Degenerated nucleus pulposus cells derived exosome carrying miR-27a-3p aggravates intervertebral disc degeneration by inducing M1 polarization of macrophages

Abstract

Background: Intervertebral disc degeneration (IVDD) is a major contributor to spinal disorders. Previous studies have indicated that the infiltration of immunocytes, specifically macrophages, plays a crucial role in the advancement of IVDD. Exosomes (exo) are believed to play a significant role in intercellular communication. This study aims to investigate the role of exosomes derived from degenerated nucleus pulposus (dNPc) in the process of macrophages M1 polarization.

Methods: Nucleus pulposus (NP) tissue and nucleus pulposus cells (NPc) were collected from patients with intervertebral disc degeneration (IVDD) and idiopathic scoliosis. Immunohistochemistry analysis was performed to determine the number of M1 macrophages in NP tissue. Subsequently, exosomes derived from degenerated NP cells (dNPc-exo) and non-degenerated NP cells (nNPc-exo) were collected and co-cultured with M0 macrophages, which were induced from THP-1 cells. The M1 phenotype was assessed using western blot, flow cytometry, immunofluorescence staining, and qRT-PCR. RNA-sequencing analysis was conducted to examine the expression levels of microRNAs in the dNPc-exo and nNPc-exo groups, and qRT-PCR was performed to investigate the effect pf different microRNA to induce macrophage polarization. Furthermore, western blot and qRT-PCR were employed to demonstrate the regulatory effect of microRNAs carried by dNPc-exo on downstream target signaling pathways in macrophages. Finally, an animal model of IVDD was utilized to investigate the impact of dNPc-exo on inducing M1 polarization of macrophages and its role in the IVDD process.

Results: In this study, we observed an increase in the number of M1 macrophages as the intervertebral disc (IVD) degraded. Additionally, we discovered that dNPc releases exosomes (dNPc-exo) could promote the polarization of macrophages towards the M1 phenotype. Notably, through RNA-sequencing analysis of dNPc-exo and nNPc-exo groups, we identified miR-27a-3p as a highly expressed miRNA in the dNPc-exo group, which significantly influences the induction of M1 polarization of macrophages. And then, we discovered that dNPc-exo has the ability to transport miR-27a-3p and target the PPARγ/NFκB/PI3K/AKT signaling pathway, thereby influencing the M1 polarization of macrophages. We conducted experiments using rat model of IVDD and observed that the exosomes carrying miR-27a-3p actually induced the M1 polarization of macrophages and exacerbated the degradation of IVD.

Conclusion: In conclusion, our findings highlight the significant role of dNPc-exo in IVDD process and provide a basis for further investigation into the mechanism of IVDD and the potential of exosome-based therapy.

Keywords: Exosome; Inflammatory regulation; Intervertebral disc degeneration; Macrophages; Nucleus pulposus.

Fig. 1

NP cells and NPc-exo isolation and characterization. (A) Representative magnetic resonance images (MRI) of idiopathic scoliosis and degenerated IVD. (B) Representative confocal images for F4/80 (green) and iNOS (M1 macrophage marker) (red). The nucleus was counterstained with DAPI (blue). (C) Quantitative analysis showed the number of iNOS positive cells were significantly increased in IVDD group. (D) Expression of Aggrecan and Col2A1 was shown in the two groups by immunohistochemistry. (E) Quantitative analysis showed that the expression of Aggrecan and Col2A1 were significantly decreased in IVDD group. (F) Representative images of the percentage of CD86 (M1 macrophage marker) positive cells in NP tissues from different IVD grade groups detected by flow cytometry analysis. (G) Schematic illustration of NPc-exo isolation using differential centrifugation. (H) Typical image of NPc-exo was captured by transmission electron microscopy (TEM). Scale bar = 200 nm. (I) Particle size distribution of NPc-exo was examined by nanoparticle trafficking analysis (NTA). (J) Western blot analysis of CD9, CD63 and TSG101 markers. (K) The M0 macrophages were incubated with PKH26-labeled dNPc-exo and observed. The red fluorescence proved cellular internalization of dNPc-exo into M0 macrophages. Scale bar = 10 μm. The data are expressed as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

