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Published on: June 23, 2013
PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis
Liguo Zhang1, Fujiao Nie1, Jingjing Zhao1
1Department of Periodontology, School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration & Shandong Provincial Clinical Research Center for Oral Diseases, Shandong University, Jinan, Shandong, 250012, China.
Background And Objective:
Progranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment.
Methods:
Immunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization.
Results:
The IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization.
Conclusions:
In summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.
Insights
Progranulin (PGRN) promotes M2 macrophage polarization in periodontitis by binding to TNFR2. This finding clarifies PGRN's immune role in periodontal disease, impacting inflammation and tissue repair.
Area of Science:
- Immunology
- Cell Biology
- Periodontology
Background:
- Progranulin (PGRN) is a growth factor with known roles in various diseases.
- Its specific immune regulatory function in periodontitis, particularly concerning macrophage polarization, remains unclear.
Purpose of the Study:
- To investigate the regulatory effects of PGRN on macrophage polarization within the periodontitis microenvironment.
- To elucidate the underlying molecular mechanisms, including the role of TNFR2.
Main Methods:
- Immunohistochemistry (IHC) and multiplex IHC on human gingival samples.
- In vitro studies using RAW264.7 cells and bone marrow-derived macrophages (BMDMs) stimulated with LPS or IL-4, with or without PGRN.
- Gene and protein expression analysis (qRT-PCR, IF, ELISA, flow cytometry).
- Co-immunoprecipitation and TNFR2 blocking assays.
Main Results:
- Macrophages accumulate in periodontitis tissues, with increased M1 and M2 markers. PGRN expression is elevated and co-localizes with M2 macrophages.
- PGRN treatment decreased M1 markers and increased M2 markers in LPS-stimulated macrophages.
- PGRN enhanced IL-4-induced M2 polarization and directly interacted with TNFR2.
- TNFR2 blockade abrogated PGRN's effect on M2 polarization.
Conclusions:
- Progranulin (PGRN) promotes M2 macrophage polarization in periodontitis.
- This effect is mediated through the binding of PGRN to its receptor, TNFR2.
- PGRN influences macrophage phenotypes in both pro- and anti-inflammatory periodontal conditions.
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