The Plasma Level of IL-1β As a Biomarker of Angiopathy in Systemic Chronic Active EBV Infection

Background and aim: Systemic chronic active Epstein-Barr virus infection (sCAEBV) is an intractable rare disease revealing persistent systemic inflammation with clonal proliferation of EBV-infected T- or NK-cells. 25% of sCAEBV patients accompany angiopathy such as aneurysm and vasculitis. (Yonese et al. Blood advances. 2020). Because angiopathy degrades patients' quality of life and is one of the main causes of death in sCAEBV, it is crucial to clarify the mechanisms of angiopathy development in sCAEBV. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) are reported to be involved in angiopathy onset. We investigated if IL-1β and TNFα play roles to induce vascular endothelial cell damage in sCAEBV with angiopathy.

Methods: sCAEBV patients were diagnosed when meeting all of the following four conditions: 1) inflammation persisting for more than 3 months, 2) increasing EBV-DNA in peripheral blood (PB) or in diseased tissue, 3) EBV infected T- or NK-cells, 4) not applying to other known diseases. We measured the plasma concentration of cytokines by high sensitivity cytokine beads assay (MILLIPLEX ^®MAP Kit). We isolated EBV-infected cells and monocytes by magnetic beads (MACS ^® Cell Separation). Ea.hy926, a human umbilical vein endothelial cell line, was used as a vascular endothelial cell model.

Results: Samples from 17 sCAEBV patients (infected cell types: CD4 in 7; CD8 in 1; and CD56 in 9) and 8 healthy donors were examined. We detected elevated levels of IL-1β in 4 out of 17 sCAEBV patient's plasma. Interestingly, among the 4, 3 had clinically associated angiopathy: 1 aneurysm, 1 vasculitis, and 1 intracranial vascular lesions with multiple cerebral infarctions. The EBV-infected cells of these 3 patients were CD4-positive cells. The TNFα concentration of patients' plasma was higher than that of healthy donors, but there was no correlation with angiopathy. The mRNA of IL-1β in the EBV-infected cells of patients with high plasma IL-1β was not enhanced compared to that of patients with undetectable plasma IL-1β. In one patient with high plasma IL-1β, the level of IL-1β mRNA of the monocytes was 17.2 times higher than the level of the same patient's EBV-infected cells. IL-1β inhibited the proliferation of Ea.hy926 cells. IL-1β upregulated the mRNA expression of Tissue factor (TF) as well as Plasminogen activator inhibitor-1 (PAI-1) and suppressed the mRNA of Thrombomodulin (TM) in Ea.hy926 cells.

Discussion: IL-1β originally exists in immunocompetent cells such as CD4-positive cells in the form of precursor protein (pro-IL-1β). In the occurrence of inflammation, pro-IL-1β is cleaved by caspase-1 activated by the inflammasome, and the production and the secretion of IL-1β is induced without mRNA elevation. The infected cells of sCAEBV patients with high level plasma IL-1β and vascular lesion were all CD4-positive. Consequently, we suspect that EBV-infected, CD4-positive cells are responsible for the production of IL-1β. Likewise, we detected high expression of IL-1β in monocytes derived from a patient with high plasma IL-1β. Based on these facts, we set two hypotheses on the production of IL-1β in sCAEBV. One is the production in EBV-infected cells by the cleavage of pro-IL-1β. The other is the production through transcription and translation in monocytes. IL-1β suppressed the proliferation of Ea.hy926 cells. In the same cells, IL-1β also induced TF and PAI-1 expression and suppressed TM expression. These results suggest that IL-1β may induce vascular damage and blood coagulation to cause angiopathy.

Conclusion: In sCAEBV, IL-1β may be a biomarker of angiopathy. IL-1β may be a therapeutic target to treat sCAEBV accompanying vascular lesions.