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Exosomal PPARγ derived from macrophages suppresses LPS-induced peritonitis by negative regulation of CD14/TLR4 axis

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Abstract

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.

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Data availability

The datasets generated during the current study are available in the ProteomeXchange Consortium via the PRIDE partner repository with the identifier PXD039203 (http://www.proteomexchange.org/). All other data generated during this study are included in this published article and its supplementary information file.

Abbreviations

CD14:

Cluster of differentiation 14

CD63:

Cluster of differentiation 63

FDR:

False discovery rate

HE:

Hematoxylin–eosin

IHC:

Immunohistochemistry

IL-1β:

Interleukin 1β

iNOS:

Inducible nitric oxide synthase

KEGG:

Kyoto encyclopedia of genes and genomes

LPS:

Lipopolysaccharide

MS:

Mass spectrometry

NTA:

Nanoparticle tracking analysis

OE-PPARγ:

Overexpression of PPARγ

OE-V:

Overexpression of vector

PD:

Peritoneal dialysis

PMCs:

Peritoneal mesothelial cells

PPARγ:

Peroxisome proliferator-activated receptor gamma

qRT-PCR:

Quantitative real-time PCR

TEM:

Transmission electron microscopy

TNF-α:

Tumor necrosis factor-alpha

TLR4:

Toll-like receptor 4

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Funding

This work was supported by National Natural Science Foundation of China (81800675; 81860143; 82200758), Medical Scientific Research Foundation of Guangdong Province of China (A2021251), Guangdong Basic and Applied Basic Research Foundation (2023A1515011221; 2023A1515010248), China Postdoctoral Science Foundation (2018M633085), Guangzhou Science and Technology Project (202201010034). Open project supported by the Key Laboratory of tumor molecular biology of the Ministry of education of Jinan University (202203).

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Author notes

  1. Meng Meng and Meizhi Lu have contributed equally to this work.

Authors and Affiliations

  1. MOE 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

    Meng Meng, Luxuan Chen, Xiaohui Liu, Langxia Liu & Xuejuan Gao

  2. Department of Nephrology, Affiliated Huadu Hospital, Southern Medical University (People’s Hospital of Huadu District), Guangzhou, China

    Meizhi Lu, Junxia Feng, Xiaoying Zhou, Ping Meng & Yunfang Zhang

  3. Department of Nephrology, The Fifth Affiliated (Zhuhai) Hospital of Zunyi Medical University, Zhuhai, 519100, China

    Xunliang Zou

  4. The Third School of Clinical Medicine, Southern Medical University, Guangzhou, China

    Yunfang Zhang

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  1. Meng Meng
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Contributions

All authors contributed to the study conception and design. MM and ML contributed to the execution of most experiments and data analysis. JF, XZ, PM, LC and XL contributed to the performance of the rest experiments. MM, LL, XG and YZ wrote the first draft of the manuscript. XZ, LL, XG and YZ contributed to the conception and design of the experiment, data analysis, drafted the manuscript and approved the final version to be published.

Corresponding authors

Correspondence to Langxia Liu, Xuejuan Gao or Yunfang Zhang.

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Conflict of interest

The authors declare no competing interests.

Ethical approval

All animal procedures were approved by the Experimental Animal Ethics Committee of Jinan University, Guangzhou, China (approval number: 100041).

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Supplementary file1 (DOCX 334 KB)

11_2023_1765_MOESM2_ESM.xlsx

Supplementary file2Table S1. Differentially secreted proteins of exosomes from the indicated groups identified in this study (XLSX 463 KB)

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Meng, M., Lu, M., Feng, J. et al. Exosomal PPARγ derived from macrophages suppresses LPS-induced peritonitis by negative regulation of CD14/TLR4 axis. Inflamm. Res. 72, 1567–1581 (2023). https://doi.org/10.1007/s00011-023-01765-5

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