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Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages
Published on: June 23, 2013
Bone marrow mesenchymal stem cells induce M2 microglia polarization through PDGF-AA/MANF signaling
Fan Yang1, Wen-Bin Li1, Ye-Wei Qu1
1Department of Neurology, The First Clinical College of Harbin Medical University, Harbin 150001, Heilongjiang Province, China.
Background:
Bone marrow mesenchymal stem cells (BMSCs) are capable of shifting the microglia/macrophages phenotype from M1 to M2, contributing to BMSCs-induced brain repair. However, the regulatory mechanism of BMSCs on microglia/macrophages after ischemic stroke is unclear. Recent evidence suggests that mesencephalic astrocyte-derived neurotrophic factor (MANF) and platelet-derived growth factor-AA (PDGF-AA)/MANF signaling regulate M1/M2 macrophage polarization.
Aim:
To investigate whether and how MANF or PDGF-AA/MANF signaling influences BMSCs-mediated M2 polarization.
Methods:
We identified the secretion of MANF by BMSCs and developed transgenic BMSCs using a targeting small interfering RNA for knockdown of MANF expression. Using a rat middle cerebral artery occlusion (MCAO) model transplanted by BMSCs and BMSCs-microglia Transwell coculture system, the effect of BMSCs-induced downregulation of MANF expression on the phenotype of microglia/macrophages was tested by Western blot, quantitative reverse transcription-polymerase chain reaction, and immunofluorescence. Additionally, microglia were transfected with mimics of miR-30a*, which influenced expression of X-box binding protein (XBP) 1, a key transcription factor that synergized with activating transcription factor 6 (ATF6) to govern MANF expression. We examined the levels of miR-30a*, ATF6, XBP1, and MANF after PDGF-AA treatment in the activated microglia.
Results:
Inhibition of MANF attenuated BMSCs-induced functional recovery and decreased M2 marker production, but increased M1 marker expression in vivo or in vitro. Furthermore, PDGF-AA treatment decreased miR-30a* expression, had no influence on the levels of ATF6, but enhanced expression of both XBP1 and MANF.
Conclusion:
BMSCs-mediated MANF paracrine signaling, in particular the PDGF-AA/miR-30a*/XBP1/MANF pathway, synergistically mediates BMSCs-induced M2 polarization.
Insights
Bone marrow mesenchymal stem cells (BMSCs) promote brain repair by shifting microglia/macrophages to an M2 phenotype via mesencephalic astrocyte-derived neurotrophic factor (MANF). The PDGF-AA/miR-30a*/XBP1/MANF pathway is crucial for this M2 polarization.
Area of Science:
- Neuroscience
- Stem Cell Biology
- Immunology
Background:
- Bone marrow mesenchymal stem cells (BMSCs) can modulate microglia/macrophages from M1 to M2 phenotypes, aiding brain repair after ischemic stroke.
- The precise regulatory mechanisms of BMSCs on microglia/macrophages post-stroke, particularly involving mesencephalic astrocyte-derived neurotrophic factor (MANF) and platelet-derived growth factor-AA (PDGF-AA), remain incompletely understood.
- MANF and PDGF-AA/MANF signaling are implicated in regulating M1/M2 macrophage polarization.
Purpose of the Study:
- To elucidate the role of MANF and the PDGF-AA/MANF signaling pathway in BMSCs-mediated M2 polarization.
- To investigate the molecular mechanisms by which BMSCs influence microglia/macrophage phenotypes after ischemic stroke.
Main Methods:
- BMSCs were engineered for MANF knockdown using small interfering RNA.
- A rat middle cerebral artery occlusion (MCAO) model and a Transwell co-culture system were used to assess the impact of MANF inhibition on microglia/macrophage phenotype.
- Western blot, quantitative reverse transcription-polymerase chain reaction, and immunofluorescence were employed to analyze M1/M2 markers.
- Microglia were transfected with miR-30a* mimics, and the expression of ATF6, XBP1, and MANF was examined following PDGF-AA treatment.
Main Results:
- Inhibition of MANF in BMSCs attenuated functional recovery and M2 marker production while increasing M1 markers in vivo and in vitro.
- PDGF-AA treatment reduced miR-30a* expression but did not affect ATF6 levels.
- PDGF-AA treatment enhanced the expression of X-box binding protein 1 (XBP1) and MANF.
Conclusions:
- BMSCs-derived MANF paracrine signaling is essential for M2 polarization.
- The PDGF-AA/miR-30a*/XBP1/MANF pathway plays a synergistic role in mediating BMSCs-induced M2 polarization.
- This pathway represents a potential therapeutic target for stroke recovery.
