Related Articles

Increased IFN-α signaling is a heritable risk factor for systemic lupus erythematosus (SLE). IFN induced with helicase C domain 1 (IFIH1) is a cytoplasmic dsRNA sensor that activates IFN-α pathway signaling. We studied the impact of the autoimmune-disease–associated IFIH1 rs1990760 (A946T) single nucleotide polymorphism upon IFN-α signaling in SLE patients in vivo. We studied 563 SLE patients (278 African-American, 179 European-American, and 106 Hispanic-American). Logistic regression models were used to detect genetic associations with autoantibody traits, and multiple linear regression was used to analyze IFN-α–induced gene expression in PBMCs in the context of serum IFN-α in the same blood sample. We found that the rs1990760 T allele was associated with anti-dsDNA Abs across all of the studied ancestral backgrounds (meta-analysis odds ratio = 1.34, p = 0.026). This allele also was associated with lower serum IFN-α levels in subjects who had anti-dsDNA Abs (p = 0.0026). When we studied simultaneous serum and PBMC samples from SLE patients, we found that the IFIH1 rs1990760 T allele was associated with increased IFN-induced gene expression in PBMCs in response to a given amount of serum IFN-α in anti-dsDNA–positive patients. This effect was independent of the STAT4 genotype, which modulates sensitivity to IFN-α in a similar way. Thus, the IFIH1 rs1990760 Tallele was associated with dsDNA Abs, and in patients with anti-dsDNA Abs this risk allele increased sensitivity to IFN-α signaling. These studies suggest a role for the IFIH1 risk allele in SLE in vivo.

Systemic lupus erythematosus (SLE) is a severe multi-system autoimmune disease which results from both genetic predisposition and environmental factors. Many lines of investigation support interferon alpha (IFN-α) as a causal agent in human lupus, and high levels of serum IFN-α are a heritable risk factor for SLE. Interferon regulatory factors (IRFs) are a family of transcription factors involved in host defense, which can induce transcription of IFN-α and other immune response genes following activation. In SLE, circulating immune complexes which contain nucleic acid are prevalent. These complexes are recognized by endosomal Toll-like receptors, resulting in activation of downstream IRF proteins. Genetic variants in the IRF5 and IRF7 genes have been associated with SLE susceptibility, and these same variants are associated with increased serum IFN-α in SLE patients. The increase in serum IFN-α related to IRF5 and 7 genotypes is observed only in patients with particular antibody specificities. This suggests that chronic stimulation of the endosomal Toll-like receptors by autoantibody immune complexes is required for IRF SLE-risk variants to cause elevation of circulating IFN-α and subsequent risk of SLE. Recently, genetic variation in the IRF8 gene has been associated with SLE and multiple sclerosis, and studies support an impact of IRF8 genotype on the IFN-α pathway. In summary, the SLE-associated polymorphisms in the IRF family of proteins appear to be gain-of-function variants, and understanding the impact of these variants upon the IFN-α pathway in vivo may guide therapeutic strategies directed at the Toll-like receptor/IRF/IFN-α pathway in SLE.

Interferon alpha (IFN-α) is a critical mediator of human systemic lupus erythematosus (SLE). This review will summarize evidence supporting the role for IFN-α in the initiation of human SLE. IFN-α functions in viral immunity at the interface of innate and adaptive immunity, a position well suited to setting thresholds for autoimmunity. Some individuals treated with IFN-α for chronic viral infections develop de novo SLE, which frequently resolves when IFN-α is withdrawn, supporting the idea that IFN-α was causal. Abnormally high IFN-α levels are clustered within SLE families, suggesting that high serum IFN-α is a heritable risk factor for SLE. Additionally, SLE-risk genetic variants in the IFN-α pathway are gain of function in nature, resulting in either higher circulating IFN-α levels or greater sensitivity to IFN-α signaling in SLE patients. A recent genome-wide association study has identified additional novel genetic loci associated with high serum IFN-α in SLE patients. These data support the idea that genetically determined endogenous elevations in IFN-α predispose to human SLE. It is possible that some of these gain-of-function polymorphisms in the IFN-α pathway are useful in viral defense, and that risk of SLE is a burden we have taken on in the fight to defend ourselves against viral infection.

Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 × 10−8) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 × 10−5). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 × 10−5) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.

Background

Overexpression of type I interferon (IFN-I)-induced genes is a common feature of systemic lupus erythematosus (SLE) and its experimental models, but the participation of endogenous overproduction of IFN-I on it is not clear. To explore the possibility that abnormally increased IFN-I receptor (IFNAR) signaling could participate in IFN-I-induced gene overexpression of SLE, we examined the phosphorylation status of the IFNAR-associated signaling partners Jak1 and STAT2, and its relation with expression of its physiologic inhibitor SOCS1 and with plasma levels of IFNα and IFN-like activity.

