Dag Leonard

IFN-α Production by Plasmacytoid Dendritic Cells Stimulated with RNA-Containing Immune Complexes Is Promoted by NK Cells via MIP-1β and LFA-1

Several systemic autoimmune diseases display a prominent IFN signature. This is caused by a continuous IFN-α production by plasmacytoid dendritic cells (pDCs), which are activated by immune complexes (ICs) containing nucleic acid. The IFN-α production by pDCs stimulated with RNA-containing IC (RNA-IC) consisting of anti-RNP autoantibodies and U1 small nuclear ribonucleoprotein particles was recently shown to be inhibited by monocytes, but enhanced by NK cells. The inhibitory effect of monocytes was mediated by TNF-α, PGE2, and reactive oxygen species, but the mechanisms for the NK cell-mediated increase in IFN-α production remained unclear. In this study, we investigated the mechanisms whereby NK cells increase the RNA-IC–induced IFN-α production by pDCs. Furthermore, NK cells from patients with systemic lupus erythematosus (SLE) were evaluated for their capacity to promote IFN-α production. We found that CD56dim NK cells could increase IFN-α production >1000-fold after RNA-IC activation, whereas CD56bright NK cells required costimulation by IL-12 and IL-18 to promote IFN-α production. NK cells produced MIP-1α, MIP-1β, RANTES, IFN-γ, and TNF-α via RNA-IC–mediated FcγRIIIA activation. The IFN-α production in pDCs was promoted by NK cells via MIP-1β secretion and LFA-mediated cell–cell contact. Moreover, NK cells from SLE patients displayed a reduced capacity to promote the RNA-IC–induced IFN-α production, which could be restored by exogenous IL-12 and IL-18. Thus, different molecular mechanisms can mediate the NK cell-dependent increase in IFN-α production by RNA-IC–stimulated pDCs, and our study suggests that the possibility to therapeutically target the NK–pDC axis in IFN-α–driven autoimmune diseases such as SLE should be investigated.

A STAT4 risk allele is associated with ischaemic cerebrovascular events and anti-phospholipid antibodies in systemic lupus erythematosus

Objective To investigate whether the risk allele for systemic lupus erythematosus (SLE) in the signal transducer and activator of transcription factor 4 (STAT4) gene, defined by the single nucleotide polymorphism (SNP) rs10181656(G), is associated with vascular events and/or presence of prothrombotic anti-phospholipid antibodies (aPL) in patients with SLE. Methods Two independent groups of unrelated patients with SLE of Swedish ethnicity (n=424 and 154) were genotyped, and occurrence of previous manifestations of ischaemic heart disease (IHD), ischaemic cerebrovascular disease (ICVD) and venous thromboembolic events (VTE) was tabulated. aPL values were measured by ELISA. Matched controls (n=492 and 194) were genotyped. Results The STAT4 risk allele was more frequent in patients with SLE with previous arterial events (combined OR (ORc)=1.5, 95% CI 1.1 to 2.0) compared to patients without such events. The association was mainly attributable to an accumulation of the risk allele among patients with ICVD (ORc=2.3, CI 1.6 to 3.3). There was no association with IHD or VTE. The presence of two or more aPLs was associated with the risk allele (ORc=1.6, 95% CI 1.2 to 2.0). In multivariable-adjusted logistic regression analyses treatment for hypertension, at least one STAT4 risk allele, older age, IgG anti-cardiolipin antibodies and longer SLE duration remained independently associated with previous ICVD (p≤0.02 for all). Conclusion Patients with SLE with the STAT4 risk allele had a strikingly increased risk of ICVD, comparable in magnitude to that of hypertension. The results imply that a genetic predisposition is an important and previously unrecognised risk factor for ICVD in SLE, and that aPLs may be one underlying mechanism.

Clinical manifestations and anti-phospholipid antibodies in 712 patients with systemic lupus erythematosus: evaluation of two diagnostic assays

OBJECTIVES To evaluate the agreement and performance of two tests for aPLs with regard to association with manifestations of the APS in patients with SLE. METHODS We investigated 712 SLE patients and 280 population controls. Cardiolipin and β(2) glycoprotein-I antibodies were measured with routine ELISA and a new automated method. Three positivity cut-offs (99%, 90% of controls and recommended cut-off by manufacturers) were used. Associations with previous thrombotic events, thrombocytopenia and, in a subgroup of patients, obstetric morbidity (n = 296) were evaluated. Results were compared with the LA test, performed in 380 patients. RESULTS Inter-test agreement was moderate (demonstrated by κ-values 0.16-0.71). Performance of the two tests was similar: at the 99th percentile cut-off, sensitivity for any thrombotic event ranged from 3.7% to 24.8%, while specificity was 84.7-97.7%. Regardless of assay, IgG isotypes were associated with venous thrombosis and ischaemic cerebrovascular disease, whereas aPLs of IgM isotype were weakly associated with ischaemic heart disease. Associations were greatly affected by aPL level. LA performed better than the specific aPL tests. LA was associated with any thrombotic event, odds ratio 5.4 (95% CI 3.1, 9.4), while the specific aPL tests ranged from non-significant to an odds ratio of 1.9 (95% CI 1.03, 3.4) using criteria cut-off. LA was also convincingly associated with other APS manifestations. CONCLUSION In relation to thrombotic manifestations, there was moderate agreement but no clear advantages when comparing a routine aPL ELISA with an automated method. APL isotype and titre as well as LA positivity are important for risk assessment in SLE patients.

