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. 2021 Jul 12:2021:7988320.
doi: 10.1155/2021/7988320. eCollection 2021.

Reduced TGF- β Expression and CD206-Positive Resident Macrophages in the Intervertebral Discs of Aged Mice

Yuji Yokozeki  1 , Ayumu Kawakubo  1 , Masayuki Miyagi  1 , Akiyoshi Kuroda  1 , Hiroyuki Sekiguchi  2 , Gen Inoue  1 , Masashi Takaso  1 , Kentaro Uchida  1   2
Affiliations

Reduced TGF- β Expression and CD206-Positive Resident Macrophages in the Intervertebral Discs of Aged Mice

Yuji Yokozeki et al. Biomed Res Int. .

Abstract

Age is a key factor in intervertebral disc (IVD) degeneration; however, the changes that occur in IVDs with age are not fully understood. Tissue-resident macrophages are critical for tissue homeostasis and are regulated by transforming growth factor- (TGF-) β. We examined changes in the proportion of resident macrophages in young versus aged mice and the role of TGF-β in regulating resident macrophages in IVDs. IVDs were harvested from 4-month (young) and 18-month-old (aged) C57BL/6J mice. The proportion of macrophages in IVDs was determined using flow cytometry (n = 5 for each time point) and the expression of Cd11b, Cd206, and Tgfb genes, which encode CD11b, CD206, and TGF-β protein, respectively, using real-time PCR. To study the role of TGF-β in the polarization of resident macrophages, resident macrophages isolated from IVDs from young and aged mice were treated with recombinant TGF-β with and without a TGF-β inhibitor (SB431542). Additionally, SB431542 was intraperitoneally injected into young and aged mice, and Cd206 expression was examined using real-time PCR (n = 10 for each time point). The proportion of CD11b+ and CD11b+ CD206+ cells was significantly reduced in aged versus young mice, as was Cd11b, Cd206, and Tgfb expression. TGF-β/IL10 stimulation significantly increased the expression of Cd206, an M2 macrophage marker, in disc macrophages from both young and aged mice. Meanwhile, administration of a TGF-β inhibitor significantly reduced Cd206 expression compared to vehicle control in both groups. Conclusion. Resident macrophages decrease with age in IVDs, which may be associated with the concomitant decrease in TGF-β. Our findings provide new insight into the mechanisms of age-related IVD pathology.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this article.

Figures

Figure 1
Figure 1
Flow cytometry analysis of the macrophage population in intervertebral discs of young and aged mice. Dot plots showing CD11b+ and CD11b+ CD206+ cells in intervertebral discs (IVDs) of (a) young (age 4 months) and (b) aged (age 18 months) mice. The x-axis indicates CD206 and the y-axis indicates CD11b. (c) Percentage of CD45+ CD11b+ cells in IVDs (n = 5). (d) Percentage of CD11b+ CD206+ cells in IVDs (n = 5). ∗P < 0.05 compared with young mice.
Figure 2
Figure 2
Expression of CD11b, Cd206, and Tgfb in intervertebral discs of young and aged mice. Expression of (a) Cd11b, (b) Cd206, and (c) Tgfb in intervertebral discs of young (age 4 months) and aged (age 18 months) mice. ∗P < 0.05.
Figure 3
Figure 3
Effect of TGF-β on M2 marker expression in vitro. Disc macrophages derived from young (age 4 months) and aged (age 18 months) mice were stimulated with α-MEM (control), 10 ng/ml mouse recombinant (mr) TGF-β+10 ng/ml IL-10 (TGF/IL10), or 10 ng/ml mrTGF-β +10 ng/ml IL-10 +10 μM SB431542 (TGF/IL10/SB431542) for 24 h (n = 5). Relative expression was determined based on expression in control samples. ∗P < 0.05 compared to vehicle.
Figure 4
Figure 4
Effect of a TGF-β inhibitor on Cd206 expression in vivo. Cd206 expression following administration of vehicle (DMSO) and TGF-β inhibitor (SB431542) to (a) young and (b) aged mice (n = 10 for each group). ∗P < 0.05 compared to vehicle.
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