Methodology/Principal Findings

Peripheral blood mononuclear cells (PBMC) from SLE patients with or without disease activity and healthy controls cultured in the presence or in the absence of IFNβ were examined by immunoprecipitation and/or western blotting for expression of the two IFNAR chains, Jak1, Tyk2, and STAT2 and their phosphorylated forms. In SLE but not in healthy control PBMC, Jak1 and STAT2 were constitutively phosphorylated, even in the absence of disease activity (basal pJak1: controls vs. active SLE p<0.0001 and controls vs. inactive SLE p = 0.0006; basal pSTAT2: controls vs. active and inactive SLE p<0.0001). Although SOCS1 protein was slightly but significantly decreased in SLE in the absence or in the presence of IFNβ (p = 0.0096 to p<0.0001), in SOCS1 mRNA levels were markedly decreased (p = 0.036 to p<0.0001). IFNβ induced higher levels of the IFN-I-dependent MxA protein mRNA in SLE than in healthy controls, whereas the opposite was observed for SOCS1. Although there was no relation to increased serum IFNα, active SLE plasma could induce expression of IFN-dependent genes by normal PBMC.

Conclusions/Significance

These findings suggest that in some SLE patients IFN-I dependent gene expression could be the result of a low IFNAR signaling threshold.

Background

The C1858T polymorphism in PTPN22 has been associated with risk of systemic lupus erythematosus (SLE), as well as multiple other autoimmune diseases. We have previously shown that high serum interferon alpha (IFN-α) activity is a heritable risk factor for SLE, and we hypothesized that the PTPN22 risk variant may shift serum cytokine profiles to higher IFN-α activity resulting in risk of disease.

Methods

IFN-α was measured in 143 SLE patients using a functional reporter cell assay, and TNF-α was measured with ELISA. The rs2476601 SNP in PTPN22 (C1858T) was genotyped in the same patients. Patients were grouped using a clustering algorithm into four cytokine groups (IFN-α predominant, IFN-α and TNF-α correlated, TNF-α predominant, and IFN-α and TNF-α both low).

Results

SLE patients carrying the risk allele of PTPN22 had higher serum IFN-α activity than patients lacking the risk allele (p=0.027). TNF-α levels were lower in risk allele carriers (p=0.030), and the risk allele was more common in patients with an IFN-α predominant or IFN-α and TNF-α correlated cytokine profile as compared to patients with TNF-α predominance or both cytokines low (p=0.002). 25% of male patients carried the risk allele, compared to 10% of female patients (p=0.02), however cytokine skewing was similar in both sexes.

Conclusions

The autoimmune disease risk allele of PTPN22 is associated with skewing of serum cytokine profiles toward higher IFN-α activity and lower TNF-α in SLE patients in vivo. This serum cytokine pattern may be relevant in other autoimmune diseases associated with the PTPN22 risk allele.

Osteopontin (OPN) is a multifunctional cytokine involved in long bone remodeling and immune system signaling. Additionally, OPN is critical for interferon-α (IFN-α) production in murine plasmacytoid dendritic cells. We have previously shown that IFN-α is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variants of OPN have been associated with SLE susceptibility, and one study suggests that this association is particular to men. In this study, the 3′ UTR SLE-risk variant of OPN (rs9138C) was associated with higher serum OPN and IFN-α in men (P = 0.0062 and P = 0.0087, respectively). In women, the association between rs9138 C and higher serum OPN and IFN-α was restricted to younger subjects, and risk allele carriers showed a strong age-related genetic effect of rs9138 genotype on both serum OPN and IFN-α (P<0.0001). In African-American subjects, the 5′ region single nucleotide polymorphisms, rs11730582 and rs28357094, were associated with anti-RNP antibodies (odds ratio (OR) = 2.9, P = 0.0038 and OR = 3.9, P = 0.021, respectively). Thus, we demonstrate two distinct genetic influences of OPN on serum protein traits in SLE patients, which correspond to previously reported SLE-risk variants. This study provides a biologic relevance for OPN variants at the protein level, and suggests an influence of this gene on the IFN-α pathway in SLE.

Objective

Genetic variants of interferon regulatory factor 5 (IRF5) are associated with susceptibility to systemic lupus erythematosus (SLE). IRF5 regulates the expression of proinflammatory cytokines and type I interferons (IFN) believed to be involved in SLE pathogenesis. The aim of this study was to determine the activation status of IRF5 by assessing its nuclear localization in immune cells of SLE patients and healthy donors, and to identify SLE triggers of IRF5 activation.