HLA-DRB1*04/*13 alleles are associated with vascular disease and antiphospholipid antibodies in systemic lupus erythematosus

Background and objectives Vascular disease is common in systemic lupus erythematosus (SLE) and patients with antiphospholipid antibodies (aPL) are at high risk to develop arterial and venous thrombosis. Since HLA class II genotypes have been linked to the presence of pro-thrombotic aPL, we investigated the relationship between HLA-DRB1 alleles, aPL and vascular events in SLE patients. Methods 665 SLE patients of Caucasian origin and 1403 controls were included. Previous manifestations of ischaemic heart disease, ischaemic cerebrovascular disease (ICVD) and venous thromboembolism (together referred to as any vascular events (AVE)) were tabulated. aPL were measured with ELISA. Two-digit HLA-DRB1 typing was performed by sequence-specific primer-PCR. Results HLA-DRB1*04 was more frequent among SLE patients with ICVD compared to unaffected patients. This association remained after adjustment for known traditional cardiovascular risk factors. HLA-DRB1*13 was associated with AVE. All measured specificities of aPL—cardiolipin IgG and IgM, β2-glycoprotein-1 IgG, prothrombin (PT) IgG and a positive lupus anticoagulant test were associated with HLA-DRB1*04—while HLA-DRB1*13 was associated with IgG antibodies (β2-glycoprotein-1, cardiolipin and PT). In patients with the combined risk alleles, HLA-DRB1*04/*13, there was a significant additive interaction for the outcomes AVE and ICVD. Conclusions The HLA-DRB1*04 and HLA-DRB1*13 alleles are associated with vascular events and an aPL positive immune-phenotype in SLE. Results demonstrate that a subset of SLE patients is genetically disposed to vascular vulnerability.

Coronary heart disease in systemic lupus erythematosus is associated with interferon regulatory factor-8 gene variants

Background—Patients with systemic lupus erythematosus have increased morbidity and mortality in coronary heart disease (CHD). We asked whether there was a genetic influence on CHD in systemic lupus erythematosus. Methods and Results—The association between single-nucleotide polymorphisms (SNPs) and CHD in 2 populations of patients with systemic lupus erythematosus was assessed. Patients were genotyped on a custom 12k Illumina Array. The allele frequencies were compared between patients with (n=66) and without (n=509) CHD. We found 61 SNPs with an association (P<0.01) to CHD, with the strongest association for 3 SNPs located in the interferon regulatory factor-8 (IRF8) gene. Comparison of the allele frequencies of these 61 SNPs in patients with (n=27) and without (n=212) CHD in the second study population revealed that 2 SNPs, rs925994 and rs10514610 in IRF8 (linkage disequilibrium, r2=0.84), were associated with CHD in both study populations. Meta-analysis of the SNP rs925994 gave an odds ratio of 3.6 (2.1–6.3), P value 1.9×10–6. The identified IRF8 allele remained as a risk factor for CHD after adjustment for traditional CHD risk factors. The IRF8 risk allele was associated with the presence of carotid plaques (P<0.001) and increased intima-media thickness (P=0.01). By electrophoretic mobility shift assays, we show weaker binding of protein to the risk allele of the highly linked SNP rs11117415, and by flow cytometry, a reduced frequency of circulating B cells was detected in patients with the IRF8 risk allele. Conclusions—There is a considerable genetic component for CHD in systemic lupus erythematosus, with IRF8 as a strong susceptibility locus.

Increased carotid intima thickness and decreased media thickness in premenopausal women with systemic lupus erythematosus: an investigation by non-invasive high-frequency ultrasound

Objective: To determine whether high-frequency ultrasound (US) yielding separate assessments of intima and media thickness gives additional information about the vascular morphology compared with the total common carotid artery intima–media thickness (CCA-IMT). Methods: Using a 22 MHz US instrument, we determined the near-wall CCA-IMT, the intima and media layers, and the intima/media (I/M) ratio in 47 premenopausal women with systemic lupus erythematosus (SLE), 20 healthy women, and 17 postmenopausal women (mean ages 37, 40, and 69 years, respectively). Results: In SLE, the carotid intima was thicker (0.19 ± 0.04 vs. 0.12 ± 0.02 mm), the media thinner (0.45 ± 0.12 vs. 0.68 ± 0.24 mm), the I/M ratio higher (0.45 ± 0.17 vs. 0.20 ± 0.07) (all p < 0.0001), and the CCA-IMT lower (0.64 ± 0.13 vs. 0.80 ± 0.25 mm, p < 0.01) compared to age-matched controls. The SLE patients had a thicker carotid intima compared to the postmenopausal women (0.19 ± 0.04 vs. 0.14 ± 0.03 mm, p < 0.0001) and a similar I/M ratio. Conclusion: Separate assessment of carotid artery wall layers demonstrated a thicker intima, thinner media, and a higher I/M ratio in women with SLE compared to healthy controls and indicated an artery wall status in SLE comparable to 30-years-older healthy women. Separate estimates of carotid intima and media layers may be preferable to CCA-IMT in SLE patients.

