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.

Nucleus Pulposus Cells Induce M2 Polarization of RAW264.7 via CX3CL1/CX3CR1 Pathway and M2 Macrophages Promote Proliferation and Anabolism of Nucleus Pulposus Cells

“…The presence of M2 macrophages in the ruptured IVD has been occasionally reported recent years [ 7 , 8 , 24 ]. It has been declared that the M2 macrophages were differentiated from resident macrophages in IVD and were regulated by TGF- β [ 24 ], while another study reported that M2a macrophages were absent in normal IVD but recruited in degenerated IVD in mice [ 7 ]. The above contrary findings indicate that the origin of M2 macrophages in IVD is still unclear.…”

Macrophages and Intervertebral Disc Degeneration

“…Levels of Tgfβ within the disc positively align with disc degeneration severity in humans [ 149 , 150 , 151 , 152 ], but it is unknown if this is a cause or consequence of disc degeneration, and the effects on specific cells within the disc are likewise unclear. Recent studies suggest that Tgfβ polarizes macrophages towards an M2 phenotype, but the change in macrophage activation in response to Tgfβ may be distinct from that elicited by IL-4/IL-13 [ 153 , 154 , 155 ]. To evaluate levels of M2 within murine intervertebral discs, Kawakubo et al utilized Cd206 as a marker for tissue resident macrophages.…”

Immune exposure: how macrophages interact with the nucleus pulposus

“…Transforming growth factor (TGF), the most common pro-fibrotic factor released by M2 macrophages, is absent in the normal NP and in the non-granulation area with obvious inflammatory response but is widely distributed across the granulation tissue area rich in M2 macrophages (71). TGF expression was noted to gradually increase with the progressive aggravation of NP degeneration; this not only promoted the polarization of M2 macrophages, but also promoted tissue fibrosis (72,73). TGF exerts extensive molecular regulatory effects on the NP.…”