Mesenchymal stromal cell-derived extracellular vesicles modulate microglia/macrophage polarization and protect the brain against hypoxia-ischemic injury in neonatal mice by targeting delivery of miR-21a-5p

Abstract

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) (MSC-EVs) exhibit protective effects in damaged or diseased tissues. However, the role of EVs secreted by MSC in hypoxia-ischemic (HI) injury in neonatal mice remains unknown. Systemic administration of MSC-EVs attenuated acute brain damage and neuroinflammation, and skewed CD11b⁺/CD45^low microglia and CD11b⁺/CD45^high brain monocyte/macrophage towards a more anti-inflammatory property as determined at 72 h post-HI. In addition, MSC-EVs remarkably improve the injury outcomes pups prior to weaning (P21), while no effect on long-term memory impairment (P42). Importantly, these effects were preceded by incorporation of MSC-EVs into a large number of neurons and microglia within HI group. Abundant levels of miR-21a-5p were present in EVs as determined with next-generation sequencing. Notably, MSC-EVs treatment further increased miR-21a-5p levels at 72 h post HI. Knockdown analyses revealed that miR-21a-5p, and its target-Timp3, were essential for this neuroprotective property of MSC-EVs following HI exposure as demonstrated in both in vitro and in vivo models. These findings suggest that a systemic administration of EVs derived from MSC, have the capacity to incorporated into neurons and microglia where they can then exert neuroprotection against HI-induced injury in neonates through the delivery of miR-21a-5p.

Introduction

Neonatal hypoxic–ischemia encephalopathy (HIE) is a common neurologic condition encountered in newborns and currently no promising therapy exists. One recent approach has involved that of cell therapy with use of mesenchymal stromal cell (MSC). This protocol has shown promise in animal models of perinatal hypoxia-ischemic (HI) brain injury, including decreases in the volume of ipsilateral hemisphere and improved functional outcomes [1,2]. The mechanisms through which MSC mediate these effects are unclear and continue to be investigated. While only a small number of transplanted MSC actually are able to engraft in host tissues, and most are not permanently retained, their therapeutic benefits, in part, appear to involve a promotion of endogenous repair processes such as inducing neurogenesis and angiogenesis and a reduction in glial scarring resulting from the release of paracrine factors [3], [4], [5], [6].

Findings from recent studies suggested that the paracrine effects of MSCadministration are largely due to extracellular vesicles (EVs) secreted by MSC [7,8]. EVs are small membrane vesicle, and classified into exosomes (30–200 nm), microvesicles (200–1000 nm) and apoptosomes (1–10 μm) depending on their size [9]. Exosome-enriched EV fractions mediate intercellular communication via transferring bio-active molecules, including DNA, messengerRNAs (mRNAs), microRNAs (miRNAs), proteins and lipids [10]; and, it has been suggested that miRNAs can be taken up by recipient cells and, in this way, influence the fate of target cells [11,12].

Results from recent studies have indicated that MSC-EVs can exert beneficial effects in ischemic injury. For example, a systemic administrations of EVs derived from MSC have been shown to offer neuroprotective effects against ischemia in adult mice [13,14]; and intravenous administration of human bone marrow-MSC-EVs prevented global HI induced functional impairment and structural injury within the preterm brain [15]. Moreover, MSC-derived EVs have the potential to prevent oxygen–glucose deprivation/reoxygenation (OGD/R)-initiated apoptosis in mouse neuroblastoma cells [16]. However, whether EVs secreted by MSC display any functional activity within neonatal HI injury remain unknown. In this study, we investigated the effects of a short-term MSC-EVs administration on acute brain injury, and on long-term neurological functioning within neonatal HI mice. In addition, we characterized some of the underlying mechanisms involved in these effects.