Mechanism Of Peroxidized PE Mediated Macrophage Phagocytosis Of Ferroptotic Cells

Research ideas:The development and stability of organisms depend on the balance of cell survival,death and regeneration in the body,and the removal of dead cells in the body is an inevitable event for cell renewal.The most classical programmed cell death include apoptosis and necrosis.In recent years,Stockwell's team has discovered a new form of programmed cell death,ferroptosis,which is characterized by iron dependent and accumulation of lipid peroxidation in the cell membrane.Different kinds of cell death have different clearance mechanisms which determines the development of the body's physiology and pathology condition.Current studies have shown that apoptotic cells can release or present find and eat-me signals to regulate their clearance by macrophages,which is an immune tolerant behavior and is beneficial to maintaining the body's stability after clearance.Necrotic cells do not have enough regulatory signals of apoptotic cells,and necrotic debris can recruit inflammatory cells and cause inflammation after clearance.However,it is not clear whether ferroptotic-cell can be removed by macrophages.When ferroptosis occurs,the cell membrane will rupture,which is similar to necrosis.At the same time,ferroptosis is regulated by a series of enzymatic reactions,similar to apoptotic characteristics.So we put forward a scientific question:Can ferroptotic-cells be removed by macrophages?If so,what is the eat-me signal molecule recognized by macrophages in ferroptotic-cells?In response to the above assumptions,this study used RSL3 as ferroptosis inducer and apoptosis inducer STS to induce ferroptosis and apoptosis in human promyelocytic acute leukemia cell HL60 and mouse leukemia cell L1210,respectively,in order to compare the difference of macrophages'phagocytosis ability and mechanism of ferroptotic-cell and apoptotic cell,and then investigate the phagocytosis signal of ferroptotic-cell clearance.Research method:1)To explore whether ferroptotic-cell can be recognized and cleared by macrophages:using HL60 cells and L1210 cells,RSL3 was given as an ferroptosis inducer to establish ferroptotic-cells model,and ferroptosis inhibitors such as Fer-1,DFO,and VE were administered to testify the occurrence of ferroptosis,using CCK-8and LDH release kit which was used to detect cell viability;flow cytometry was used to detect the phagocytosis of two cell death by macrophage:a concentration-dependent phagocytosis curve was established to determine the concentration of RSL3 and STS inducing the same phagocytosis index.The GPX4^-/-Pfa1 cell model was used to verify the phagocytosis of ferroptotic-cells.2)To explore the difference of phagocytosis-promoting signals between the two death modes:choose the concentrations of RSL3 and STS at the same phagocytosis rate.The known eat-me signals of apoptotic-cells expression,including ICAM3,GAS6,MFG-E8,CRT,PS and and don't eat-me signal CD47 were investigated by means of qRT-PCR,western blotting and flow cytometry.The difference of lipid peroxidation between the two death modes was detected by flow cytometry and confocal laser scanning microscopy to investigate whether there was specific phagocytosis signal in ferroptosis.3)LC-MS based lipidomics was used to study the lipid peroxidation of ferroptosis.The lipid synthesis and oxidation pathway were intervened by exogenous incubating of arachidonic acid?AA?combined with RSL3 was compared with STS,or exogenous incubating of PE-O-OH.After that phagocytosis was detected by flow cytometry to elucidate the mechanism.Finally,SAPE was biotinylated and used for in situ pull down test to screen receptors that recognize phagocytic signals,meanwhile SAPE was used as an example to molecular docking with the known receptor which can recognize"eat-me signal"and at the same timeResearch result:The results showed that RSL3 and STS induced ferroptosis and apoptosis of HL60and L1210 cells,respectively,in a concentration-dependent manner.Multiple experiments using macrophages co-cultured with dead cells and specific labeling of macrophages indicate that the ferroptotic-cell can be engulfed like apoptotic-cell,but their eat-me signal was different from apoptotic cells:there were bearly difference in ICAM3,GAS6,MFG-E8,CRT gene or protein expression and PS ectropion in ferroptotic-cells.Lipid peroxidation is one of the key steps in ferroptosis,and the results of LC-MS showed that the degree of oxidation of various lipids on the cell membrane was different when ferroptosis occurred.Peroxidized phosphatidylethanolamine?SAPE-O-OH?was the most obvious one;it was also the marker of ferroptosis.Exogenous incubation of AA can synergize with RSL3 to promote macrophage phagocytosis of ferroptotic-cells,and we found that exogenously added SAPE-O-OH can achieve the same phagocytosis index level as RSL3.These results suggest that lipid peroxidation during ferroptosis is the most important factor affecting phagocytosis,and PE-O-OH is a key signal molecule recognized by macrophages.In order to further explore the macrophage receptor recognition of the phagocytic signal PE-O-OH,we used virtual molecular docking to obtain highly related proteins with SAPE,and at the same time synthesized biotin-labeled SAPE.The protein fishing of biotinylated probe was analyzed by mass spectrometry,and the target protein TLR2 was screened by comparing with the results of molecular docking.In summary,our results show that oxidized lipid SAPE-O-OH on ferroptotic-cell membrane can be recognized as eat-me signal by macrophage receptor TLR2 to promote macrophage clearance of ferroptotic-cells.Next research,we will use a variety of biological models and interventions to explore this mechanism in depth.This topic is expected to lay a theoretical foundation for exploring the clearance mechanism of ferroptosis,and provide a new target for the development of new drugs in ferroptosis-related diseases,and provide a new understanding of the role of ferroptosis in the physiological and pathological development of the body.Conclusion:1)Ferroptotic cells can be recognized and engulfed by macrophages,but their phagocytic mechanisms are different from apoptotic cells;2)The oxidized lipid PE-O-OH on ferroptotic-cell membrane can be recognized as the eat-me signal by the macrophage receptor TLR2,thereby promoting the clearance of ferroptotic-cells by macrophages.