YAP1 Regulates LPS-induced M1 Macrophage Polarization And Mesenchymation

Objective: Macrophages have a great functional diversity and play an important role in the normal development of the body,homeostasis,tissue repair and immune response to pathogens.At the same time,macrophage is one of the most plastic cells in the hematopoietic system.In the process of monocyte induced macrophage formation in vitro,the cell polarity is enhanced.There are two main subtypes of macrophages,M1 and M2.M1 macrophage participates in the inflammatory response and plays a key role in host defense against bacterial and viral infection.M2 macrophage is associated with anti-inflammatory response,parasite infection,tissue remodeling,fibrosis and tumor disease development.Hippo pathway is a highly conserved signal pathway in recent years.YAP1,as an important co transcriptional activator,plays a regulatory role in tumor development.However,its function in immune cells is still rarely reported.Based on previous studies,our study used lipopolysaccharide(LPS)to induce M1 macrophages polarization,to explore the effect of YAP1 on M1 polarization and polarity changes of macrophages.Method: 1.The M1 macrophage polarization model was induced by LPS in vitro.The cytoskeletal changes were detected by rhodamine-phalloidin staining,and the changes of M1 macrophage markers were detected by qRT-PCR;2.GSEA analyzes the enrichment of nine classical signaling pathways during M1 macrophage polarization;3.Western Blot was used to detect the YAP1 expression changes during polarization,and qRT-PCR was used to detect the transcription of YAP1 and its downstream target genes;4.Immunofluorescence was used to detect the nuclear localization of YAP1 after LPS induction.5.Nuclear and plasma proteins were isolated and the expression of YAP1 in cytoplasm and nucleus was detected by Western Blot.6.qRT-PCR was used to detect the effect of YAP1 deletion on LPS induced expression of M1 macrophage markers;7.Western blot was used to detect the change of protein level of mesenchymal markers during M1 macrophage polarization;8.Western blot was used to detect the effect of YAP1 deficiency on the mesenchymation;9.Western blot was used to detect the expression of stemness related factors during M1 macrophage polarization;10.Bioinformatics was used to analyze the correlation between YAP1 and stemness related factors;11.The binding of YAP1 and SOX2 was detected by Co-immunoprecipitation(Co-IP);12.The localization of YAP1 and SOX2 during macrophage polarization was detected by immunofluorescence.Result: 1.The establishment of M1 macrophage polarization model;2.Bioinformatics methods were used to analyze the signal pathways that affect the M1 macrophages polarization;3.The expression of YAP1 increased during M1 polarization;4.During M1 macrophage polarization,YAP1 moved to the nucleus;5.YAP1 regulates the expression of M1 macrophage polarization markers;6.The polarization process of M1 macrophage is accompanied by the cell mesenchymal markers increasing;7.YAP1 regulates the expression of mesenchymal markers in M1 macrophage;8.Snail regulates the expression of mesenchymal markers in M1 macrophage;9.The polarization of M1 macrophage is accompanied by the increase of the expression of stem cell markers;10.During the polarization of M1 macrophage,the YAP1/SOX2 complex moves into the nucleus.Conclusion: 1.YAP1 can regulate M1 polarization and mesenchymation of macrophages;2.The M1 polarization and mesenchymation of macrophages were parallel;3.Intracellular heterotopia of YAP1/SOX2 complex during M1 polarization of macrophages.