IFNγ).

Results

IFNα‐regulated genes were induced by SLE plasma samples, but not by most of the RA or healthy control plasma samples. The activity in SLE plasma was inhibited >90% by anti‐IFNα antibody, but not by anti‐IFNβ or anti‐IFNγ antibodies. The expression of each IFNα target gene induced by SLE plasma correlated with the expression of that gene studied ex vivo in PBMCs from the same patients and with the titer of anti–RNA binding protein (anti‐RBP)–specific autoantibodies. Plasma activity paralleled PBMC expression of IFNα‐inducible genes over time.

Conclusion

IFNα in SLE plasma is a major stimulus of IFN target gene expression and is related to expression of those genes in PBMCs from SLE patients and to the titer of anti‐RBP autoantibodies. These data provide additional support for the view that IFNα mediates immune system activation and dysregulation in SLE.

     

Statins,inhibitors of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase,function as inhibitors of cellular and molecular components involved in type I interferon production

Objective

Statins, which are used as cholesterol‐lowering agents, have pleiotropic immunomodulatory properties. Although beneficial effects of statins have been reported in autoimmune diseases, the mechanisms of these immunomodulatory effects are still poorly understood. Type I interferons (IFNs) and plasmacytoid dendritic cells (PDCs) represent key molecular and cellular pathogenic components in autoimmune diseases such as systemic lupus erythematosus (SLE). Therefore, PDCs may be a specific target of statins in therapeutic strategies against SLE. This study was undertaken to investigate the immunomodulatory mechanisms of statins that target the IFN response in PDCs.

Methods

We isolated human blood PDCs by flow cytometry and examined the effects of simvastatin and pitavastatin on PDC activation, IFNα production, and intracellular signaling.

Results

Statins inhibited IFNα production profoundly and tumor necrosis factor α production modestly in human PDCs in response to Toll‐like receptor ligands. The inhibitory effect on IFNα production was reversed by geranylgeranyl pyrophosphate and was mimicked by either geranylgeranyl transferase inhibitor or Rho kinase inhibitor, suggesting that statins exert their inhibitory actions through geranylgeranylated Rho inactivation. Statins inhibited the expression of phosphorylated p38 MAPK and Akt, and the inhibitory effect on the IFN response was through the prevention of nuclear translocation of IFN regulatory factor 7. In addition, statins had an inhibitory effect on both IFNα production by PDCs from SLE patients and SLE serum–induced IFNα production.

Conclusion

Our findings suggest a specific role of statins in controlling type I IFN production and a therapeutic potential in IFN‐related autoimmune diseases such as SLE.

     

Lupus‐like disease and high interferon levels corresponding to trisomy of the type I interferon cluster on chromosome 9p

Objective

Systemic lupus erythematosus (SLE) is associated with type I interferons (IFNs) and can be induced by IFNα treatment. This study looked for evidence of autoimmunity in a pedigree consisting of 4 family members with a balanced translocation 9;21 and 2 members with an unbalanced translocation resulting in trisomy of the short (p) arm and part of the long (q) arm of chromosome 9. These latter 2 subjects had 3 copies of the IFN gene cluster.

Methods

Subjects were evaluated clinically and serologically for autoimmune disease. Expression levels of IFNα4, IFNβ, the type I IFN–inducible gene Mx1, the type I IFN receptor, interleukin‐6, and tumor necrosis factor α were determined by real‐time polymerase chain reaction. Circulating plasmacytoid dendritic cells, the main IFN‐producing cells, were quantified by flow cytometry.

Results

Both subjects with trisomy of chromosome 9p had a lupus‐like syndrome with joint manifestations and antinuclear antibodies: one had anti‐RNP and antiphospholipid autoantibodies, and the other had anti–Ro 60. The 3 family members with a balanced translocation 9;21 had no clinical or serologic evidence of autoimmunity, similar to that in relatives who were unaffected by the chromosomal translocation. In the 2 subjects with trisomy of 9p, high levels of IFNα/β (comparable with those found in patients with SLE), increased signaling through the IFN receptor (as indicated by high Mx1 expression), and low levels of circulating plasmacytoid dendritic cells (as observed in patients with SLE) were evident. These abnormalities were not seen in individuals with a balanced translocation.