A single nucleotide polymorphism in the NCF1 gene leading to reduced oxidative burst is associated with systemic lupus erythematosus

Objectives Ncf1 polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases in animal models. The human NCF1 gene is very complex with both functional and non-functional gene copies and genotyping requires assays specific for functional NCF1 genes. We aimed at investigating association and function of the missense single nucleotide polymorphism (SNP), rs201802880 (here denoted NCF1-339) in NCF1 with systemic lupus erythematosus (SLE). Methods We genotyped the NCF1-339 SNP in 973 Swedish patients with SLE and 1301 controls, using nested PCR and pyrosequencing. ROS production and gene expression of type 1 interferon-regulated genes were measured in isolated cells from subjects with different NCF1-339 genotypes. Results We found an increased frequency of the NCF1-339 T allele in patients with SLE, 11% compared with 4% in controls, OR 3.0, 95% CI 2.4 to 3.9, p=7.0×10−20. The NCF1-339 T allele reduced extracellular ROS production in neutrophils (p=0.004) and led to an increase expression of type 1 interferon-regulated genes. In addition, the NCF1-339 T allele was associated with a younger age at diagnosis of SLE; mean age 30.3 compared with 35.9, p=2.0×1−6. Conclusions These results clearly demonstrate that a genetically controlled reduced production of ROS increases the risk of developing SLE and confirm the hypothesis that ROS regulate chronic autoimmune inflammatory diseases.

Activated T cells enhance interferon-α production by plasmacytoid dendritic cells stimulated with RNA-containing immune complexes

Objectives Patients with systemic lupus erythematosus (SLE) have an ongoing interferon-α (IFN-α) production by plasmacytoid dendritic cells (pDCs). We investigated whether T cells can promote IFN-α production by pDCs. Methods Human pDCs were stimulated with immune complexes (ICs) containing U1 small nuclear ribonucleic proteins particles and SLE-IgG (RNA-IC) in the presence of T cells or T cell supernatants. T cells were activated by anti-CD3/CD28 antibodies or in a mixed leucocyte reaction. IFN-α and other cytokines were determined in culture supernatants or patient sera with immunoassays. The effect of interleukin (IL) 3 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) on pDCs was examined by the use of antibodies, and the expression of CD80/CD86 was determined using flow cytometry. Results Activated T cells and supernatants from activated T cells increased IFN-α production by >20-fold. The stimulatory effect of T cell supernatants was reduced after depletion of GM-CSF (81%) or by blocking the GM-CSF receptor (55%–81%). Supernatant from activated T cells, furthermore, increased the frequency of CD80 and CD86 expressing pDCs stimulated with RNA-IC from 6% to 35% (p<0.05) and from 10% to 26% (p<0.01), respectively. Activated SLE T cells enhanced IFN-α production to the same extent as T cells from healthy individuals and a subset of patients with SLE had increased serum levels of GM-CSF. Conclusions Activated T cells enhance IFN-α production by RNA-IC stimulated pDCs via GM-CSF and induce pDC maturation. Given the increased serum levels of GM-CSF in a subset of patients with SLE, these findings suggest that activated T cells may upregulate type I IFN production in SLE.

Novel risk genes for systemic lupus erythematosus predicted by random forest classification

Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individual’s SLE risk we designed a random forest classifier using SNP genotype data generated on the “Immunochip” from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes (ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells.

Direct and indirect costs for systemic lupus erythematosus in Sweden. A nationwide health economic study based on five defined cohorts

OBJECTIVES The main objectives of this study were to calculate total costs of illness and cost-driving disease features among patients with systemic lupus erythematosus (SLE) in Sweden. METHODS Five cohorts of well-defined SLE patients, located in different parts of the country were merged. Incident and prevalent cases from 2003 through 2010 were included. The American College of Rheumatology (ACR) classification criteria was used. From the local cohorts, data on demographics, disease activity (SLEDAI 2K), and organ damage (SDI) were collected. Costs for inpatient care, specialist outpatient care and drugs were retrieved from national registries at the National Board of Health and Welfare. Indirect costs were calculated based on sickness leave and disability pensions from the Swedish Social Insurance Agency. RESULTS In total, 1029 SLE patients, 88% females, were included, and approximately 75% were below 65 years at the end of follow-up, and thus in working age. The mean number of annual specialist physician visits varied from six to seven; mean annual inpatient days were 3.1-3.6, and mean annual sick leave was 123-148 days, all per patient. The total annual cost was 208,555 SEK (