Methods

IRF5 nuclear localization in subpopulations of peripheral blood mononuclear cells (PBMC) from 14 genotyped SLE patients and 11 healthy controls was assessed using imaging flow cytometry. IRF5 activation and function were examined after ex vivo stimulation of healthy donor monocytes with SLE serum or components of SLE serum. Cellular localization was determined by ImageStream and cytokine expression by Q-PCR and ELISA.

Results

IRF5 was activated in a cell type-specific manner; monocytes of SLE patients had constitutively elevated levels of nuclear IRF5 compared to NK and T cells. SLE serum was identified as a trigger for IRF5 nuclear accumulation; however, neither IFNα nor SLE immune complexes could induce nuclear localization. Instead, autoantigens comprised of apoptotic/necrotic material triggered IRF5 nuclear accumulation in monocytes. Production of cytokines IFNα, TNFα and IL6 in monocytes stimulated with SLE serum or autoantigens was distinct yet correlated with the kinetics of IRF5 nuclear localization.

Conclusion

This study provides the first formal proof that IRF5 activation is altered in monocytes of SLE patients that is in part contributed by the SLE blood environment.

Background

Recent studies demonstrated an association of STAT4 variants with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), indicating that multiple autoimmune diseases share common susceptibility genes. We therefore investigated the influence of STAT4 variants on the susceptibility and phenotype of inflammatory bowel diseases (IBD) in a large patient and control cohort.

Methodology/Principal Findings

Genomic DNA from 2704 individuals of Caucasian origin including 857 patients with Crohn's disease (CD), 464 patients with ulcerative colitis (UC), and 1383 healthy, unrelated controls was analyzed for seven SNPs in the STAT4 gene (rs11889341, rs7574865, rs7568275, rs8179673, rs10181656, rs7582694, rs10174238). In addition, a detailed genotype-phenotype analysis was performed. Our analysis revealed an association of the STAT4 SNP rs7574865 with overall decreased susceptibility to CD (p = 0.047, OR 0.86 [95% CI 0.74–0.99]). However, compared to CD patients carrying the wild type genotype, the STAT4 SNP rs7574865 was significantly associated with early CD onset (p = 0.021) and colonic CD (p = 0.008; OR = 4.60, 95% CI 1.63–12.96). For two other STAT4 variants, there was a trend towards protection against CD susceptibility (rs7568275, p = 0.058, OR 0.86 [95% CI 0.74–1.00]; rs10174238, p = 0.057, OR 0.86 [95% CI 0.75–1.00]). In contrast, we did not observe any association with UC susceptibility. Evidence for weak gene-gene interaction of STAT4 with the IL23R SNP rs11209026 was lost after Bonferroni correction.

Conclusions/Significance

Our results identified the STAT4 SNP rs7574865 as a disease-modifying gene variant in colonic CD. However, in contrast to SLE and RA, the effect of rs7574865 on CD susceptibility is only weak.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organ systems. Previous studies have suggested that interferon-lambda 1 (IFN-λ1), a type III interferon, plays an immunomodulatory role. In this study we investigated its role in SLE, including its correlation with disease activity, organ disorder and production of chemokines.

Methods

We determined levels of IFN-λ1 mRNA in peripheral blood mononuclear cells (PBMC) and serum protein levels in patients with SLE using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunoassay (ELISA). Further, we detected the concentration of IFN-inducible protein-10 (IP-10), monokine induced by IFN-γ (MIG) and interleukin-8 (IL-8) secreted by PBMC under the stimulation of IFN-λ1 using ELISA.

Results

IFN-λ1 mRNA and serum protein levels were higher in patients with SLE compared with healthy controls. Patients with active disease showed higher IFN-λ1 mRNA and serum protein levels compared with those with inactive disease as well. Serum IFN-λ1 levels were positively correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), anti-dsDNA antibody, C-reactive protein (CRP) and negatively correlated with complement 3. Serum IFN-λ1 levels were higher in SLE patients with renal involvement and arthritis compared with patients without the above-mentioned manifestations. IFN-λ1 with different concentrations displayed different effects on the secretion of the chemokines IP-10, MIG and IL-8.

Conclusions

These findings indicate that IFN-λ1 is probably involved in the renal disorder and arthritis progression of SLE and associated with disease activity. Moreover, it probably plays an important role in the pathogenesis of SLE by stimulating secretion of the chemokines IP-10, MIG and IL-8. Thus, IFN-λ1 may provide a novel research target for the pathogenesis and therapy of SLE.

Background

Systemic lupus erythematosus (SLE) patients have a striking increase in cardiovascular (CV) comorbidity not fully explained by the Framingham risk score. Recent evidence from in vitro studies suggests that type I interferons (IFN) could promote premature CV disease (CVD) in SLE. We assessed the association of type I IFN signatures with functional and anatomical evidence of vascular damage, and with biomarkers of CV risk in a cohort of lupus patients without overt CVD.

