Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a poorly understood preclinical stage of immune dysregulation and symptom accrual. Accumulation of antinuclear autoantibody (ANA) specificities is a hallmark of impending clinical disease. Yet, many ANA-positive individuals remain healthy, suggesting that additional immune dysregulation underlies SLE pathogenesis. Indeed, we have recently demonstrated that interferon (IFN) pathways are dysregulated in preclinical SLE. To determine if other forms of immune dysregulation contribute to preclinical SLE pathogenesis, we measured SLE-associated autoantibodies and soluble mediators in samples from 84 individuals collected prior to SLE classification (average timespan = 5.98 years), compared to unaffected, healthy control samples matched by race, gender, age (± 5 years), and time of sample procurement. We found that multiple soluble mediators, including interleukin (IL)-5, IL-6, and IFN-γ, were significantly elevated in cases compared to controls more than 3.5 years pre-classification, prior to or concurrent with autoantibody positivity. Additional mediators, including innate cytokines, IFN-associated chemokines, and soluble tumor necrosis factor (TNF) superfamily mediators increased longitudinally in cases approaching SLE classification, but not in controls. In particular, levels of B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL) were comparable in cases and controls until less than 10 months pre-classification. Over the entire pre-classification period, random forest models incorporating ANA and anti-Ro/SSA positivity with levels of IL-5, IL-6, and the IFN-γ-induced chemokine, MIG, distinguished future SLE patients with 92% (± 1.8%) accuracy, compared to 78% accuracy utilizing ANA positivity alone. These data suggest that immune dysregulation involving multiple pathways contributes to SLE pathogenesis. Importantly, distinct immunological profiles are predictive for individuals who will develop clinical SLE and may be useful for delineating early pathogenesis, discovering therapeutic targets, and designing prevention trials.

Objective Antinuclear antibodies (ANA) are detected in approximately 18% of females, yet only 5–8% develop autoimmune disease. Immunological differences between ANA-positive (ANA+) healthy individuals and SLE patients may elucidate the regulatory mechanisms by which ANA+ individuals avoid transition to clinical autoimmune disease. Methods Healthy individuals (n=790) were screened for autoantibodies specific for 11 lupus, systemic sclerosis and Sjögren antigens. From this screen, 31 European-American ANA+ healthy individuals were selected and demographically matched to ANA− controls and SLE patients. Serum cytokine profiles, leukocyte subset frequency and reactivity were analyzed by multiplex assays, immunophenotyping and phosphoflow cytometry. Results Of 790 individuals screened, 57(7%) were ANA+. The majority of pro-inflammatory cytokines, including IFNγ, TNFα, IL-17 and G-CSF, exhibited a stepwise increase in serum levels from ANA− controls to ANA+ healthy individuals to SLE patients (P<0.0001). IFNα, IFNβ, IL-12p40 and SCF/c-kit ligand were increased in SLE patients only (P<0.05). BLyS was elevated in SLE patients, but decreased in ANA+ individuals (P<0.001). Further, IL-1RA was down-regulated in SLE patients only (P<0.0001). ANA+ individuals had elevated frequencies of monocytes, memory B cells and plasmablasts and increased levels of pSTAT1 and pSTAT3 following IFNα stimulation compared to ANA− controls (P<0.05). Conclusion ANA+ healthy individuals exhibit dysregulation in multiple immune pathways yet differ from SLE patients by the absence of elevated interferons, BLyS, IL-12p40 and SCF/c-kit ligand. Further, severely decreased levels of IL-1RA in SLE patients compared to ANA+ individuals may contribute to disease development. These results highlight the importance of IFN-related pathways and regulatory elements in SLE pathogenesis.

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