Conclusion

Trisomy of the type I IFN cluster of chromosome 9p was associated with lupus‐like autoimmunity and increased IFNα/β and IFN receptor signaling. The data support the idea that abnormal regulation of type I IFN production is involved in the pathogenesis of SLE.

     

Activation of the interferon‐α pathway identifies a subgroup of systemic lupus erythematosus patients with distinct serologic features and active disease

Objective

Gene‐expression studies have demonstrated increased expression of interferon (IFN)–inducible genes (IFIGs) in peripheral blood mononuclear cells (PBMCs) of many patients with systemic lupus erythematosus (SLE), with a predominant effect of type I IFN. This study examined the hypothesis that increased disease severity and activity, as well as distinct autoantibody specificities, characterize SLE patients with activation of the type I IFN pathway.

Methods

Freshly isolated PBMCs from 77 SLE patients, 22 disease controls, and 28 healthy donors were subjected to real‐time polymerase chain reaction for 3 IFIGs that are preferentially induced by IFNα, and the data were used to derive IFNα scores for all individuals. Expression of IFIGs was significantly higher in SLE patients compared with disease controls or healthy donors. SLE patients with high and low IFNα scores were compared for clinical manifestations of disease, disease severity, disease activity, serologic features, and potential confounders, by bivariate and multivariate analyses.

Results

SLE patients with a high IFNα score had a significantly higher prevalence of renal disease, a greater number of American College of Rheumatology criteria for SLE, and a higher Systemic Lupus International Collaborating Clinics damage index (SDI) score than did SLE patients with low IFNα scores. Patients with high scores showed increased disease activity, as measured by lower C3 levels, hemoglobin levels, absolute lymphocyte counts, and albumin levels, and a higher anti–double‐stranded DNA (dsDNA) titer, erythrocyte sedimentation rate, and SLE Disease Activity Index 2000 score. The presence of antibodies specific for Ro, U1 RNP, Sm, and dsDNA, but not phospholipids, was significantly associated with a high IFNα score. Logistic regression analysis confirmed that renal disease, higher SDI scores, low complement levels, and presence of anti–RNA binding protein (RBP) autoantibodies were associated with a high IFNα score.

Conclusion

Activation of the IFNα pathway defines a subgroup of SLE patients whose condition is characterized by increased disease severity, including renal disease, increased disease activity, reflected in complement activation, and autoreactivity to RBP.

     

A possible mechanism for endogenous activation of the type I interferon system in myositis patients with anti–Jo‐1 or anti–Ro 52/anti–Ro 60 autoantibodies

Objective

To investigate type I interferon (IFN) system activation and its correlation with autoantibodies and organ manifestations in polymyositis (PM), dermatomyositis (DM), and inclusion body myositis.

Methods

Sera from 30 patients and 16 healthy controls, or purified IgG, were combined with material released from necrotized cells to stimulate IFNα production by peripheral blood mononuclear cells (PBMCs) from healthy blood donors. Muscle biopsy specimens from 25 patients and 7 healthy controls were investigated for blood dendritic cell antigen 2 (BDCA‐2)–positive plasmacytoid dendritic cells (PDCs) and IFNα/β‐inducible myxovirus resistance 1 (MX‐1) protein.

Results

Sera from 13 patients who were positive for anti–Jo‐1 or anti–Ro 52/anti–Ro 60 autoantibodies induced IFNα production in PBMCs when combined with necrotic cell material. In addition, IgG prepared from anti–Jo‐1–positive PM sera induced IFNα with necrotic material, but not when the latter was treated with RNase. BDCA‐2 expression in PDCs in muscle tissue was increased in PM patients with anti–Jo‐1 autoantibodies, while MX‐1 staining in capillaries was increased in DM patients, compared with healthy individuals. IFNα‐inducing capacity correlated with interstitial lung disease, while MX‐1 expression in the capillaries correlated with DM.

Conclusion

Immune complexes containing anti–Jo‐1 or anti–Ro 52/anti–Ro 60 autoantibodies and RNA may act as endogenous IFNα inducers that activate IFNα production in PDCs. These PDCs could be of importance for inducing myositis, whereas in DM patients without autoantibodies the presence of MX‐1 protein in capillaries suggests another cellular IFNα source and induction mechanism. Consequently, the type I IFN system may be of importance in both PM and DM, but via different pathways.

