Down-regulation miR-146a-5p in Schwann cell-derived exosomes induced macrophage M1 polarization by impairing the inhibition on TRAF6/NF-κB pathway after peripheral nerve injury.
Both Schwann cell-derived exosomes (SC-Exos) and macrophagic sub-phenotypes are closely related to the regeneration and repair after peripheral nerve injury (PNI). However, the crosstalk between them is less clear.
We aim to investigate the roles and underlying mechanisms of exosomes from normoxia-condition Schwann cell (Nor-SC-Exos) and from post-injury oxygen-glucose-deprivation-condition Schwann cell in regulating macrophagic sub-phenotypes and peripheral nerve injury repair.
Both Nor-SC-Exos and OGD-SC-Exos were extracted through ultracentrifugation, identified by transmission electron microscopy (TEM), Nanosight tracking analysis (NTA) and western blotting. High-throughput sequencing was performed to explore the differential expression of microRNAs in both SC-Exos. In vitro, RAW264.7 macrophage was treated with two types of SC-Exos, M1 macrophagic markers (IL-10, Arg-1, TGF-β1) and M2 macrophagic markers (IL-6, IL-1β, TNF-α) were detected by enzyme-linked Immunosorbent Assay (ELISA) or qRT-PCR, and the expression of CD206, iNOS were detected via cellular immunofluorescence (IF) to judge macrophage sub-phenotypes. Dorsal root ganglion neurons (DRGns) were co-cultured with RAW264.7 cells treated with Nor-SC-Exos and OGD-SC-Exos, respectively, to explore their effect on neuron growth. In vivo, we established a sciatic nerve crush injury rat model. Nor-SC-Exos and OGD-SC-Exos were locally injected into the injury site. The mRNA expression of M1 macrophagic markers (IL-10, Arg-1, TGF-β1) and M2 macrophagic markers (IL-6, IL-1β, TNF-α) were detected by qRT-PCR to determine the sub-phenotype of macrophages in the injury site. IF was used to detect the expression of MBP and NF200, reflecting the myelin sheath and axon regeneration, and sciatic nerve function index (SFI) was measured to evaluate function repair.
In vitro, Nor-SC-Exos promoted macrophage M2 polarization, increased anti-inflammation factors secretion, and facilitated axon elongation of DRGns. OGD-SC-Exos promoted M1 polarization, increased pro-inflammation factors secretion, and restrained axon elongation of DRGns. High-throughput sequencing and qRT-PCR results found that compared with Nor-SC-Exos, a shift from anti-inflammatory (pro-M2) to pro-inflammatory (pro-M1) of OGD-SC-Exos was closely related to the down-regulation of miR-146a-5p and its decreasing inhibition on TRAF6/NF-κB pathway after OGD injury. In vivo, we found Nor-SC-Exos and miR-146a-5p mimic promoted regeneration of myelin sheath and axon, and facilitated sciatic function repair via targeting TRAF6, while OGD-SC-Exos and miR-146a-5p inhibitor restrained them.
Our study confirmed that miR-146a-5p was significantly decreased in SC-Exos under the ischemia-hypoxic microenvironment of the injury site after PNI, which mediated its shift from promoting macrophage M2 polarization (anti-inflammation) to promoting M1 polarization (pro-inflammation), thereby limiting axonal regeneration and functional recovery.
Sun J
，Liao Z
，Li Z
，Li H
，Wu Z
，Chen C
，Wang H
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Exosomes from LPS-preconditioned bone marrow MSCs accelerated peripheral nerve regeneration via M2 macrophage polarization: Involvement of TSG-6/NF-κB/NLRP3 signaling pathway.
Lipopolysaccharide (LPS)-preconditioned mesenchymal stem cells (MSCs) possessed strong immunomodulatory and anti-inflammatory functions by secreting exosomes as major paracrine effectors. However, the specific effect of exosomes from LPS pre-MSCs (LPS pre-Exos) on peripheral nerve regeneration has yet to be documented. Here, we established a sciatic nerve injury model in rats and an inflammatory model in RAW264.7 cells to explore the potential mechanism between LPS pre-Exos and peripheral nerve repair. The local injection of LPS pre-Exos into the nerve injury site resulted in an accelerated functional recovery, axon regeneration and remyelination, and an enhanced M2 Macrophage polarization. Consistent with the data in vivo, LPS pre-Exos were able to shift the pro-inflammation macrophage into a pro-regeneration macrophage. Notably, TNF stimulated gene-6 (TSG-6) was found to be highly enriched in LPS pre-Exos. We obtained si TSG-6 Exo by the knockdown of TSG-6 in LPS pre-Exos to demonstrate the role of TSG-6 in macrophage polarization, and found that TSG-6 served as a critical mediator in LPS pre-Exos-induced regulatory effects through the inhibition of NF-ΚΒ and NOD-like receptor protein 3 (NLRP3). In conclusion, our findings suggested that LPS pre-Exos promoted macrophage polarization toward an M2 phenotype by shuttling TSG-6 to inactivate the NF-ΚΒ/NLRP3 signaling axis, and could provide a potential therapeutic avenue for peripheral nerve repair.
