Inhibition of IFNα secretion in cells from patients with juvenile dermatomyositis under TBK1 inhibitor treatment revealed by single-molecular assay technology

Detection of IFNαs proteins secreted by cells from JDM patients opens new perspective for drug discovery. In the present study, we evaluated the ability of anti-inflammatory drugs, particularly the TBK1 inhibitor BX795, to control IFNα secretion from cells of JDM patients. Previous studies have shown that TBK1 inhibition controls disease activity in a mouse model of SLE, and interferon signalling in fibroblasts from lupus patients [4] and PBMCs isolated from patients with gain-of-function of STING [3]. TBK1 inhibitors are currently in preclinical evaluation for their use in inflammatory diseases such as SLE. We first demonstrated that our Simoa assay allowed the detection of IFNα secretion by unstimulated peripheral blood mononuclear cells (PBMCs) isolated from JDM patients: culture of frozen PBMCs from all seven patients tested demonstrated elevated levels of IFNα (median 1319 fg/ml [range: 211.7– 5940 fg/ml]) in comparison to controls (median 0.65 fg/ml [0.65–45.05 fg/ml]; P = 0.0025) (Fig. 1A). In order to evaluate the ability of TBK1 to control IFNα secretion by PBMCs isolated from JDM patients, we cultured PBMCs overnight with or without 2 µM of BX795. Inhibition of TBK1 by BX795 resulted in a strong decrease of spontaneous secretion of IFNα in all tested patients (Fig. 1B). As in our previous study on cells from STING-mutated patients [3], BX795 did not affect cell viability at this concentration (Fig. 1C). We have also previously shown that myogenic precursor cells (MPCs) from JDM patients can produce type I-IFN [5]. This may be explained in part by the overexpression of nucleic acid sensors, including MDA5 [6] (Fig. 1D). Thus, in order to mimic disease-related inflammation, we stimulated MPCs derived from JDM patients by transfection of the MDA5 ligand poly I:C. After 24 h, IFNα was measured in the supernatant by Simoa, and cell viability was evaluated by ATP measurement. Upon stimulation, MPCs from 11 of 15 patients demonstrated increased levels of IFNα (median non-stimulated 0.20 fg/ml [0.005–1.9 fg/ml]; median stimulated 1.03 fg/ml [0.015–137.8 fg/ml] P = 0.017 (Fig. 1E and F). IFNα production by MPCs under poly I:C stimulation was decreased by treatment with BX795 (median 1.03 fg/ml [0.015–137.8 fg/ml]; median 0.36 fg/ml [0.003–4.69 fg/ml] P = 0.007) (Fig. 1F) with no or minor effect on MPCs viability (Fig. 1G). The increased sensitivity to detect IFNα protein by Simoa technology has allowed us to reveal that circulating leukocytes from JDM patients demonstrate spontaneous ex vivo production of IFNα, which can be controlled by TBK1 inhibition. Similarly, we were able to measure, for the first time, the production of IFNα by stimulated primary MPCs of JDM patients, and to evaluate the inhibition of this secretion by a TBK1 inhibitor. As such, our data demonstrate that it is now possible to use IFNα secretion as a read-out to evaluate the modulation of interferon by JDM patient-derived material, and to interrogate the effect of potential therapeutic agents. Thus, we envision that Simoa technology will inform drug discovery and personalized approaches to therapy. On the other hand, how type I IFNs are involved in JDM pathogenesis remains an open question. Notably, the sources of the interferon in JDM remain unknown, as does the impact of chronic exposure to interferon, even at very low levels, on endothelial and muscular cells within the tissues. The increased sensitivity to detect IFNα proteins by Simoa should help to identify the interferon-producing cells and paves the way for a better understanding of JDM physiopathology. TBK1 inhibition blocks IFNα secretion by JDM primary cells (A) IFNα secreted by PBMCs of seven JDM patients can be detected by Simoa. (B) Treatment with a TBK1 inhibitor strongly reduces IFNα secretion. (C) Evaluation of TBK1 inhibition on cell survival. Results from two independent experiments on PBMCs from P6 and P7 show no toxic effect. (D) MPCs from JDM patients express more IFIH1 transcript than controls. (E) Stimulation of patient MPCs with poly I:C induced secretion of IFNα that can be measured by Simoa. (F) TBK1 inhibition controls Poly I:C induced IFNα secretion by MPCs, (G) with no or minor effect on MPCs viability. TR: transfection reagent; Wilcoxon matched-pairs rank test: *P<0.05; **P<0.01. D.D. and Y.J.C acknowledge Immunoqure for provision of mAbs for Simoa assays. Funding: This work was supported by by the Agence National de la Recherche sur le SIDA et les Hépatites ANRS (AAP 2017–166 to J.-P.H. and N.B.), SATT idfinnov (Grant No. 303 to J.-P.H.) and Agence National de Recherche ANR (CE17001002 to Y.J.C. and D.D.). Disclosure statement: The authors have declared no conflicts of interest.