Macrophage polarization regulates intervertebral disc degeneration by modulating cell proliferation, inflammation mediator secretion, and extracellular matrix metabolism

Abstract

Macrophage infiltration and polarization have been increasingly observed in intervertebral disc (IVD) degeneration (IDD). However, their biological roles in IDD are still unrevealed. We harvested conditioned media (CM) derived from a spectrum of macrophages induced from THP-1 cells, and examined how they affect nucleus pulposus cells (NPCs) in vitro, by studying cell proliferation, extracellular matrix (ECM) synthesis, and pro-inflammation expression; and in vivo by injection CM in a rat IDD model. Then, high-throughput sequencing was used to detect differentially expressed genes (DEGs). Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks were used to further analysis. Higher CCR7+ (M1 marker) and CD206+ (M2 marker) cell counts were found in the degenerated human IVD tissues as compared with the control. Furthermore, the cell co-culture model showed M1CM attenuated NPC proliferation, downregulated the expression of ECM anabolic genes encoding aggrecan and collagen IIα1, upregulated the expression of ECM catabolic genes encoding MMP-13, and inflammation-related genes encoding IL-1β, IL-6, and IL-12, while M2CM showed contrasting trends. In IDD model, higher histological scores and lower disc height index were found following M1CM treatment, while M2CM exhibited opposite results. M1CM injection decreased ECM anabolic and increased ECM catabolic, as well as the upregulation of inflammation-related genes after 8 weeks treatment, while M2CM slowed down these trends. Finally, a total of 637 upregulated and 655 downregulated genes were detected in M1CM treated NPCs, and 975 upregulated genes and 930 downregulated genes in the M2CM groups. The top 30 GO terms were shown and the most significant KEGG pathway was cell cycle in both groups. Based on the PPI analysis, the five most significant hub genes were PLK1, KIF20A, RRM2, CDC20, and UBE2C in the M1CM groups and RRM2, CCNB1, CDC20, PLK1, and UBE2C in the M2CM groups. In conclusion, macrophage polarization exhibited diverse roles in IDD progression, with M1CM exacerbating cell proliferation suppression and IVD degeneration, while M2CM attenuated IDD development. These findings may facilitate the further elucidation of the role of macrophage polarization in IDD, and provide novel insights into the therapeutic potential of macrophages.

Keywords: inflammation; intervertebral disc degeneration (IDD); low back pain; macrophage polarization; musculoskeletal disorder; nucleus pulposus cells (NPCs).

Figure 1

Macrophage accumulation and polarization in human IVD tissues. (A) Representative MR images showing normal (Pfirrmann grade I, n = 5) and IDD samples (Pfirrmann grades III-V, n = 8), respectively. (B) Representative images showing the immunolocalization of CCR7-positive cells in human normal IVD tissues; (C) Comparison of the proportion of positive cells corresponding to normal and degenerated IVD samples. (D) Representative images showing the immunolocalization of CD206-positive cells in human IDD tissues, and (E) Comparison of positive cells corresponding to normal and IDD samples. The data were analyzed using Mann–Whiney U test. **p < 0.01; ***p < 0.001. Data are shown as mean ± SD. IVD, intervertebral disc; IDD, intervertebral disc degeneration. (F) Flow diagram of harvesting conditioned medium (CM) and grouping process of CM in vitro and in vivo.

Figure 2

| Effect of macrophage polarization on the proliferation of NPCs in the TNF-α-treated environment. (A) Representative images showing EDU incorporation staining of NPCs in different groups. (B) Comparison of EDU positive staining ratios corresponding to different test compounds at different time points. (C) Comparison of OD values corresponding to different test compounds at different time points based on cell proliferation assays. Data are expressed as the mean ± SD (n = 4). ^*, significant difference between the control and TNF-α-treated groups at P < 0.05; ^$, significant difference between the TNF-α- and M1CM+ TNF-α-treated groups at P < 0.05; ^&, significant difference between the TNF-α- and M2CM+ TNF-α-treated groups at P < 0.05. NPCs, nucleus pulposus cells; TNF-α, tumor necrosis factor-α.

Figure 3

Effect of macrophage polarization on ECM synthesis and pro-inflammatory mediator secretion in TNF-α-treated NPCs. (A) Representative images showed collagen II immunofluorescence staining results corresponding to NPCs in different groups. (B) Comparison of the OD values corresponding to different test compounds after 7 days of treatment. (C, D) WB analysis of anabolic aggrecan and catabolish MMP-13in NPCs treated with different CMs for 7 days. (E–L) Gene expression of ECM components (aggrecan, collagen IIα1, and collagen Iα1), ECM-modifying enzymes (MMP-13), and pro-inflammatory mediators (IL-1β, IL-6, IL-8, and IL-12) in TNF-α-treated NPCs after 3 days based on qPCR. All data are expressed as the mean ± SD, n = 3, *P < 0.05, **P < 0.01. NPCs, nucleus pulposus cells; TNF-α, tumor necrosis factor-α; CM, condition medium; ECM, extracellular matrix; OD, optical density.

