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Updated: Sep 22, 2025
Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
Origin of M2 Mϕ and its macrophage polarization by TGF-β in a mice intervertebral injury model
Ayumu Kawakubo1, Masayuki Miyagi1, Yuji Yokozeki1
1Department of Orthopedic Surgery, 38088Kitasato University School of Medicine, Sagamihara, Japan.
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.
Insights
Resident macrophages may be the source of M2 macrophages after intervertebral disc injury. Transforming growth factor-beta (TGF-β) plays a key role in regulating M2 macrophage polarization in injured discs.
Area of Science:
- Immunology
- Regenerative Medicine
- Orthopedics
Background:
- Macrophages, including M1 and M2 subtypes, are present in injured intervertebral discs (IVDs).
- The origin and regulatory factors of M2 macrophages in IVDs remain unclear.
- Transforming growth factor-beta (TGF-β) is known to regulate M2 polarization in other tissues.
Purpose of the Study:
- To investigate the origin of M2 macrophages following intervertebral disc injury.
- To determine the role of TGF-β in the M2 polarization of macrophages in injured IVDs.
Main Methods:
- Utilized GFP chimeric mice created via bone marrow transplantation to track macrophage origin.
- Analyzed M1 (CD86) and M2 (CD206) macrophage markers by flow cytometry post-IVD puncture.
- Administered a TGF-β inhibitor (SB431542) to assess its effect on M2 polarization via flow cytometry and real-time PCR.
Main Results:
- Increased CD86+ (M1) macrophages were observed from day 1 to 14 post-injury.
- CD206+ (M2) macrophages significantly increased on days 7 and 14 post-injury, with a higher proportion originating from GFP- populations (suggesting resident origin).
- TGF-β inhibition reduced CD206+ cells and Cd206 gene expression at 7 days post-injury.
Conclusions:
- M2 macrophages observed after intervertebral disc injury likely originate from resident macrophages.
- TGF-β is identified as a crucial factor regulating M2 macrophage polarization in the context of IVD injury.
