Lack of strong innate immune reactivity renders macrophages alone unable to control productive Varicella-Zoster Virus infection in an isogenic human iPSC-derived neuronal co-culture model.

With Varicella-Zoster Virus (VZV) being an exclusive human pathogen, human induced pluripotent stem cell (hiPSC)-derived neural cell culture models are an emerging tool to investigate VZV neuro-immune interactions. Using a compartmentalized hiPSC-derived neuronal model allowing axonal VZV infection, we previously demonstrated that paracrine interferon (IFN)-α2 signalling is required to activate a broad spectrum of interferon-stimulated genes able to counteract a productive VZV infection in hiPSC-neurons. In this new study, we now investigated whether innate immune signalling by VZV-challenged macrophages was able to orchestrate an antiviral immune response in VZV-infected hiPSC-neurons. In order to establish an isogenic hiPSC-neuron/hiPSC-macrophage co-culture model, hiPSC-macrophages were generated and characterised for phenotype, gene expression, cytokine production and phagocytic capacity. Even though immunological competence of hiPSC-macrophages was shown following stimulation with the poly(dA:dT) or treatment with IFN-α2, hiPSC-macrophages in co-culture with VZV-infected hiPSC-neurons were unable to mount an antiviral immune response capable of suppressing a productive neuronal VZV infection. Subsequently, a comprehensive RNA-Seq analysis confirmed the lack of strong immune responsiveness by hiPSC-neurons and hiPSC-macrophages upon, respectively, VZV infection or challenge. This may suggest the need of other cell types, like T-cells or other innate immune cells, to (co-)orchestrate an efficient antiviral immune response against VZV-infected neurons.

A glimpse into the future of systemic lupus erythematosus.

This viewpoint article on a forecast of clinically meaningful changes in the management of systemic lupus erythematosus (SLE) in the next 10 years is based on a review of the current state of the art. The groundwork has been laid by a robust series of classification criteria and treatment recommendations that have all been published since 2019. Building on this strong foundation, SLE management predictably will take significant steps forward. Assessment for lupus arthritis will presumably include musculoskeletal sonography. Large-scale polyomics studies are likely to unravel more of the central immune mechanisms of the disease. Biomarkers predictive of therapeutic success may enter the field; the type I interferon signature, as a companion for use of anifrolumab, an antibody against the common type I interferon receptor, is one serious candidate. Besides anifrolumab for nonrenal SLE and the new calcineurin inhibitor voclosporin in lupus nephritis, both of which are already approved in the United States and likely to become available in the European Union in 2022, several other approaches are in advanced clinical trials. These include advanced B cell depletion, inhibition of costimulation via CD40 and CD40 ligand (CD40L), and Janus kinase 1 (Jak1) and Tyrosine kinase 2 (Tyk2) inhibition. At the same time, essentially all of our conventional therapeutic armamentarium will continue to be used. The ability of patients to have successful SLE pregnancies, which has become much better in the last decades, should further improve, with approaches including tumor necrosis factor blockade and self-monitoring of fetal heart rates. While we hope that the COVID-19 pandemic will soon be controlled, it has highlighted the risk of severe viral infections in SLE, with increased risk tied to certain therapies. Although there are some data that a cure might be achievable, this likely will remain a challenge beyond 10 years from now.

The heterogeneity of systemic lupus erythematosus: Looking for a molecular answer / La heterogeneidad del lupus eritematoso sistémico: buscando una respuesta molecular

ABSTRACT The heterogeneity of SLE is a major limitation when designing clinical trials and understand ing the mechanisms of the disease. The analyses conducted before the new technologies for the identification of the single cell transcriptome focused on the detection of molecular patterns such as interferon signature in total blood or through the analysis of major sepa rate cell populations, such as CD4+ T cells. The analyses of molecular patterns have mainly focused on the transcriptome and DNA methylation changes. The first studies on single cell transcriptomics have now been published for mononuclear blood cells and tissues or the knowledge derived from them, total kidney, tubules and skin keratinocytes. The latter have defined patterns of nonresponse to treatment. However, much work still needs to be done to be able to use these methods in clinical practice.

