The Effcets Of FB₁ On The Maturation And Immune Function Of BMDCs

Fumonisin B1(FB1)is one kind of mycotoxins that has the neurotoxicity,carcinogenicity,hepatotoxicity and immunotoxicity produced by the fungus Fusarium verticillioides,which commonly infects corn and otherProductions and is harmful to animal and human health upon consumption of FB1-contaminated feed or food.However,the mechanism of immunotoxicity,especially the immunosuppresion induced by FB1 is still unclear.The most pivotal cells in the induction of immune responses are dendritic cells(DCs).To our knowledge,the effects of FB1 on BMDCs have never been reported before.In this study,we used murine bone marrow-derived dendritic cells(BMDCs)as a model system to elucidate the effect of FB1 on the immune function of BMDCs through biological methods.And the morphology,endocytosis,T-cell stimulatory capacity,expresion of surface molecules,cytokine and NO production were studied.Test I Culture of murine bone marrow-derived dendritic cells in vitroThe BMDCs induced by cytokine GM-CSF(10 ng/mL)and IL-4(10 ng/mL)cultured for 7 days.After the clusters of DC and many dendritie-like were observed under light microscope.The purity of BMDCs can reach above 80%by flow cytometry,which achieve the following research requirements.Test II Effect of FB1 on the phenotype and the morphological changes of murine bone marrow derived dendritic cellsThe cultured cells were treated in experiment.Immature BMDCs were exposed for 6 h with increasing concentrations(250,500,1000 ng/mL)of FB1 before adding LPS(1 ug/mL)for further 18 h.In each experiment,the untreated BMDCs and BMDCs incubated with LPS alone were used as negative and positive control,respectively.Data are representative of three independent experiments.In vitro cytotoxicity of FB1 on BMDCs was determined by CCK-8 assay and the morphological changes of BMDCs were observed by transmitted electron microscopy(TEM)and scanning electron microscope(SEM).The expression of key surface molecules on BMDCs were confirmed by Flow cytometry(FCM).The results showed that FB1 reduced the viability of BMDCs.LPS and 250ng/mL FB1 did not exert any significant cytotoxic effects on BMDCs(P>0.05).However,500 and 1000 ng/mL of FB1 significantly reduced BMDCs viability comPared to the positive control(P<0.05).DCs can sample the antigens by extending their dendrites and use endocytic receptors to present antigens.There were many phagosomes and dendrites in control group.With the proces of maturation of BMDCs,the number of dendrites increased markedly in the LPS-activated control.However,the number of phagosomes decreased.After pre-treatment with 1000 ng/mL of FB1,the number of dendrites decreased markedly compared to the LPS-treated control,but the number of phagosomes increased.These observations indicated that 1000 ng/mL of FB1 was able to reverse the morp hological changes of LPS-activated BMDCs.The expressions of CD80,CD86 and MHC Ⅱ were up-regulated after exposure to LPS(1 ug/mL)for 18h.However,when pre-treatment with 1000 ng/mL of FB1,the expressions of CD80,CD86 and MHC Ⅱ were significantly reduced compared to positive control(P<0.05).Meanwhile,the expresions of CD80,CD86 and MHC II in 250 and 500 ng/mL group were not significantly decreased compared to positive control(P>0.05).Test Ⅲ Effect of FB1 on immune function of murine bone marrow-derived dendritic cellsThe cultured cells were treated in experiment.Immature BMDCs were exposed for 6 h with increasing concentrations(250,500,1000 ng/mL)of FB1 before adding LPS(lug/mL)for further 18 h.In each experiment,the untreated BMDCs and BMDCs incubated with LPS alone were used as negative and positive control,respectively.Data are representative of three independent experiments.The phagocytosis of BMDCs were analyzed by FCM.Cytokine assay were analyzed by by ELISA and NO production were analyzed by Nitric oxide(NO)assay.Besidesthe T-cell stimulatory capacity of BMDCs were evaluated by mixed lymphocyte reaction(MLR).The results showed that the immature DCs progressively lose their antigen uptake function until the mature stage.After exposure to LPS(1 ug/mL)for 18h,the percentage of double positive cells(CDllc xdextran-FITC)was decreased from 47.70 to 15.90.However,FB1 increased the endocytosis capability of LPS-stimulated BMDCs.After explore the 1000 ng/mL of FB1,the percentage of double positive cells(CDllcxdextran-FITC)was increased to 26.00;IL-6 and IL-10 were significantly down-regulated after pre-treatment with 500 and 1000 ng/mL of FB,comPared to the positive control(P<0.01).Similarly,IL-12 was also significantly decreased when pre-treatment with three different concentrations of FB1(P<0.01).However,TNF-a was significantly up-regulated after pre-incubated with FB1(250 and 500 ng/mL)compared to the positive control(P<0.01);NO productionwere significantly down-regulated after pre-treatment with 250.500 and 1000 ng/mL of FB1 compared to the positive control(P<0.01).500 and 1000 ng/mL of FB1 significantly inhibited the ability of LPS-induced BMDCs to induce the proliferation of T cells at ratios of 1:10,1:20 and 1:40(P<0.01).It was considered that the immunosuppresive effects of FB1 were mainly caused by changing the morphology and interfering with the process of antigen uptake,processing and presentation.We demonstrated that FB1 had the capacity to modulate the immune responses of BMDCs,especially the adaptive immunity.