Figure 4

Effect of macrophage polarization on the radiographic changes and histological scores corresponding to rat coccygeal IVDs. (A) Representative general view, HE staining and Safranin-O/Fast Green staining images of rat coccygeal IVDs corresponding to different treatment groups (Coronal position). (B) Histological grading score showing changes in IVD at 8 weeks after initial puncture and different treatments. (C) Representative X-ray images showing rat coccygeal IVDs in different treatment groups and (D), %DHI values showing changes in IVD at 8 weeks after initial puncture for different treatments. All data are expressed as the mean ± SD. n = 6, *P < 0.05, **P < 0.01. IVD, intervertebral disc; TNF-α, tumor necrosis factor-α; CM, condition medium; HE, Hematoxylin and eosin; DHI, disc height index.

Figure 5

Effect of macrophage polarization on ECM metabolism and pro-inflammatory mediator secretion in rat coccygeal IVDs. (A) Representative immunohistochemical images showing rat coccygeal IVDs in different groups. (B, C) Semi-quantification analysis of collagen II and aggrecan staining in rat coccygeal IVDs 8 weeks after initial puncture and different treatments. (D–I) Effect of macrophage polarization on the gene expression of ECM components (aggrecan and collagen IIα1), ECM-modifying enzymes (MMP-13), and pro-inflammatory mediators (IL-1 and IL-12) in rat coccygeal IVDs 8 weeks after puncture operation and different treatments. All data are expressed as the mean ± SD, n = 6, *P < 0.05, **P < 0.01. IVD, intervertebral disc; ECM, extracellular matrix; TNF-α, tumor necrosis factor-α; CM, condition medium.

Figure 6

Cluster analysis and volcano plot showing differentially expressed genes (DEGs) in M1CM or M2CM treated NPCs. (A, B) Cluster analysis heat map of DEGs, where each small square represents each mRNA, and its color represents the amount of mRNA expression. The greater the amount of mRNA expression, the darker is the color (red indicates high expression, blue indicates low expression). The first line indicates sample grouping, blue indicates treatment samples, red indicates control samples. Each row represents the expression of each mRNA in different samples, and each column represents the expression of all differential mRNAs in each sample. The tree on the left shows the cluster analysis results of different mRNAs from different samples. (C, D) Volcano map of DEGs, where each dot represents an mRNA. A red dot indicates that the gene expression is upregulated, a blue dot indicates that the mRNA expression is down regulated, and a gray dot indicates that there is no significant difference in the mRNA levels.

Figure 7

GO functional and KEGG pathway enrichment analysis of differentially expressed genes (DEGs) in M1CM or M2CM treated NPCs. The GO terms and KEGG pathway names are shown on the y-axis. Top 30 most significantly enriched GO terms of NPCs treated with M1CM (A) or M2CM (C) are displayed in a GO functional enrichment strip map. The length of the bars on the x-axis represents the gene counts, and the GO terms are shown on the y-axis. The bubble chart of the top 10 most significantly enriched GO terms of NPCs treated with M1CM (B) or M2CM (D) are displayed in terms of BPs, MF and CCs respectively. The x-axis represents the gene proportion in the annotation pathway, and the y-axis represents the GO terms. The color is determined by the P value, and the size is determined by the number of genes in the annotation pathway. (E, F) KEGG function enrichment bubble chart. The x-axis represents the ratio of genes in the annotation pathway, and the y-axis represents the KEGG pathway, the color is determined by the P value, and the size is determined by the number of genes in the annotation pathway.

Figure 8

Network interaction diagram of hub genes in M1CM (A) and M2CM (B) groups. The nodes represent the hub genes, and the edges represent the protein protein interaction. The color is determined by the results of the six indicators from large to small, and the greater the score, the darker is the color.

Figure 9

The role and potential mechanism of M1 and M2 macrophages in the process of IDD. Macrophages take part in IDD mainly in M1 and M2 polarization rather than macrophage themselves. Specifically, M1CM inhibits cell proliferation and exacerbates IVD degeneration, while M2CM promote cell proliferation and attenuates IDD development. A total of 1038 DEGs were identified in M1CM treated NPCs, and 1905 DEGs in the M2CM group. Cell cycle was the most significant pathway enriched in KEGG enrichment, and organelle fission, nuclear division, and chromosome segregation were highly enriched in GO functional enrichment in both treatment NPCs. Finally, the five most significant genes in the M1CM group were PLK1, KIF20A, RRM2, CDC20, UBE2C and those in the M2CM groups were RRM2, CCNB1, CDC20, PLK1, and UBE2C. IDD, intervertebral disc degeneration; IVD, intervertebral disc.