Dysregulation of innate and adaptive serum mediators precedes systemic lupus erythematosus classification and improves prognostic accuracy of autoantibodies

Abstract

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.

Keywords: Autoantibodies; Biomarkers; Cytokines; Disease progression; Forecasting; Systemic lupus erythematosus.

Figure. 1. Individuals moving toward SLE classification have distinct preclinical soluble mediator profiles compared to healthy controls

Heat map color type and intensity were determined by median normalized fluorescence intensity values in cases vs. race, gender, age (± 5 years), and time of sample procurement-matched healthy controls at four different quartile periods relative to SLE classification. Blue is lower expression and red is higher expression.

Figure 2. Select soluble mediators increase in cases as they approach SLE classification, but not in healthy controls

(A) Number of positive soluble mediators over time in patients prior to SLE classification (red), vs. race, gender, age (± 5 years) and time of sample procurement-matched healthy controls (blue). P-values for the fixed effect of disease status are shown. Normalized FI of IP-10 (B), MIG (C), IL-2 (D), IL-5 (E), and IL-21 (F), with pg/ml concentration of BLyS (G), and APRIL (H) are compared in cases (red) vs. controls (blue) over time relative to SLE classification by mixed linear regression models. Slope of line for cases (red) vs. matched healthy controls (blue) is presented in Table 2.

Figure 3. Dysregulation of Innate and Th-type mediators occurs prior to or concurrent with lupus-associated autoantibodies during early SLE pathogenesis

Kaplan-Meier plots demonstrating proportion of cases positive for serum cytokines IL-5 (green dotted line), IL-6 (blue dotted line), MIG (orange dotted line), IL-4 (green dotted line), and IFN-γ (teal dotted line) vs. SLE-associated autoantibody specificities against Ro/SSA (red solid line), RNP (blue solid line), chromatin (black solid line), Sm (grey solid line), dsDNA (orange solid line), and La/SSB (pink solid line) relative to time of SLE classification (arrow) are shown. Hazard ratios are presented in Table 3.

Figure 4. ANA Negative and ANA/Anti-Ro/SSA Positive, pre-clinical SLE patients show elevated IL-5 and IFN-γ >3.5 years before disease classification

(A) Scatter plots, showing individual cases (red dots) and matched healthy controls (blue dots) as separate points, were generated using multidimensional reduction analysis of the random forest proximity matrix. ANA positivity (B), IL-5 levels (C), IFN-γ levels (D), and anti-Ro/SSA positivity (E) are shown. SLE cluster 1 (red circle), made up of ANA⁻/IL-5^Hi/IFN-γ^Hi cases, and SLE cluster 2 (blue circle), made up of Anti-Ro⁺/IL-5^Hi/IFN-γ^Lo cases, are highlighted.

Figure 5. Proposed model of immune dysregulation leading to pathogenic autoimmunity and SLE classification

(A). Temporal relationship among dysregulated Th-type immune mediators associated with pre-clinical SLE is shown. (B) Hypothesis model of pre-clinical SLE pathogenesis: Genetic predisposition affecting apoptotic clearance, antigen-presentation, and lymphocyte responses may contribute to the appearance and maintenance of autoreactive cells (B1), leading to aberrant elevation of T helper (Th)-type cytokines, providing further co-stimulatory signals for the expansion of auto-reactive cells and potentiating the accrual of lupus-associated autoantibodies (B2). Immune dysregulation results in tissue damage and further exposure to intracellular auto-antigens, which may result in hyperactivation of innate immune cells (B3), leading to further dysregulation of soluble mediators that contribute to enhanced apoptosis and intracellular auto-antigen exposure, perpetuating the cycle of autoimmunity (B4).