The Effects Of CMPK2 On Macrophage Polarization And Anti-tumor Immunity

Macrophages are important immune cells that spread throughout the body.Macrophages can phagocytize and present antigens,remove pathogens and cell debris from the body,and participate in adaptive immunity and innate immunity.Macrophages are involved in many physiological and biochemical processes,such as organ homeostasis,tissue development and repair,pathogen resistance,chronic inflammation,fibrosis and cancer development.Macrophages are highly heterogeneous and plastic.The M1 and M2 polarization of macrophages is the process in which macrophages change to specific phenotypic and get functional responses to stimuli and signals in their own environments.The activation of macrophages is reversible and the popμlation of macrophages is dynamic,as the polarization of macrophages is continuous.The dichotomy of macrophage polarization is an simplification of the complex process of continuous changes in the functional state.Classically activated macrophages,M1 type macrophages,appear in an inflammatory environment dominated by Toll-like receptors(TLR)and interferon signal transduction.They participate in the body’s immune response against bacteria and intracellular pathogens.They usually secrete pro-inflammatory cytokines,like IL-1β、IL-12 and TNF-α.Alternative activated macrophages,M2 type macrophages,are usually induced by Th2response-dominated environments such as worm infections,asthma and allergies.They produce and secrete more IL-10 and TGFβ and other cytokines,which participate in the elimination and removal of parasites and suppression of inflammation.CMPK2 is a monophosphate kinase located in mitochondria,which participates in the Salvage Synthetic pathway of mitochondrial DNA,using ATP as a phosphate donor to phosphorylate pyrimidine nucleoside such as CMP、UMP、d CMP、d UMP and dd C、d Fd C、ara C 、 BVDU.There are several articals showing that CMPK2 have founction in the resistance of virus.CMPK2 affects the transcription and translation of viral genes by participating in the antiviral response of interferon,and then regμlates the innate immune response of pathogen infection.In a recent study,scientists demonstrated that CMPK2-dependent mt DNA synthesis is required in the activation of NLRP3 inflammasome.CMPK2 in mice was first obtained from a c DNA library isolated and prepared from RAW264.7 after LPS treatment,and was named Tyki.After stimulated by LPS or proinflammatory cytokines(such as TNF-α 、 IFN-γ 、 IL-1β and IFN-α),theexpression of CMPK2 was significantly up-regμlated.In order to explore whether CMPK2 affects the polarization of macrophages,we prepared CMPK2-deleted macrophages from mice.On the other hand,we constructed CMPK2 overexpressing plasmid and obtained Raw264.7 cells with stable overexpression of CMPK2 by plasmid transfection and G418 drug screening.Macrophages polarized in different directions were induced by LPS/IFN-γ or IL4 stimulation.The m RNA expression of polarization-related genes such as i NOS and Arg1 were detected by Quantitative Real-time PCR.The results showed that CMPK2 deletion increased the expression of M2 related genes and suppressed the expression of M1 related genes in primary macrophages.In the CMPK2-overexpressing cell lines,the results were consistent with that obtained in CMPK2-deletion cells.The cytokines secretion in polarized macrophage was detected by ELISA.And it’s showed that the secretion of IL12、 IL6 and TNF-α was decreased in M1 macrophages,while the IL10 secretion was increased in M2 macrophages,when CMPK2 was depeleted.The state of macrophages polarization was detected by flow cytometry.The deletion of CMPK2 inhibited the expression of MHCII in M1 macrophages and increased the expression of CD206 in M2 macrophages.To investgate the changes in polarization-related signal pathways,we used Western Blot to detect the activation state and expression of related proteins.After CMPK2 was knocked out,LPS/IFN-γ-induced phosphorylation of STAT1 、 p38 and AKT were reduced.Meanwhile,in CMPK2 overexpressing cells,LPS/IFN-γ-induced phosphorylation of STAT1,p38 and AKT were increased.In IL4-stimulated macrophages,CMPK2 deficiency increased GSK3 phosphorylation and CMPK2 overexpression inhibited GSK3 phosphorylation.According to the experimental results,we concluded that CMPK2 participates in the polarization of macrophages by regulating STAT1 and GSK3 phosphorylation.Tumor-associated macrophage(TAM)is involved in the progression and development of tumor,metastasis,prognosis and other aspects.In most tumors,such as breast cancer,cutaneous melanoma and lung cancer,TAM shows M2-like macrophage phenotypes,and is related to poor prognosis.We searched tumor related databases and found that CMPK2’s expression is different in a variety of tumors.We selected melanoma as the research object and found that CMPK2 promotes the infiltration of CD4+T cells,CD8+T cells,neutrophils,macrophages and dendritic cells in melanoma,which is related to the prognosis of melanoma.Combining the results of the bio-information analysis and the results above,wepredict that the CMPK2 may affect anti-tumour immunity against melanoma by regulating the polarization of TAM.We used tumor-bearing model by subcutaneously injecting B16F10 into mice to monitor the the effect of CMPK2 on tumor growth.The experimental results showed that the CMPK2 knockout mice had larger tumors than the control wild type,consisting with our previous prediction that knockout CMPK2 promotes tumor growth.We isolated the tumor tissues of tumor-bearing mice and inspected the distribution and states of immune cells in the tumor by flow cytometry.We observed that the TAM in CMPK2 knockout mice,compared with that control mice,have more M2 polarization markers expressed.The ratio of CD4 and CD8 in tumor-infiltrating T cells were reduced.In brief,we found that CMPK2 can regulate the phosphorylation of STAT1 and GSK3,affect the polarization of macrophages,and affect tumor growth by regulating the tumor immune microenvironment.