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Uptake of Fluorescent Labeled Small Extracellular Vesicles <em>In Vitro</em> and in Spinal Cord

Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, ...

Drexel University College of Medicine

Extracellular RNA in circulation mediates intercellular communication in normal and pathological processes. One mode of circulating miRNA transport in bodily fluids is within 30-150 nm small extracellular vesicles (sEVs) or exosomes. Uptake of sEVs can regulate gene expression in recipient cells enabling circulating miRNAs to exert paracrine and systemic effects. Complex regional pain syndrome (CRPS) is a debilitating pain disorder characterized by chronic inflammation. Our previous investigations identified a significant decrease of hsa-miR-939 in whole blood from CRPS patients compared to control; we also observed that overexpression of miR-939 can negatively regulate several proinflammatory genes . Though downregulated in whole blood, miR-939 was significantly upregulated in sEVs isolated from patient serum. Here we investigated miR-939 packaging into sEVs  under inflammation induced by monocyte chemoattractant protein-1 (MCP-1), a chemokine that is upregulated in CRPS patients. Stimulation of THP-1 monocytes by MCP-1 led to elevated levels of miR-939 in sEVs, which was abrogated using inhibitors of exosome secretion. miRNAs loaded into exosomes largely contain short miRNA sequence motifs called EXOmotifs. Mutation analysis of miR-939 showed that EXOmotif is one of the possible cellular mechanisms responsible for packaging miR-939 into sEVs. We confirmed gene expression changes in recipient cells following the uptake of sEVs enriched in miR-939 using RNA sequencing. Additionally, our data from primary immune cell-derived sEVs of CRPS patients and controls demonstrate that while the relative expression of miR-939 is higher in sEVs derived from B cells, T cells and NK cells relative to monocyte-derived sEVs in controls, only the B cell-derived sEVs showed a significantly higher level of miR-939 in CRPS patients. Differential miRNA sorting into exosomes and its functional impact on recipient cells may contribute to the underlying pathophysiology of CRPS.

Inflammation potentiates miR-939 expression and packaging into small extracellular vesicles.

Small extracellular vesicles (sEVs) derived from antigen-presenting cells such as macrophages can induce therapeutically relevant immune responses. Anti-inflammatory miRNAs are elevated in sEVs secreted by RAW 264.7 mouse macrophages after lipopolysaccharide (LPS) stimulation. We observed uptake of these sEVs by primary mouse cortical neurons, microglia and astrocytes followed by downregulation of proinflammatory miRNA target genes in recipient cells. Pre-treating primary microglia with these sEVs decreased pro-inflammatory gene expression. A single intrathecal injection of sEVs derived from LPS stimulated RAW 264.7 cells attenuated mechanical hyperalgesia in the complete Freund's adjuvant (CFA) mouse model of inflammatory pain and formalin induced acute pain. Importantly, sEVs did not alter the normal pain threshold in control mice. RNA sequencing of dorsal horn of the spinal cord showed sEVs-induced modulation of immune regulatory pathways. Further, a single prophylactic intrathecal injection of sEVs two weeks prior, attenuated CFA-induced pain hypersensitivity and was ineffective in formalin model. This indicates that prophylactic sEVs administration can be beneficial in attenuating chronic pain without impacting responses to the protective physiological and acute inflammatory pain. Prophylactic administration of sEVs could form the basis for a safe and novel vaccine-like therapy for chronic pain or as an adjuvant, potentially reducing the dose of drugs needed for pain relief.

Zhucheng Lin

Department of Pharmacology & Physiology

Drexel University College of Medicine

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Zhucheng Lin

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmacology & Physiology

Drexel University College of Medicine

Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Department of Pharmacology & Physiology