Jessica M. Dorschner

Genetic analysis of the pathogenic molecular sub-phenotype interferon-alpha identifies multiple novel loci involved in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by inflammation of multiple organ systems and dysregulated interferon responses. SLE is both genetically and phenotypically heterogeneous, greatly reducing the power of case-control studies in SLE. Elevated circulating interferon-alpha (IFN-α) is a stable, heritable trait in SLE, which has been implicated in primary disease pathogenesis. About 40–50% of patients have high IFN-α, and high levels correspond with clinical differences. To study genetic heterogeneity in SLE, we performed a case−case study comparing patients with high vs low IFN-α in over 1550 SLE cases, including genome-wide association study and replication cohorts. In meta-analysis, the top associations in European ancestry were protein kinase, cyclic GMP-dependent, type I (PRKG1) rs7897633 (PMeta=2.75 × 10−8) and purine nucleoside phosphorylase (PNP) rs1049564 (PMeta=1.24 × 10−7). We also found evidence for cross-ancestral background associations with the ankyrin repeat domain 44 (ANKRD44) and pleckstrin homology domain containing, family F member 2 gene (PLEKHF2) loci. These loci have not been previously identified in case-control SLE genetic studies. Bioinformatic analyses implicated these loci functionally in dendritic cells and natural killer cells, both of which are involved in IFN-α production in SLE. As case-control studies of heterogeneous diseases reach a limit of feasibility with respect to subject number and detectable effect size, the study of informative pathogenic sub-phenotypes becomes an attractive strategy for genetic discovery in complex disease.

CD11b activation suppresses TLR-dependent inflammation and autoimmunity in systemic lupus erythematosus

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-&bgr;, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Increased pretreatment serum IFN-β/α ratio predicts non-response to tumour necrosis factor α inhibition in rheumatoid arthritis

OBJECTIVE Studies suggest that circulating type I interferon (IFN) may predict response to biological agents in rheumatoid arthritis (RA). Prediction of response prior to initiating therapy would represent a major advancement. METHODS We studied sera from a test set of 32 patients with RA from the Auto-immune Biomarkers Collaborative Network Consortium and a validation set of 92 patients with RA from the Treatment Efficacy and Toxicity in Rheumatoid Arthritis Database and Repository registry. The test set included those with good response or no response to tumour necrosis factor (TNF) inhibitors at 14 weeks by European League Against Rheumatism criteria. The validation set included subjects with good, moderate or no response at 12 weeks. Total serum type I IFN activity, IFN-α and IFN-β activity were measured using a functional reporter cell assay. RESULTS In the test set, an increased ratio of IFN-β to IFN-α (IFN-β/α activity ratio) in pretreatment serum associated with lack of response to TNF inhibition (p=0.013). Anti-cyclic citrullinated peptide antibody titre and class of TNF inhibitor did not influence this relationship. A receiver-operator curve supported a ratio of 1.3 as the optimal cut-off. In the validation set, subjects with an IFN-β/α activity ratio >1.3 were significantly more likely to have non-response than good response (OR=6.67, p=0.018). The test had 77% specificity and 45% sensitivity for prediction of non-response compared with moderate or good response. Meta-analysis of test and validation sets confirmed strong predictive capacity of IFN-β/α activity ratio (p=0.005). CONCLUSIONS Increased pretreatment serum IFN-β/α ratio strongly associated with non-response to TNF inhibition. This study supports further investigation of serum type I IFN in predicting outcome of TNF inhibition in RA.

Lupus-Associated Functional Polymorphism in PNP Causes Cell Cycle Abnormalities and Interferon Pathway Activation in Human Immune Cells

Systemic lupus erythematosus (SLE) is frequently characterized by activation of the type I interferon (IFN) pathway. We previously observed that a missense single‐nucleotide polymorphism (rs1049564) in the purine nucleoside phosphorylase (PNP) gene was associated with high levels of IFN in SLE. PNP is a key enzyme involved in purine metabolism. In this study, we performed functional follow‐up of this polymorphism in human cells.