Methodology/Principal Findings

Serum type I IFN activity (induction of five IFN-inducible genes; IFIGs) from 95 SLE patient and 38 controls was quantified by real-time PCR. Flow mediated dilatation (FMD) of the brachial artery and carotid intima media thickness (CIMT) were quantified by ultrasound, and coronary calcification by computed tomography. Serum vascular biomarkers were measured by ELISA. We evaluated the effect of type I IFNs on FMD, CIMT and coronary calcification by first applying principal components analysis to combine data from five IFIGs into summary components that could be simultaneously modeled. Three components were derived explaining 97.1% of the total IFIG variation. Multivariable linear regression was utilized to investigate the association between the three components and other covariates, with the outcomes of FMD and CIMT; zero-inflated Poisson regression was used for modeling of coronary calcification. After controlling for traditional CV risk factors, enhanced serum IFN activity was significantly associated with decreased endothelial function in SLE patients and controls (p<0.05 for component 3), increased CIMT among SLE patients (p<0.01 for components 1 and 2), and severity of coronary calcification among SLE patients (p<0.001 for component 3).

Conclusions

Type I IFNs are independently associated with atherosclerosis development in lupus patients without history of overt CVD and after controlling for Framingham risk factors. This study further supports the hypothesis that type I IFNs promote premature vascular damage in SLE.

A hallmark of SLE is the production of high-titer, high-affinity, isotype-switched IgG autoantibodies directed against nucleic acid–associated antigens. Several studies have established a role for both type I IFN (IFN-I) and the activation of TLRs by nucleic acid–associated autoantigens in the pathogenesis of this disease. Here, we demonstrate that 2 IFN-I signaling molecules, IFN regulatory factor 9 (IRF9) and STAT1, were required for the production of IgG autoantibodies in the pristane-induced mouse model of SLE. In addition, levels of IgM autoantibodies were increased in pristane-treated Irf9–/– mice, suggesting that IRF9 plays a role in isotype switching in response to self antigens. Upregulation of TLR7 by IFN-α was greatly reduced in Irf9–/– and Stat1–/– B cells. Irf9–/– B cells were incapable of being activated through TLR7, and Stat1–/– B cells were impaired in activation through both TLR7 and TLR9. These data may reveal a novel role for IFN-I signaling molecules in both TLR-specific B cell responses and production of IgG autoantibodies directed against nucleic acid–associated autoantigens. Our results suggest that IFN-I is upstream of TLR signaling in the activation of autoreactive B cells in SLE.

Many patients with systemic autoimmune diseases have signs of a continuous production of type I interferon (IFN) and display an increased expression of IFN-α-regulated genes. The reason for the on-going IFN-α synthesis in these patients seems to be an activation of plasmacytoid dendritic cells (pDCs) by immune complexes (ICs), consisting of autoantibodies in combination with DNA or RNA-containing autoantigens. Such interferogenic ICs are internalized via the FcγRIIa expressed on pDCs, reach the endosome, and stimulate Toll-like receptor (TLR)-7 or -9, which subsequently leads to IFN-α gene transcription. Variants of genes involved in both the IFN-α synthesis and response have been linked to an increased risk to develop systemic lupus erythematosus (SLE) and other autoimmune diseases. Among these autoimmunity risk genes are IFN regulatory factor 5 (IRF5), which is involved in TLR signaling, and the signal transducer and activator of transcription 4 (STAT4) that interacts with the type I IFN receptor. Several other gene variants in the IFN signaling pathway also confer an increased risk to develop an autoimmune disease. The observations that IFN-α therapy can induce autoimmunity and that many autoimmune conditions have an on-going type I IFN production suggest that the type I IFN system has a pivotal role in the etiopathogenesis of these diseases. Possible mechanisms behind the dysregulated type IFNsystem in autoimmune diseases and how the IFN-α produced can contribute to the development of an autoimmune process will be reviewed.

