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M1 Macrophage-Derived Interleukin-6 Promotes the Osteogenic Differentiation of Ligamentum Flavum Cells
Xiaochen Qu1,2,3, Gang Xu1,2, Xiaofei Hou4
1Department of Orthopedics, First Affiliated Hospital of Dalian Medical University, Dalian, PR China.
Study Design:
Basic experimental study.
Objective:
The aim of this study was to clarify the role of macrophages (Mφs) in the osteogenic differentiation of ligamentum flavum (LF) cells.
Summary Of Background Data:
Mφs and secreted factors are involved in the regulation of cell osteogenic differentiation, and play an important role in the process of heterotopic ossification. Whether Mφs are involved in the development of ossification of the ligamentum flavum (OLF) have not been reported.
Methods:
The expression of CD68+ Mφs in ossified LF tissue was identified by immunohistochemical staining. THP-1 cells were polarized to M1 and M2, and identified by flow cytometry and immunofluorescence. The alkaline phosphatase activity and osteogenic differentiation-related gene expression in LF cells were evaluated following incubation with each Mφs conditioned medium (CM). Enzyme-linked immunosorbent assay was used to detect the pro-inflammatory cytokines in the supernatants, and qPCR was used to detect the expression of the corresponding receptors in the LF cells after incubation with the CM. LF cells were induced with CM-M1 in the presence of neutralizing antibodies to further test whether cytokines secreted by M1 Mφs impacted their osteogenic differentiation.
Results:
CD68+ Mφs were found on the OLF samples. THP-1 cells were polarized into M1 and M2, and both M1 and M2 Mφs promoted the osteogenic differentiation of LF cells. The concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-1 β, and IL-6 in M1 Mφ supernatants were greater than those in M2, and greater levels of these cytokine receptors were observed in LF cells induced with CM-M1 than those with CM-M2. Osteogenic differentiation of LF cells induced by CM-M1 decreased after IL-6 was neutralized; however, not after IL-1β and TNF-α were neutralized.
Conclusion:
M1 Mφ-derived IL-6 promotes the osteogenic differentiation of LF cells, which may be a pathway in which Mφs regulate the osteogenic differentiation of LF cells.
Insights
Macrophages, specifically M1 type, promote ligamentum flavum cell osteogenic differentiation via IL-6. This finding clarifies a key pathway in ossification of the ligamentum flavum, offering insights into heterotopic ossification mechanisms.
Area of Science:
- Biomedical research
- Cell biology
- Orthopedics
Background:
- Macrophages (Mφs) and their secreted factors regulate osteogenic differentiation and heterotopic ossification.
- The role of Mφs in ossification of the ligamentum flavum (OLF) remains unelucidated.
Purpose of the Study:
- To investigate the role of macrophages (Mφs) in the osteogenic differentiation of ligamentum flavum (LF) cells.
Main Methods:
- Immunohistochemical staining identified CD68+ Mφs in ossified LF tissue.
- THP-1 cells were polarized into M1 and M2 subtypes and used to generate conditioned media (CM).
- LF cells were cultured with CM, and osteogenic differentiation markers, cytokine levels, and receptor expression were analyzed. Neutralizing antibodies were used to confirm cytokine roles.
Main Results:
- Both M1 and M2 Mφs promoted LF cell osteogenic differentiation.
- M1 Mφ CM contained higher levels of TNF-α, IL-1β, and IL-6 compared to M2 Mφ CM.
- Neutralization of IL-6 significantly reduced CM-M1-induced osteogenic differentiation in LF cells, while IL-1β and TNF-α neutralization had no significant effect.
Conclusions:
- Interleukin-6 (IL-6) secreted by M1 macrophages promotes osteogenic differentiation of LF cells.
- This IL-6 mediated pathway represents a mechanism by which Mφs contribute to LF cell osteogenic differentiation and potentially OLF.