A Novel ELANE Mutation Associated with Inflammatory Arthritis, Defective NETosis, and Recurrent Parvoviral Infection

We describe a 38‐year‐old woman who presented with a history of inflammatory arthritis, rash, and daily fevers. She was noted to have chronic parvovirus infection with persistently detectable viral titers and a novel mutation in the ELANE gene. ELANE encodes neutrophil elastase, a neutrophil serine protease with important antimicrobial effects, and is found as part of neutrophil extracellular trap (NET) complexes. Pathogenic ELANE mutations have been identified in forms of hereditary neutropenia. However, our patient never had neutropenia. Because of the striking clinical scenario, we investigated this mutation functionally.

Associations between type I interferon and antiphospholipid antibody status differ between ancestral backgrounds

Objective The type I interferon pathway is activated in many patients with systemic lupus erythematosus (SLE), and anti-double-stranded DNA (dsDNA) and anti-RNA binding protein autoantibodies are correlated with high interferon-α (IFNα) activity. We studied whether antiphospholipid (APL) antibodies, which should not stimulate Toll-like receptors, are also associated with high levels of IFNα activity. Methods Serum IFNα activity was measured in patients with SLE using the WISH cell bioassay. IgG APL, anti-RBP and anti-dsDNA antibodies were measured in the clinical laboratory, and standard clinical cut-offs were used to define the positive results. Results High IFNα activity was associated with anti-RBP and anti-dsDNA antibodies in all three ancestral backgrounds. Strikingly, African-American subjects with a positive APL antibody test had higher IFNα activity than those without IgG APL antibodies. This was not shared with other ancestral backgrounds. This finding was independent of other autoantibody profiles, and clinical features did not differ between IgG APL antibody positive versus negative African-American patients. Conclusion The difference in association between IFNα activity and IgG APL status between ancestral backgrounds supports differences in molecular pathogenesis. This may suggest B cell hyperactivity in the setting of type I IFN in African-Americans and could suggest ways to individualise therapy.

PS1:1 High type i interferon activity is associated with active class 3/4 lupus nephritis in european-american lupus patients independent of anti-dsdna antibodies

Background/purpose Lupus nephritis (LN) is one of the most severe types of organ involvement in systemic lupus erythematosus (SLE), despite the recent advances in immunosuppressive therapies. High type 1 interferon (IFN) is a heritable risk for SLE, and some previous studies have suggested a link between high IFN and lupus nephritis. However, little is known about the relationships between high levels of IFN and the subtypes of LN, and whether IFN is more associated with anti-dsDNA antibodies or with clinical nephritis. Methods We studied 244 European-American (EA) SLE patients and measured type 1 IFN in sera by performing WISH IFN bioassay as described previously. Subtypes of LN were confirmed by renal biopsy review. Complements, anti-dsDNA and other auto-antibodies were measured in the clinical laboratory, and standard clinical cut-offs were used to define a positive result. Non-parametric analyses were used to compare IFN data with the clinical data. Results IFN level and SLEDAI score was positively correlated (r=0.26, p<0.0001, Spearman) in our cross-sectional evaluation. EA subjects with a high levels of IFN (IFN score >2) were more likely to have renal manifestations compared to the subjects with a low levels of IFN (IFN score <2) (p<0.001, OR=3.0, Fisher’s exact test). In addition, the incidence rate of class 3/4 LN was significantly higher among patients with a high levels of IFN compared to the patients with low levels of IFN (p<0.01, OR=5.5, Fisher’s exact test). Notably, IFN level was significantly higher in active class 3/4 LN compared to inactive class 3/4 LN (p<0.05 Mann-Whitney U) and this was not observed in non-class 3/4 LN populations. Positivity of ds-DNA antibody did not show significant difference between inactive class 3/4 LN and active class 3/4 LN. Conclusion Our data support an association between type 1 IFN and class 3/4 nephritis that is independent of overall SLEDAI and anti-dsDNA antibodies, suggesting that IFN is involved in renal pathogenesis. These data also suggest that IFN could predict renal disease activity or the future risk of developing LN, especially class 3/4 LN in EA SLE patients.

