LPS-preconditioned mesenchymal stromal cells modify macrophage polarization for resolution of chronic inflammation via exosome-shuttled let-7b

摘要

Background Within the last few years, it has become evident that LPS-preconditioned mesenchymal stromal cells (LPS pre-MSCs) show enhanced paracrine effects, including increased trophic support and improved regenerative and repair properties. MSCs may release large amounts of exosomes for cell-to-cell communication and maintain a dynamic and homeostatic microenvironment for tissue repair. The present study assesses the therapeutic efficacy and mechanisms of LPS-preconditioned MSC-derived exosomes (LPS pre-Exo) for chronic inflammation and wound healing. Methods We extracted exosomes from the supernatant of LPS pre-MSCs using a gradient centrifugation method. In vitro, THP-1 cells were cultured with high glucose (HG, 30?mM) as an inflammatory model and treated with LPS pre-Exo for 48?h. The expression of inflammation-related cytokines was detected by real-time RT-PCR, and the distribution of macrophage subtype was measured by immunofluorescence. Next, the miRNA expression profiles of LPS pre-Exo were evaluated using miRNA microarray analysis. The molecular signaling pathway responsible for the regenerative potential was identified by western blotting. In vivo, we established a cutaneous wound model in streptozotocin-induced diabetic rats, and LPS pre-Exo were injected dispersively into the wound edge. The curative effects of LPS pre-Exo on inflammation and wound healing were observed and evaluated. Results LPS pre-Exo have a better ability than untreated MSC-derived exosomes (un-Exo) to modulate the balance of macrophages due to their upregulation of the expression of anti-inflammatory cytokines and promotion of M2 macrophage activation. Microarray analysis of LPS pre-Exo identified the unique expression of let-7b compared with un-Exo, and the let-7b/TLR4 pathway served as potential contributor to macrophage polarization and inflammatory ablation. Further investigation of the mechanisms that control let-7b expression demonstrated that a TLR4/NF-κB/STAT3/AKT regulatory signaling pathway plays a critical role in the regulation of macrophage plasticity. Knockdown of AKT in THP-1 cells similarly abolished the immunomodulatory effect of LPS pre-Exo. In vivo, LPS pre-Exo greatly alleviated inflammation and enhanced diabetic cutaneous wound healing. Conclusion LPS pre-Exo may have improved regulatory abilities for macrophage polarization and resolution of chronic inflammation by shuttling let-7b, and these exosomes carry much immunotherapeutic potential for wound healing.