RESUMEN La heterogeneidad del lupus es una limitante al momento de diseñar estudios clínicos, así como también para nuestra facultad de comprender los mecanismos de la enfermedad. Los análisis previos a las nuevas tecnologías para la detección del transcriptoma de célula única trabajaron en la identificación de patrones moleculares, como la firma del interferón en sangre total, o a través del análisis de poblaciones celulares principales separadas, como son las células T CD4+. Los análisis de patrones moleculares se han enfocado primordialmente en el transcriptoma y en los cambios de metilación del ADN. Ya se han publicado los primeros estudios de transcriptoma de célula única para células sanguíneas mononucleares y para tejidos, riñón total, túbulos y queratinocitos de piel. Estos últimos han definido patrones de no-respuesta al tratamiento. Aún falta mucho para que los métodos o los conocimientos derivados de los mismos sean de utilidad en la práctica clínica.

Continental-scale genomic analysis suggests shared post-admixture adaptation in the Americas.

American populations are one of the most interesting examples of recently admixed groups, where ancestral components from three major continental human groups (Africans, Eurasians and Native Americans) have admixed within the last 15 generations. Recently, several genetic surveys focusing on thousands of individuals shed light on the geography, chronology and relevance of these events. However, even though gene flow could drive adaptive evolution, it is unclear whether and how natural selection acted on the resulting genetic variation in the Americas. In this study, we analysed the patterns of local ancestry of genomic fragments in genome-wide data for ~ 6000 admixed individuals from 10 American countries. In doing so, we identified regions characterized by a divergent ancestry profile (DAP), in which a significant over or under ancestral representation is evident. Our results highlighted a series of genomic regions with DAPs associated with immune system response and relevant medical traits, with the longest DAP region encompassing the human leukocyte antigen locus. Furthermore, we found that DAP regions are enriched in genes linked to cancer-related traits and autoimmune diseases. Then, analysing the biological impact of these regions, we showed that natural selection could have acted preferentially towards variants located in coding and non-coding transcripts and characterized by a high deleteriousness score. Taken together, our analyses suggest that shared patterns of post admixture adaptation occurred at a continental scale in the Americas, affecting more often functional and impactful genomic variants.

The Role of BANK1 in B Cell Signaling and Disease.

The B cell scaffold protein with ankyrin repeats (BANK1) is expressed primarily in B cells and with multiple but discrete roles in B cell signaling, including B cell receptor signaling, CD40-related signaling, and Toll-like receptor (TLR) signaling. The gene for BANK1, located in chromosome 4, has been found to contain genetic variants that are associated with several autoimmune diseases and also other complex phenotypes, in particular, with systemic lupus erythematosus. Common genetic variants are associated with changes in BANK1 expression in B cells, while rare variants modify their capacity to bind efferent effectors during signaling. A BANK1-deficient model has shown the importance of BANK1 during TLR7 and TLR9 signaling and has confirmed its role in the disease. Still, much needs to be done to fully understand the function of BANK1, but the main conclusion is that it may be the link between different signaling functions within the B cells and they may act to synergize the various pathways within a cell. With this review, we hope to enhance the interest in this molecule.

An elevated polyclonal free light chain level reflects a strong interferon signature in patients with systemic autoimmune diseases.

High amount of polyclonal free light chains (FLC) are reported in systemic autoimmune diseases (SAD) and we took advantage of the PRECISESADS study to better characterize them. Serum FLC levels were explored in 1979 patients with SAD (RA, SLE, SjS, Scl, APS, UCTD, MCTD) and 614 healthy controls. Information regarding clinical parameters, disease activity, medications, autoantibodies (Ab) and the interferon α and/or γ scores were recorded. Among SAD patients, 28.4% had raised total FLC (from 12% in RA to 30% in SLE and APS) with a normal kappa/lambda ratio. Total FLC levels were significantly higher in SAD with inflammation, active disease in SLE and SjS, and an impaired pulmonary functional capacity in SSc, while independent from kidney impairment, infection, cancer and treatment. Total FLC concentrations were positively correlated among the 10/17 (58.8%) autoantibodies (Ab) tested with anti-RNA binding protein Ab (SSB, SSA-52/60 kDa, Sm, U1-RNP), anti-dsDNA/nucleosome Ab, rheumatoid factor and negatively correlated with complement fractions C3/C4. Finally, examination of interferon (IFN) expression as a potential driver of FLC overexpression was tested showing an elevated level of total FLC among patients with a high IFNα and IFNγ Kirou's score, a strong IFN modular score, and the detection in the sera of B-cell IFN dependent factors, such as TNF-R1/TNFRSF1A and CXCL10/IP10. In conclusion, an elevated level of FLC, in association with a strong IFN signature, defines a subgroup of SAD patients, including those without renal affectation, characterized by increased disease activity, autoreactivity, and complement reduction.

