Neutrophil Surface GlycoRNA Regulates Immune Cell Recruitment
RNA always functions in the nucleus or cytoplasm. Even if it appears on the cell membrane, it is considered to have only weak and unstable interactions with the cell membrane. In 2020, researchers found that RNA does exist on the cell membrane of human monocytes, and the corresponding Oligonucleotide fragments antagonized the function of RNA, which would affect the adhesion between monocytes and endothelium.
In 2021, Carolyn R. Bertozzi's team from Harvard University found that some microRNAs on the surface of cancer cell lines and embryonic stem cells were N-glycosylated, and named this type of RNA glycoRNA. The occurrence of glycosylation is related to several enzymes that cause protein glycosylation.
As an emerging research field, the specific mechanism of GlycoRNA production and its physiological effects at the in vivo level are still largely unknown. On January 22, 2024, the team of Jun Lu and Dianqing Wu from Yale University published an article titled "Cell surface RNAs control neutrophil recruitment" in Cell. This work took neutrophils as the research object and found that glycoRNA on their surface affects their adhesion to endothelial cells, thereby affecting their chemotaxis to the site of inflammation. At the same time, the researchers also found that glycoRNA interacts with P-selectin (Selp) on endothelial cells, is produced inside the cells, and is transported to the cell surface via Sidt.
The researchers first used the method reported by Bertozzi's team in 2021 to verify whether glycoRNA exists on the surface of immortalized myeloid cells (HOXB8), neutrophils differentiated from the myeloid cells, and primary mouse neutrophils. In simple terms, the researchers first labeled the sugar moiety with Ac4ManNAz, and after extracting the total RNA of the cells, they used DBCO-PEG4-Biotin to label the azide group of Ac4ManNAz, and detected the biotin signal to determine the presence of glycoRNA. The results showed that biotin signals were present in all three cells, and the signal was sensitive to RNaseA. Further experiments found that RNaseA treatment would also cause the biotin signal to disappear without damaging the cell membrane, and when it was stained, it was found that the signal was mainly present on the cell surface, further verifying the cell membrane localization of glycoRNA.
Does glycoRNA on the surface of neutrophils affect their biological functions in vivo? Usually, when inflammation occurs in the body, neutrophils are recruited to the site of inflammation, and go through a series of processes such as rolling, adhesion, and invasion on the surface of vascular endothelial cells to reach the site of inflammation. After inducing a Mouse Peritonitis Model with thioglycolate (TG), the researchers injected RNaseA-treated and non-RNaseA-treated neutrophils into their blood at a ratio of 1:1. After 2.5 hours, flow cytometry was used to detect the ratio of the two types of cells that reached the peritoneum. The results showed that the ratio of cells treated with RNaseA to the peritonitis site was significantly reduced.
Fig. 1 Removing glycoRNA on the cell surface affects the migration of neutrophils to inflammatory sites. (Zhang, et al., 2024)
When using the Transwell experiment for verification, the researchers found that after RNaseA removed cell surface glycoRNA, the ability of neutrophils to migrate through endothelial cells to the lower layer was significantly weakened under the stimulation of the chemotactic signal fMLP. In the in vivo experiment, a mixture of RNaseA-treated neutrophils and mock-treated neutrophils was injected into the mouse carotid artery, and the fluorescence signals of the two types of cells were detected in the blood vessels of the cremaster muscle. It was found that after RNaseA treatment, the ratio of neutrophils that changed from free flow to rolling on endothelial cells was significantly reduced.
In the process of neutrophil adhesion to endothelial cells, integrins on the surface of neutrophils or selectins on the surface of endothelial cells usually play a key role. RNaseA treatment did not significantly change the levels of integrins Cd11a and Cd11b on the surface of neutrophils, nor did it change their interaction with the ligand ICAM-1. However, after blocking P-selectin with antibodies, the binding between glycoRNA and endothelial cells was partially weakened (E-selectin did not show the corresponding phenomenon). In subsequent animal experiments, Knockout of Selp also led to a weakening of neutrophil rolling on vascular endothelial cells.
Previous studies on glycoRNA believed that glycoRNA was released by dead cells into the extracellular environment and then captured by cells on the cell surface. However, there may be another answer to this question, that is, the glycoRNA on the cell surface is synthesized by the cell itself. To clarify this issue, the researchers used Ac4ManNAz to label glycans and labeled cells with green fluorescence, while glycan-unlabeled cells were given red fluorescence, and the two types of cells were co-cultured. The results showed that Biotin signals could only be detected in cells labeled with Ac4ManNAz and green fluorescence, while cells carrying red fluorescence did not capture RNA carrying the corresponding label, suggesting that glycoRNA is likely produced by the cells themselves and transported to the cell surface. When the homologous protein of C. elegans RNA transporter Sid-1 in mammals was knocked out, the content of glycoRNA on the cell surface was significantly reduced, and the migration ability of neutrophils was significantly weakened.
Fig. 2 Experimental design ideas for exploring the source of glycoRNA. (Zhang, et al., 2024)
RNA Glycosylation is an important supplement to traditional glycobiology based on proteins and lipids. Given the ever-changing forms and types of RNA, its biological functions should be far more than this, and more details of the story are still waiting to be added by future research.
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