Secretome of adipose-derived mesenchymal stem cells establish anti-inflammatory milieu by developing M2b/c macrophage

Abstract

Abstract Mesenchymal stem cells (MSCs) express immunomodulatory and anti-inflammatory effect toward immune system with several mechanisms such as secreting bioactive molecules. Distinct mechanisms of their immune actions, however, are not fully understood. As one of the important cells in immune system, macrophages play a vital role in innate immunity with two functional phenotypes, classically activated (M1) and alternatively activated (M2) macrophages. In this study, immunomodulatory ability of adipose-derived MSCs (AD-MSCs) secretome toward peritoneal macrophage was studied. Secretome was concentrated from human AD-MSCs using 3K TFF membrane. Murine peritoneal macrophages were cultured for 24 hours with secretome of AD-MSCs and 4 hours with LPS. Macrophages were analyzed by flow cytometry, ELISA, and real-time PCR. LC-MS/MS proteomics analysis of secretome was performed and acquired protein sets were analyzed by DAVID Bioinformatics. Secretome of AD-MSCs increased cell viability of peritoneal macrophages. Secretome treated peritoneal macrophages resulted in reduced M1 markers such as CD86, iNOS, and TNFα and increased M2 markers such as CD206, Arginase-1, and IL-10. Also, peritoneal macrophage expressed upregulation of mRNA level of LIGHT and MerTK, M2b and M2c markers, respectively. 83 proteins were identified in secretome according to the proteomics analysis. Several proteins among them were selected as candidates after confirmation of inhibiting the effect of secretome with neutralizing antibodies. In conclusion, secretome of AD-MSCs induced polarization of peritoneal macrophages from M1 macrophages to M2 macrophages and made them create anti-inflammatory milieu by several key secreted proteins. Supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (2021R1F1A1049630) and the Ministry of Health and Welfare, Republic of Korea (HI14C1324)