Presence of an interferon signature in individuals who are anti-nuclear antibody positive lacking a systemic autoimmune rheumatic disease diagnosis

Abstract

Background: Elevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody-positive (ANA⁺) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA⁺ individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question.

Methods: Healthy ANA^- control subjects and ANA⁺ titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score.

Results: The mean IFN5 scores were increased in all ANA⁺ participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA⁺ and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA⁺ subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA⁺ subset, this score also correlated with the ANA titre, whereas in the other ANA⁺ subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores.

Conclusions: An IFN signature is seen in a significant proportion of ANA⁺ individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.

Keywords: Anti-nuclear antibodies; Interferon; Pre-clinical; Systemic autoimmune rheumatic disease.

Fig. 1

Levels of interferon (IFN)-induced gene expression in the participant subsets, stratified by diagnosis. a IFN-induced gene expression was quantified in whole peripheral blood RNA using NanoString technology and the normalized levels of five ubiquitously expressed IFN-induced genes summed to produce an IFN5 score. Results to the left of the figure are shown for healthy control subjects (Control), individuals who were asymptomatic anti-nuclear antibody–positive (ANA⁺), and patients who had undifferentiated connective tissue disease (UCTD) and early systemic autoimmune rheumatic disease (SARD). Significant differences from healthy control subjects are indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001. Results to the right of the figure show the IFN5 scores for the different early SARD patient subsets with the significant differences between groups indicated. b IFN5 scores for different ANA⁺ subject subsets, stratified by ethic group. c Expression levels of two IFN-β-induced genes (EIF2AK2 and PLSCR1) and BAFF in whole peripheral blood. Significant differences from healthy control subjects are indicated. d Correlation between EIF2AK2 and BAFF levels and IFN5 score for the different ANA⁺ subsets. Solid lines indicate linear regression curves. For all panels, dashed lines represent 2 SD above the mean for healthy control subjects. e Correlation between serum IFN-α levels as measured by enzyme-linked immunosorbent assay and IFN5 scores for the different ANA⁺ subsets. Solid lines indicate linear regression curves. SSc Systemic sclerosis, SS Sjögren’s syndrome, SLE Systemic lupus erythematosus, DM Dermatomyositis, MCTD Mixed connective tissue disease

Fig. 2

Association between anti-nuclear antibody (ANA) titre and interferon (IFN)-induced gene expression. a ANA titres in asymptomatic ANA⁺ individuals, patients with undifferentiated connective tissue disease (UCTD) and patients with early systemic autoimmune rheumatic disease (SARD). Significant differences from patients with early SARD are indicated as *** p < 0.001. b Association between IFN5 score and ANA titre in participants with non-SARD (asymptomatic ANA⁺ and UCTD) and those with early SARD. c Association between IFN5 scores and ANA titre for asymptomatic ANA⁺ participants and patients with UCTD. Significant associations are indicated

Fig. 3

Association between the number of different anti-nuclear antibody (ANA) specificities detected and interferon (IFN)-induced gene expression. a The number of different ANA specificities detected using the BioPlex® 2200 system in asymptomatic ANA⁺ individuals, participants with undifferentiated connective tissue disease (UCTD), and participants with early systemic autoimmune rheumatic disease (SARD). Significant differences from patients with early SARD are indicated as * p < 0.05, *** p < 0.001. Association between IFN5 score and the number of autoantibodies detected in (b) non-SARD (asymptomatic ANA⁺ and UCTD) and early SARD participants and in (c) asymptomatic ANA⁺ individuals and participants with UCTD. Significant associations are indicated

Fig. 4

Correlation between interferon (IFN)-induced gene expression and the presence of specific anti-nuclear antibodies (ANA). The levels of specific ANA were measured using the BioPlex® 2200 ANA screening system, and participants were stratified on the basis of presence or absence of anti-Ro and/or anti-La (Ro/La), anti-Smith (anti-Sm) and/or anti-Sm/ribonuclear protein (RNP) and/or anti-RNP (Sm/RNP), or anti-double-stranded (anti-dsDNA) and/or anti-chromatin (DNA). Correlation between each cluster of autoantibodies and the IFN5 score for (a) all ANA⁺ participants and the non-systemic autoimmune rheumatic disease (SARD) and early SARD subsets and (b) asymptomatic ANA⁺ individuals and participants with undifferentiated connective tissue disease (UCTD). Significant correlations are indicated. Dashed lines in each figure represent 2 SD above the mean for healthy control subjects