Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotective effects in a 3xTg model of Alzheimer's disease

Abstract

The critical role of neuroinflammation in favoring and accelerating the pathogenic process in Alzheimer's disease (AD) increased the need to target the cerebral innate immune cells as a potential therapeutic strategy to slow down the disease progression. In this scenario, mesenchymal stem cells (MSCs) have risen considerable interest thanks to their immunomodulatory properties, which have been largely ascribed to the release of extracellular vesicles (EVs), namely exosomes and microvesicles. Indeed, the beneficial effects of MSC-EVs in regulating the inflammatory response have been reported in different AD mouse models, upon chronic intravenous or intracerebroventricular administration. In this study, we use the triple-transgenic 3xTg mice showing for the first time that the intranasal route of administration of EVs, derived from cytokine-preconditioned MSCs, was able to induce immunomodulatory and neuroprotective effects in AD. MSC-EVs reached the brain, where they dampened the activation of microglia cells and increased dendritic spine density. MSC-EVs polarized in vitro murine primary microglia toward an anti-inflammatory phenotype suggesting that the neuroprotective effects observed in transgenic mice could result from a positive modulation of the inflammatory status. The possibility to administer MSC-EVs through a noninvasive route and the demonstration of their anti-inflammatory efficacy might accelerate the chance of a translational exploitation of MSC-EVs in AD.

Keywords: Alzheimer's disease; dendritic spines; extracellular vesicles; inflammation; mesenchymal stem cells; microglia.

FIGURE 1

Cytokine preconditioning of MSCs (p6) in a SF‐medium (SF+CYT) causes the upregulation of the immunomodulatory markers and preserves their stemness potential. A, MSCs were committed toward osteogenic (O) or adipogenic (A) lineages after the preconditioning protocol. Calcium deposits (O, in red) are visualized by Alizarin Red, while fat droplets (A, in red) are stained by Oil Red, indicating osteocytic and adipocytic differentiation, respectively. CTRL‐O and CTRL‐A: controls of MSCs grown in the absence of osteogenic (O) and adipogenic (A) inducing differentiation media. Images were acquired by phase contrast microscopy. Magnification ×20. Scale bars = 50 μm. B, Immunoblotting evaluation of the expression of typical stemness (CD90 and CD73) and immunoregulatory markers (COX2 and IDO) by MSCs subjected to SF + cytokine (SF+CYT) or SF preconditioning (CTRL: control; SF: serum‐free; CYT: cytokines, TNFα and IFNγ). C, Histograms relative to the quantification of the immunomodulatory marker bands in (B). For the comparison between groups (SF+CYT at 24 and 48 hours) unpaired, two‐tailed Student's t test was used. All the data are expressed as mean ± SEM (n = 3). MSC, mesenchymal stem cell; SF, serum‐free

FIGURE 2

pMSC‐derived EV characterization. A, Size‐distribution curve generated by NS300 NanoSight NTA (data are obtained by mean of three tracking video files, for each experiment): a main pick at a range size of 100 to 500 nm (mean size: 201.1 nm) is visible. B, Frame picture from NTA video, visualizing light scattering EVs derived from cytokine‐preconditioned MSCs. Note the presence of vesicles of different sizes. C, EV characterization by WB: the expression of EV markers after serum deprivation (SF) in the presence (CYT+EVs) or in the absence (SF‐EVs) of cytokines. EVs were positive for all the three typical markers analyzed. For each lane, 20 μg of proteins derived from the EV pellet or their respective supernatants (SF Sup, SF+CYT Sup.), derived from the last ultracentrifugation before the wash passage (see Section 2) were loaded. EVs, extracellular vesicles; MSCs, mesenchymal stem cells; NTA, nanoparticle tracking analysis; pMSC, preconditioned mesenchymal stem cell; SF, serum‐free

