Endothelial progenitor cell-derived exosomes promote anti-inflammatory macrophages via SOCS3/JAK2/STAT3 axis and improve the outcome of spinal cord injury.

Macrophage in the spinal cord injury (SCI) area imparts a chronic pro-inflammation effect that challenges the recovery of SCI. Previously, endothelial progenitor cell-produced exosomes (EPC-EXOs) have been noticed to facilitate revascularization and inflammation control after SCI. However, their effects on macrophage polarization remained unclear. This study aimed to investigate the EPC-EXOs' role in macrophage polarization and reveal its underlying mechanism. We extracted the macrophages and EPC from the bone marrow suspension of C57BL/L mice by centrifugation. After cell identification, the EPC-EXOs were collected by ultra-high-speed centrifugation and exosome extraction kits and identified by transmission electron microscopy and nanoparticle tracking analysis. Then, macrophages were cultured with EPC-EXOs in different concentrations. We labeled the exosome to confirm its internalization by macrophage and detected the macrophage polarization marker level both in vitro and in vivo. We further estimated EPC-EXOs' protective effects on SCI by mice spinal cord tissue H&E staining and motor behavior evaluation. Finally, we performed RT-qPCR to identify the upregulated miRNA in EPC-EXOs and manipulate its expression to estimate its role in macrophage polarization, SOCS3/JAK2/STAT3 pathway activation, and motor behavior improvement. We found that EPC-EXOs decreased the macrophages' pro-inflammatory marker expression and increased their anti-inflammatory marker expression on the 7 and 14 days after SCI. The spinal cord H&E staining results showed that EPC-EXOs raised the tissue-sparing area rate significantly after 28 days of SCI and the motor behavior evaluation indicated an increased BMS score and motor-evoked potential by EPC-EXOs treatment after SCI. The RT-qPCR assay identified that miR-222-3P upregulated in EPC-EXOs and its miRNA-mimic also decreased the pro-inflammatory macrophages and increased the anti-inflammatory macrophages. Additionally, miR-222-3P mimic activated the SOCS3/JAK2/STAT3 pathway, and SOCS3/JAK2/STAT3 pathway inhibition blocked miR-2223P's effects on macrophage polarization and mouse motor behavior. Comprehensively, we discovered that EPC-EXOs-derived miR-222-3p affected macrophage polarization via SOCS3/JAK2/STAT3 pathway and promoted mouse functional repair after SCI, which reveals EPC-EXOs' role in modulation of macrophage phenotype and will provide a novel interventional strategy to induce post-SCI recovery.

Yuan F ，Peng W ，Yang Y ，Xu J ，Liu Y ，Xie Y ，Huang T ，Shi C ，Ding Y ，Li C ，Qin T ，Xie S ，Zhu F ，Lu H ，Huang J ，Hu J ... -  《Journal of Neuroinflammation》

IPSC-NSCs-derived exosomal let-7b-5p improves motor function after spinal cord Injury by modulating microglial/macrophage pyroptosis.

Following spinal cord injury (SCI), the inflammatory storm initiated by microglia/macrophages poses a significant impediment to the recovery process. Exosomes play a crucial role in the transport of miRNAs, facilitating essential cellular communication through the transfer of genetic material. However, the miRNAs from iPSC-NSCs-Exos and their potential mechanisms leading to repair after SCI remain unclear. This study aims to explore the role of iPSC-NSCs-Exos in microglia/macrophage pyroptosis and reveal their potential mechanisms. iPSC-NSCs-Exos were characterized and identified using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. A mouse SCI model and a series of in vivo and in vitro experiments were conducted to investigate the therapeutic effects of iPSC-NSCs-Exos. Subsequently, miRNA microarray analysis and rescue experiments were performed to confirm the role of miRNAs in iPSC-NSCs-Exos in SCI. Mechanistic studies were carried out using Western blot, luciferase activity assays, and RNA-ChIP. Our findings revealed that iPSC-NSCs-derived exosomes inhibited microglia/macrophage pyroptosis at 7 days post-SCI, maintaining myelin integrity and promoting axonal growth, ultimately improving mice motor function. The miRNA microarray showed let-7b-5p to be highly enriched in iPSC-NSCs-Exos, and LRIG3 was identified as the target gene of let-7b-5p. Through a series of rescue experiments, we uncovered the connection between iPSC-NSCs and microglia/macrophages, revealing a novel target for treating SCI. In conclusion, we discovered that iPSC-NSCs-derived exosomes can package and deliver let-7b-5p, regulating the expression of LRIG3 to ameliorate microglia/macrophage pyroptosis and enhance motor function in mice after SCI. This highlights the potential of combined therapy with iPSC-NSCs-Exos and let-7b-5p in promoting functional recovery and limiting inflammation following SCI.