dNPc-exo induces M1 polarization of macrophages via miR-27a-3p. (A) Representative images of immunofluorescence staining of F4/80 and iNOS in M0 macrophages after dNPc-exo and nNPc-exo incubation. Scale bar = 100 μm. (B) Quantitative analysis of the percentage of iNOS⁺ macrophages in immunofluorescence staining. (C) RT-qPCR analysis of iNOS gene expression in macrophages with nNPc-exo and dNPc-exo treatment. (D) Western blot analysis of iNOS expression in macrophages with nNPc-exo and dNPc-exo treatment. (E) Quantitative analysis of iNOS expression in western blot. (F) Flow cytometry analysis of the macrophages. (G) Quantitative analysis of the iNOS⁺ macrophages percentage in flow cytometry. (H) Heatmap of nNPc-exo group and dNPc-exo group. (I) Cluster analysis of RNA-Seq between nNPc-exo group and dNPc-exo group. (J) A volcanic map of the differentially expressed genes between nNPc-exo group and dNPc-exo group. (K) Pathway enrichment factor map of differential genes (Top20). (L) RT-qPCR analysis of seven miRNAs that highly expressed in dNPc-exo group. (M) RT-qPCR analysis of iNOS gene expression in M0 macrophages with different miRNAs mimic treatment. The data are expressed as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant difference

Fig. 3

dNPc-exo induces M1 polarization of macrophages via miR-27a-3p. (A) Representative images of immunofluorescence staining of Cy3-labelled miR-27a-3p in M0 macrophages. Scale bar = 10 μm. (B) Representative images of immunofluorescence staining of macrophages after cultured with dNPc-exo, miR-27a-3p mimic and miR-27a-3p inhibitor (blue, nucleus; green, F4/80; red, iNOS). Scale bar = 100 μm. (C) Quantitative analysis of the iNOS⁺ macrophages percentage in macrophages after cultured with dNPc-exo, miR-27a-3p mimic and miR-27a-3p inhibitor. (D) RT-qPCR analysis of the miR-27a-3p expression in macrophages of control, nNPc-exo and dNPc-exo group. (E) Western blot analysis of iNOS expression in M0 macrophages after treatment of dNPc-exo, miR-27a-3p mimic and miR-27a-3p inhibitor. (F) Quantitative analysis of iNOS expression in western blot of M0 macrophages after treatment of dNPc-exo, miR-27a-3p mimic and miR-27a-3p inhibitor. The data are expressed as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant difference

Fig. 4

dNPc-exo-carried miR-27a-3p accelerates the M1 polarization of macrophages via regulation of the PPARγ/NFκB/PI3K/AKT pathway. (A, B) A bioinformatics analysis predicted the binding sites between miR-27a-3p and PPARγ, confirmed by dual-Luciferase reporter assay. (C) The loading efficiency of miR-27a-3p of exosomes. (D, F, G) RT-qPCR and western blot analysis of PPARγ, NFκB, PI3K, AKT, iNOS and TNF-α gene expression in M0 macrophages after treatment with PPARγ plasmid and siRNA. (E, H, I) RT-qPCR and western blot analysis of PPARγ, NFκB, PI3K, AKT, iNOS, TNF-α and miR-27a-3p gene expression in M0 macrophages after treatment with miR-27a-3p mimic and inhibitor. The data are expressed as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant difference

Fig. 5

Comprehensive verification of the molecular mechanism of the PPARγ/NFκB/PI3K/AKT regulatory axis of dNPc-exo-carried miR-27a-3p. (A) RT-qPCR analysis of PPARγ, NFκB, PI3K, AKT and iNOS gene expression in M0 macrophages after cultured with dNPc-exo. (B) RT-qPCR analysis of PPARγ, NFκB, PI3K, AKT, iNOS and miR-27a-3p gene expression in M0 macrophages after cultured with dNPc-exo mimic and inhibitor. (C) Western blot analysis of PPARγ, NFκB, PI3K, AKT, iNOS and and TNF-α expression in M0 macrophages after cultured with nNPc-exo and dNPc-exo. (D) Quantitative analysis of PPARγ, NFκB, PI3K, AKT, iNOS and TNF-α expression in western blot after cultured with nNPc-exo and dNPc-exo. The data are expressed as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant difference

Fig. 6

dNPc-exo-carried miR-27a-3p induces M1 polarization of macrophages in IVDD animal model. (A) X-ray images of rat spines. (B) Quantification of the DHI%. (C) MRI images of rat spines. (D) Quantification of the Pffirmann grade. (E) Micro-CT and 3D reconstruction images of rat spines. (F) Representative images of H & E staining of experimental groups. Scale bar = 500 μm. (G) The histological scores of different groups after treatment. (H) Representative images of immunofluorescence staining of different groups. Scale bar = 100 μm. (I) Quantitative analysis of the iNOS⁺ macrophages percentage in immunofluorescence staining. (J) Schematic diagram of co-culturing of nNPc and M1 macrophages. (K) Western blot analysis of Col2A1, MMP3 and TNF-α expression in nNPc after co-culturing with M1 macrophages. (L) Quantitative analysis of Col2A1, MMP3 and TNF-α expression in nNPc after co-culturing with M1 macrophages. The data are expressed as the mean ± SEM. n = 3. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, non-significant difference