A Proinflammatory Cytokine Network Profile in Th1/Type 1 Effector B Cells Delineates a Common Group of Patients in Four Systemic Autoimmune Diseases.

OBJECTIVE: The effector T cell and B cell cytokine networks have been implicated in the pathogenesis of systemic autoimmune diseases, but the association of these cytokine networks with the heterogeneity of clinical manifestations and immune profiles has not been carefully examined. This study was undertaken to examine whether cytokine profiles can delineate distinct groups of patients in 4 systemic autoimmune diseases (systemic lupus erythematosus, Sjögren's syndrome, rheumatoid arthritis, and systemic sclerosis). METHODS: A total of 179 patients and 48 healthy volunteers were enrolled in the multicenter cross-sectional PRECISE Systemic Autoimmune Diseases (PRECISESADS) study. Multi-low-dimensional omics data (cytokines, autoantibodies, circulating immune cells) were examined. Coculture experiments were performed to test the impact of the cytokine microenvironment on T cell/B cell cross-talk. RESULTS: A proinflammatory cytokine profile defined by high levels of CXCL10, interleukin-6 (IL-6), IL-2, and tumor necrosis factor characterized a distinct group of patients in the 4 systemic autoimmune diseases. In each disease, this proinflammatory cluster was associated with a specific circulating immune cell signature, more severe disease, and higher levels of autoantibodies, suggesting an uncontrolled proinflammatory Th1 immune response. We observed in vitro that B cells reinforce Th1 differentiation and naive T cell proliferation, leading to the induction of type 1 effector B cells and IgG production. This process was associated with an increase in CXCL10, IL-6, IL-2, and interferon-γ production. CONCLUSION: This composite analysis brings new insights into human B cell functional heterogeneity based on T cell/B cell cross-talk, and proposes a better stratification of patients with systemic autoimmune diseases, suggesting that combined biomarkers would be of great value for the design of personalized treatments.

Comparative analysis of affected and unaffected areas of systemic sclerosis skin biopsies by high-throughput proteomic approaches.

BACKGROUND: Pathogenesis and aetiology of systemic sclerosis (SSc) are currently unclear, thus rendering disease prognosis, diagnosis and treatment challenging. The aim of this study was to use paired skin biopsy samples from affected and unaffected areas of the same patient, in order to compare the proteomes and identify biomarkers and pathways which are associated with SSc pathogenesis. METHODS: Biopsies were obtained from affected and unaffected skin areas of SSc patients. Samples were cryo-pulverised and proteins were extracted and analysed using mass spectrometry (MS) discovery analysis. Differentially expressed proteins were revealed after analysis with the Progenesis QIp software. Pathway analysis was performed using the Enrichr Web server. Using specific criteria, fifteen proteins were selected for further validation with targeted-MS analysis. RESULTS: Proteomic analysis led to the identification and quantification of approximately 2000 non-redundant proteins. Statistical analysis showed that 169 of these proteins were significantly differentially expressed in affected versus unaffected tissues. Pathway analyses showed that these proteins are involved in multiple pathways that are associated with autoimmune diseases (AIDs) and fibrosis. Fifteen of these proteins were further investigated using targeted-MS approaches, and five of them were confirmed to be significantly differentially expressed in SSc affected versus unaffected skin biopsies. CONCLUSION: Using MS-based proteomics analysis of human skin biopsies from patients with SSc, we identified a number of proteins and pathways that might be involved in SSc progression and pathogenesis. Fifteen of these proteins were further validated, and results suggest that five of them may serve as potential biomarkers for SSc.

