IL‐1β/DNA complex elevation distinguishes autoinflammatory disorders from autoimmune and infectious diseases

IL-1-mediated autoinflammatory diseases (AID) consist of a heterogeneous group of innate immunity disorders [1]. Despite increasing awareness and research, differential diagnosis, particularly from other febrile conditions, allowing the timely initiation of appropriate therapy remains challenging because no diagnostic assay suitable for everyday clinical practice exists. In this context, several reports have shown that circulating levels of free IL-1 are neither informative nor correlated with disease causality and/or flare [2]. However, previous studies indicated that the release of neutrophil extracellular traps (NETs) carrying IL-1β on their DNA structure is a prominent feature of inflammatory attacks in familial Mediterranean fever and other AID [3, 4]. Despite the established role of microscopy methods to study NETs [5], we sought to develop a simple assay, not dependent on freshly isolated neutrophils, which could easily determine the amount of IL-1β being in complex with extracellular DNA in circulation and evaluate its diagnostic utility in AID. First, we confirmed by immunofluorescence microscopy that neutrophils, ex vivo isolated from representative patients suffering from inflammatory attacks of a typical AID, were characterized by spontaneous formation of IL-1β-enriched NETs (Fig. 1A–F). These results were consistent with the high amounts of IL-1β detected in complex with circulating DNA, as determined by a novel "in-house" IL-1β/DNA complex-specific ELISA assay in plasma samples concurrently isolated from the same patients (Fig. 1G). Additionally, the mean fluorescence intensity of microscopy sections correlated well with IL-1β/DNA complex values (Fig. 1H). Next, considering that circulating IL-1β/DNA complex levels were consistent with IL-1β-bearing NETs, we extended our study by evaluating the IL-1β/DNA complex assay in different control groups of well-characterized patients with inflammatory disorders. Significantly higher values of IL-1β/DNA complexes in AID flare, compared to active autoimmune rheumatic diseases (ARD), acute infections (INF), and healthy individuals were observed (Fig. 1I). These results were further supported by immunofluorescence microscopy in randomly selected patients from the control groups (Fig. S1). A cut-off of >0.050 arbitrary units was sufficient to distinguish AID from either INF or ARD with a sensitivity of 92.0% and a specificity of 86.4% (AUC of the ROC 0.922 ± 0.042; p < 10−6) (Fig. 1J, Table S1). Of note, levels of circulating free IL-1β and cell-free DNA, as measured simultaneously by a commercially available IL-1β ELISA and SYTOX-green dye, respectively, did not differ among groups (Fig. 1K,L). Moreover, in the total number of samples, IL-1β/DNA complex values did not correlate with free IL-1β (Fig. 1M) or cell-free DNA levels (Fig. 1N). These data suggest that the IL-1β/DNA complex assay can selectively detect the amount of IL-1β bound to extracellular DNA and is not affected by the total circulating DNA or free IL-1β (whether high or low). Finally, in a group of AID patients, IL-1β/DNA complexes were significantly reduced after successful treatment with the IL-1 inhibitors anakinra or canakinumab (Fig. 1O). Details regarding the studied groups, the principles of the IL-1β/DNA complex assay, including the in vitro inhibition studies performed using canakinumab or DNase to assess its reliability, as well as information on the methods and statistics employed, are provided in the Supporting Information section. In conclusion, we provide novel evidence that the assessment of IL-1β/DNA complex levels in plasma by ELISA is a sensitive and specific tool with high diagnostic utility, assisting the differential diagnosis of AID from other acute inflammatory disorders. It may also serve as a method to evaluate treatment response in patients receiving IL-1 inhibitors. These initial results pave the way for larger, multi-center studies, which are important to further validate the diagnostic/prognostic utility of the assay in heterogeneous inflammatory disorders. Conceptualization; supervision: Konstantinos Ritis and Panagiotis Skendros. Methodology: Anastasia-Maria Natsi, Efstratios Gavriilidis, Vasileios Papadopoulos, and Dimitrios Tsilingiris. Investigation: Anastasia-Maria Natsi, Efstratios Gavriilidis, Christina Antoniadou, Evangelos Papadimitriou, Victoria Tsironidou, Dimitris Anastasios Palamidas, Loukas Chatzis, Eleni Sertaridou, Charalampos Papagoras, Dimitrios T. Boumpas, and Athanasios G. Tzioufas. Visualization: Anastasia-Maria Natsi, Efstratios Gavriilidis, Christina Antoniadou, and Vasileios Papadopoulos. Funding acquisition; project administration: Panagiotis Skendros. Writing—original draft: Anastasia-Maria Natsi, Efstratios Gavriilidis, Christina Antoniadou, Vasileios Papadopoulos, Konstantinos Ritis, and Panagiotis Skendros. Writing—review and editing: Anastasia-Maria Natsi, Efstratios Gavriilidis, Christina Antoniadou, Vasileios Papadopoulos, Dimitrios Tsilingiris, Dimitrios T. Boumpas, Athanasios G. Tzioufas, Charalampos Papagoras, Konstantinos Ritis, and Panagiotis Skendros. The authors declare that they have no conflicts of interest. Greek General Secretariat for Research and Innovation (GSRI) Research and Innovation Programme CytoNET, Grant Number: MIS-5048548; Special Account for Research Funds (SARF), Democritus University of Thrace, Grant Number: 80895 All study participants provided written informed consent in accordance with the principles expressed in the Declaration of Helsinki. Patients' records were anonymized and de-identified prior to analysis to ensure anonymity and confidentiality. The study protocol was approved by the Scientific and Ethics Committee of the University Hospital of Alexandroupolis (Approval Number 803/23-09-2019). The data that support the findings of this study are available from the corresponding author upon reasonable request. Table S1: Sensitivity and specificity of proposed IL-1β/DNA complex cutoffs Table S2: Characteristics of HI group Table S3: Characteristics of AID group Table S4: Characteristics of ARD group Table S5: Characteristics of INF group Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.