     

Correlation of interferon‐inducible chemokine plasma levels with disease severity in systemic sclerosis

Objective

To measure interferon (IFN)–inducible chemokines in the plasma of patients with systemic sclerosis (SSc) and investigate whether the chemokine levels are correlated with disease severity.

Methods

Plasma levels of the IFN‐inducible chemokines IFNγ‐inducible protein 10 (IP‐10/CXCL10), IFN‐inducible T cell α chemoattractant (I‐TAC/CXCL11), and monocyte chemoattractant protein 1 (CCL2) were measured in SSc patients and examined for correlation with the IFN gene expression signature. A composite IFN‐inducible chemokine score was generated for chemokines showing a correlation with the IFN gene signature (IP‐10 and I‐TAC), and this score was compared between 266 patients with SSc enrolled in the Genetics versus Environment in Scleroderma Outcome Study (GENISOS) cohort and 97 matched control subjects. Subsequently, the correlation between the IFN‐inducible chemokine score at baseline and markers of disease severity was assessed. In addition, the course of the IFN‐inducible chemokine score over time was examined.

Results

The plasma IFN‐inducible chemokine score correlated with the IFN gene expression signature, and this score was higher in SSc patients compared to controls. The IFN‐inducible chemokine score was also associated with the absence of anti–RNA polymerase III antibodies and presence of anti–U1 RNP antibodies, but not with disease duration, disease type, or other autoantibodies. The chemokine score correlated with concomitantly obtained scores on the Medsger Severity Index for muscle, skin, and lung involvement in SSc, as well as the forced vital capacity, diffusing capacity for carbon monoxide, and creatine kinase levels. The association of the chemokine score with disease severity was independent of the presence of anti–U1 RNP or other potential confounders (age, sex, ethnicity, disease duration, and treatment with immunosuppressive agents). Finally, there was not a significant change in the IFN‐inducible chemokine score over time.

Conclusion

The IFN‐inducible chemokine score is a stable serologic marker of a more severe form of SSc and may be useful for risk stratification of patients, regardless of disease type (limited or diffuse) or duration of disease.

     

Induction of interferon‐α production in plasmacytoid dendritic cells by immune complexes containing nucleic acid released by necrotic or late apoptotic cells and lupus IgG

Objective

To investigate the release of interferon‐α (IFNα)–inducing material by necrotic or apoptotic cells, its properties, and the necessity of autoantibodies from systemic lupus erythematosus (SLE) patients for the interferogenic activity.

Methods

U937 monocytic leukemia cells or peripheral blood mononuclear cells (PBMCs) were rendered necrotic by freeze‐thawing or apoptotic by treatment with ultraviolet light. Cell culture supernatants from these cells and IgG from SLE patients (SLE IgG) were added to cultures of normal PBMCs or purified plasmacytoid dendritic cells (PDCs). The importance of nucleic acids for IFNα induction was investigated by RNase and DNase treatment. The IFNα levels were measured by immunoassay.

Results

Both necrotic and apoptotic U937 cells released material that, combined with SLE IgG, induced IFNα production in PDCs. The release from apoptotic cells occurred with a 16‐hour delay, in late apoptosis. Also, normal PBMCs released IFNα‐inducing material, but only during necrosis. The interferogenic activity of the necrotic material required the presence of RNA, while both RNA and DNA were important in the apoptotic material. In both cases, the presence of SLE IgG was necessary, and its activity correlated with the presence of antibodies to RNA‐binding proteins, but not anti‐DNA antibodies.

Conclusion

Necrotic and late apoptotic cells release material that, combined with SLE IgG, induces production of IFNα in PDCs. The IFNα inducers probably consist of immune complexes (ICs) containing RNA and possibly DNA as essential interferogenic components. The presence of such interferogenic ICs could explain the ongoing production of IFNα in SLE and could be of etiopathogenic importance.

     

Regulation of the interferon‐α production induced by RNA‐containing immune complexes in plasmacytoid dendritic cells

Objective

Interferon‐α (IFNα) is produced in several autoimmune diseases, including systemic lupus erythematosus (SLE), and may be important in their pathogenesis. We undertook this study to investigate how IFNα production induced by RNA‐containing immune complexes (ICs) in plasmacytoid dendritic cells (PDCs) is regulated.

