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 ... -  《-》

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 ... -  《-》

OTULIN of exosomes derived from Schwann cells promotes peripheral nerve injury repair by regulating macrophage polarization via deubiquitination of ERBB2.

Wang Y ，Wan Y ，Zhou X ，Zhang P ，Zhang J ... -  《-》

Exosomes derived from LPS-preconditioned bone marrow-derived MSC modulate macrophage plasticity to promote allograft survival via the NF-κB/NLRP3 signaling pathway.

This study investigated whether exosomes from LPS pretreated bone marrow mesenchymal stem cells (LPS pre-MSCs) could prolong skin graft survival. The exosomes were isolated from the supernatant of MSCs pretreated with LPS. LPS pre-Exo and rapamycin were injected via the tail vein into C57BL/6 mice allografted with BALB/c skin; graft survival was observed and evaluated. The accumulation and polarization of macrophages were examined by immunohistochemistry. The differentiation of macrophages in the spleen was analyzed by flow cytometry. For in vitro, an inflammatory model was established. Specifically, bone marrow-derived macrophages (BMDMs) were isolated and cultured with LPS (100 ng/ml) for 3 h, and were further treated with LPS pre-Exo for 24 h or 48 h. The molecular signaling pathway responsible for modulating inflammation was examined by Western blotting. The expressions of downstream inflammatory cytokines were determined by Elisa, and the polarization of macrophages was analyzed by flow cytometry. LPS pre-Exo could better ablate inflammation compared to untreated MSC-derived exosomes (BM-Exo). These loaded factors inhibited the expressions of inflammatory factors via a negative feedback mechanism. In vivo, LPS pre-Exo significantly attenuated inflammatory infiltration, thus improving the survival of allogeneic skin graft. Flow cytometric analysis of BMDMs showed that LPS pre-Exo were involved in the regulation of macrophage polarization and immune homeostasis during inflammation. Further investigation revealed that the NF-κB/NLRP3/procaspase-1/IL-1β signaling pathway played a key role in LPS pre-Exo-mediated regulation of macrophage polarization. Inhibiting NF-κB in BMDMs could abolish the LPS-induced activation of inflammatory pathways and the polarization of M1 macrophages while increasing the proportion of M2 cells. LPS pre-Exo are able to switch the polarization of macrophages and enhance the resolution of inflammation. This type of exosomes provides an improved immunotherapeutic potential in prolonging graft survival.

Zhang P ，Wu P ，Khan UZ ，Zhou Z ，Sui X ，Li C ，Dong K ，Liu Y ，Qing L ，Tang J ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Mesenchymal Stem Cell-Derived Exosomes Promote Recovery of The Facial Nerve Injury through Regulating Macrophage M1 and M2 Polarization by Targeting the P38 MAPK/NF-Κb Pathway.

Xue R ，Xie M ，Wu Z ，Wang S ，Zhang Y ，Han Z ，Li C ，Tang Q ，Wang L ，Li D ，Wang S ，Yang H ，Zhao RC ... -  《Aging and Disease》