The vasoconstrictor adenosine 5'-tetraphosphate is a danger signal that induces IL-1β

The endogenous nucleotide adenosine 5'-tetraphosphate (Ap4) is a potent vasoconstrictor. Despite its structural similarity to the danger signal adenosine 5'-triphosphate (ATP), the immunomodulatory effects of Ap4 remain unclear.

Modulation of interleukin (IL)-1β secretion by Ap4 was studied in both immune cells lines (THP-1, U937) and primary immune cells. Genetic and pharmacological approaches were used to characterize signaling. Cytokine production was measured using ELISA and multiplex assays, while cell viability was determined by MTT and LDH assays. Calcium influx and YO-PRO-1 uptake were assessed via microplate assays and flow cytometry, respectively. RNA sequencing and Western blotting were performed to analyze global gene expression and protein levels.

We demonstrate that Ap4 stimulates IL-1β release in primed immune cells without affecting the levels of other cytokines, suggesting specificity in its immunomodulatory actions. Mechanistically, Ap4-induced IL-1β release was partially modulated by the P2X7 receptor, a key mediator of inflammation. However, unlike canonical inflammasome activators, this process was independent of potassium efflux, the NLRP3 inflammasome, and caspase-1. Ap4 specifically increased LDH release in macrophages irrespective of priming. Furthermore, Ap4-mediated calcium influx, crucial for immune cell activation, predominantly occurred through P2Y receptors rather than P2X7 receptors. Transcriptomic analysis highlighted Ap4-induced upregulation of metallothioneins, implicating metal ion homeostasis in Ap4-mediated responses.

Collectively, our findings suggest Ap4 as a novel pro-inflammatory mediator capable of inducing IL-1β release in innate immune cells through distinct mechanisms from classical NLRP3 inflammasome activators, shedding light on its potential role in inflammatory diseases and vascular disorders.