Origin of M2 Mϕ and its macrophage polarization by TGF-β in a mice intervertebral injury model

Abstract

Introduction: Studies have identified the presence of M1 and M2 macrophages (Mϕ) in injured intervertebral discs (IVDs). However, the origin and polarization-regulatory factor of M2 Mϕ are not fully understood. TGF-β is a regulatory factor for M2 polarization in several tissues. Here, we investigated the source of M2 Mϕ and the role of TGF-β on M2 polarization using a mice disc-puncture injury model.

Methods: To investigate the origin of M2 macrophages, 30 GFP chimeric mice were created by bone marrow transplantation. IVDs were obtained from both groups on pre-puncture (control) and post-puncture days 1, 3, 7, and 14 and CD86 (M1 marker)- and CD206 (M2 marker)-positive cells evaluated by flow cytometry (n = 5 at each time point). To investigate the role of TGF-β on M2 polarization, TGF-β inhibitor (SB431542) was also injected on post-puncture days (PPD) 5 and 6 and CD206 expression was evaluated on day 7 by flow cytometry (n = 5) and real time PCR (n = 10).

Results: The proportion of CD86⁺ Mϕ within the GFP+ population was significantly increased at PPD 1, 3, 7, and 14 compared to control. CD206-positive cells in GFP-populations were significantly increased on PPD 7 and 14. In addition, the percentage of CD206-positive cells was significantly higher in GFP-populations than in GFP+ populations. TGF-β inhibitor reduced CD206-positive cells and Cd206 expression at 7 days after puncture.

Conclusion: Our findings suggest that M2 Mϕ following IVD injury may originate from resident Mϕ. TGF-β is a key factor for M2 polarization of macrophages following IVD injury.

Keywords: Macrophages; TGF-β; resident.

Figure 1.

Flow cytometry analysis of the M1 and M2 macrophage population after intervertebral injury. Percentage of Mφ (CD11b+F4/80+) (a), CD86+Mφ (b), and CD206+Mφ (c) in IVDs. Ratio of CD206+/CD86+ (d). All data show the mean ± standard error (n = 5). *, p < .05 in comparison with control (Con).

Figure 2.

Ratio of M1 and M2 macrophages in GFP- and GFP+ cells. A–E. Dot plots of GFP+ and GFP− CD86⁺ cells in the intervertebral discs (IVDs). The X-axis indicates CD11b and the y-axis indicates green fluorescence protein (GFP). F. Percentage of CD86⁺ GFP-negative (white) and -positive (gray) cells in IVDs. G-K. Dot plots of GFP+ and GFP− CD206+ cells in IVDs. L. Percentage of CD206+ GFP-negative (white) and -positive (gray) cells in IVDs. All data represent mean ± standard error (n = 5). A, p < .05 compared with control (Con); b, p < .05 compared with the time-matched GFP− population.

Figure 3.

Expression of TGF-β after IVD injury. Tgfb expression in IVDs (a). TGF-β protein concentration in IVDs (b). All data represent mean ± standard error (n = 10).*, p < .05 compared with control (Con).

Figure 4.

Localization of TGF-β following IVD injury. Hematoxylin and eosin-stained tissue sections of IVDs in the control (a) and injury groups (b) at post injury day 7. Immunostaining for TGFb in the control (c) and injury groups (d) at post injury day 7. Negative control section without primary antibody in the control (e) and injury groups (f). Scale bar = 100 µm.

Figure 5.

Effect of TGF-β on Smad pathway in vitro. Effects of TGF-β and TGF-β inhibitor on phosphorylation of smad2, smad3, smad4, and M2 maker (Fizz1 and Cd206) mRNA expression. Disc macrophages (DMs) were exposed to α-MEM (control), 10 ng/mL mouse recombinant (mh) TGF-β + 10 ng/mL IL-10 (TGF/IL-10), or 10 ng/mL rhTGF-β + 10 ng/mL IL-10 +10 μM SB431542 (TGF/IL-10/SB) for 30 min before protein extraction and western blot. Grouping of gels/blots cropped from different parts of the same gel or obtained from different gels. Western blotting for phosphorylated smad2 (p-smad2), smad2, p-smad3, smad3, smad4, and GAPDH (a). Densitometry of western blot protein bands for p-smad2 (b), smad2 (c), p-smad3 (d), smad3 (e), and smad4 (f) were normalized to the expression of GAPDH. Data indicate mean ± SE (n = 5).*p < .05 in comparison with control.

Figure 6.

Effect of TGF-β on M2 maker expression in vitro. RT-PCR for Cd206 (A) and Fizz1 (B). DMs were exposed to the α-MEM control, TGF/IL-10, or TGF/IL-10/SB for 24 h. Relative expression was evaluated with regard to expression in the control samples. The data show mean ± SE (n = 5).*p < .05 in comparison with control.

Figure 7.

Effect of TGF-β inhibitor on M2 marker expression in vivo. Dot plots of CD11b+CD206+ cells in vehicle (DMSO)- and TGF-β inhibitor (SB435124)-treated groups (a). Ratio of CD11b+CD206+ cells in vehicle (DMSO)- and TGF-β inhibitor (SB435124)-treated groups (n = 5 for each group) (b). Expression of Cd206 (c) and Fizz1 (d) expression in vehicle (DMSO)- and TGF-β inhibitor (SB435124)-treated groups (n = 10 for each group). The data show mean ± standard error.*p < .05 in comparison with vehicle.