Systemic lupus erythematosus (SLE), an autoimmune disease, develops at a female-to-male ratio of 10:1. Increased serum levels of type I interferons (IFN-α/β) and induction of “IFN-signature” genes are associated with an active SLE disease in patients. Moreover, SLE patients exhibit three- to four-fold increase in the risk of developing malignancies involving B cells, including non-Hodgkin lymphoma (NHL) and Hodgkin's lymphoma (HL). Interestingly, homozygous mice expressing a deletion mutant (the proline-rich domain deleted) of the p53 develop various types of spontaneous tumors, particularly of B-cell origin upon aging. The deletion is associated with defects in transcriptional activation of genes by p53 and inhibition of DNA damage-induced apoptosis. Notably, increased levels of the p202 protein, which is encoded by the p53-repressible interferon-inducible Ifi202 gene, in B cells of female mice are associated with defects in B cell apoptosis, inhibition of the p53-mediated transcription of pro-apoptotic genes, and increased lupus susceptibility. In this review we discuss how increased levels of the p202 protein (and its human functional homologue IFI16 protein) in B cells increase lupus susceptibility and are likely to increase the risk of developing certain B cell malignancies. A complete understanding of the molecular mechanisms that regulate B cell homeostasis is necessary to identify SLE patients with an increased risk to develop B cell malignancies.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a breakdown of tolerance to nuclear antigens and the development of immune complexes. Genomic approaches have shown that human SLE leukocytes homogeneously express type I interferon (IFN)–induced and neutrophil-related transcripts. Increased production and/or bioavailability of IFN-α and associated alterations in dendritic cell (DC) homeostasis have been linked to lupus pathogenesis. Although neutrophils have long been shown to be associated with lupus, their potential role in disease pathogenesis remains elusive. Here, we show that mature SLE neutrophils are primed in vivo by type I IFN and die upon exposure to SLE-derived anti-ribonucleoprotein antibodies, releasing neutrophil extracellular traps (NETs). SLE NETs contain DNA as well as large amounts of LL37 and HMGB1, neutrophil proteins that facilitate the uptake and recognition of mammalian DNA by plasmacytoid DCs (pDCs). Indeed, SLE NETs activate pDCs to produce high levels of IFN-α in a DNA- and TLR9 (Toll-like receptor 9)–dependent manner. Our results reveal an unsuspected role for neutrophils in SLE pathogenesis and identify a novel link between nucleic acid–recognizing antibodies and type I IFN production in this disease.

Objective

In contrast to rheumatoid arthritis (RA), Jaccoud arthritis (JA) joint inflammation in systemic lupus erythematosus (SLE) is nonerosive. Although the mechanism responsible is unknown, the anti-osteoclastogenic cytokine interferon-alpha (IFN-α), whose transcriptome is present in SLE monocytes, may be responsible. To test this, we examined effects of IFN-α versus lupus disease on osteoclasts and erosion in the NZBxNZW F1 SLE mouse model with K/BxN serum-induced arthritis (SIA).

Methods

Elevated systemic IFN-α levels were obtained by administration of an adenoviral vector expressing IFN-α (Ad-IFN-α). SLE disease was marked by anti-dsDNA antibody titer and proteinuria, and Ifi202 and Mx1 expression represented the IFN-α transcriptome. Micro-CT was used to evaluate bone erosions. Flow cytometry for CD11b and CD11c was used to evaluate the frequency of circulating osteoclast precursors (OCP) and myeloid dendritic cells (mDC) in blood.

Results

Administration of Ad-IFN-α to NZBxNZW F1 mice induced osteopetrosis. Pre-autoimmune NZBxNZW F1 mice are fully susceptible to focal erosions in the setting of SIA. However, NZBxNZW F1 mice with high anti-dsDNA antibody titers and the IFN-α transcriptome were protected against bone erosions. Ad-IFN-α pre-treatment of NZW mice before K/BxN serum administration also resulted in protection against bone erosion (r2=0.4720, p<0.01), which was associated with a decrease in circulating CD11b+CD11c− OCP, and a concomitant increase in CD11b+CD11c+ cells (r2=0.6330, p<0.05) that are phenotypic of mDC.

Conclusion

These findings suggest that IFN-α in SLE shifts monocyte development toward mDC at the expense of osteoclastogenesis thereby resulting in decreased bone erosion.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple genetic risk factors, high levels of interferon alpha (IFN-α), and the production of autoantibodies against components of the cell nucleus. Interferon regulatory factor 5 (IRF5) is a transcription factor which induces the transcription of IFN-α and other cytokines, and genetic variants of IRF5 have been strongly linked to SLE pathogenesis. IRF5 functions downstream of Toll-like receptors and other microbial pattern-recognition receptors, and immune complexes made up of SLE-associated autoantibodies seem to function as a chronic endogenous stimulus to this pathway. In this paper, we discuss the physiologic role of IRF5 in immune defense and the ways in which IRF5 variants may contribute to the pathogenesis of human SLE. Recent data regarding the role of IRF5 in both serologic autoimmunity and the overproduction of IFN-α in human SLE are summarized. These data support a model in which SLE-risk variants of IRF5 participate in a “feed-forward” mechanism, predisposing to SLE-associated autoantibody formation, and subsequently facilitating IFN-α production downstream of Toll-like receptors stimulated by immune complexes composed of these autoantibodies.

Background

Systemic lupus erythematosus (SLE), an autoimmune disease, predominantly affects women of childbearing age. Moreover, increased serum levels of interferon-α (IFN-α) are associated with the disease. Although, the female sex hormone estrogen (E2) is implicated in sex bias in SLE through up-regulation of IFN-γ expression, the molecular mechanisms remain unknown. Here we report that activation of IFN (α or γ)-signaling in immune cells up-regulates expression of estrogen receptor-α (ERα; encoded by the Esr1 gene) and stimulates expression of target genes.

