MIF Regulates The Immune Microenvironment Of Brain Metastases From Lewis Lung Cancer By Inhibiting The Phenotypic Transformation Of M2 Macrophages

ObjectiveOur previous studies showed that inhibition of MIF/CD74 signaling pathway can promote the transformation of intracranial macrophages after radiotherapy activation from m2 to M1 type,and play a role in radiosensitization.It is reported that M2 macrophages are the main components of tumor immunosuppression microenvironment,and the surface marker of the macrophage,arg-1,can decompose the essential arginine in the growth and development of T cells.Therefore,this paper will focus on the mechanism of inhibiting MIF through regulating macrophages to influence tumor infiltrating lymphocytes in radiotherapy of brain metastasis.MethodsMIF-Knockdown in Lewis cells were constructed by lentiviral sh RNA interference technique.The secretion of MIF in Lewis cell supernatant was detected by ELISA kit.The expression levels of MIF in Lewis and i NOS(M1 type marker),Arg-1(M2 type marker)in BV2 cells were detected by Western Blotting.The RNA expression levels of M1 and M2 tags were detected by qPCR.The expression of i NOS,Arg-1 and Iba-1in lung cancer BMs model mice was detected by immunofluorescence staining.The content of arginine in tumor tissue and cell supernatant was detected with Arginine kit.The proportion of TILs in vivo and in vitro was measured by flow cytometry.The proliferation of primary T cells was tested by CFSE.BMs tumor growth in mice was observed by Small Animals Imaging Technology.Result1.In animal experiments,WB、PCR and IF results showed that MIF knockdown(sh-MIF)combined with whole brain irradiation(IR)can raise the expression of i NOS,while the Arg-1 lessend,suggesting that downregulation of MIF can promote M1 phenotype and reduce M2 phenotype.Flow cytometry results showed that sh-MIF combined with IR increased the proportion of CD8+T/CD4+T and CD8+IFN-r+T/CD4+Foxp3+T cells.Small Animals Imaging results showed that inhibition of MIF enhanced the shrinkage of tumor induced by whole brain irradiation.After clearance of macrophage,Flow cytometry showed no difference between the sh-MIF group and the control group(NC).Small Animals Imaging results also showed that the inhibition of tumor growth by sh-MIF was less significant than macrophages presence.It is indicated that down-regulation of MIF expression combined with irradiation promote T cell infiltration and inhibit tumor may be mediated by macrophages.Arginine kit results showed that the content of arginine increased after IR or sh-MIF,while it increased overall after macrophage clearance,but the difference between each groups was not significant,suggesting that IR or sh-MIF could increase the content of arginine by reducing M2-type macrophages.Flow cytometry and animals imaging results showed that the CD8+/CD4+TILs ratio was increased and tumor growth was inhibited after the addition of exogenous arginine.In conclusion,inhibition of MIF can reduce M2-type macrophages,then increase the content of arginine to improve the proportion of TILs,and enhance the killing effect of IR on BMs.2.In cellular experiments,sh-MIF Lewis cell co-culture with BV2 cells activated by radiation of 24 h.The results of WB,PCR demonstrate that compared with the NC,inhibition of MIF can increased the i NOS expression significantly,decreased Arg-1expression markedly.Arginine kit is used to measure the above groups of cells supernatant,the results showed the change of arginine content is opposite to the Arg-1protein expression.It is suggested that inhibition of MIF can reduce the expression of Arg-1+M2 macrophages and increase the extracellular arginine level.The results of flow cytometry showed that,after treating primary T cells extracted from BMs with co-cultured supernatants,both sh-MIF and irradiation could increase the ratio of CD8+T/CD4+T cells,and the effect of combination was more significant,while the addition of arginine was the best.CFSE results showed that both sh-MIF and irradiation could promote the proliferation of T cells,the combination of the two is even better,while the exogenous arginine supplementation has the most obvious effect.ConclusionInhibitory MIF expression can reduce the expression of Arg-1 from M2 type macrophages in TME,thus raise the content of arginine,promote the infiltration of lymphocytes in tumors,improve the immune microenvironment,achieve radiotherapy sensitization finally.