Detection of interferon alpha protein reveals differential levels and cellular sources in disease

Abstract

Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation.

Figure 1.

Specificity, sensitivity, and reproducibility of the Simoa IFNα assay. (A) Simoa IFNα assay reactivity with IFNα17, IFNβ, IFNλ1, IFNλ2, IFNω, and IFNγ recombinant proteins. Lowest concentration is the blank. (B) Simoa IFNα assay cross-reactivity with IFNα1, IFNα1 (Val¹¹⁴), IFNα2a, IFNα2b, IFNα2c, IFNα4a, IFNα4b, IFNα5, IFNα6, IFNα7, IFNα8, IFNα10, IFNα14, IFNα16, IFNα17, and IFNα21. (C) Simoa IFNα competition assay; measurement of IFNα in five SLE patient plasma samples after preincubation with the human anti-IFNα capture antibody for 30 min before analysis. (D) Reproducibility testing for each concentration, acquired as duplicates across three independent runs. Dashed line represents the LOD, defined by mean blank average enzyme per bead (AEB) + 3 SD of all runs. (E) 22 plasma samples (8 SLE, 8 JDM, 3 AGS, and 3 STING) were analyzed with two independently prepared lots of beads by different users and at different times. Spearman correlation is reported. (F) Correlation of IFNα protein measured by Simoa in paired plasma and serum samples from 10 AGS patients. Spearman correlation is reported.

Figure 2.

Quantification of plasma, serum, and CSF IFNα in patient cohorts. (A) Plasma from healthy controls (n = 20) and patients with RVCL (n = 30), SLE (n = 72), JDM (n = 43), and molecularly defined interferonopathies (n = 27) were assayed by Simoa for IFNα protein. Values were assessed by one-way ANOVA test (Kruskal–Wallis) and Dunn’s multiple comparison testing between groups. (B) CSF samples from acute meningitis (n = 9), acute encephalitis (n = 9), acute meningoencephalitis (n = 1), and RVCL (n = 12) were assayed by Simoa for IFNα protein. Values were assessed by Mann–Whitney T test. ***, P < 0.001; ****, P < 0.0001; n.s., not significant; horizontal lines indicate the median.

Figure 3.

Comparison of IFNα concentration with antiviral activity and ISG expression. (A) Correlation of Simoa IFNα protein measurement with IFN activity measured by a cytopathic assay for interferonopathy (n = 10), JDM (n = 26), and JSLE (n = 2) patients. (B) Correlation of Simoa IFNα protein measurement with IFN activity measured by a cytopathic assay for CSF samples from acute viral meningitis (n = 9), acute viral encephalitis (n = 9), acute viral meningoencephalitis (n = 1), AGS (n = 1), and RVCL (n = 12). (C–E) Correlation of Simoa IFNα concentration with the ISG score in SLE (C; n = 21), JDM (D; n = 23), and molecularly defined interferonopathy patients (E; n = 29). Spearman correlations were calculated for each patient group, excluding samples where both the ISG score and the IFNα concentration were negative (SLE n = 10, JDM n = 1).

Figure 4.

Disease associations of serum IFNα in SLE patients. (A–D) Higher serum IFNα levels associate with higher SLEDAI (P < 0.001; A), ESR (P < 0.01; B), lower CH50 activity (P < 0.001; C), and number of specific autoantibodies against ribonucleoproteins (anti-Ro, La, Sm, RNP; P < 0.0001; D). IFNα <10 fg/ml: n = 25; IFNα = 10–300 fg/ml: n = 14; IFNα >300 fg/ml: n = 8; one-way ANOVA (Kruskal–Wallis) p-values are reported. (E) Profile of autoantibodies directed against ribonucleoproteins in patients with low, intermediate, and high IFN levels. Green, <25 U/ml; yellow, 25–50 U/ml; orange, 50–100 U/ml; red, >100 U/ml. A positive result is >25 U/ml. The total number of autoantibodies against ribonucleoproteins (anti-Ro, La, Sm, RNP) is significantly increased in patients with the highest levels of serum IFNα (two-way ANOVA, P < 0.0001). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., not significant; horizontal lines indicate median.

Figure 5.

Identification of circulating IFNα-producing cells in STING patients. IFNα protein levels presented as median attograms per cell in sorted CD4 and CD8 T cells, NK cells, B cells, monocytes, and pDCs from STING mutation (red, n = 3), JDM (blue, n = 3), AGS (green, n = 4), and SLE (purple n = 3) patients. The black line on each plot represents the median of four control healthy donors.