Abstract
Background Identification of non-invasive differentially regulated markers in SLE are valuable to improve diagnosis, prediction of response to treatment(s) or classification of disease subpopulations. Our goal was to assess a broad range of serum protein markers in a large cohort of SLE patients and further characterize the role of autoantibodies in serum of these patients for their functional capacity as specific immune complexes to activate the IFN axis.
Methods In this multicenter study, we have characterised a large cohort of mostly pediatric SLE patients (n=150) with longitudinal sera samples (total n=269). Serum cytokines/chemokines levels and their extractable nuclear antigen antibody (ENA) and antinuclear antibody (ANA) profiles measured from SLE patients and healthy subjects using a 30-panel Multiplex and individual enzyme-linked immunosorbent assays. Patient sera containing autoantibodies and specific antigens activating the IFN pathway quantified on human primary cells using AlphaLisa technology. Statistics for significance Mann-Whitney (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).
Results Differentially regulated cytokines/chemokines were elevated in sera of SLE patients compared to healthy individuals. Notably, IFN-related proteins (IFNalpha*, IFNgamma* and IP10****), B-cell and monocytes chemokines including CXCL13**** and MCP-1***, as well as Th1/17 activating cytokines (IL-12****, IL-17**) and those involved in antibody production by B lymphocytes (IL-10**, IL-13**, IL-6*) being more significant. Collectively, these peripheral markers may serve to help inform diagnosis and support the choice treatment in the clinic. We established a miniaturized functional assay and show that the vast majority of SLE patient sera containing common ENAs (against RNP/Sm, SS-A) and ANAs (dsDNA) could specifically activate the type I IFN pathway as an immune-complex on healthy peripheral blood mononuclear cells (PBMCs) (figure 1). We demonstrate that SLE containing immune-complexes (with specific antigens) can trigger IFN release potentially via nucleic-acid RNA/DNA sensors. Clustering patient autoantibody ENAs profiles reveal that most of the SLE patients in this cohort display a stronger IFN functional activation profile.
SLE patient sera activate the type I IFN pathway as an immune-complex
Conclusions Given the high incidence of elevated IFN signatures in SLE patients, it is not surprising that increased IFN-related proteins and ENA/ANAs that trigger IFN responses are more evident in SLE patient serum. The prevalence of ENA/ANAs in patient sera that have the functional capacity to activate IFN downstream of nucleic acid sensors may help to define subpopulations of SLE.
Funding Source(s): National Council for Scientific and Technological Development (CNPq 157534/2015–4)
PBMCs were prepared from fresh healthy human blood, stimulated with individual patient SLE serum in combination with defined antigens corresponding to their ENA or ANA titer profile. After 20 hour, IFNalpha protein levels were quantified and expressed relative to agonist ssRNA40. Data shown are from 2 more independent donors with total SLE patients numbers indicated in parenthesis. ****p<0.0001 (unpaired t-test).
