The Function And Mechanism Study Of Gpr54 Mediated Macrophage Polarization In Tumor Formation And Development

Tumor associated macrophages?TAMs?in tumor tissues belong to innate immune cells,of which the functional phenotype is highly heterogeneous and plastic.TAMs are generally polarized into an immunosuppressive phenotype that promotes tumor growth in the tumor microenvironment.Therefore,it has become a hot spot and direction in the field of tumor immunotherapy by adjusting the functional state of macrophages to reprogam the tumor microenvironment and to improve the therapeutic effect of tumor.As the largest member of membrane receptor families in cells,G protein coupled receptors?GPCRs?regulate the physiological functions of macrophages by perceiving various signals in the extracellular environment.However,for such a large receptor family,the study on the regulation of macrophage function has just started.In order to explore the regulation of GPCR on macrophage function,we use RNAseq method to screen the differential expression of GPCR in the process of macrophage polarization.We found that G protein coupling receptor54?Gpr54,also known as Kiss1r?that involved in the development of puberty presented the opposite tendency in the M1/M2 state of macrophages,that is,expression of Gpr54 was upregulated in IFN-?induced M1 macrophage,but decreased in IL-4 induced M2 macrophages.Furthermore,we demonstrated that Gpr54 could significantly promote the expression of M1 related genes in macrophages by Gpr54 knockout and overexpression,ligand stimulation and signal transduction pathway that polarized macrophage toward M1 phenotype.However,we found that Gpr54 had no significant effect on the M2 polarization of macrophage.Then we detected the activation of the downstream JAK-STAT signaling pathway in IFN-?stimulated bone marrow derived macrophages from the Gpr54^+/+and Gpr54^-/-mice,respectively.Results showed that the knockout of Gpr54 greatly inhibited the JAK1-STAT1 signaling pathway activated by IFN-?.Taken together,our results indicate that Gpr54 promotes macrophage M1 polarization by enhancing the IFN-JAK1-STAT1 signaling pathway.In order to reveal the important role and significance of Gpr54 in the regulation of macrophage M1 polarization in the occurrence and development of tumor,we inoculated subcutaneously with murine melanoma cell line B16F10 to Gpr54^+/+and Gpr54^-/-mice to establish the model of melanoma.By monitoring and comparing the size of the tumor and the survival rate of mice,we found that the tumors of Gpr54^-/-mice were significantly greater than that of the control group and showed a lower survival rate accordingly.We then used immunofluorescence staining and flow cytometry to analyze the infiltration of the F4/80⁺MHC II⁺M1type TAM and the CD45⁺CD8⁺killer T cells in the tumor tissues.Results showed that the deletion of Gpr54 could change the equilibrium of M1/M2functional state of TAM in melanoma tumor tissue,that is,Gpr54 can promote the proportion of M1 type TAM and cytotoxic cells in tumor tissues.It will further enhance the effective anti-tumor immune response and inhibit the development of tumors.With the absence of Gpr54,the degree of infiltration of these cells in the tumor tissue will be greatly reduced,thus enhancing the proliferation of tumor cells and ultimately promoting the development of melanoma tumor tissue.Taken together,Our study reveals the important regulatory functions of the G protein coupled receptor Gpr54 in the functional regulation of macrophage,and proves that this regulatory function can have a far-reaching impact on the development of melanoma.Most importantly,our study reveals the new function of Gpr54 in the immune system that serves previously as a sex hormone regulating receptor.Our study may lead to the expension of therapeutic strategies of tumor immunotherapy and provide important research basis and theoretical reference for the establishment of the internal relationship between endocrine regulation and tumor occurrence and development.