A Flare Risk Index Informed by Select Immune Mediators in Systemic Lupus Erythematosus.

OBJECTIVE: Systemic lupus erythematosus (SLE) is marked by immune dysregulation linked to varied clinical disease activity. Using a unique longitudinal cohort of SLE patients, this study sought to identify optimal immune mediators informing an empirically refined flare risk index (FRI) reflecting altered immunity prior to clinical disease flare. METHODS: Thirty-seven SLE-associated plasma mediators were evaluated by microfluidic immunoassay in 46 samples obtained in SLE patients with an imminent clinical disease flare (preflare) and 53 samples obtained in SLE patients without a flare over a corresponding period (pre-nonflare). SLE patients were selected from a unique longitudinal cohort of 106 patients with classified SLE (meeting the American College of Rheumatology 1997 revised criteria for SLE or the Systemic Lupus International Collaborating Clinics 2012 revised criteria for SLE). Autoantibody specificities, hybrid SLE Disease Activity Index (hSLEDAI) scores, clinical features, and medication usage were also compared at preflare (mean ± SD 111 ± 47 days prior to flare) versus pre-nonflare (99 ± 21 days prior to nonflare) time points. Variable importance was determined by random forest analysis with logistic regression subsequently applied to determine the optimal number and type of analytes informing a refined FRI. RESULTS: Preflare versus pre-nonflare differences were not associated with demographics, autoantibody specificities, hSLEDAI scores, clinical features, nor medication usage. Forward selection and backward elimination of mediators ranked by variable importance resulted in 17 plasma mediator candidates differentiating preflare from pre-nonflare visits. A final combination of 11 mediators best informed a newly refined FRI, which achieved a maximum sensitivity of 97% and maximum specificity of 98% after applying decision curve analysis to define low, medium, and high FRI scores. CONCLUSION: We verified altered immune mediators associated with imminent disease flare, and a subset of these mediators improved the FRI to identify SLE patients at risk of imminent flare. This molecularly informed, proactive management approach could be critical in prospective clinical trials and the clinical management of lupus.

IRF5 haplotypes demonstrate diverse serological associations which predict serum interferon alpha activity and explain the majority of the genetic association with systemic lupus erythematosus.

OBJECTIVE: High serum interferon α (IFNα) activity is a heritable risk factor for systemic lupus erythematosus (SLE). Auto-antibodies found in SLE form immune complexes which can stimulate IFNα production by activating endosomal Toll-like receptors and interferon regulatory factors (IRFs), including IRF5. Genetic variation in IRF5 is associated with SLE susceptibility; however, it is unclear how IRF5 functional genetic elements contribute to human disease. METHODS: 1034 patients with SLE and 989 controls of European ancestry, 555 patients with SLE and 679 controls of African-American ancestry, and 73 patients with SLE of South African ancestry were genotyped at IRF5 polymorphisms, which define major haplotypes. Serum IFNα activity was measured using a functional assay. RESULTS: In European ancestry subjects, anti-double-stranded DNA (dsDNA) and anti-Ro antibodies were each associated with different haplotypes characterised by a different combination of functional genetic elements (OR>2.56, p<1.9×10(-14) for both). These IRF5 haplotype-auto-antibody associations strongly predicted higher serum IFNα in patients with SLE and explained >70% of the genetic risk of SLE due to IRF5. In African-American patients with SLE a similar relationship between serology and IFNα was observed, although the previously described European ancestry-risk haplotype was present at admixture proportions in African-American subjects and absent in African patients with SLE. CONCLUSIONS: The authors define a novel risk haplotype of IRF5 that is associated with anti-dsDNA antibodies and show that risk of SLE due to IRF5 genotype is largely dependent upon particular auto-antibodies. This suggests that auto-antibodies are directly pathogenic in human SLE, resulting in increased IFNα in cooperation with particular combinations of IRF5 functional genetic elements. SLE is a systemic autoimmune disorder affecting multiple organ systems including the skin, musculoskeletal, renal and haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population. These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness and are likely pathogenic in SLE.

Serologic markers of Epstein-Barr virus reactivation are associated with increased disease activity, inflammation, and interferon pathway activation in patients with systemic lupus erythematosus.

