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

Abstract

Objectives: This study investigated whether exosomes from LPS pretreated bone marrow mesenchymal stem cells (LPS pre-MSCs) could prolong skin graft survival.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Allograft; Exosome; LPS preconditioning; Macrophage polarization; Mesenchymal stromal cells.

Fig. 1

Identification of Exo. A TEM images of Exo and LPS pre-Exo. B The diameter of exosomes were detected by NTA analysis (n = 3). C The enhanced secretion of exosomes detected by BCA assay after LPS pretreatment (n = 3). Data are presented as mean ± SD.*P < 0.05. D The markers (HSP90B1, CD63, TSG101, CD9) of exosomes analyzed by Western blot. (Full-length blots are presented in Additional file 1: Fig. S2). E Macrophage phagocytosis of Exo and LPS Pre-Exo was observed under a fluorescence microscope. F, G Representative image of exosome (PKH26⁺) Uptake by Macrophages (F4/80.⁺) with flow cytometry analysis. Values are presented as the mean ± SD (n = 3). *P < 0.05

Fig. 2

Effects of LPS pre-Exo on the survival of allogeneic skin grafts in vivo. A Schematic diagram of allogeneic skin grafting. The allografts Skin from BALB/c were transplanted into C57 recipient mice. B, C The necrosis of allograft skins was observed immediately after operation and at postoperative day 7, 14, 21, 23. Necrosis was observed at post-transplanted day 7 in the control group. The survival time of transplanted skins was prolonged by both rapamycin (1 mg/kg) and Exo (50 mg/kg), while LPS pre-Exo(50 mg/kg) was more effective (n = 5). B The percentage of allograft skin necrosis after transplantation (n = 5). D, E Representative H&E staining images of skin graft (day 7) in various groups. E Quantification of allograft rejection score in transplanted skin tissue in each group (n = 7). *P < 0.05, ****P < 0.0001

Fig. 3

Effects of LPS pre-Exo on the polarization of macrophages in transplanted skin. The allogeneic skins were harvested at postoperative day 7. A Representative images of immunohistochemical staining of F4/80, iNOS and CD206 of transplanted skin in various groups. B Representative immunofluorescence images of F4/80 and iNOS in the transplanted skin tissues were obtained from each group of recipient mice. C Representative images of immunofluorescence of F4/80 and CD206 in allografted skin tissues of recipient mice in each group. D, E The data of positive cells in immunofluorescence images are shown as mean ± SD with One-way ANOVA analyses (n = 5). ns > 0.05, * P < 0.05, ** P < 0.005. F4/80: green; iNOS: red;CD206: magenta; DAPI: blue. Scale bars: 50 μm

Fig. 4

Effects of LPS pre-Exo on macrophage polarization in allograft recipients. A Gating strategy for detection of spleen macrophages by flow cytometry. B, C Representative images of CD86⁺and CD206⁺ at postoperative day 7 in various groups of spleen macrophages analyzed by flow cytometry. Values are presented as the mean ± SD (n = 3). ns > 0.05, *P < 0.05, **P < 0.005, **** P < 0.0001. D, E Representative images of CD86⁺and CD206.⁺ at postoperative day 14 in various groups of spleen macrophages analyzed by flow cytometry. Values are presented as the mean ± SD (n = 3). ns > 0.05, *P < 0.05, **P < 0.005, **** P < 0.0001

Fig. 5

Effects of LPS pre-Exo on macrophage polarization in vitro. A, B BMDM cells were stimulated with LPS for 3 h, followed by Exo or LPS pre-Exo treatment for 24 h. IL-10 were added to the experimental groups for 24 h(n = 3). C, D The effect of the NF-κB inhibitor, BAY 11–8072 on the polarization of macrophages. B, D Representative images of CD86⁺ and CD206.⁺in different groups of BMDM cells analyzed by flow cytometry. Values are presented as the mean ± SD (n = 3). ns > 0.05, **P < 0.005, *** P < 0.0005, **** P < 0.0001

Fig. 6

Effects of LPS pre-Exo on protein expression levels in macrophages. A The concentrations of cytokine (IL-2, IL-6, IL-1β, IL-18) were detected by Elisa in the macrophage supernatant of each group.(n = 4). B, C BMDM cells were pretreated with LPS (100 ng/m) for 3 h before culturing with LPS pre-Exo for 24 h. Protein expressions of NF-κB, NLRP3, Procaspase-1 and ASC in macrophages of each group analyzed by western blot. (Full-length blots are presented in Additional file 1: Fig. S3) The effect of LPS pre-Exo on protein expression in the macrophages compared with bm-Exo (n = 3). D, E BMDM cells were pretreated with LPS (100 ng/m) for 3 h before culturing with LPS pre-Exo for 48 h. Protein expression of NF-κB, NLRP3, Procaspase-1 and ASC in the macrophages of each group analyzed by Western blot. (Full-length blots are presented in Additional file 1: Fig. S4) The effect of LPS pre-Exo on protein expression in the macrophages compared with bm-Exo. (n = 3). A, C, E Data are presented as mean ± SD. *P < 0.05, **P < 0.005, *** P < 0.0005, **** P < 0.0001

Fig. 7

The expression of MiRNA-222-3p in LPS pre-Exos was sharply increased and it can regulate macrophages through exosomal transfer. A The heatmap showed the differential expression of miRNAs between Bm-Exo group and LPS pre-Exo group (n = 3). (Fold change > 1.0; P < 0.05). B The volcano plot demonstrated the altered expression levels of miRNAs between Bm-Exos and LPS pre-Exos. C The KEGG analysis of target genes associated with differentially expressed miRNAs showed that 30 signaling pathways were significant enriched (P < 0.05). D Gene Ontology (GO) analysis was conducted on the target genes of differentially expressed miRNAs (P < 0.05). E The qPCR analysis demonstrated the differential expression level of miR-486, miR-206-3p, miR-409a-3p, miR-543-3p, miR-144, miR-222-3p, miR-1b, and miR-411-3p in Bm-Exo group and LPS pre-Exo group. Data are presented as mean ± SD (n = 3). *** P < 0.0005

Fig. 8

The simulation of signaling pathway by which Exo/LPS pre-Exo modulates the immune response to allografts. Step 1: LPS was recognized by macrophages and induced upregulation of NLRP3, IL-1β and IL-18 expression through the NF-κB signaling pathway. Step 2: After being endocytosed, LPS pre-Exo interacted with the NF-κB for preventing its activation and inhibited the expression of NLRP3 with suppressing the synthesis of NLRP3 inflammasome and subsequent production of IL-1β and IL-18. Step 3: In the subsequent immune response, following administration of LPS pre-Exo which induced alternation in the polarization of macrophages for expanding the ratio of M2 with CD86⁺ macrophages. Taken together, the functional outcome is to suppress the innate immune response