Monocytes shape the neuroprotective and immunomodulatory effects of mesenchymal stromal cell-derived extracellular vesicles

Abstract BACKGROUND Mesenchymal stromal cell–derived extracellular vesicles (MSC-EVs) exert neuroprotective effects in ischemic stroke largely through immunomodulatory mechanisms. Monocytes are first-line responders to MSC-EVs. Their contribution to MSC-EV-induced neuroprotection remains poorly understood. This study investigated the role of monocytes in shaping neuroprotective responses to MSC-EVs after ischemic stroke. METHODS Male C57BL/6J mice were exposed to transient middle cerebral artery occlusion (MCAO). Monocytes were depleted using pharmacological (clodronate liposomes), immunological (anti-CCR2), or genetic ( Mrp8-Cre +/– Nr4a1 fl/fl ) approaches removing total, CCR2 + , or Ly6C low monocytes, respectively. In additional cohorts, neutrophils and T cells were simultaneously depleted by anti-Ly6G or anti-CD4/CD8 antibodies. Small EVs from clonally expanded immortalized MSCs were administered intravenously. Neurological deficits, ischemic injury, and immune responses were analyzed up to 72 hours post-MCAO. Complementary ex vivo studies were performed, in which MSC-EVs were administered to monocyte-depleted or non-depleted peripheral blood mononuclear cells (PBMCs) obtained from acute ischemic stroke patients. RESULTS In ischemic mice with intact monocyte compartment, MSC-EVs reduced neurological deficits, infarct volume, neuronal injury, and brain leukocyte infiltrates. These protective effects were abolished in monocyte-depleted mice, particularly following CCR2 + monocyte depletion. Under these conditions, MSC-EV treatment exacerbated neurological deficits, ischemic injury, and leukocyte infiltration, accompanied by neutrophil and T cell expansion and overactivation. Depletion of neutrophils or T cells prevented the EV-induced worsening of stroke outcome in monocyte-deficient mice. Ly6C low monocytes played a crucial role in orchestrating immune responses to MSC-EVs. Their depletion abolished EV-induced neuroprotection. In stroke patient PBMCs, MSC-EVs induced phenotypic reprogramming of monocytes, whereas they promoted CD4 + and CD8 + T cell activation in the absence of monocytes. CONCLUSIONS Monocytes shape the immunomodulatory actions of MSC-EVs. In their absence, MSC-EVs trigger neutrophil and T cell overactivation that worsens stroke outcome. These findings highlight the importance of monocyte- and T cell-related potency assays for the clinical translation of MSC-EV therapies.