Distinct Functions of Autoantibodies Against Interferon in Systemic Lupus Erythematosus: A Comprehensive Analysis of Anticytokine Autoantibodies in Common Rheumatic Diseases

Abstract

Objective: Anticytokine autoantibodies occur across a range of hematologic, pulmonary, and infectious diseases. However, systematic investigation of their presence and significance in autoimmune diseases is lacking. This study was undertaken to examine the distinct functions of anticytokine autoantibodies in patients with systemic lupus erythematosus (SLE) compared to patients with other rheumatic diseases and healthy controls.

Methods: Serum samples from patients with SLE (n = 199), patients with primary Sjögren's syndrome (SS) (n = 150), patients with rheumatoid arthritis (RA) (n = 149), and healthy controls (n = 200) were screened for 24 anticytokine autoantibodies using a multiplex bead-based assay. To evaluate the biologic activity of anticytokine autoantibodies, their ability to block cytokine-induced signal transduction or protein expression was measured. RNA sequencing was performed on whole blood in a subset of healthy controls and patients with SLE.

Results: Patients with SLE and those with SS had a striking excess of autoantibodies against interferons and the interferon-responsive chemokine interferon-inducible protein 10 (IP-10). Only autoantibodies against type I interferon, interleukin-12 (IL-12), and IL-22 exhibited neutralizing activity. In SLE, the presence of anti-interferon-γ autoantibodies was correlated with more severe disease activity, higher levels of anti-double-stranded DNA antibodies, and elevated expression of interferon-α/β-inducible genes. Conversely, in SLE patients with blocking anti-interferon-α autoantibodies, the type I interferon gene expression signature was normalized. Anti-type III interferon autoantibodies (λ2, λ3) and anti-IP-10 autoantibodies were newly recognized in SLE patient serum, and autoantibodies against macrophage-colony stimulating factor, IL-4, IL-7, IL-17, and IL-22, none of which have been previously identified in rheumatic conditions, were discovered.

Conclusion: Anticytokine autoantibodies are associated with distinct patterns of disease in SLE, SS, and RA. Anti-interferon autoantibodies are overrepresented in patients with SLE and those with SS, and fall into distinct functional classes, with only a subset of anti-type I interferon antibodies exhibiting neutralizing activity. Anti-interferon-γ autoantibodies are correlated with increased disease activity and interferon-related gene expression, suggesting that such autoantibodies may contribute to the pathogenesis of SLE.

Figure 1. Spectrum of Anticytokine Autoantibodies

Dot plots of the distribution of all anticytokine autoantibodies measured by multiplex particle-based technology in patients with (A) Systemic Lupus Erythematosus (B) Primary Sjögren’s Syndrome and (C) Rheumatoid Arthritis and compared with two standard deviations (2SD) of the mean for healthy controls (box and whiskers). (D) Frequency of patients with 1, 2, 3 or ≥4 anticytokine autoantibodies. (E) Heatmap based on unsupervised hierarchal clustering of the prevalence of anticytokine autoantibodies in SLE, SS and RA patients. Key on bottom left indicates percent prevalence. (F) Distribution of anti-interferon autoantibodies based on type in SLE patients. Venn-diagram represents the overlap of autoantibodies against type I, II and III interferons in SLE patients with any anti-interferon autoantibody (N=43). Calculated using http://www.bioinformatics.lu/venn.php. Concentrations above two standard deviations (2SD) of mean of healthy controls were classified as positive. Anti-TNF^*, anti-LTα^*: patients who received anti-TNF biologics are shaded in purple in RA cohort dot plot and were analyzed as anti-TNF or anti-LTa negative for the heatmap. G-CSF: granulocyte-colony stimulating factor; GM-CSF: granulocyte monocyte-colony stimulating factor; M-CSF: macrophage-colony stimulating factor; TNF: tumor necrosis factor; LTα: lymphotoxin-α; IFN: interferon; IL: interleukin; IP-10: interferon-inducible-protein-10

Figure 2. Examples of Neutralizing Anticytokine Autoantibodies

Representative examples of blocking antibodies demonstrated using (A) Normal PBMC incubated with healthy control or patient serum, left unstimulated or stimulated with interferon-α, interferon-β or interferon-ω. Interferon-induced STAT-1 phosphorylation was measured by flow cytometry. (B) Normal PBMC-derived lymphoblasts incubated with healthy control serum, healthy control serum spiked with 2μg/mL commercial monoclonal antibody against IL-12, high titer anti-IL-12 autoantibody patient serum, or serum from patient with low titer anti-IL-12-autoantibody, left unstimulated or stimulated with IL-12 and examined for phosphoSTAT-4 production. (C) A549 cells in presence of healthy control serum, healthy control serum spiked with commercial monoclonal antibody to IL-22 and sera from patients with high or low titer anti-IL-22 autoantibodies were left unstimulated or stimulated with IL-22. aab: autoantibody; +ve: positive; IFN: interferon; IL: interleukin

Figure 3. Serum IgG Fraction Contains All the Interferon Neutralizing Activity

Total IgG, was captured on protein G (GE Healthcare) and the flow through collected prior to eluting the IgG fraction. Representative examples show the ability of each fraction to inhibit (A) interferon-α; (B) interferon-β or (C) interferon-ω induced STAT-1 phosphorylation in normal PBMC as measured by flow cytometry. aab: autoantibody; +ve: positive; IFN: interferon

Figure 4. Interferon-α/β-inducible Genes and Anti-interferon Autoantibodies in SLE Patients

Median fold induction of 21 interferon-α/β-inducible genes in the NIH cohort of SLE patients (n=95) when compared to 47 healthy controls (dashed line). Patient subsets are compared based on presence or absence of specific autoantibodies. The horizontal bars represent the median with interquartile ranges. P-value determined by using Mann-Whitney test. ^#: one patient with anti-interferon-γ autoantibody also had blocking anti-interferon-α autoantibody and is shaded black and excluded from the analysis. aab: autoantibody; +ve: positive; IFN: interferon