Exosomes derived from umbilical cord mesenchymal stem cells reduce microglia-mediated neuroinflammation in perinatal brain injury

Abstract

Background: Preterm newborns are at high risk of developing neurodevelopmental deficits caused by neuroinflammation leading to perinatal brain injury. Human Wharton's jelly mesenchymal stem cells (hWJ-MSC) derived from the umbilical cord have been suggested to reduce neuroinflammation, in part through the release of extracellular vesicle-like exosomes. Here, we studied whether exosomes derived from hWJ-MSC have anti-inflammatory effects on microglia-mediated neuroinflammation in perinatal brain injury.

Methods: Using ultracentrifugation, we isolated exosomes from hWJ-MSC culture supernatants. In an in vitro model of neuroinflammation, we stimulated immortalized BV-2 microglia and primary mixed glial cells with lipopolysaccharide (LPS) in the presence or absence of exosomes. In vivo, we introduced brain damage in 3-day-old rat pups and treated them intranasally with hWJ-MSC-derived exosomes.

Results: hWJ-MSC-derived exosomes dampened the LPS-induced expression of inflammation-related genes by BV-2 microglia and primary mixed glial cells. The secretion of pro-inflammatory cytokines by LPS-stimulated primary mixed glial was inhibited by exosomes as well. Exosomes interfered within the Toll-like receptor 4 signaling of BV-2 microglia, as they prevented the degradation of the NFκB inhibitor IκBα and the phosphorylation of molecules of the mitogen-activated protein kinase family in response to LPS stimulation. Finally, intranasally administered exosomes reached the brain and reduced microglia-mediated neuroinflammation in rats with perinatal brain injury.

Conclusions: Our data suggest that the administration of hWJ-MSC-derived exosomes represents a promising therapy to prevent and treat perinatal brain injury.

Keywords: BV-2; Exosomes; Extracellular vesicles; Hypoxia-ischemia; Intranasal; Mesenchymal stem cells; Microglia; Neuroinflammation; Perinatal brain damage; Preterm birth; Umbilical cord; White matter injury.

Fig. 1

Rat model of perinatal brain injury. a Schematic representation of the experimental outline. Perinatal brain injury was induced in postnatal day 2 (P2) rat pups by i.p. injection of LPS, followed by left common carotid artery cauterization 2 h later and exposure to hypoxia for 65 min. Hyaluronidase was administered into both nostrils 30 min before i.n. exosome or PBS application and the latter was done immediately before the cauterization of the left common carotid. b Detailed overview of the three experimental groups. ELISA enzyme-linked immunosorbent assay, Exo exosomes, i.p. intraperitoneal, i.n. intranasal, n number of animals, P2 postnatal day 2, RT-PCR reverse transcription polymerase chain reaction

Fig. 2

Characterization of human Wharton’s jelly mesenchymal stem cells (hWJ-MSC) and hWJ-MSC-derived exosomes. a Representative bright field microscopy image of hWJ-MSC. b Representative flow cytometry histograms of hWJ-MSC at passage 6. c Representative electron microscopy image of hWJ-MSC-derived exosomes (d) revealing a median diameter of 43 nm. e Representative Exo-Check antibody array of isolated exosomes

Fig. 3

Anti-inflammatory effects of internalized human Wharton’s jelly mesenchymal stem cell-derived exosomes on BV-2 cells. a Representative confocal image after the co-culture of exosomes with BV-2 cells for 6 h. Exosomes were labeled with the fluorescent membrane dye PKH26 (red). BV-2 cells were stained with β-tubulin (green), and their nuclei were counterstained with 4′,6-diamidino-2′-phenylindole-dihydrochloride (DAPI) (blue). b PKH26-labeled exosomes (1 μg/ml) were co-cultured with BV-2 cells for 15 min, 30 min, 3 h, 6 h, or 8 h and analyzed by flow cytometry. c Quantification of Tnf, Il6, and Il1b mRNA expressed by BV-2 cells either left untreated or stimulated with LPS and/or co-incubated with 1 μg/ml exosomes for 6 h. d Quantification of TNFα and IL-6 secretion by BV-2 cells either left untreated or stimulated with LPS and/or co-incubated with 1 μg/ml exosomes for 6 or 24 h. Error bars illustrate mean ± 95% CI of n = 3 (b), n = 10 (c), and n = 7 (d) biological replicates. *p < 0.05 **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. non-significant, CI confidence interval, Exo exosomes, Il1b/Il-1β interleukin 1 beta, Il6/IL-6 interleukin-6, LPS lipopolysaccharide, Tnf/TNFα tumor necrosis factor α

