Study On The Mechanism Of IL-33/ST2 Axis Promoting Tumor Growth By Regulating M2 Polarization Of Macrophages

Tumor associated macrophage(Tumor associated macrophage,TAM),which mainly exhibits the M2 type macrophage phenotype,is the cell component with the highest proportion of immune cells in most tumor microenvironments.Anti-tumor therapy targeting TAM come to receive more and more attention in recent years.TAM activates the immunosuppressive response,induces tumor invasion and angiogenesis,and promotes tumor growth and metastasis.The polarization of M2macrophages is regulated by a variety of cytokines in the microenvironment.Studies have shown that IL-33(Interleukin 33)interacts with its receptor ST2(Suppression of tumorigenicity 2).It promotes the polarization of M2 type macrophages.Our previous studies also proved that the IL-33/ST2 axis is essential for the activation of LPS-stimulated macrophages.Therefore,the discovery of the effect and mechanism of IL-33/ST2 axis on macrophage polarization will provide new possible targets for the phenotypic transformation of macrophages,and further provide theoretical basis and feasible strategies for new tumor immunotherapies targeting macrophages.There are significant differences in the energy metabolism of macrophages with different phenotypes,and different ways of energy metabolism determine the different phenotypes and functions of macrophages,showing different effects on tumor growth.M1 macrophages with anti-tumor effects mainly use aerobic glycolysis for rapid energy supply(similar to the"Warburg effect"of tumor cells),while M2macrophages with tumor-promoting effects mainly rely on mitochondrial oxidative phosphorylation function.Studies have shown that IL-4 enhances mitochondrial oxidative phosphorylation by activating STAT6 and PGC1?to promote the polarization of M2,suggesting that energy metabolism is an important factor in regulating the polarization of macrophages.In order to maintain normal energy metabolism and life activities of cells,when damaged or under stress conditions,mitophagy often occurs to ensure the quality of mitochondria,maintain mitochondrial function homeostasis,redox balance,and meet the needs of mitochondrial metabolism.For macrophages,the level of mitophagy will be adjusted according to the energy metabolism requirements of different functions,so as to promote their conversion to different phenotypes.Studies have shown that when tissues are damaged,macrophages will reduce the occurrence of mitophagy and promote the transformation to M2 to meet the needs of tissue repair.Therefore,elucidating the regulation mechanism of energy metabolism on macrophage polarization is the basis for probing the mechanism of macrophage polarization affecting tumor growth.As a pleiotropic cytokine,IL-33 can promote the polarization of M2 type macrophages,but the mechanism is not clear.The connection between IL-33 and macrophages is very important for all stages of the immune response,including the initiation,maintenance and final resolution stages.It is reported in the literature that IL-33 may change the phenotype of macrophages through the classic ST2/MYD88/IRAK1/4 pathway,or through the combination of full-length IL-33 and transcription factors.Our previous research has shown that energy metabolism is a core participant in macrophage polarization.IL-33 is a member of the IL-1 family that affects the activation and polarization of macrophages.The signal axis of IL-33 and its receptor ST2 regulates mitochondrial biosynthesis-related PGC1?(peroxisome proliferator-activated receptor?coactivator-1?,)affects the energy metabolism mode of macrophages,thereby enhancing the LPS(Lipopolysaccharide)response of macrophages,suggesting there is a close relationship with the IL-33/ST2 axis,macrophage energy metabolism and macrophage polarization.In addition,the IL-33/ST2 pathway is involved in many metabolic processes,which can enhance the glucose uptake of non-small cell lung cancer cells by up-regulating the glucose transporter 1(Glucose transporter 1,GLUT1).Therefore,from the perspective of energy metabolism model reprogramming,the IL-33/ST2 axis can explain the regulatory effect and mechanism of the M2-type polarization of macrophages.It provides new ideas for proving that IL-33/ST2 axis regulates macrophage polarization and affects tumor growth.In this study,we examined the in vivo growth of