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Updated: Jul 23, 2025
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
Exosomal PPARγ derived from macrophages suppresses LPS-induced peritonitis by negative regulation of CD14/TLR4 axis
Meng Meng1, Meizhi Lu2, Junxia Feng2
1MOE Key Laboratory of Tumor Molecular Biology and Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou, 510632, China.
Background:
Intercellular communication between macrophages and peritoneal mesothelial cells (PMCs) has been suggested as a key factor regulating peritonitis development. Here, we explored whether PPARγ (peroxisome proliferator-activated receptor gamma) can be packaged into macrophage exosomes to mediate intercellular communication and regulate peritonitis.
Methods:
Macrophage exosomes were isolated by ultracentrifugation and identified by nanoparticle tracking analysis and transmission electron microscopy. Proteomic analysis of macrophage-derived exosomes was performed using mass spectrometry. Co-culture models of supernatants or exosomes with PMCs, as well as a mouse peritonitis model induced by lipopolysaccharide (LPS), were employed.
Results:
In this study, using stable Raw264.7 cells overexpressing GFP-FLAG-PPARγ (OE-PPARγ), we found that PPARγ inhibited LPS-induced inflammatory responses in Raw264.7 cells and that PPARγ was incorporated into macrophage exosomes during this process. Overexpression of PPARγ mainly regulated the secretion of differentially expressed exosomal proteins involved in the biological processes of protein transport, lipid metabolic process, cell cycle, apoptotic process, DNA damage stimulus, as well as the KEGG pathway of salmonella infection. Using co-culture models and mouse peritonitis model, we showed that exosomes from Raw264.7 cells overexpressing PPARγ inhibited LPS-induced inflammation in co-cultured human PMCs and in mice through downregulating CD14 and TLR4, two key regulators of the salmonella infection pathway. Pretreatment of the PPARγ inhibitor GW9662 abolished the anti-inflammatory effect of exosomes from Raw264.7 OE-PPARγ cells on human PMCs.
Conclusions:
These results suggested that overexpression of PPARγ largely altered the proteomic profile of macrophage exosomes and that exosomal PPARγ from macrophages acted as a regulator of intercellular communication to suppress LPS-induced inflammatory responses in vitro and in vivo via negatively regulating the CD14/TLR4 axis.
Insights
Macrophage exosomes carrying peroxisome proliferator-activated receptor gamma (PPARγ) suppress inflammation. These exosomal PPARγ molecules regulate intercellular communication, reducing peritonitis by downregulating CD14 and TLR4.
Area of Science:
- Cell biology
- Immunology
- Molecular biology
Background:
- Peritonitis involves intercellular communication between macrophages and peritoneal mesothelial cells (PMCs).
- Peroxisome proliferator-activated receptor gamma (PPARγ) role in peritonitis is unclear.
- Investigating PPARγ packaging into macrophage exosomes for intercellular communication.
Purpose of the Study:
- To determine if PPARγ is packaged into macrophage exosomes.
- To explore the role of exosomal PPARγ in intercellular communication.
- To investigate the therapeutic potential of exosomal PPARγ in peritonitis.
Main Methods:
- Macrophage exosome isolation via ultracentrifugation.
- Exosome characterization using nanoparticle tracking analysis and transmission electron microscopy.
- Proteomic analysis via mass spectrometry.
- In vitro co-culture models and in vivo mouse peritonitis models.
Main Results:
- Overexpression of PPARγ in macrophages led to its packaging into exosomes.
- Exosomal PPARγ inhibited lipopolysaccharide (LPS)-induced inflammatory responses in macrophages and PMCs.
- PPARγ-containing exosomes downregulated CD14 and TLR4, key regulators in inflammation.
- Inhibition of PPARγ abolished the anti-inflammatory effects of the exosomes.
Conclusions:
- Macrophage-derived exosomes can package PPARγ.
- Exosomal PPARγ mediates intercellular communication to suppress inflammation.
- Exosomal PPARγ exerts anti-inflammatory effects by downregulating the CD14/TLR4 axis in peritonitis.
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