Li C
，Li X
，Shi Z
，Wu P
，Fu J
，Tang J
，Qing L
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Exosomes produced by adipose-derived stem cells inhibit schwann cells autophagy and promote the regeneration of the myelin sheath.
Peripheral nerve injury (PNI) is encountered relatively commonly in the clinic and often results in long-term functional deficits. Research to develop methods to improve regeneration following nerve injury is ongoing. Numerous studies have shown that adipose-derived stem cells (ADSCs) promote the regeneration of peripheral nerve injury; however, the mechanism is unclear. Autophagy, a highly conserved intracellular process responsible for maintaining cellular homeostasis, and Schwann cells (SCs), play important roles in regeneration after PNI. In the present study, we explored the effect and mechanism of exosomes produced by adipose-derived stem cells (ADSC-Exos) on autophagy of SCs in PNI, as well as their effect on the regeneration of the nerve myelin sheath. The levels of autophagy and the expression of karyopherin subunit alpha 2 (Kpna2) in SCs increased markedly after the sciatic nerve was injured in SCs (SNI-SCs). The enhanced autophagy and the upregulated Kpna2 in SNI-SCs were inhibited after treatment with ADSC-Exos in vivo and in vitro. The effect of ADSC-Exos on inhibiting SC autophagy was blocked by overexpression of Kpna2 in SNI-SCs. Using quantitative real-time reverse transcription PCR, ADSC-Exos were demonstrated to contain a large amount of miRNA-26b, which was predicted to regulate Kpna2 on the TargetScan website. The effect of ADSC-Exos on inhibiting SCs autophagy was blocked after the silencing of miRNA-26b. Moreover, ADSC-Exos promoted the regeneration of the myelin sheath by inhibiting SC autophagy in rat SNI models. In conclusion, our results indicated that ADSC-Exos promote the regeneration of the myelin sheath by moderately reducing autophagy of injured SCs via miRNA-26b downregulation of Kpna2.
Yin G
，Yu B
，Liu C
，Lin Y
，Xie Z
，Hu Y
，Lin H
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Exosomal OTULIN from M2 macrophages promotes the recovery of spinal cord injuries via stimulating Wnt/β-catenin pathway-mediated vascular regeneration.
Vascularization following spinal cord injury (SCI) provides trophic support for rebuilding up and maintaining the homeostasis of neuronal networks, and the promotion of angiogenesis is beneficial for functional recovery after SCI. M2 macrophages have been reported to exhibit powerful pro-angiogenic functions during tissue repair. Exosomes are important paracrine mediators of their parent cells and play critical roles in tissue regeneration. However, the role of M2 macrophage-derived exosomes (M2-Exos) in SCI is still largely unknown. In the present study, we determined that M2-Exos could augment the angiogenic activities of spinal cord microvascular endothelial cells (SCMECs) in vitro. Hydrogel-mediated sustained release of M2-Exos significantly promoted vascular regeneration and functional recovery in mice after SCI. Furthermore, proteomics analysis showed that ubiquitin thioesterase otulin (OTULIN) protein was highly enriched in M2-Exos. Functional assays demonstrated that OTULIN protein was required for the M2-Exos-induced pro-angiogenic effects in SCMECs, as well as positive effects on vascular regeneration, cell proliferation, and functional recovery in the mouse model of SCI. Mechanically, OTULIN from M2-Exos could activate the Wnt/β-catenin signaling by increasing the protein level of β-catenin via inhibiting its ubiquitination and trigger the expression of angiogenesis-related genes that are reported to be the downstream targets of Wnt/β-catenin signaling. Inhibition of the Wnt/β-catenin signaling by ICG001 markedly attenuated the pro-angiogenic activities of M2-Exos in vitro/vivo. Our findings indicate that M2-Exos positively modulate vascular regeneration and neurological functional recovery after SCI by activating Wnt/β-catenin signaling through the transfer of OTULIN protein. STATEMENT OF SIGNIFICANCE: M2 macrophages have been identified to promote vascular regeneration, cell proliferation and tissue growth after spinal cord injury (SCI), which is beneficial to the functional recovery. Exosomes are essential paracrine mediators involved in cell-to-cell communication and play important roles in tissue regeneration. In the present study, we revealed that M2 macrophages-derived exosomes (M2-Exos) could promote functional recovery post SCI by targeting angiogenesis. We demonstrated for the first time that OTULIN protein from M2-Exos mediated the angiogenic effects through activating Wnt/β-catenin signaling and triggering the expression of angiogenic-related genes in spinal cord microvascular endothelial cells (SCMECs). The hydrogel-M2-Exos sustained released system provides potential therapeutic clues of local cell-free interventions for the treatment of SCI.
Luo Z
，Peng W
，Xu Y
，Xie Y
，Liu Y
，Lu H
，Cao Y
，Hu J
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ResearchGate
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