Methods

Normal PDCs purified from peripheral blood mononuclear cells (PBMCs) were cocultivated with other cell populations isolated from healthy individuals or SLE patients. IFNα production was induced by RNA‐containing ICs, which consisted of anti‐RNP autoantibodies and U1 small nuclear RNP particles, and the effects of prostaglandin E₂ (PGE₂), reactive oxygen species (ROS), or the cytokines IFNα2b, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), interleukin‐10 (IL‐10), or tumor necrosis factor α (TNFα) were explored.

Results

Monocytes inhibited IFNα production by PDCs in PBMC cultures, while natural killer (NK) cells were stimulatory. The monocytes had little effect on IFNα production by pure PDCs but inhibited its stimulation by NK cells. Monocytes from SLE patients were less inhibitory. Exposure of PBMCs or PDCs to IFNα2b/GM‐CSF increased their IFNα production. RNA‐containing ICs caused production of ROS, PGE₂, and TNFα, especially in monocytes. These mediators and IL‐10 suppressed IFNα production in PBMC cultures, with ROS and PGE₂ also inhibiting IFNα production by purified PDCs. Inhibition by all of these agents, except for ROS, was abolished by IFNα2b/GM‐CSF. The inhibitory effect of monocytes was significantly counteracted by the ROS scavengers serotonin and catalase.

Conclusion

IFNα production induced by RNA‐containing ICs in PDCs is regulated by a network of interactions between monocytes, NK cells, and PDCs, involving several pro‐ and antiinflammatory molecules. This should be considered when designing and applying new therapies.

     

Epstein‐Barr virus promotes interferon‐α production by plasmacytoid dendritic cells

Objective

Epstein‐Barr virus (EBV) infection has been linked to systemic lupus erythematosus (SLE), as demonstrated by the presence of increased seroprevalence and elevated viral loads, but the mechanism of this linkage has not been elucidated. Increased interferon‐α (IFNα) levels and signatures, which are associated with innate immune responses, have been found in patients with SLE. Plasmacytoid dendritic cells (PDCs) are innate immune cells that mediate viral immunity by producing large quantities of IFNα, but the role they play during infection with EBV remains unclear. To address this issue, we investigated the ability of EBV to promote IFNα production by PDCs in healthy subjects.

Methods

Human PDCs were sorted and cultured in the presence of EBV, EBV‐encoded RNA, and EBV double‐stranded DNA. IFNα production by PDCs was measured by enzyme‐linked immunosorbent assay, with the activation of these cells measured by flow cytometry.

Results

We found that EBV DNA and RNA promoted IFNα production by human PDCs through engagement of Toll‐like receptor 9 (TLR‐9) and TLR‐7, respectively, with the initial viral recognition by PDCs mediated by binding to class II major histocompatibility complex (MHC) molecules.

Conclusion

These data demonstrate that class II MHC–specific engagement by virus, with subsequent viral nucleic acid recognition, mediates IFNα production by PDCs. Our results suggest that elevated levels of IFNα found in SLE patients may be a result of aberrantly controlled chronic viral infection.

     

Interferon‐regulated chemokines as biomarkers of systemic lupus erythematosus disease activity: A validation study

Objective

Systemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by unpredictable flares of disease activity and irreversible damage to multiple organ systems. An earlier study showed that SLE patients carrying an interferon (IFN) gene expression signature in blood have elevated serum levels of IFN‐regulated chemokines. These chemokines were associated with more‐severe and active disease and showed promise as SLE disease activity biomarkers. This study was designed to validate IFN‐regulated chemokines as biomarkers of SLE disease activity in 267 SLE patients followed up longitudinally.

Methods

To validate the potential utility of serum chemokine levels as biomarkers of disease activity, we measured serum levels of CXCL10 (IFNγ‐inducible 10‐kd protein), CCL2 (monocyte chemotactic protein 1), and CCL19 (macrophage inflammatory protein 3β) in an independent cohort of 267 SLE patients followed up longitudinally over 1 year (1,166 total clinic visits).