Methodology/Principal Findings

We found that treatment of mouse splenic cells and mouse cell lines with IFN (α or γ) increased steady-state levels of ERα mRNA and protein. The increase in the ERα mRNA levels was primarily due to the transcriptional mechanisms and it was dependent upon the activation of signal transducer and activator of transcription-1 (STAT1) factor by IFN. Moreover, the IFN-treatment of cells also stimulated transcription of a reporter gene, expression of which was driven by the promoter region of the murine Esr1 gene. Notably, splenic cells from pre-autoimmune lupus-prone (NZB × NZW)F1 female mice had relatively higher steady-state levels of mRNAs encoded by the IFN and ERα-responsive genes as compared to the age-matched males.

Conclusions/Significance

Our observations identify a novel mutually positive regulatory feedback loop between IFNs and ERα in immune cells in mice and support the idea that activation of this regulatory loop contributes to sex bias in SLE.

The type I IFNs (IFN-α and IFN-β), which are crucial in antiviral defense and immune regulation, signal via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway with activation of STAT1 and STAT2. Here, the function of STAT2 was studied in transgenic mice (termed GIFN/STAT2–/–) with CNS production of IFN-α. Surprisingly, GIFN/STAT2–/–, but not GIFN/STAT1-null, transgenic mice, with CNS production of IFN-α, died prematurely with medulloblastoma. An immune response also induced in the brain of the GIFN/STAT2–/– mice was associated with IFN-γ gene expression by CD3+ T cells and the activation of the STAT1, STAT3, STAT4, and STAT5 molecules. Expression of the Sonic hedgehog (Shh) and the downstream transcriptional factor Gli-1 genes, implicated in the pathogenesis of medulloblastoma, was found to be significantly increased and cotranscribed in cerebellar granule neurons of the GIFN/STAT2–/– mice. IFN-γ, but not IFN-α, induced STAT1-dependent expression of the Shh gene in cultured cerebellar granule neurons. Thus, there is an unexpected and extraordinarily adverse biological potency of IFN-α in the CNS when the primary signal transduction molecule STAT2 is absent. Moreover, a hitherto unknown role is indicated for the immune system in the pathogenesis of developmental disorders and tumorigenesis of the CNS via dysregulated Shh signaling mediated by IFN-γ.

Background

Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disorder that affects multiple organ systems and is characterized by unpredictable flares of disease. Recent evidence indicates a role for type I interferon (IFN) in SLE pathogenesis; however, the downstream effects of IFN pathway activation are not well understood. Here we test the hypothesis that type I IFN-regulated proteins are present in the serum of SLE patients and correlate with disease activity.

Methods and Findings

We performed a comprehensive survey of the serologic proteome in human SLE and identified dysregulated levels of 30 cytokines, chemokines, growth factors, and soluble receptors. Particularly striking was the highly coordinated up-regulation of 12 inflammatory and/or homeostatic chemokines, molecules that direct the movement of leukocytes in the body. Most of the identified chemokines were inducible by type I IFN, and their levels correlated strongly with clinical and laboratory measures of disease activity.

Conclusions

These data suggest that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed in human SLE. Furthermore, the levels of serum chemokines may serve as convenient biomarkers for disease activity in lupus.

A comprehensive survey of the serologic proteome in human SLE suggests that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed.

Editors' Summary

Background.

The term “lupus,” meaning wolf in Latin, is often used as an abbreviation for the disease systemic lupus erythematosus (SLE). The name may have been given because some people with SLE have a rash that slightly resembles a wolf's face. The condition affects around 50 to 100 people per 100,000, and is much more common in women than men. SLE is a complicated disease that comes about when antibodies inappropriately attack the body's own connective tissues, although it is not known why this happens. Symptoms vary between different people; the disease may get better and then worse, without explanation; and can affect many different organs including the skin, joints, kidneys, blood cells, and brain and nervous system. SLE is difficult for doctors to diagnose. Although the disease cannot be cured, patients who are diagnosed with SLE can be treated for their symptoms, and the right management can slow progress of the disease. One area of SLE research focuses on finding “molecular markers” (e.g., proteins or other compounds) that could be tested for in the blood. Researchers hope this would help doctors to more accurately diagnose SLE initially, and then also help to track progress in a patient's condition.

Why Was This Study Done?