FIGURE 3

EVs switch microglia toward an anti‐inflammatory phenotype. A, Cells in control conditions displayed a morphology characterized by thin and long processes (CTRL, EVs). After cytokine treatment, microglia acquire a reactive phenotype defined by bigger soma and amoeboid‐like morphology (CYT). The presence of EVs did not alter microglial morphology neither in control (EVs) nor in inflammatory conditions (CYT+EVs). Images were acquired by phase contrast microscopy. Magnification ×10. Scale bars = 100 μm. B, Representative WB bands (left column) and relative quantification histograms (right column) of microglial markers. Cytokine treatment caused a significative upregulation, when compared to the controls, of the activation of M1 markers such as Iba‐1, iNOS, and CD68, while downregulated the M2 marker CD206. EV treatment did not significantly affect the expression of none of the markers after the inflammatory challenge (Iba‐1, CD68: n = 3; iNOS: n = 4, CD206: n = 4). β‐actin was used as loading control for Iba‐1 and iNOS expression analysis, while CD68 and CD206 expression was normalized on total protein (Ponceau staining). Comparison between groups (eg, CYT vs CYT+EVs, CTRL vs CYT) used paired, one‐tailed Student's t test. C, EV treatment switched microglia toward an anti‐inflammatory phenotype. Histograms show the quantification of microglia release of IL‐6, IL‐1β, and IL‐10 by ELISA. In TNFα‐IFNγ activated microglia, EVs induced the release of the anti‐inflammatory cytokine IL‐10 (n = 6) and negatively modulated the secretion of the pro‐inflammatory mediators IL‐6 (n = 4) and IL‐1β (n = 5). (CTRL: control; EVs: extracellular vesicles; CYT: cytokines). For the comparison between groups (eg, CYT vs CYT+EVs, CTRL vs CYT, CTRL vs EVs) paired, one‐tailed Student's t test was used; *P < .05, **P < .01. All the data are expressed as ±SEM. ELISA, enzyme‐linked immunosorbent assay; EVs, extracellular vesicles; iNOS, inducible nitric oxide synthase

FIGURE 4

IN administration of EVs reduces the density of Iba‐1⁺ cells in 3xTg AD mice. A, Representative images of the distribution of Iba‐1⁺ (green) microglia in the CA1 medial hippocampus (CA1), entorhinal cortex (EC), and prefrontal cortex (PC) of control (CTRL) and EV‐treated mice (EVs). B, Histograms compare the number of microglial cells in the same areas of (A). Note that the animals receiving EVs (EVs) displayed a reduced number of Iba‐1⁺ cells compared to animals from the control group that received Phosphate Buffered Saline (PBS). For the comparison between groups (n = 4), unpaired two‐tailed Student's t test was used; *P < .05, **P < .01. Data are expressed as arbitrary unit (a.u.) mean ± SD. Scale bars = 100 μm. AD, Alzheimer's disease; EVs, extracellular vesicles

FIGURE 5

IN administration of EVs reduces cell soma size of Iba‐1⁺ cells. A, Representative image of microglia cells stained for Iba‐1 in medial hippocampus CA1 of control (CTRL) and EV‐treated (EVs) 3xTg AD mice. Scale bars = 30 μm. B, Histograms comparing the reduction (shown as a.u.) of microglial cell body size in hippocampus (CA1), entorhinal cortex (EC), prefrontal cortex (PC) of control and treated mice. Average cell body size was quantified by means of ImageJ software (see the thresholding method description in Supporting Information). Comparison between groups (n = 4) used unpaired, two‐tailed Student's t test; *P < .05. Data are expressed as mean ± SD. EVs, extracellular vesicles

FIGURE 6

pMSC‐derived EVs reduce Iba‐1 and CD68 expression in microglia of AD mice. A,C, Representative confocal images of the hippocampal CA1 region showing the effect of EVs in lowering the expression of Iba‐1 (A, green) and CD68 (C, red) in microglial cells of EV‐treated (EVs) compared to control mice. Scale bars (A, C) = 30 μm; C1 and 2: magnified views of the boxed regions in (C) = 10 μm. Note in C1 and C2, the yellow/orange dots representing CD68 and Iba‐1 colocalization. B,D, Histograms comparing the quantification of the fluorescence intensity of Iba‐1 (B, n = 4) and CD68 (D, n = 4) in CA1 region of the medial hippocampus (CA1), entorhinal cortex (EC), and prefrontal cortex (PC) of control (CTRL) and EV‐treated mice (EVs). Comparison between untreated and treated groups (CTRL vs EVs) used unpaired, two‐tailed Student's t test; *P < .05, **P < .01, and ***P < .001. Data are expressed as mean ± SD. AD, Alzheimer's disease; EVs, extracellular vesicles; pMSCs, preconditioned mesenchymal stem cells

FIGURE 7

MSC‐derived EVs increase dendritic spine density in 3xTg mice. A, Representative photomicrographs of Golgi‐Cox stained dendritic segments from hippocampal CA1 pyramidal neuron (CA1), entorhinal cortex (EC), and prefrontal cortex (PC) neurons, of control (CTRL) and EV‐treated mice (EVs). Scale bars = 5 μm. B, Histograms show the quantification of dendritic spine density (spines/10 μm) in the same areas. Animals treated with EVs (EVs) display a significative higher number of dendritic spines compared to the nontreated group (CTRL). *P < .05; **P < .01. EVs, extracellular vesicles; MSCs, mesenchymal stem cells