M1 macrophage-derived exosomes promote intervertebral disc degeneration by enhancing nucleus pulposus cell senescence through LCN2/NF-κB signaling axis

Abstract

Intervertebral disc degeneration (IVDD) is the primary factor contributing to low back pain (LBP). Unlike elderly patients, many young IVDD patients usually have a history of trauma or long-term abnormal stress, which may lead to local inflammatory reaction causing by immune cells, and ultimately accelerates degeneration. Research has shown the significance of M1-type macrophages in IVDD; nevertheless, the precise mechanism and the route by which it influences the function of nucleus pulposus cell (NPC) remain unknown. Utilizing a rat acupuncture IVDD model and an NPC degeneration model induced by lipopolysaccharide (LPS), we investigated the function of M1 macrophage-derived exosomes (M1-Exos) in IVDD both in vivo and in vitro in this study. We found that M1-Exos enhanced LPS-induced NPC senescence, increased the number of SA-β-gal-positive cells, blocked the cell cycle, and promoted the activation of P21 and P53. M1-Exos derived from supernatant pretreated with the exosome inhibitor GW4869 reversed this result in vivo and in vitro. RNA-seq showed that Lipocalin2 (LCN2) was enriched in M1-Exos and targeted the NF-κB pathway. The quantity of SA-β-gal-positive cells was significantly reduced with the inhibition of LCN2, and the expression of P21 and P53 in NPCs was decreased. The same results were obtained in the acupuncture-induced IVDD model. In addition, inhibition of LCN2 promotes the expression of type II collagen (Col-2) and inhibits the expression of matrix metalloproteinase 13 (MMP13), thereby restoring the equilibrium of metabolism inside the extracellular matrix (ECM) in vitro and in vivo. In addition, the NF-κB pathway is crucial for regulating M1-Exo-mediated NPC senescence. After the addition of M1-Exos to LPS-treated NPCs, p-p65 activity was significantly activated, while si-LCN2 treatment significantly inhibited p-p65 activity. Therefore, this paper demonstrates that M1 macrophage-derived exosomes have the ability to deliver LCN2, which activates the NF-κB signaling pathway, and exacerbates IVDD by accelerating NPC senescence. This may shed new light on the mechanism of IVDD and bring a fresh approach to IVDD therapy.

Keywords: Cellular senescence; Exosome; Intervertebral disc degeneration; LCN2; Macrophage.

Fig. 1

M1-dominated macrophage infiltration was present in IVDD. A Single cell expression matrix reduction of 8 degenerated intervertebral disc tissues. B Heat maps of specific high-expression genes in the top 5 of 15 cell subsets. C Relative expression levels of intervertebral disc cell markers in single cell expression matrix. D The relative expression levels of immune cell markers and macrophage markers in single cell expression matrix. E Final annotation results of single cell expression matrix. F UMAP dimension reduction results of macrophage reclassification. G The relative expression levels of M1 and M2 macrophage markers in the macrophage expression matrix

Fig. 2

Extraction and identification of M1 macrophage-derived exosomes and their effects on IVDD. A Representative TEM images of M1-Exos and M0-Exos. Scale bars, 200 nm. B Representative chart of exosome size distribution measured by dynamic light scattering method. C WB analysis of Exos markers CD63 and TSG101 in the supernatant (Sup) and exosomes (Exos). D, E The expression and quantification of IVDD-specific proteins. F Representative fluorescence images of IVDD-specific markers. Scale bars, 50 μm. G The expression of IVDD-specific genes. ns: no significance, *P < 0.05, **P < 0.01

Fig. 3

Targeted regulation of M1-Exos can affect the senescence of NPCs. A Representative fluorescence images of senescence markers after addition of M1-Exos. Scale bars, 50 μm. B SA-β-gal staining and quantitative analysis results after addition of M1-Exos. Scale bars, 100 μm. C The expression and quantification of senescence proteins after addition of M1-Exos. D Representative fluorescent images of senescence markers after GW4869 intervention. Scale bars, 50 μm. E SA-β-gal staining and quantitative analysis results after GW4869 intervention. Scale bars, 100 μm. F The expression and quantification of senescence proteins after GW4869 intervention. ns: no significance, *P < 0.05, **P < 0.01

Fig. 4

Inhibition of M1 macrophage-derived exosomes can delay the senescence of nucleus pulposus cells in vivo. A X-ray examination of rat caudal at 1 week after surgery. B T2-weighted MRI scans of rat caudal at 1 week after surgery. C H&E and safranin-O staining of rat caudal IVD and histological grade. Scale bars, 1000 μm. D Representative fluorescent images of senescence markers after GW4869 intervention. Scale bars, 50 μm. E Representative fluorescent images of IVDD-specific markers after GW4869 intervention. Scale bars, 50 μm. ns: no significance, *P < 0.05, **P < 0.01

Fig. 5

M1-Exos aggravates IVDD via LCN2. A Volcano plots of all differentially expressed genes (> 1.3 times) after M1-Exos and M0-Exos intervention with NPCs. B Heatmaps of all differentially expressed genes (> 1.3 times) after M1-Exos and M0-Exos intervention with NPCs. C The enriched KEGG pathways. D Representative fluorescent images of LCN2. Scale bars, 50 μm. E The expression and quantification of LCN2. F The mRNA levels of LCN2. ns: no significance, *P < 0.05, **P < 0.01

Fig. 6

M1-Exos transport LCN2 promotes senescence of NPCs through NF-κB signaling pathway. A Representative fluorescence images of senescence markers after transfection with si-LCN2. Scale bars, 50 μm. B SA-β-gal staining and quantitative analysis results after transfection with si-LCN2. Scale bars, 100 μm. C The expression and quantification of senescence proteins after transfection with si-LCN2. D Representative fluorescent images of LCN2 after transfection with si-LCN2. Scale bars, 50 μm. E The expression and quantification of LCN2. F Representative fluorescence images of IVDD-specific markers after transfection with si-LCN2. Scale bars, 50 μm. G The expression and quantification of IVDD-specific proteins after transfection with si-LCN2. H The expression and quantification of NF-κB signaling pathway proteins after transfection with si-LCN2. ns: no significance, *P < 0.05, **P < 0.01

Fig. 7

Inhibition of LCN2 can delay the senescence of nucleus pulposus cells in vivo. A X-ray examination of rat caudal at 1 week after surgery. B T2-weighted MRI scans of rat caudal at 1 week after surgery. C H&E and safranin-O staining of rat caudal IVD and histological grade. Scale bars, 1000 μm. D Representative fluorescent images of senescence markers after si-LCN2 intervention. Scale bars, 50 μm. E Representative fluorescent images of LCN2 after si-LCN2 intervention. Scale bars, 50 μm. F Representative fluorescent images of IVDD-specific markers after si-LCN2 intervention. Scale bars, 50 μm. ns: no significance, *P < 0.05, **P < 0.01

Fig. 8

Schematic illustration of the mechanism related to LCN2 delivery by M1 macrophage-derived exosomes to promote nucleus pulposus cell senescence