Opioid-mediated Neutrophil Microparticle Production and NLRP3 Inflammasome Activation Cause Vascular Damage

Abstract

Background Opioid-receptors are found on leukocytes within and outside the central nervous system. Inflammation and interleukin (IL)-1β, in particular, play major roles in addiction. Because IL-1β must be packaged within extracellular vesicles for transport, we hypothesized that opioids stimulate production of microparticles (MPs). Methods Human and murine neutrophil MPs production were assessed by flow cytometry in response to morphine and hydromorphone ex vivo , and mice were injected with drugs to assess in vivo responses. Results Human and murine neutrophils generate MPs high in IL-1β by a complex oxidative stress response involving mitochondria, NADPH oxidase and nitric oxide synthase-2, similar to that seen with other stimuli (FRBM 106:406, 2017). After 1 hour incubation of 550 murine neutrophils, 0, 0.1, 1, 5 and 10 µM morphine triggered generation of, respectively (mean + SE, n=3, *p + 5, 296 + 34, 1351 + 179*, 2560 + 413* and 3113 + 982* MPs. IL-1β content of MPs was similarly increased such that control MPs had 1.6 + 0.6 pg/million MPs whereas those generated due to 10 µM morphine had 92.8 + 8.1 (p . 01) pg/million MPs. Morphine responses were inhibited by 1 µM naloxone (opioid-receptor antagonist). When injected into mice, morphine and hydromorphone increase blood-borne MPs with surface proteins indicative of production by neutrophils (Ly6G+), microglia (P2Y12 and CD45+) and endothelium (CD31+/CD41-dim). Time-course and dose-responses demonstrated diffuse capillary leak similar to that seen with other stimuli (J Appl Physiol 123:297, 2017) likely due to neutrophil MPs, spinal microglia activation assessed as CD68 expression by immunohistochemistry, and progressive elevations of circulating microglia MPs with repetitive dosing. Conclusion Opioid-receptor stimulation triggers neutrophil oxidative stress, MPs production and NLRP3 inflammasome activation. Circulating MPs cause vascular injuries and may play a critical role in opioid addiction.