BANK1 interacts with TRAF6 and MyD88 in innate immune signaling in B cells.

Evidence supports a possible role of BANK1 in innate immune signaling in B cells. In the present study, we investigated the interaction of BANK1 with two key mediators in interferon and inflammatory cytokine production, TRAF6 and MyD88. We revealed by coimmunoprecipitation (CoIP) analyses the binding of BANK1 with TRAF6 and MyD88, which were mediated by the BANK1 Toll/interleukin-1 receptor (TIR) domain. In addition, the natural BANK1-40C variant showed increased binding to MyD88. Next, we demonstrated in mouse splenic B cells that BANK1 colocalized with Toll-like receptor (TLR) 7 and TLR9 and that after stimulation with TLR7 and TLR9 agonists, the number of double-positive BANK1-TLR7, -TLR9, -TRAF6, and -MyD88 cells increased. Furthermore, we identified five TRAF6-binding motifs (BMs) in BANK1 and confirmed by point mutations and decoy peptide experiments that the C-terminal domain of BANK1-full-length (-FL) and the N-terminal domain of BANK1-Delta2 (-D2) are necessary for this binding. Functionally, we determined that the absence of the TIR domain in BANK1-D2 is important for its lysine (K)63-linked polyubiquitination and its ability to produce interleukin (IL)-8. Overall, our study describes a specific function of BANK1 in MyD88-TRAF6 innate immune signaling in B cells, clarifies functional differences between the two BANK1 isoforms and explains for the first time a functional link between autoimmune phenotypes including SLE and the naturally occurring BANK1-40C variant.

Trans-Ethnic Mapping of BANK1 Identifies Two Independent SLE-Risk Linkage Groups Enriched for Co-Transcriptional Splicing Marks.

BANK1 is a susceptibility gene for several systemic autoimmune diseases in several populations. Using the genome-wide association study (GWAS) data from Europeans (EUR) and African Americans (AA), we performed an extensive fine mapping of ankyrin repeats 1 (BANK1). To increase the SNP density, we used imputation followed by univariate and conditional analysis, combined with a haplotypic and expression quantitative trait locus (eQTL) analysis. The data from Europeans showed that the associated region was restricted to a minimal and dependent set of SNPs covering introns two and three, and exon two. In AA, the signal found in the Europeans was split into two independent effects. All of the major risk associated SNPs were eQTLs, and the risks were associated with an increased BANK1 gene expression. Functional annotation analysis revealed the enrichment of repressive B cell epigenomic marks (EZH2 and H3K27me3) and a strong enrichment of splice junctions. Furthermore, one eQTL located in intron two, rs13106926, was found within the binding site for RUNX3, a transcriptional activator. These results connect the local genome topography, chromatin structure, and the regulatory landscape of BANK1 with co-transcriptional splicing of exon two. Our data defines a minimal set of risk associated eQTLs predicted to be involved in the expression of BANK1 modulated through epigenetic regulation and splicing. These findings allow us to suggest that the increased expression of BANK1 will have an impact on B-cell mediated disease pathways.

Moving towards a molecular taxonomy of autoimmune rheumatic diseases.

Autoimmune rheumatic diseases pose many problems that have, in general, already been solved in the field of cancer. The heterogeneity of each disease, the clinical similarities and differences between different autoimmune rheumatic diseases and the large number of patients that remain without a diagnosis underline the need to reclassify these diseases via new approaches. Knowledge about the molecular basis of systemic autoimmune diseases, along with the availability of bioinformatics tools capable of handling and integrating large volumes of various types of molecular data at once, offer the possibility of reclassifying these diseases. A new taxonomy could lead to the discovery of new biomarkers for patient stratification and prognosis. Most importantly, this taxonomy might enable important changes in clinical trial design to reach the expected outcomes or the design of molecularly targeted therapies. In this Review, we discuss the basis for a new molecular taxonomy for autoimmune rheumatic diseases. We highlight the evidence surrounding the idea that these diseases share molecular features related to their pathogenesis and development and discuss previous attempts to classify these diseases. We evaluate the tools available to analyse and combine different types of molecular data. Finally, we introduce PRECISESADS, a project aimed at reclassifying the systemic autoimmune diseases.

