Study On The Effect Of Babesia Microti Against Melanoma Growth

Babesia microti is an obligate intraerythrocytic protozoan transmitted by Ixodes tick,it mainly infects small rodents and can also infect humans.Experimental infected mice usually eliminated the parasite on day 17 post-infection,and the animal will eventually survive and shows no clinic symptom,which is pretty similar with the infection in health human.According to the study of protozoa and tumors,it has been found that some protozoa have antitumor effects by activating immune response,inhibiting tumor cell proliferation and angiogenesis.Some protozoa such as Plasmodium yoelii and Toxoplasma gondii have been confirmed to have inhibitory effects on various tumors such as melanoma,ovarian cancer and lung cancer in mice.The infection of B.microti will stimulate complex immune response in host body.Th1-type immune response plays a leading role in eliminating Babesia,and macrophages are also an indispensable key factor in infection control.Th1-type immune response and activation of macrophages also take a part in the regulation of the occurrence and development of tumors.Melanoma is a malignant tumor caused by malignant transformation of melanocytes,and melanoma is one of the most sensitive tumors to immune regulation.Therefore,in this study,B.microti was used to study the development and effect of melanoma in mice.In this study,mice were first injected with B16 cells for melanoma tumor,B.microti was used to treat the tumor-bearing mice at the same time.The results demonstrated that the occurrence and growth of melanoma did not affect the parasitemia of B.microti,but B.microti could significantly inhibit the growth of melanoma and prolong the survival time of tumor-bearing mice.On the 6th day after inoculation with B.microti,the spleen of the mice was significantly enlarged.The splenocyte of the 3rd,6th,12 th,and 17 th days after the inoculation were analyzed by flow cytometry.It was found that on the 3rd day,the proportion of CD4+ T cells,CD8+ T cells,macrophages and NK cells in splenocytes did not change.On the 6th day,the proportion of CD4+ T cells,CD8+ T cells,and NK cells in splenocytes all decreased,but with the course of infection,the percentages of CD4+and CD8+ T cells gradually returned to normal,while the percentages of NK cells remained lower than normal,and the percentages of macrophages increased significantly.These results indicate that B.microti infection significantly stimulated the immune system of mice,and the number of immune cells in the spleen increased significantly,especially the increase in the proportion and number of macrophages.At the 17 th day of infection,the expression of CD31 and the proportion of CD4 + T cells in the tumor appeared significantly decreased,and the proportion of macrophages did not change significantly,but the expression of MHC-Ⅱ in macrophages was elevated,and the expression of CD206 was decreased,which suggested that B.microti infection inhibits tumor angiogenesis,alters the tumor immune microenvironment,recruits CD4 +T cells to the tumor,and reduces M2 macrophage numbers.A total of 255 differential genes were screened by transcriptome analysis of tumor at the 12 th day after inoculation,including 93 up-regulated genes and 162 down-regulated genes.The pathway enrichment analysis showed that the differential genes were mainly concentrated in signal transduction,metabolism,immune system and other related pathways.Transcriptomics data revealed that B.microti infection may inhibit the growth of melanoma by affecting the host’s immune system and metabolic processes.The phagocytosis of macrophages was determined by indirect immunofluorescence assay,the results showed that macrophages could eliminate the parasites by phagocytosis.In the meanwhile,q PCR results indicated that B.microti and its in vitro culture supernatant could induce the high expression of M1 type polarization markers(TNF-α,i NOS,IL-6)in both mouse peritoneal macrophages and RAW264.7 cells.In addition,the culture supernatant of B.microti can convert IL-4 induced M2 type macrophages into M1 type.In summary,B.microti was used to treat melanoma mice,the results showed that the growth inhibitory effect of B.microti can significantly inhibit tumor size and prolong the survive period of mice.Flow cytometry,immunohistochemistry and parasite-stimulated macrophage polarization assay suggested that B.microti infection can activate the immune response,change the spleen and tumor immune environment,stimulate the macrophages to M1 type and reverse the M2 type macrophages to M1 type.These provide new ideas and references for further study of the immune response of B.microti in host and antitumor microbial immunotherapy.