SLE is a clinically heterogeneous disease characterized by an unpredictable relapsing-remitting disease course. Although the etiology and mechanisms of SLE flares remain elusive, Epstein-Barr virus (EBV) reactivation is implicated in SLE pathogenesis. This study examined the relationships between serological measures of EBV reactivation, disease activity, and interferon (IFN)-associated immune pathways in SLE patients. Sera from adult SLE patients (n = 175) and matched unaffected controls (n = 47) were collected and tested for antibodies against EBV-viral capsid antigen (EBV-VCA; IgG and IgA), EBV-early antigen (EBV-EA; IgG), cytomegalovirus (CMV; IgG), and herpes simplex virus (HSV-1; IgG). Serological evidence of EBV reactivation was more common in SLE patients compared to controls as demonstrated by seropositivity to EBV-EA IgG (39% vs 13%; p = 0.0011) and EBV-VCA IgA (37% vs 17%; p = 0.018). EBV-VCA, CMV1, and HSV-1 IgG seropositivity rates did not differ between SLE patients and controls. Furthermore, concentrations of EBV-VCA (IgG and IgA) and EBV-EA (IgG) were higher in SLE patients. SLE patients with high disease activity had increased concentrations of EBV-VCA IgA (mean ISR 1.34 vs. 0.97; p = 0.041) and EBV-EA IgG levels (mean ISR 1.38 vs. 0.90; p = 0.007) compared with those with lower disease activity. EBV reactivation was associated with enhanced levels of the IFN-associated molecule IP-10 (p < 0.001) and the soluble mediators BLyS (p < 0.001) and IL-10 (p = 0.0011). In addition, EBV-EA IgG responses were enriched in two previously defined patient clusters with robust expression of IFN and inflammatory or lymphoid and monocyte responses. Patients in these clusters were also more likely to have major organ involvement, such as renal disease. This study supports a possible role for EBV reactivation in SLE disease activity.

Lupus patient decisions about clinical trial participation: a qualitative evaluation of perceptions, facilitators and barriers.

Objective: Although SLE disproportionately affects minority racial groups, they are significantly under-represented in clinical trials in the USA. This may lead to misleading conclusions in race-based subgroup analyses. We conducted focus groups to evaluate the perceptions of diverse patients with lupus about clinical trial participation. Methods: A qualitative research design employed three 90 min focus groups led by a trained moderator and guided by the Theory of Planned Behaviour. Open-ended questions about trial participation included advantages and disadvantages (behavioural beliefs), approving and disapproving significant others (normative beliefs), and participation enhancers and barriers (control beliefs). Discussions were recorded, transcribed and analysed to identify emerging themes. Results: Patients with SLE (n=23) aged 21-72, with increased proportion of minority groups (65%), participated. Reported advantages of trial participation included altruism and personal benefit. Disadvantages included uncertainties, disappointment, information burden, and life-health balance. Although some patients had discussed research participation with approving or disapproving family or friends, self-approval superseded external approval. Barriers included logistics and time, and facilitators included flexibility in scheduling, advance notice of studies, streamlined forms, and hope for SLE improvement. Conclusions: Knowledge about potential benefits of clinical trial participation was high. Minority patients demonstrated confidence in making their own informed decisions, but major barriers for all participants included burdensome forms, travel, childcare, and work. These suggest a major impact on minority and all recruitment from behavioural and control aspects, which should be considered in the logistics of trial design. This does not minimise the potential importance of improved access and education about clinical research.

Adults with systemic lupus exhibit distinct molecular phenotypes in a cross-sectional study.

BACKGROUND: The clinical and pathologic diversity of systemic lupus erythematosus (SLE) hinders diagnosis, management, and treatment development. This study addresses heterogeneity in SLE through comprehensive molecular phenotyping and machine learning clustering. METHODS: Adult SLE patients (n = 198) provided plasma, serum, and RNA. Disease activity was scored by modified SELENA-SLEDAI. Twenty-nine co-expression module scores were calculated from microarray gene-expression data. Plasma soluble mediators (n = 23) and autoantibodies (n = 13) were assessed by multiplex bead-based assays and ELISAs. Patient clusters were identified by machine learning combining K-means clustering and random forest analysis of co-expression module scores and soluble mediators. FINDINGS: SLEDAI scores correlated with interferon, plasma cell, and select cell cycle modules, and with circulating IFN-α, IP10, and IL-1α levels. Co-expression modules and soluble mediators differentiated seven clusters of SLE patients with unique molecular phenotypes. Inflammation and interferon modules were elevated in Clusters 1 (moderately) and 4 (strongly), with decreased T cell modules in Cluster 4. Monocyte, neutrophil, plasmablast, B cell, and T cell modules distinguished the remaining clusters. Active clinical features were similar across clusters. Clinical SLEDAI trended highest in Clusters 3 and 4, though Cluster 3 lacked strong interferon and inflammation signatures. Renal activity was more frequent in Cluster 4, and rare in Clusters 2, 5, and 7. Serology findings were lowest in Clusters 2 and 5. Musculoskeletal and mucocutaneous activity were common in all clusters. INTERPRETATION: Molecular profiles distinguish SLE subsets that are not apparent from clinical information. Prospective longitudinal studies of these profiles may help improve prognostic evaluation, clinical trial design, and precision medicine approaches. FUNDING: US National Institutes of Health.