SLE redefined on the basis of molecular pathways.

The implementation of precision medicine requires the recruiting of patients in statistically enough numbers, the possibility of obtaining enough materials, and the integration of data from various platforms, which are all real limitations. These types of studies have been performed extensively in cancer but barely on systemic lupus erythematosus (SLE) or other rheumatic diseases. To consider the practical use of the information obtained from such studies, we have to take into account the best biological fluid to use, the ease to perform the analysis in clinical practice, and its relevance to clinical practice. Here we review the most relevant studies that have performed analyses that attempt to classify or stratify SLE. We focus on two types of studies: those that stratify individuals diagnosed with SLE and those that compare SLE with other autoimmune diseases, defining differences and similarities that may be clinically relevant in the future.

Effects of Amerindian Genetic Ancestry on Clinical Variables and Therapy in Patients with Rheumatoid Arthritis.

OBJECTIVE: To define whether Amerindian genetic ancestry correlates with clinical and therapeutic variables in admixed individuals with rheumatoid arthritis (RA) from Latin America. METHODS: Patients with RA (n = 1347) and healthy controls (n = 1012) from Argentina, Mexico, Chile, and Peru were included. Samples were genotyped for the Immunochip v1 using the Illumina platform. Clinical data were obtained through interviews or the clinical history. RESULTS: Percentage of Amerindian ancestry was comparable between cases and controls. Morning stiffness (p < 0.0001, OR 0.05), rheumatoid factor (RF; p < 0.0001, OR 0.22), radiographic changes (p < 0.0001, OR 0.05), and higher number of criteria were associated with lower Amerindian ancestry after Bonferroni correction. Higher Amerindian ancestry correlated only with weight loss (pBonferroni < 0.0001, OR 2.85). Increased Amerindian ancestry correlated with higher doses of azathioprine (p < 0.0001, OR 163.6) and sulfasalazine (p < 0.0001, OR 48.6), and inversely with methotrexate (p = 0.001, OR 0.35), leflunomide (p = 0.001, OR 0.16), and nonsteroidal antiinflammatory drugs (pBonferroni = 0.001, OR 0.37). Only the presence of RF and weight loss were modified after confounders adjustment. CONCLUSION: Amerindian ancestry protects against most major clinical criteria of RA, but regarding the association of RF with increased European ancestry, age, sex, and smoking are modifiers. Ancestry also correlates with the therapeutic profiles.

Metagene projection characterizes GEN2.2 and CAL-1 as relevant human plasmacytoid dendritic cell models.

MOTIVATION: Plasmacytoid dendritic cells (pDC) play a major role in the regulation of adaptive and innate immunity. Human pDC are difficult to isolate from peripheral blood and do not survive in culture making the study of their biology challenging. Recently, two leukemic counterparts of pDC, CAL-1 and GEN2.2, have been proposed as representative models of human pDC. Nevertheless, their relationship with pDC has been established only by means of particular functional and phenotypic similarities. With the aim of characterizing GEN2.2 and CAL-1 in the context of the main circulating immune cell populations we have performed microarray gene expression profiling of GEN2.2 and carried out an integrated analysis using publicly available gene expression datasets of CAL-1 and the main circulating primary leukocyte lineages. RESULTS: Our results show that GEN2.2 and CAL-1 share common gene expression programs with primary pDC, clustering apart from the rest of circulating hematopoietic lineages. We have also identified common differentially expressed genes that can be relevant in pDC biology. In addition, we have revealed the common and differential pathways activated in primary pDC and cell lines upon CpG stimulatio. AVAILABILITY AND IMPLEMENTATION: R code and data are available in the supplementary material. CONTACT: pedro.carmona@genyo.es or concepcion.maranon@genyo.es. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Tartrate-Resistant Acid Phosphatase Deficiency in the Predisposition to Systemic Lupus Erythematosus.