“Gene expression” is a term meaning the process by which a gene's DNA sequence is converted into the structures and functions of a cell. These investigators had found in previous studies that certain genes were more “highly expressed” in the blood cells of patients with SLE. Some of these genes were already known to be regulated by interferons (a group of proteins, produced by certain blood cells, that are important in helping to defend against viral infections). The investigators performing this study wanted to understand more clearly the role of interferon in SLE and to see whether the genes that are more highly expressed in patients with SLE go on to produce higher levels of protein, which might then provide useful markers for monitoring the condition.

What Did the Researchers Do and Find?

This research project was a “case-control” study, in which the researchers compared the levels of certain proteins in the blood of people who had SLE with the levels in people who did not have the condition. Thirty people were recruited as cases, from a group of patients with SLE who have been under evaluation at Johns Hopkins School of Medicine since 1987. Fifteen controls were recruited from a group of healthy people of similar age and sex as the patients with SLE; everyone involved in the study gave their consent to take part. Blood samples were taken from each individual, and the serum (liquid component of blood) was separated out. The serum levels of 160 different blood proteins were then measured. When comparing levels of blood proteins between the groups, the researchers found that 30 specific proteins were present at higher or lower levels in the SLE-affected patients. Many of these proteins are cytokines, which are regulated by interferons and are involved in the process of “signaling” within the immune system. A few proteins were found at lower levels. Levels of the interferon-regulated proteins were, on average, seen at higher levels in people whose condition was more severe.

What Do These Findings Mean?

These results suggest that patients with SLE are likely to have a very different pattern of regulation of certain proteins within the blood, particularly the proteins involved in signaling within the immune system. The authors propose that these proteins may be involved in the progression of the disease. There is also the possibility that some of these proteins may prove useful in diagnostic tests, or in tests for monitoring how the disease progresses. However, before any such tests could be used in clinical practice, they would need to be further developed and then thoroughly tested in clinical trials.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030491

Patient information from the UK National Health Service on systemic lupus erythematosus

Patient handout from the US National Institutes of Health

MedlinePLUS encyclopedia entry on lupus

Information on lupus from the UK Arthritis Research Campaign

The best-characterized type 1 interferon (IFN) signaling pathway depends on signal transducer and activator of transcription 1 (STAT1) and STAT2. The cytokines can, however, conditionally activate all STATs. Regulation of their access to particular signaling pathways is poorly understood. STAT4 is important for IFN-γ induction, and NK cells are major producers of this cytokine. We report that NK cells have high basal STAT4 levels and sensitivity to type 1 IFN–mediated STAT4 activation for IFN-γ production. Increases in STAT1, driven during viral infection by either type 1 IFN or IFN-γ, are associated with decreased STAT4 access. Both STAT1 and STAT2 are important for antiviral defense, but STAT1 has a unique role in protecting against sustained NK cell IFN-γ production and resulting disease. The regulation occurs with an NK cell type 1 IFN receptor switch from a STAT4 to a STAT1 association. Thus, a fundamental characteristic of NK cells is high STAT4 bound to the type 1 IFN receptor. The conditions of infection result in STAT1 induction with displacement of STAT4. These studies elucidate the critical role of STAT4 levels in predisposing selection of specific signaling pathways, define the biological importance of regulation within particular cell lineages, and provide mechanistic insights for how this is accomplished in vivo.

Introduction

Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE), a complex autoimmune disease. Comprehensively quantifying basal STATs phosphorylation and their signaling response to cytokines should help us to better understand the etiology of SLE.

Methods

Phospho-specific flow cytometry was used to measure the basal STAT signaling activation in three immune cell types of peripheral-blood mononuclear cells from 20 lupus patients, 9 rheumatoid arthritis (RA) patients and 13 healthy donors (HDs). A panel of 27 cytokines, including inflammatory cytokines, was measured with Bio-Plex™ Human Cytokine Assays. Serum Prolactin levels were measured with an immunoradiometric assay. STAT signaling responses to inflammatory cytokines (interferon α [IFNα], IFNγ, interleukin 2 [IL2], IL6, and IL10) were also monitored.

Results

We observed the basal activation of STAT3 in SLE T cells and monocytes, and the basal activation of STAT5 in SLE T cells and B cells. The SLE samples clustered into two main groups, which were associated with the SLE Disease Activity Index 2000, their erythrocyte sedimentation rate, and their hydroxychloroquine use. The phosphorylation of STAT5 in B cells was associated with cytokines IL2, granulocyte colony-stimulating factor (G-CSF), and IFNγ, whereas serum prolactin affected STAT5 activation in T cells. The responses of STAT1, STAT3, and STAT5 to IFNα were greatly reduced in SLE T cells, B cells, and monocytes, except for the STAT1 response to IFNα in monocytes. The response of STAT3 to IL6 was reduced in SLE T cells.

Conclusions

The basal activation of STATs signaling and reduced response to cytokines may be helpful us to identify the activity and severity of SLE.