Fig. 4

The anti-inflammatory effects of human Wharton’s jelly mesenchymal stem cell-derived exosomes are mediated via the Toll-like receptor 4 pathway. BV-2 cells were either left untreated or stimulated with LPS and/or co-incubated with exosomes for 15, 30 or 60 min. Western blot analysis of the expression of a NFκB inhibitor IκBα and the phosphorylation of MAPK family molecules b ERK, c JNK, and d p38. Error bars illustrate mean ± 95% CI of n = 4 biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 CI confidence interval, Exo exosome, p phosphorylation, IκBα nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha MAPK mitogen-activated protein kinase, LPS lipopolysaccharide, NFκB nuclear factor kappa-light-chain-enhancer of activated B cells

Fig. 5

Anti-inflammatory effects of internalized human Wharton’s jelly mesenchymal stem cell-derived exosomes on primary mixed glial cells. a Representative confocal image after the co-culture of exosomes with primary mixed glial cells for 6 h. Exosomes were labeled with the fluorescent membrane dye PKH26 (red). Primary mixed glial cells were stained with β-tubulin (green) and their nuclei were counterstained with 4′,6-diamidino-2′-phenylindole-dihydrochloride (DAPI) (blue). b PKH26-labeled exosomes (1 μg/ml) were co-cultured with mixed glial cells for 15 min, 30 min, 3 h, 6 h, or 8 h and analyzed by flow cytometry. c Quantification of Tnf, Il6, and Il1b mRNA expressed by mixed glial cells either left untreated or stimulated with LPS and/or co-incubated with exosomes for 6 h. d Quantification of TNFα and IL-1β secretion by mixed glial cells either left untreated or stimulated with LPS and/or co-incubated with exosomes for 6 or 24 h. Error bars illustrate mean ± 95% CI of n = 3 (b), n = 9 (c), and n = 6 (d) biological replicates. *p < 0.05, ****p < 0.0001, n.s. non-significant, CI confidence interval, Exo exosomes, Il1b/Il-1β interleukin 1 beta, Il6/IL-6 interleukin-6, LPS lipopolysaccharide, Tnf/TNFα tumor necrosis factor α

Fig. 6

Anti-inflammatory effects of human Wharton’s jelly mesenchymal stem cell-derived exosomes in perinatal brain injury. a Quantification of Tnf, Il6, Il1b, Cxcl10, Il18, and Cxcl2 mRNA expressed in the brain of healthy rats and of rats 24 h after injury with and without exosome treatment. b Quantification of TNFα and IL-1β expression in the brain parenchyma of healthy rats and of rats 24 h after brain injury with and without exosome treatment. Error bars illustrate mean ± 95% CI of at least four different animals. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001, n.s. non-significant, CI confidence interval, Cxcl2 C-X-C motif chemokine ligand 2, Cxcl10 C-X-C motif chemokine ligand 10, Exo exosomes, Il1b/Il-1β interleukin 1 beta, Il18 interleukin 18, Il6 interleukin-6, Tnf/TNFα tumor necrosis factor alpha

Fig. 7

Prevention of microgliosis by human Wharton’s jelly mesenchymal stem cell-derived exosomes in perinatal brain injury. a Representative chromogenic immunohistochemistry images and quantification of Iba1⁺ cells in the corpus callosum of healthy rats and of rats 24 h post brain injury with and without exosome treatment. b Representative fluorescent immunohistochemistry images and quantification of CD68⁺ cells in the corpus callosum of healthy rats and of rats 24 h after brain injury with and without exosome treatment. Bottom row images are higher magnifications (40x) of the top row images (10x). Error bars illustrate mean ± 95% CI of at least three different animals. *p < 0.05, ***p < 0.001, CC corpus callosum, CI confidence interval, CD68 cluster of differentiation 68, DAPI 4′,6-diamidino-2′-phenylindole-dihydrochloride, Exo exosomes, Iba1 ionized calcium-binding adaptor molecule