Single-cell gene expression patterns in lupus monocytes independently indicate disease activity, interferon and therapy

Objectives Important findings can be masked in gene expression studies of mixed cell populations. We examined single-cell gene expression in SLE patient monocytes in the context of clinical and immunological features. Methods Monocytes were purified from patients with SLE and controls, and individually isolated for single-cell gene expression measurement. A panel of monocyte-related transcripts were measured in individual classical (CL) and non-classical (NCL) monocytes. Results Analyses of both CL and NCL monocytes demonstrated that many genes had a lower expression rate in SLE monocytes than in controls. Unsupervised hierarchical clustering of the CL and NCL data sets demonstrated independent clusters of cells from the patients with SLE that were related to disease activity, type I interferon (IFN) and medication use. Thus, each of these factors exerted a different impact on monocyte gene expression that could be identified separately, and a number of genes correlated uniquely with disease activity. We found within-cell correlations between genes directly induced by type I IFN-induced and other non–IFN-induced genes, suggesting the downstream biological effects of type I IFN in individual human SLE monocytes which differed between CLs and NCLs. Conclusions In summary, single-cell gene expression in monocytes was associated with a wide range of clinical and biological features in SLE, providing much greater detail and insight into the cellular biology underlying the disease than previous mixed-cell population studies.

10 Single cell expression quantitative trait LOCI (EQTL) analsis of established lupus-risk loci in patient monocytes

Background and aims While most of the confirmed SLE-risk loci are in or near genes with immune system function, a major unanswered question is how these loci influence diverse immune cell subsets. Methods CD14++CD16- classical monocytes (CL) and CD14dimCD16+ non classical (NCL) monocytes from SLE patients were purified by magnetic separation. The Fluidigm C1 System was used for single cell capture and target gene pre-amplification and equal numbers of classical and non-classical monocytes were studied. 90 monocyte-related genes and 7 SLE-risk SNPs were included in eQTL analyses. Results The SLE-associated SNPs demonstrated more eQTLs in NCLs as compared to CLs (p=2.5x10-8). For a given SNP, the associated transcripts differed between cell types (p<0.001 for all 7 SNPs for discordance), suggesting that the same SNP resulted in different cellular events between the two monocyte subsets. Loci which shared a significant proportion of eQTL associations with each other in NCLs included TNFAIP3, IRF5, IRF7, PTPN22, and SPP1. In CLs, TNFAIP3 shared a large number of eQTLs with SPP1 and ITGAM, although SPP1 and ITGAM showed more limited overlap with each other. Thus, SLE-associated risk loci exert coordinated effects on gene expression within individual human monocytes, and the risk loci interact in different ways in different cell types. Conclusions Our study revealed striking differences in the occurrence and interaction between of SLE risk associated eQTLs within different but closely related cell types. This suggests pleiotropic effects from each locus across various immune cell types, and a high degree of complexity when considering how these loci impact the immune system.

Association of IRF5 polymorphisms with increased risk for systemic lupus erythematosus in population of Crete, a southern-eastern European Greek island

Interferon regulatory factor 5 (IRF5) regulates type I interferon (IFN)-responsive genes, and has been one of the most consistently associated genes with systemic lupus erythematosus (SLE). We sought to investigate whether IRF5 haplotypes are associated with risk for SLE in the genetically homogeneous Greek population of the island of Crete, as well as whether these haplotypes are associated with increased type I IFN. 322 SLE patients and 247 healthy controls from Crete were genotyped for rs2004640, rs3807306, rs10488631 and rs2280714 SNPs of IRF5 gene by using Taqman primer-probe sets. Type I IFN levels were measured using a functional reporter cell assay. All IRF5 SNPs examined were found to be associated with SLE in univariate case-control analysis. The 4 SNPs formed 5 major haplotypes and the Neanderthal-derived TACA risk haplotype was present in Crete and enriched in the SLE cases (OR=2.01, P=0.0003). Serum IFN levels were measured in a subset of the SLE patients, and carriage of the TACA haplotype was associated with higher circulating type I IFN levels (P=0.037). This study demonstrates the association of IRF5 with an increased susceptibility for SLE in the population of Crete and emphasizes the association of the Neanderthal-derived IRF5 haplotype with SLE susceptibility. Patients carrying allele the Neanderthal allele C had greater type I IFN, supporting a functional consequence of this polymorphism.