Extracellular vesicles derived from CD73 modified human umbilical cord mesenchymal stem cells ameliorate inflammation after spinal cord injury

Abstract

Background: Spinal cord injury (SCI) is an inflammatory condition, and excessive adenosine triphosphate (ATP) is released into the extracellular space, which can be catabolized into adenosine by CD73. Extracellular vesicles have been designed as nano drug carriers in many diseases. However, their impacts on delivery of CD73 after SCI are not yet known. We aimed to construct CD73 modified extracellular vesicles and explore the anti-inflammatory effects after SCI.

Methods: CD73 engineered extracellular vesicles (CD73+ hucMSC-EVs) were firstly established, which were derived from human umbilical cord mesenchymal stem cells (hucMSCs) transduced by lentiviral vectors to upregulate the expression of CD73. Effects of CD73+ hucMSC-EVs on hydrolyzing ATP into adenosine were detected. The polarization of M2/M1 was verified by immunofluorescence. Furthermore, A2aR and A_2bR inhibitors and A2bR knockdown cells were used to investigate the activated adenosine receptor. Biomarkers of microglia and levels of cAMP/PKA were also detected. Repetitively in vivo study, morphology staining, flow cytometry, cytokine analysis, and ELISA assay, were also applied for verifications.

Results: CD73+ hucMSC-EVs reduced concentration of ATP and promoted the level of adenosine. In vitro experiments, CD73+ hucMSC-EVs increased macrophages/microglia M2:M1 polarization, activated adenosine 2b receptor (A2bR), and then promoted cAMP/PKA signaling pathway. In mice using model of thoracic spinal cord contusion injury, CD73+ hucMSC-EVs improved the functional recovery after SCI through decreasing the content of ATP in cerebrospinal fluid and improving the polarization from M1 to M2 phenotype. Thus, the cascaded pro-inflammatory cytokines were downregulated, such as TNF-α, IL-1β, and IL-6, while the anti-inflammatory cytokines were upregulated, such as IL-10 and IL-4.

Conclusions: CD73+ hucMSC-EVs ameliorated inflammation after spinal cord injury by reducing extracellular ATP, promoting A2bR/cAMP/PKA pathway and M2/M1 polarization. CD73+ hucMSC-EVs might be promising nano drugs for clinical application in SCI therapy.

Keywords: CD73; Extracellular vesicles; Inflammation; Mesenchymal stem cell; Spinal cord injury.

Fig. 1

Characteristics of hucMSC-EVs and CD73 +hucMSC-EVs. A Representative images of hucMSCs and CD73+ hucMSCs are observed under microscopy. B Representative images of hucMSC-EVs and CD73+ hucMSC-EVs are observed under transmission electron microscopy (TEM). C Size distribution of extracellular vesicle is measured by nanoparticle tracking analysis (NTA, ZetaView, Particle Metrix Inc., German). D Western-blotting analysis of indicated proteins is detected, including the modified protein of CD73, exosomal positive biomarkers of CD9, CD63, CD81, TSG101, and ALIX, and exosomal negative biomarker of calnexin. (E and F) The ATP and AMP hydrolytic activities of the indicated dose of hucMSC-EVs or CD73+hucMSC-EVs with or without 100 μM APCP are measured in vitro (n = 3)

Fig. 2

CD73+ ucMSC-EVs promote M2 polarization and inhibit M1 polarization in vitro. A The effects of 30 ng/ml CD73, 30 μg/ml hucMSC-EVs and 30 μg/ml CD73+hucMSC-EVs with or without 100 μM APCP on the change of M1 subsets in BV2 cells treated with 1 μg/ml LPS are determined by immunofluorescences. B The effects of 30 ng/ml CD73, 30 μg/ml hucMSC-EVs and 30 μg/ml CD73+ hucMSC-EVs with or without 100 μM APCP on the change of M2 subsets in BV2 cells treated with 5 μg/ml IL-4 are determined by immunofluorescences. C and D Fluorescent intensities are normalized to fluorescent levels in LPS group or IL-4 group (*p < 0.05 versus LPS/IL-4, #p < 0.05 versus LPS/IL-4 + CD73+hucMSC-EVs, n = 3)