Liu J ，Kong G ，Lu C ，Wang J ，Li W ，Lv Z ，Tong J ，Liu Y ，Xiong W ，Li H ，Fan J ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Analysis of miR-203a-3p/SOCS3-mediated induction of M2 macrophage polarization to promote diabetic wound healing based on epidermal stem cell-derived exosomes.

The development of therapeutic strategies to improve wound healing in individual diabetic patients remains challenging. Stem cell-derived exosomes represent a promising nanomaterial, and microRNAs (miRNAs) can be isolated from them. It is important to identify the potential therapeutic role of specific miRNAs, given that miRNAs can play a therapeutic role. qPCR, flow cytometry, and western blotting were used to verify the effect of epidermal stem cell-derived exosomes (EpiSC-EXOs) on M2 macrophage polarization and SOCS3 expression. By screening key miRNAs targeting SOCS3 in EpiSC-EXOs by high-throughput sequencing, we verified the mechanism in vitro. Finally, an animal model was used to verify the effect of promoting healing. The use of EpiSC-EXOs reduced SOCS3 expression and promoted M2 macrophage polarization. The abundant miR-203a-3p present in the EpiSC-EXOs specifically bound to SOCS3 and activated the JAK2/STAT3 signaling pathway to induce M2 macrophage polarization. Treatment of the db/db mouse wound model with miR-203a-3p agomir exerted a pro-healing effect. Our results demonstrated that the abundant miR-203a-3p present in EpiSC-EXOs can promote M2 macrophage polarization by downregulating SOCS3 and suggested that diabetic wounds can obtain better healing effects through this mechanism.

Yang H ，Xu H ，Wang Z ，Li X ，Wang P ，Cao X ，Xu Z ，Lv D ，Rong Y ，Chen M ，Tang B ，Hu Z ，Deng W ，Zhu J ... -  《-》

Peripheral Macrophage-derived Exosomes promote repair after Spinal Cord Injury by inducing Local Anti-inflammatory type Microglial Polarization via Increasing Autophagy.

Treatment for spinal cord injury (SCI) remains a challenge worldwide, and inflammation is a major cause of secondary injury after SCI. Peripheral macrophages (PMs) have been verified as a key factor that exert anti-inflammatory effects after SCI, but the mechanism is unidentified. As local macrophages, microglia also exert significant effects after SCI, especially polarization. Exosomes show source cell-like biological functions to target cells and have been the subject of much research in recent years. Thus, we hypothesized the PM-derived exosomes (PM-Exos) play an important role in signal transmission with local microglia and can be used therapeutic agents for SCI in a series of in vivo and in vitro studies. For the in vivo experiment, three groups of Sprague-Dawley (SD) rats subjected to spinal cord contusion injury were injected with 200 µg/ml PM-Exos, 20 µg/ml PM-Exos or PBS via the tail vein. Recovery of the rats and of spinal cord function were observed. In vitro, we investigated the potential anti-inflammatory mechanism of PM-Exos and evaluated microglial autophagy, anti-inflammatory type microglia polarization and the upstream signaling pathway. The results showed that spinal cord function and recovery were better in the PM-Exo groups than the control group. In the in vitro study, microglial autophagy levels and the expression of anti-inflammatory type microglia were higher in the experimental groups than the control group. Moreover, the expression of proteins related to the PI3K/AKT/mTOR autophagic signaling pathway was suppressed in the PM-Exo groups. PM-Exos have a beneficial effect in SCI, and activation of microglial autophagy via inhibition of the PI3K/AKT/mTOR signaling pathway, enhancing the polarization of anti-inflammatory type microglia, that may play a major role in the anti-inflammatory process.

Zhang B ，Lin F ，Dong J ，Liu J ，Ding Z ，Xu J ... -  《International Journal of Biological Sciences》

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 ... -  《-》