OBJECTIVE: Mutations in the ACP5 gene, which encodes tartrate-resistant acid phosphatase (TRAP), cause the immuno-osseous disorder spondyloenchondrodysplasia, which includes as disease features systemic lupus erythematosus (SLE) and a type I interferon (IFN) signature. Our aims were to identify TRAP substrates, determine the consequences of TRAP deficiency in immune cells, and assess whether ACP5 mutations are enriched in sporadic cases of SLE. METHODS: Interaction between TRAP and its binding partners was tested by a yeast 2-hybrid screening, confocal microscopy, and immunoprecipitation/Western blotting. TRAP knockdown was performed using small interfering RNA. Phosphorylation of osteopontin (OPN) was analyzed by mass spectrometry. Nucleotide sequence analysis of ACP5 was performed by Sanger sequencing or next-generation sequencing. RESULTS: TRAP and OPN colocalized and interacted in human macrophages and plasmacytoid dendritic cells (PDCs). TRAP dephosphorylated 3 serine residues on specific OPN peptides. TRAP knockdown resulted in increased OPN phosphorylation and increased nuclear translocation of IRF7 and P65, with resultant heightened expression of IFN-stimulated genes and IL6 and TNF following Toll-like receptor 9 stimulation. An excess of heterozygous ACP5 missense variants was observed in SLE compared to controls (P = 0.04), and transfection experiments revealed a significant reduction in TRAP activity in a number of variants. CONCLUSION: Our findings indicate that TRAP and OPN colocalize and that OPN is a substrate for TRAP in human immune cells. TRAP deficiency in PDCs leads to increased IFNα production, providing at least a partial explanation for how ACP5 mutations cause lupus in the context of spondyloenchondrodysplasia. Detection of ACP5 missense variants in a lupus cohort suggests that impaired TRAP functioning may increase susceptibility to sporadic lupus.

Regulation of Fn14 Receptor and NF-κB Underlies Inflammation in Meniere's Disease.

Meniere's disease (MD) is a rare disorder characterized by episodic vertigo, sensorineural hearing loss, tinnitus, and aural fullness. It is associated with a fluid imbalance between the secretion of endolymph in the cochlear duct and its reabsorption into the subarachnoid space, leading to an accumulation of endolymph in the inner ear. Epidemiological evidence, including familial aggregation, indicates a genetic contribution and a consistent association with autoimmune diseases (AD). We conducted a case-control study in two phases using an immune genotyping array in a total of 420 patients with bilateral MD and 1,630 controls. We have identified the first locus, at 6p21.33, suggesting an association with bilateral MD [meta-analysis leading signal rs4947296, OR = 2.089 (1.661-2.627); p = 1.39 × 10-09]. Gene expression profiles of homozygous genotype-selected peripheral blood mononuclear cells (PBMCs) demonstrated that this region is a trans-expression quantitative trait locus (eQTL) in PBMCs. Signaling analysis predicted several tumor necrosis factor-related pathways, the TWEAK/Fn14 pathway being the top candidate (p = 2.42 × 10-11). This pathway is involved in the modulation of inflammation in several human AD, including multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. In vitro studies with genotype-selected lymphoblastoid cells from patients with MD suggest that this trans-eQTL may regulate cellular proliferation in lymphoid cells through the TWEAK/Fn14 pathway by increasing the translation of NF-κB. Taken together; these findings suggest that the carriers of the risk genotype may develop an NF-κB-mediated inflammatory response in MD.

Genetics of systemic lupus erythematosus and Sjögren's syndrome: an update.

PURPOSE OF REVIEW: To describe the recent studies on the genetics of systemic lupus erythematosus (SLE) and Sjögren's syndrome. RECENT FINDINGS: We overview the most recent findings on the genetic susceptibility of the diseases and provide information on their genetic similarities and differences. SUMMARY: SLE and Sjögren's syndrome are two closely related systemic autoimmune diseases that share multiple clinical and molecular aspects, including a significant number of susceptibility genes. Several genome-wide association studies were recently published in different populations that provide a better picture of their molecular mechanisms. It is becoming clear that their genetic architecture is quite well established, but more information is required on expression quantitative trait loci, epigenetic genome-wide analyses, gene × gene interactions and the role of rare variants.

BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation.

The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease.

Preferential association of a functional variant in complement receptor 2 with antibodies to double-stranded DNA.

OBJECTIVES: Systemic lupus erythematosus (SLE; OMIM 152700) is characterised by the production of antibodies to nuclear antigens. We previously identified variants in complement receptor 2 (CR2/CD21) that were associated with decreased risk of SLE. This study aimed to identify the causal variant for this association. METHODS: Genotyped and imputed genetic variants spanning CR2 were assessed for association with SLE in 15 750 case-control subjects from four ancestral groups. Allele-specific functional effects of associated variants were determined using quantitative real-time PCR, quantitative flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-PCR. RESULTS: The strongest association signal was detected at rs1876453 in intron 1 of CR2 (pmeta=4.2×10(-4), OR 0.85), specifically when subjects were stratified based on the presence of dsDNA autoantibodies (case-control pmeta=7.6×10(-7), OR 0.71; case-only pmeta=1.9×10(-4), OR 0.75). Although allele-specific effects on B cell CR2 mRNA or protein levels were not identified, levels of complement receptor 1 (CR1/CD35) mRNA and protein were significantly higher on B cells of subjects harbouring the minor allele (p=0.0248 and p=0.0006, respectively). The minor allele altered the formation of several DNA protein complexes by EMSA, including one containing CCCTC-binding factor (CTCF), an effect that was confirmed by ChIP-PCR. CONCLUSIONS: These data suggest that rs1876453 in CR2 has long-range effects on gene regulation that decrease susceptibility to lupus. Since the minor allele at rs1876453 is preferentially associated with reduced risk of the highly specific dsDNA autoantibodies that are present in preclinical, active and severe lupus, understanding its mechanisms will have important therapeutic implications.

Identification of a Systemic Lupus Erythematosus Risk Locus Spanning ATG16L2, FCHSD2, and P2RY2 in Koreans.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder whose etiology is incompletely understood, but likely involves environmental triggers in genetically susceptible individuals. Using an unbiased genome-wide association (GWA) scan and replication analysis, we sought to identify the genetic loci associated with SLE in a Korean population. METHODS: A total of 1,174 SLE cases and 4,246 population controls from Korea were genotyped and analyzed with a GWA scan to identify single-nucleotide polymorphisms (SNPs) significantly associated with SLE, after strict quality control measures were applied. For select variants, replication of SLE risk loci was tested in an independent data set of 1,416 SLE cases and 1,145 population controls from Korea and China. RESULTS: Eleven regions outside the HLA exceeded the genome-wide significance level (P = 5 × 10(-8) ). A novel SNP-SLE association was identified between FCHSD2 and P2RY2, peaking at rs11235667 (P = 1.03 × 10(-8) , odds ratio [OR] 0.59) on a 33-kb haplotype upstream of ATG16L2. In the independent replication data set, the SNP rs11235667 continued to show a significant association with SLE (replication meta-analysis P = 0.001, overall meta-analysis P = 6.67 × 10(-11) ; OR 0.63). Within the HLA region, the SNP-SLE association peaked in the class II region at rs116727542, with multiple independent effects observed in this region. Classic HLA allele imputation analysis identified HLA-DRB1*1501 and HLA-DQB1*0602, each highly correlated with one another, as most strongly associated with SLE. Ten previously established SLE risk loci were replicated: STAT1-STAT4, TNFSF4, TNFAIP3, IKZF1, HIP1, IRF5, BLK, WDFY4, ETS1, and IRAK1-MECP2. Of these loci, previously unreported, independent second risk effects of SNPs in TNFAIP3 and TNFSF4, as well as differences in the association with a putative causal variant in the WDFY4 region, were identified. CONCLUSION: Further studies are needed to identify true SLE risk effects in other loci suggestive of a significant association, and to identify the causal variants in the regions of ATG16L2, FCHSD2, and P2RY2.