Fig. 3

CD73+ ucMSC-EVs augment M2/M1 polarization via A_2b adenosine receptor activation. A and B BV2 cells are treated with 1 μg/ml LPS in the presence or absence of 30 μg/ml CD73+hucMSC-EVs, and together with 1 μM MRS1706 (A_2bR inhibitor) or 1 μM SCH58261 (A_2aR inhibitor). A Intracellular cAMP levels are measured after 10 min stimulation (n = 5). B PKA protein expression in BV2 cells is detected (n = 3). (*p < 0.05 versus control, #p < 0.05 versus CD73+ hucMSC-EVs) C–F BV2 cells are treated with 1 μg/ml LPS or 5 μg/ml IL-4 in the presence or absence of 30 μg/ml CD73+hucMSC-EVs, and together with the indicated dose of MRS1706 or SCH58261. The iNOS/Arg-1 protein expression in BV2 cells was either investigated. G The mRNA relative expression level of M1 phase is detected, including TNF-α, IL-1β, iNOS, and CD86. H The mRNA relative expression level of M2 phase is detected, including arginase 1, IL-10, and CD206. (*p < 0.05 versus LPS/IL-4, #p < 0.05 versus LPS/IL-4 + CD73+hucMSC-EVs, n = 3)

Fig. 4

A_2bR knockdown cells and PKA inhibitor reverse the stimulatory effect of CD73+hucMSC-EVs. A–F BV2 cells are permanently transfected with scrambled shRNA or A_2bR shRNA. Cells are then treated with 1 μg/ml LPS or 5 μg/ml IL-4 in the presence or absence of 30 μg/ml CD73+ hucMSC-EVs. A and B Intracellular cAMP levels are measured after 10 min stimulation (n = 5). PKA protein expression is detected (n = 3). C and D The iNOS/Arg-1 protein expression in BV2 cells is investigated. E and F The mRNA relative expression level of M1/M2 phase is either detected. (*p < 0.05 versus shRNA scrambled/A_2bR + LPS, #p < 0.05 versus shRNA scrambled + CD73 + hucMSC-EVs). G and H The iNOS/Arg-1 protein expression in BV2 cells is investigated in presence or absence of 10 μM H-89 (PKA inhibitor)

Fig. 5

CD73+hucMSC-EVs ameliorate SCI and decrease intracellular cAMP levels in mice. (A and B) In vivo biodistribution of DiR-labeled CD73+hucMSC-EVs. Mice are analyzed at 24 h after A subcutaneous injection of EVs around injured spinal cord and B intraperitoneal injection of EVs. C BMS scores at different time-point after spinal cord injury. D BBB scores at different time-point after spinal cord injury. E cAMP levels from cerebrospinal fluid at different time-point after spinal cord injury. F Histological images (H&E staing), G Nissl staining and (H) TUNEL staining of longitudinal sections of injured spinal cords on day 21

Fig. 6

CD73+hucMSC-EVs regulate M1/M2 polarization of microglia in mice. A and B Changes of arginase-1 and iNOS are determined by immunofluorescence in different groups at × 5 magnification. C Fluorescent intensities are normalized to the sham group. (*p < 0.05 versus SCI group, #p < 0.05 versus SCI + CD73+hucMSC-EVs group, n = 5). D and E Representative dot spot of flow cytometry for microglia/macrophage subsets is shown. CD206 and CD86 are selected as biomarkers of M2 and M1 microglia, respectively. The data are calculated as M2:M1. (*p < 0.05 versus SCI group, #p < 0.05 versus SCI + CD73+hucMSC-EVs group, n = 5)

Fig. 7

CD73+hucMSC-EVs regulate the production of proinflammatory cytokines after SCI. A Cytokines of spinal samples from SCI mice are quantitative analyzed by Bio-Plex system 3d after SCI and hucMSC-EVs/CD73+hucMSC-EVs treatment. B–H The protein levels analyzed by Bio-Plex system, such as IL-1β, IL-6, TNF-α, IFN-γ, MCP-1 and MIP-1β are significantly decreased, while IL-4 and IL-10 are significantly increased. I–L The protein levels, including IL-1β, IL-6, TNF-α, and IL-10, are also analyzed by ELISA. (*p < 0.05 versus SCI group, #p < 0.05 versus SCI+CD73+hucMSC-EVs group, n = 5)

Fig. 8

Schematic diagram showing the effects of CD73+hucMSC-EVs on microglia