YTHDF3 Regulates Macrophage Activation: Investigation of the Mechanisms Involved

PENG Keren, YIN Qimin, TONG Jiyu

Abstract

To investigate the role and the underlying mechanisms of N6-methyladenosine (m6A) reader YTHDF3 in macrophages activation.

Methods 

shRNA-mediated Ythdf3 knockdown in RAW264.7 cells was performed and these RAW264.7 cells were stimulated with LPS. Then, changes in the pro-inflammatory and anti-tumor functions, including cytokine production, phagocytosis, and tumoricidal ability were evaluated. The effect of Ythdf3 knockdown on the activation of the Toll-like receptor 4 (TLR4) downstream MAPK and NF-κB pathways was assessed by immunoblotting. After Ythdf3 knockdown, the expression levels and mRNA stability of key junction proteins and signaling molecules of the TLR4 signaling pathway were analyzed to identify YTHDF3 target genes and investigate the underlying regulatory mechanism.

Results 

After LPS stimulation of wild-type RAW264.7 cells, the level of pro-inflammatory factors increased and then decreased. However, the level of YTHDF3 showed the opposite trend to that of pro-inflammatory factors, suggesting that YTHDF3 might play a role in the negative regulation of macrophage activation. shRNA-mediated Ythdf3 knockdown in RAW264.7 cells significantly increased the expression of pro-inflammatory factors, nitric oxide (NO) production, and phagocytosis. In addition, Ythdf3 knocked-down RAW264.7 cells co-cultured with tumor cells exhibited enhanced tumor killing ability. The findings suggested that YTHDF3 deletion could promote LPS-induced activation of RAW264.7 cells and enhance their production of pro-inflammatory factors and tumor killing function. further investigation into the underlying mechanisms revealed that Ythdf3 knockdown inhibited the degradation of Cd36, Irak1, Tab1/2, and Tirap mRNAs, which were key junction proteins and signaling molecules in the TLR4 pathway, which in turn, enhanced the phosphorylation of p38, a downstream key kinase and the activation of macrophages.

Conclusion 

By targeting the mRNA of the key junction proteins and signaling molecules of the TLR4 pathway, YTHDF3 accelerates their rapid degradation and suppresses macrophage activation. Ythdf3 knockdown significantly promotes macrophage activation and enhances the tumor killing activities of macrophages.

 

Keywords: YTHDF3, Macrophage activation, TLR4 signaling pathway

 

Full Text:

PDF


References


FRANKEN L, SCHIWON M, KURTS C. Macrophages: sentinels and regulators of the immune system. Cell Microbiol, 2016, 18(4): 475-487. doi: 10.1111/cmi.12580.

ZHU X, TANG H, YANG M, et al. N6-methyladenosine in macrophage function: a novel target for metabolic diseases. Trends Endocrinol Metab, 2023, 34(2): 66-84. doi: 10.1016/j.tem.2022.12.006.

LIU C P, ZHANG X, TAN Q L, et al . NF-κB pathways are involved in M1 polarization of RAW 2647 macrophage by polyporus polysaccharide in the tumor microenvironment. PLoS ONE, 2017, 12(11): e0188317. doi: 10.1371/journal.pone.0188317.

DOMINISSINI D, MOSHITCH-MOSHKOVITZ S, SCHWARTZ S, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature, 2012, 485(7397): 201-206. doi: 10.1038/nature11112.

ZHAO W, QI X, LIU L, et al. Epigenetic regulation of m6A modifications in human cancer. Mol Ther Nucleic Acids, 2020, 19: 405-412. doi: 10. 1016/j.omtn.2019.11.022.

YANG C, HU Y, ZHOU B, et al. The role of m6A modification in physiology and disease. Cell Death Dis, 2020, 11(11): 960. doi: 10.1038/s41419-020-03143-z.

LI H B, TONG J, ZHU S, et al. m6A mRNA methylation controls T cell homeostasis by targeting the IL-7/STAT5/SOCS pathways. Nature, 2017, 548(7667): 338-342. doi: 10.1038/nature23450.

DONG L, CHEN C, ZHANG Y, et al. The loss of RNA N6-adenosine methyltransferase Mettl14 in tumor-associated macrophages promotes CD8(+) T cell dysfunction and tumor growth. Cancer Cell, 2021, 39(7): 945-957. doi: 10.1016/j.ccell.2021.04.016.

TONG J, WANG X, LIU Y, et al. Pooled CRISPR screening identifies m(6)A as a positive regulator of macrophage activation. Sci Adv, 2021, 7(18): eabd4742. doi: 10.1126/sciadv.abd4742.

