Macrophages Polarized By Expression Of ToxoGRA15_â…¡ Inhibit Growth Of Hepatic Carcinoma

Background Hepatocellular Carcinoma(HCC)is one of the most common maliganceies in the world with a morality rate which ranks second.In recent years,tumor immunotherapy,which aims to improve the body’s own immune function by breaking the body or tumor local immune tolerance or immunosuppression,has become a new concept of tumor bearing living and one of the important treatment strategy.Tumor immunotherapy can activate the body’s immune system,reconstruct of normal immune warning function,has many other merits such as accurate targeting,low side effects and immunological memory.In the tumor inflammatory microenvironment,tumor-associated macrophages(TAMs)are closely related to the occurrence and development of solid tumors.Studies have shown that the classically activated macrophages phenotype(M1)has a high antigenpresenting ability,play a positive immune regulation and cytotoxicity,while alternatively activated macrophages phenotype(M2)has weaker antigen-presenting ability,and involves in the negative immune regulation.Toxoplasma gondii is an obligatory intracellular parasite that affects any warm-blood animals including humans.Recent studies have showed that T.gondii strains have a rich genetic diversity in geographical regions around the world.Type I(RH,GT1),type II(PRU,ME49),and type III(CTG)are widely distributed in Europe and North America.In the process of anti-Toxoplasma infection,macrophages are the first barrier,and closely related to parasite proliferation and spreading,and the consequences of infection.Macrophages infected with type II strain of Toxoplasma are classically activated.This isdue to its dense granule protein GRA15 II,which directly activates nuclear factor(NF)-κB,drives macrophage to M1 polarization and evokes Th1 type immune response.Objective To explore the bias of tumor-associated macrophages(TAMs)to classically activated macrophages(M1)induced by Toxoplasma derived polymorphic effector(Toxo GRA15II)in vitro and its inhibitory impact on the proliferation,invasion,and metastasis of hepatic carcinoma in tumor-bearing mice.Methods Toxo GRA15 II coding sequence was amplified from T.gondii PRU strain(genotype II)and recombinant plasmids p EGFP-gra15 II was constructed.The plasmids were subcloned into lentivirus(Lv)vector,with which RAW264.7 cells were infected.In vitro,transwell co-culture of hepatic cancer cell line Hepa1-6 was performed with Lvgra15Ⅱ-Macs(group A),vector Lv-Macs as mock(group B)and macrophages without viral infection as control(group C).Proliferation,metastasis,and invasion of cocultured Hepa1-6 cells and expression of MMP-9 and MMP-2 were examined.In vivo,seventy-five C57BL/6 mice(4-5w,female)were subjected to tumor cells plantation in right groin.The animals were randomly divided into 3 groups after 3 days of tumorigenesis,with 25 in each.Mice of group A received transfusion of 2×106 Lvgra15Ⅱ-Macs in 150μl saline on 3d and 5d via tail vein;similarly,animals of group B were injected with 2×106 Lv-Macs;and group C were given equal volume of normal saline.All animals were sacrificed under euthanasia 10 d after subdermal plantation of Hepa1-6.We tracked the fate of the transferred macrophages in vivo and oberserved the effect of transfused macrophages on the spleen.Tumor and spleen tissues were removed for detections of size,TAMs infiltration,cytokine expression and angiogenesis byimmunohistochemistry;q RT-PCR was run for m RNA expression of i NOS,TGF-β,VEGF and IL-10;IL-6 m RNA and protein expression was examined by q RT-PCR and ELISA,respectively.Results Toxo GRA15 II drove RAW264.7 cells to M1 polarization.Macrophages infected with Lv-gra15Ⅱ produced high levels of PD-L1,CD80,TNF-α,IL-12p40,i NOS and nitric oxide(NO)determined by either FCM or culture supernatants,(P <0.05).In vivo experiments revealed that transfusion of Lv-gra15Ⅱ-Macs attenuated infiltration of TAMs and induced bias of TAMs towards M1,resulting in alleviated growth of the planted tumors(P <0.05).The results of frozen sections showed that the transfected macrophages circulated into solid tumor and survived for at least 72 h.Additionally,immunohistochemistry of solid tumors showed a down-expression of IL-10,TGF-β and VEGF,and an up-expression of MHC II and i NOS examined by q RT-PCR(P <0.05).Particularly,IL-6 expression,which is closely associated with the cancer malignant behaviors,was significantly dampened in tumor tissues(P <0.05).The results of spleen frozen sections indicated that Toxo GRA15 II activated macrophages transfusion may restrict tumor growth via enhancing systemic immunity of the tumor-bearing host.Spleen q PCR test showed that mice injected with LV-gra15II-Mφ exhibited notable increase in m RNA transcription of TNF-α(P < 0.01)and IL-12(P < 0.05),and decrease in IL-6(P < 0.01)and IL-10(P < 0.01),in comparison to the animals injected with LVMφ.Hepa1-6 cells co-cultured with Lv-gra15Ⅱ-Macs showed remarkable inhibition of migration and invasion,and decreased expression of MMP-9 and MMP-2,without notable apoptosis of Hepa1-6 cells.Compared with groups B and C,mice of group A gave rise to a decreased level of MMP-9 and MMP-2 tested by q RT-PCR and Western blotting(P <0.05).Conclusion Our results indicate that the effector molecule of Toxo GRA15 II is able to induce macrophage polarization to M1 that has a restrictive effect on tumor growth via its related cytokines profile in tumor and spleen tissues.Besides,Toxo GRA15 II,due to its early activation of specified cell population and non-toxicity to mammalians,has a potential value for a novel therapeutic strategy of enhancing host innate immunity against tumor development.