transplanted tumors in transgenic mice,as well as the phenotypic transformation,energy metabolism changes,and mitophagy of bone marrow-derived macrophage(BMDMs)derived from transgenic mice.The expression and localization of related proteins,and the role and mechanism of IL-33/ST2 axis regulating M2 polarization to promote tumor growth through energy metabolism reprogramming were discussed,in order to clarify the role of the mechanism of IL-33/ST2 axis regulating macrophage polarization and provide the theoretical basis and feasible strategies of anti-tumor therapy targeting macrophages.Methods:(1)Subcutaneously inoculate mouse melanoma cells(B16)and mouse liver cancer cells(H22)on the back of wild-type(Wild-type,WT),ST2 knockout (ST2^-/-)and IL-33 overexpression(IL-33 overexpression)mice,construct a transplanted tumor model to verify the effect of IL-33/ST2 pathway on tumor growth in vivo;tissue immunofluorescence staining detects the expression of M2 macrophage markers F4/80 and CD206 in tumor tissue,and evaluates the infiltration of M2 macrophages.(2)Detect the expression of M2 type marker genes in BMDMs of WT,ST2^-/-and IL-33 overexpression mice by q PCR to evaluate the M2 polarization of BMDMs.Detect glucose uptake and lactic acid production,extracellular oxygen consumption rate and lactic acid acidification rate of BMDMs to evaluate glucose metabolism indexes of BMDMs in WT,ST2^-/-and IL-33 overexpression mice.(3)Use Mito Tracker Red flow cytometry staining and fluorescence staining to detect the changes in the number of mitochondria in BMDMs of WT,ST2^-/-and IL-33 overexpression mice.VDAC1,Cytc,COX?,LC3?/LC3?,PINK1,Parkin and p62 protein expression were detected by Western blot.The co-localization of Parkin and VDAC1 was detected by immunofluorescence to evaluate the occurrence of mitophagy in BMDMs.(4)Use JC-1 and Mito SOX staining to detect the mitochondrial membrane potential(MMP)and mitochondrial ROS(mt ROS)production of BMDMs in WT,ST2^-/-and IL-33 overexpression mice;Detect p70s6k and P-p70s6k protein expression by Western blot to evaluate the activation of mTOR.Results:(1)Compared with WT mice,the average tumor volume and weight of melanoma xenograft tumors in IL-33 overexpression mice have increased,and M2 macrophage infiltration increased;The average volume and weight of melanoma and liver cancer xenograft tumors in ST2^-/-mice are reduced,and the infiltration of M2 macrophages is reduced.The IL-33/ST2 axis promotes tumor growth by promoting the polarization of M2 macrophages.(2)Compared with BMDMs in WT mice,the expression of M2 marker genes in ST2^-/-mice BMDMs decreased,and the expression of M2 marker genes in BMDMs in IL-33 overexpression mice increased.The IL-33/ST2 axis promoted BMDMs to M2 type polarization.(3)Compared with BMDMs in WT mice,BMDMs in ST2^-/-mice have increased mitophagy and decreased mitochondrial numbers.The energy metabolism of BMDMs is more dependent on glycolysis.After chloroquine(CQ)is used to inhibit lysosomal function,the glycolysis of ST2^-/-BMDMs is reduced;IL-33 overexpression mice BMDMs reduces mitophagy and increases the number of mitochondria.The energy metabolism of BMDMs depends on oxidative phosphorylation.(4)Compared with BMDMs in WT mice,there was no significant difference in the production of MMP and mt ROS in ST2^-/-or IL-33 overexpression mice BMDMs;the ratio of mTOR downstream protein P-p70s6k/p70s6k in IL-33 overexpression mice BMDMs increase,the result in ST2^-/-BMDMs is opposite.Conclusions:(1)The IL-33/ST2 axis promotes the growth of transplanted tumors in mice by increasing the infiltration of M2 macrophages.(2)The IL-33/ST2 axis regulates energy metabolism reprogramming,and promotes the polarization of bone marrow-derived macrophages(BMDMs)to M2 by enhancing oxidative phosphorylation.(3)The IL-33/ST2 axis may change the level of mitophagy by regulating the activation of mTOR,thereby maintaining mitochondrial homeostasis and mediating the energy metabolism reprogramming of BMDMs.In summary,this study clarified the role and mechanism of IL-33/ST2 axis by inhibiting mitophagy to promote the oxidative phosphorylation of BMDMs in mice and increase the polarization of BMDMs to M2 to promote tumor growth.It provides theoretical basis and feasible strategies for elucidating the mechanism of IL-33/ST2axis regulating macrophage polarization and anti-tumor therapy targeting macrophages.