WANG H, HU X, HUANG M, et al. Mettl3-mediated mRNA m6A methylation promotes dendritic cell activation. Nat Commun, 2019, 10(1): 1898. doi: 10.1038/s41467-019-09903-6.

MA S, YAN J, BARR T, et al. The RNA m6A reader YTHDF2 controls NK cell antitumor and antiviral immunity. J Exp Med, 2021, 218(8): e20210279. doi: 10.1084/jem.20210279.

WANG Y, LI L, LI J, et al. The emerging role of m6A modification in regulating the immune system and autoimmune diseases. Front Cell Dev Biol, 2021, 9: 755691. doi: 10.3389/fcell.2021.755691.

PATIL D P, PICKERING B F, JAFFREY S R. Reading m6A in the Transcriptome: m6A-Binding Proteins. Trends Cell Biol, 2018, 28(2): 113- 127. doi: 10.1016/j.tcb.2017.10.001.

LASMAN L, KRUPALNIK V, VIUKOV S, et al. Context-dependent functional compensation between Ythdf m6A reader proteins. Genes Dev, 2020, 34(19-20): 1373-1391. doi: 10.1101/gad.340695.120.

ZACCARA S, JAFFREY S R. A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA. Cell, 2020, 181(7): 1582-1595. doi: 10.1016/j.cell.2020.05.012.

LI A, CHEN Y S, PING X L, et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res, 2017, 27(3): 444-447. doi: 10.1038/cr.2017.10.

SHI H, WANG X, LU Z, et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res, 2017, 27(3): 315-328. doi: 10.1038/cr.2017.15.

XIAN G Y, CHEN W S, ZHANG Z J, et al. Construction of a stable Atg5 gene knockdown cell line in RAW 264.7 cells by lentivirus infection. Chin Jo Tissue Engin Res, 2021, 25(1): 84-89. doi: 10.3969/j.issn.2095-4344.2149.

WYNN T A, CHAWLA A, POLLARD J W. Macrophage biology in development, homeostasis and disease. Nature, 2013, 496(7446): 445-455. doi: 10.1038/nature12034.

YIN H, ZHANG X, YANG P, et al. RNA m6A methylation orchestrates cancer growth and metastasis via macrophage reprogramming. Nat Commun, 2021, 12(1): 1394. doi: 10.1038/s41467-021-21514-8. ARTHUR J S, LEY S C. Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol, 2013, 13(9): 679-692. doi: 10.1038/nri3495.

CAI Y, YU R, ZHANG Z, et al. Mettl3/Ythdf2 regulate macrophage inflammation and ROS generation by controlling Pyk2 mRNA stability. Immunol Lett, 2023, 264: 64-73. doi: 10.1016/j.imlet.2023.11.004.

CARTY M, GOODBODY R, SCHRöDER M, et al. The human adaptor SARM negatively regulates adaptor protein TRIF–dependent Toll-like receptor signaling. Nat Immunol, 2006, 7(10): 1074-1081. doi: 10.1038/ni1382.

SUN H, GONG S, CARMODY R J, et al. TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis. Cell, 2008, 133(3): 415-426. doi: 10.1016/j.cell.2008.03.026.

SHARIF M N, SOSIC D, ROTHLIN C V, et al. Twist mediates suppression of inflammation by type I IFNs and Axl. The Journal of experimental medicine, 2006, 203(8): 1891-1901. doi: 10.1084/jem. 20051725.

KYRIAKIS J M, AVRUCH J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiological reviews, 2001, 81(2): 807-869. doi: 10.1152/physrev.2001.81.2.807.

LEE J C, KASSIS S, KUMAR S, et al. p38 mitogen-activated protein kinase inhibitors--mechanisms and therapeutic potentials. Pharmacol Ther, 1999, 82(2-3): 389-397. doi: 10.1016/s0163-7258(99)00008-x.

YU G, YU H, YANG Q, et al. Vibrio harveyi infections induce production of proinflammatory cytokines in murine peritoneal macrophages via activation of p38 MAPK and NF-κB pathways, but reversed by PI3K/AKT pathways. Dev Comp Immunol, 2022, 127: 104292. doi: 10.1016/j.dci. 2021.104292.

WANG Y W, YAGN H, WANG Z H, et al. Proanthocyanidins alleviate lipopolysaccharide-induced inflammatory response by up-regulating SIRT1 expression and inhibiting NF-κB pathway in mouse RAW264. 7 macrophages., 2023, 39(10): 878-883. doi: 10. 13423/j.cnki.cjcmi.009640.

YU R, LI Q, FENG Z, et al. m6A reader YTHDF2 regulates LPS-Induced inflammatory response. Int J Mol Sci, 2019, 20(6): 1323. doi: 10.3390/ijms20061323.


Refbacks

  • There are currently no refbacks.