Introduction: Extracellular Vesicles (EVs) are regarded as cell-cell communication. EVs cargo miRNAs, regulating processes including inflammasome activation. Thus, miRNAs serve as new biomarkers. Exposure to ATP is one way of EV release, accompanied by P2X7 pore formation and cell death, depending on the amount of ATP (Leeson et al., 2019).
Objective: In this study, expression of inflammation-related miRNAs (miR-21, miR-146a, miR-155) was determined in EVs from blood-derived macrophages of healthy donors (HDs, n=5) and patients (n=16) with infections, pain syndromes and malignancies.
Methods: Macrophage supernatants were collected before and after stimulation with 3mM ATP. EVs were isolated by differential centrifugation and characterized by light scattering and electron microscopy. P2X7 activation was determined by patch clamping using 1mM ATP. Macrophages were tested for mitochondrial oxygen radical formation (MitoSOX), mitochondrial membrane potential (MitoTracker), and cell death (PI) using FACS. MiRNAs were determined by RT-qPCR.
Results: Eight ATP-treated cultures of both, patients and HDs released elevated amounts of EVs, but seven macrophage cultures responded with EVs‘ decrease. Extracellular calcium concentrations lowered after ATP stimulation (p<0.0001), indicating pore formation. Independently, patients showed elevated levels of miR-21 (P=0.0041) and miR-146 (P=0.2398) compared to HDs. Fourteen ATP-treated cultures showed lower levels of miR-146a, eight were lower in miR-146a and miR-21 contents. Four patients had increased miR-21 and miR-146a. The latter were congruent with a more moderate calcium decrease, glucose consumption and/or lowered extracellular lactate (<2mM).
Conclusion: Macrophages of patients and HDs responded to ATP with increased EV release. Patient-derived EVs show higher amounts of inflammation-related miRNAs, further upregulated in M1-polarized macrophages. Together, our model supports EV release after ATP exposure, with miRNA contents reflecting regulatory functions by the respective macrophage cultures.