Specialized Pro-Resolving Mediator loaded Extracellular Vesicles Mitigate Pulmonary Inflammation

Abstract

Specialized pro-resolving mediators (SPMs), including lipoxins derived from arachidonic acid and resolvins, protectins, and maresins derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), orchestrate the active resolution of inflammation. These SPMs are biosynthesized through the coordinated interaction of various cells in a process known as transcellular biosynthesis, involving the sequential action of cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), 12-lipoxygenase (12-LOX), and/or 15-lipoxygenase (15-LOX) enzymes. Additionally, Aspirin-triggered Resolvins are produced by acetylated COX-2, along with various lipoxygenases. Although SPMs regulate various cellular processes to actively resolve inflammation, their in vivo levels are typically low. To address this limitation, we engineered a multigene expression vector that co-expresses COX-2, 5-LOX, and 15-LOX, potentiating the synthesis of various SPMs. HEK293T cells transfected with this vector and cultured with fatty acid-free BSA-complexed DHA, EPA, and aspirin, successfully mimicked both transcellular and aspirin-triggered biosynthesis of Resolvins. These Resolvins are packaged into extracellular vesicles, which significantly inhibited neutrophil adhesion to endothelial cells, preserved endothelial monolayer barrier integrity, suppressed NF-{kappa}B reporter activity, and enhanced macrophage efferocytosis in vitro. Notably, post-injury administration of Resolvin-loaded EVs mitigated pulmonary inflammation in LPS-treated mice without causing systemic or pulmonary toxicity. In summary, we report a novel cell-based platform for generating Resolvin-loaded EVs that mitigate pulmonary inflammation in mouse models, underscoring their potential for treating other acute inflammatory diseases.