Results

Serum chemokine levels correlated with lupus activity at the current visit (P = 2 × 10⁻¹⁰), rising at the time of SLE flare (P = 2 × 10⁻³) and decreasing as disease remitted (P = 1 × 10⁻³); they also performed better than the currently available laboratory tests. Chemokine levels measured at a single baseline visit in patients with a Systemic Lupus Erythematosus Disease Activity Index of ≤4 were predictive of lupus flare over the ensuing year (P = 1 × 10⁻⁴).

Conclusion

Monitoring serum chemokine levels in SLE may improve the assessment of current disease activity, the prediction of future disease flares, and the overall clinical decision‐making.

     

Genetic variation at the IRF7/PHRF1 locus is associated with autoantibody profile and serum interferon‐α activity in lupus patients

Objective

Interferon‐α (IFNα) is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variation near IRF7 is implicated in SLE susceptibility. SLE‐associated autoantibodies can stimulate IFNα production through the Toll‐like receptor/IRF7 pathway. This study was undertaken to determine whether variants of IRF7 act as risk factors for SLE by increasing IFNα production and whether autoantibodies are important to this phenomenon.

Methods

We studied 492 patients with SLE (236 African American, 162 European American, and 94 Hispanic American subjects). Serum levels of IFNα were measured using a reporter cell assay, and single‐nucleotide polymorphisms (SNPs) in the IRF7/PHRF1 locus were genotyped.

Results

In a joint analysis of European American and Hispanic American subjects, the rs702966 C allele was associated with the presence of anti–double‐stranded DNA (anti‐dsDNA) antibodies (odds ratio [OR] 1.83, P = 0.0069). The rs702966 CC genotype was only associated with higher serum levels of IFNα in European American and Hispanic American patients with anti‐dsDNA antibodies (joint analysis P = 4.1 × 10⁻⁵ in anti‐dsDNA–positive patients and P = 0.99 in anti‐dsDNA–negative patients). In African American subjects, anti‐Sm antibodies were associated with the rs4963128 SNP near IRF7 (OR 1.95, P = 0.0017). The rs4963128 CT and TT genotypes were associated with higher serum levels of IFNα only in African American patients with anti‐Sm antibodies (P = 0.0012). In African American patients lacking anti‐Sm antibodies, an effect of anti‐dsDNA–rs702966 C allele interaction on serum levels of IFNα was observed, similar to the other patient groups (overall joint analysis P = 1.0 × 10⁻⁶). In European American and Hispanic American patients, the IRF5 SLE risk haplotype showed an additive effect with the rs702966 C allele on IFNα level in anti‐dsDNA–positive patients.

Conclusion

Our findings indicate that IRF7/PHRF1 variants in combination with SLE‐associated autoantibodies result in higher serum levels of IFNα, providing a biologic relevance for this locus at the protein level in human SLE in vivo.

     

B lymphocytes enhance interferon‐α production by plasmacytoid dendritic cells

Objective

The type I interferon (IFN) system and B cells are activated in many autoimmune diseases, such as systemic lupus erythematosus (SLE). The IFNα produced by plasmacytoid dendritic cells (PDCs) stimulates several B cell functions, including autoantibody production. However, not much is known about how B cells influence PDC function. The aim of this study was to investigate the regulatory effect of B cells on IFNα production by PDCs.

Methods

PDCs and B cells isolated from peripheral blood mononuclear cells from healthy blood donors were stimulated with RNA‐containing immune complexes (ICs) consisting of U1 small nuclear RNP and SLE IgG, herpes simplex virus, or oligonucleotide (ODN) 2216, alone or in cocultures. IFNα, several other cytokines, and PDC‐ or B cell–associated surface molecules were analyzed using immunoassays or flow cytometry.

Results

B cells enhanced IFNα production by PDCs up to 47‐fold, and the effect was most pronounced for PDCs stimulated with RNA‐containing ICs. Anti‐CD31 antibody reduced RNA‐containing IC–induced IFNα production by 80% but had no effect on IFNα production when ODN 2216 was used as an inducer. Supernatants from ODN 2216–stimulated B cells promoted IFNα production by PDCs, while supernatants from RNA‐containing IC–stimulated B cells did not.

Conclusion

Our results showed that a novel function of B cells is enhancement of type I IFN production by PDCs. Because B cells are activated by type I IFN, this PDC–B cell cross‐talk might be of fundamental importance in the etiopathogenesis of SLE and contribute to long‐term immune activation in SLE and other systemic rheumatic diseases.