Author Correction: Immunologic findings precede rapid lupus flare after transient steroid therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunologic findings precede rapid lupus flare after transient steroid therapy.

Systemic lupus erythematosus (SLE) flares elicit progressive organ damage, leading to disability and early mortality. This study evaluated clinical and immunologic factors associated with impending flare in the Biomarkers of Lupus Disease study. Autoantibodies and 32 soluble mediators were measured by multiplex assays, immune pathway activation by gene expression module scores, and immune cell subset frequencies and activation states by flow cytometry. After providing baseline samples, participants received transient steroids to suppress disease and were followed until flare. Flare occurred early (within 60 days of baseline) in 21 participants and late (90-165 days) in 13. At baseline, compared to the late flare group, the early flare group had differential gene expression in monocyte, T cell, interferon, and inflammation modules, as well as significantly higher frequencies of activated (aCD11b+) neutrophils and monocytes, and activated (CD86hi) naïve B cells. Random forest models showed three subgroups of early flare patients, distinguished by greater baseline frequencies of aCD11b+ monocytes, or CD86hi naïve B cells, or both. Increases in these cell populations were the most accurate biomarkers for early flare in this study. These results suggest that SLE flares may arise from an overlapping spectrum of lymphoid and myeloid mechanisms in different patients.

Preliminary test of the LFA rapid evaluation of activity in lupus (LFA-REAL): an efficient outcome measure correlates with validated instruments.

OBJECTIVE: Current disease activity measures for systemic lupus erythematosus (SLE) are difficult to score or interpret and problematic for use in clinical practice. Lupus Foundation of America (LFA)-Rapid Evaluation of Activity in Lupus (REAL) is a pilot application composed of anchored visual analogue scores (0-100 mm each) for each organ affected by lupus. This study evaluated the use of LFA-REAL in capturing SLE disease activity. METHODS: In a preliminary test of LFA-REAL, this simplified, organ-based system was compared with the most widely used outcome measures in clinical trials, the British Isles Lupus Assessment Group 2004 Index (BILAG), the SLE Disease Activity Index (SLEDAI) and the Safety of Estrogens in Lupus Erythematosus National Assessment (SELENA) SLEDAI Physician's Global Assessment (SS-PGA). The level of agreement was analysed using Spearman rank correlations. RESULTS: 91 patients with SLE with mild to severe disease activity were evaluated, their median SLEDAI score was 4.0 (range 0-28) and BILAG score 8.0 (0-32). The median SS-PGA was 38 mm (4-92) versus the total REAL 50 mm (0-268), which expands in range by additive organ scores. Thirty-three patients had moderate to severe disease activity (≥1.5 on SS-PGA landmarks). The median SS-PGA score of this group was 66 mm (50-92) versus median REAL score of 100 mm (59-268), confirming ability to detect a wider distribution of scores at higher disease activity. Total REAL correlated with SLEDAI, BILAG and SS-PGA (correlation coefficient=0.816, 0.933 and 0.903, respectively; p<0.001 for all). Individual LFA-REAL organ scores for musculoskeletal and mucocutaneous also correlated with corresponding BILAG domain scores (correlation coefficient=0.925 and 0.934, p<0.001). CONCLUSIONS: In this preliminary exercise, there were strong correlations between LFA-REAL and validated lupus disease activity indices. Further development may be valuable for consistent scoring in clinical trials, grading optimal assessment of change in disease activity and reliable monitoring of patients in practice.