Introduction

Recent studies identified STAT4 (signal transducers and activators of transcription-4) as a susceptibility gene for systemic lupus erythematosus (SLE). STAT1 is encoded adjacently to STAT4 on 2q32.2-q32.3, upregulated in peripheral blood mononuclear cells from SLE patients, and functionally relevant to SLE. This study was conducted to test whether STAT4 is associated with SLE in a Japanese population also, to identify the risk haplotype, and to examine the potential genetic contribution of STAT1. To accomplish these aims, we carried out a comprehensive association analysis of 52 tag single nucleotide polymorphisms (SNPs) encompassing the STAT1-STAT4 region.

Methods

In the first screening, 52 tag SNPs were selected based on HapMap Phase II JPT (Japanese in Tokyo, Japan) data, and case-control association analysis was carried out on 105 Japanese female patients with SLE and 102 female controls. For associated SNPs, additional cases and controls were genotyped and association was analyzed using 308 SLE patients and 306 controls. Estimation of haplotype frequencies and an association study using the permutation test were performed with Haploview version 4.0 software. Population attributable risk percentage was estimated to compare the epidemiological significance of the risk genotype among populations.

Results

In the first screening, rs7574865, rs11889341, and rs10168266 in STAT4 were most significantly associated (P < 0.01). Significant association was not observed for STAT1. Subsequent association studies of the three SNPs using 308 SLE patients and 306 controls confirmed a strong association of the rs7574865T allele (SLE patients: 46.3%, controls: 33.5%, P = 4.9 × 10-6, odds ratio 1.71) as well as TTT haplotype (rs10168266/rs11889341/rs7574865) (P = 1.5 × 10-6). The association was stronger in subgroups of SLE with nephritis and anti-double-stranded DNA antibodies. Population attributable risk percentage was estimated to be higher in the Japanese population (40.2%) than in Americans of European descent (19.5%).

Conclusions

The same STAT4 risk allele is associated with SLE in Caucasian and Japanese populations. Evidence for a role of STAT1 in genetic susceptibility to SLE was not detected. The contribution of STAT4 for the genetic background of SLE may be greater in the Japanese population than in Americans of European descent.

Objective

The type I interferon (IFN) pathway is activated in many patients with systemic lupus erythematosus (SLE), and high serum levels of IFN are associated with anti-SSA/Ro autoantibodies. To investigate the clinical features associated with type I IFN production in vivo, we compared serum IFN activity in individuals with anti-SSA/Ro antibodies who were asymptomatic with that in individuals with clinical manifestations of SLE or Sjögren's syndrome (SS).

Methods

Antibody-positive sera from 84 mothers of children with manifestations of neonatal lupus were studied for type I IFN activity, using a functional reporter cell assay. Maternal health status was characterized as asymptomatic, SS, SLE, pauci-SLE, or pauci-SS, based on a screening questionnaire, telephone interview, and review of medical records. The prefix “pauci-” indicates symptoms insufficient for a formal classification of the disease.

Results

Only 4% of asymptomatic mothers had high serum type I IFN activity, compared with 73% with pauci-SLE (P = 5.7 × 10−5), 35% with SLE (P = 0.011), and 32% of patients with SS (P = 0.032). One of the 4 patients with pauci-SS had high levels of IFN. The majority of patients for whom longitudinal data were available had stable type I IFN activity over time, and changes in IFN activity were not clearly accompanied by changes in the clinical diagnosis.

Conclusion

Patients with SLE, patients with pauci-SLE, and patients with SS are more likely to have high serum IFN activity than asymptomatic individuals with SSA/Ro autoantibodies, suggesting that these autoantibodies are insufficient for activation of the type I IFN pathway, and that disease-specific factors are important for type I IFN generation in vivo.

Systemic lupus erythematosus (SLE) is a complex systemic autoimmune disease associated with multiple immunologic abnormalities. Prominent among these is upregulation of type I interferon (IFN)—a powerful immune adjuvant. IFN is, in part, produced in SLE in response to autoantigens in the form of self-nucleic acids and their associated nuclear proteins. Sources of these autoantigens include apoptotic and necrotic cells as well as neutrophils undergoing a specific form of cell death called NETosis. Although plasmacytoid dendritic cells are the main producers of IFN-a, other cells are important regulators of this process. Both genetic and environmental risk factors play a role in the development and pathogenesis of SLE. Further highlighting the importance of IFN, candidate gene and genome-wide association studies have identified a number of genes involved in type I IFN pathways associated with SLE. In this review, 3 monogenic deficiencies that result in lupus-like phenotypes and several polygenic variants that have been consistently associated with SLE are highlighted, and the relationship of these genes to IFN-a production is discussed. Clinical associations of the type I IFN pathway and the use of IFN-blocking agents as therapeutic agents in SLE are also reviewed.