| Objective: The present study was designed to evaluate whether and how itraconazole(ITZ)can affect the macrophage polarization and its phagocytic capacity to Candida albicans(C.albicans).Methods: Mouse peritoneal macrophage-derived RAW264.7 cell line was used in all the experiment below.Groups with itraconazole 0.25,0.5,1 treated were set as experimental groups,and without the itraconazole treated group was set as control.The group without lipopolysaccharides(LPS),interleukin-4(IL-4)or C.albicans and itraconazole treated was set as negative control.(1)Cell toxicity was measured using Cell counting kit-8(CCK-8)assay with 0.0625,0.125,0.25,0.5,1,2,4,8,16 ?M itraconazole and cell vitality was detected in the time point of 12,24 and 48 h.(2)UV was used to kill Candida albicans.(3)RAW264.7 cells: fluorescein isothiocyanate(FITC)dyed Candida albicans at ratio 1:4 and 1:8 were used for the quantitative detection of itraconazole on RAW264.7 cells phagocytosis by flow cytometry.(4)Qualitative detection of different concentrations of itraconazole on RAW264.7 cells phagocytosis by confocal laser scanning microscopy(CLSM)using red fluorescent cell link kits PKH 26 dyed RAW264.7 cell membrane and FITC dyed Candida albicans.(5)Levels of RAW264.7 cells secreted cytokines induced by LPS,IL-4 or C.albicans and treated with itraconazole were measured by Luminex or Cytometric Bead Array(CBA).(6)The expression of inducible nitric oxide synthase(i NOS)and arginase-1(Arg-1)were determined by western blot.Results:(1)In comparison to the control,itraconazole significantly inhibited the growth of the cells in both a time-and a dose-dependent manner as confirmed by CCK-8: the concentration of itraconazole at 1 ?M 12 h,there was no obvious cytotoxicity to RAW264.7 cells,but in the condition of 2 ?M 12 h the inhibitory effect is 14.57 ± 6.96% and 1 ?M 24 h showed 53.61 ±1.19% inhibition.After the treatment of 1 ?M 48 h and 2 ?M 48 h,the inhibitory effect was more than 90%,so 0.25,0.5,1 ?M 12 h were selected as the following experimental condition.(2)Itraconazole significantly enhanced the phagocytosis of RAW264.7 cells to C.albicans in the concentration of 0.5 and 1 ?M(P < 0.05)which were detected by FCM.(3)CLSM observation found that itraconazole significantly enhanced phagocytic capacity of RAW264.7 cells to C.albicans.(4)Itraconazole enhanced the secretion of interleukin-6(IL-6)and tumor necrosis factor-?(TNF-?)in LPS induced RAW264.7 cells.(5)In the IL-4 induced RAW264.7 cells,itraconazole increased IL-6,TNF-?,and IL-1? while the secretion of IL-10 decreased.(6)In Candida albicans stimulated RAW264.7 cells,the secretion of IL-6 and TNF-? increased while IL-4 and IL-10 were inhibited.(7)Western blot results showed that itraconazole increased i NOS and moderately inhibited Arg-1 expression under three different stimulis.Conclusion: High concentration of itraconazole has a toxic effect on macrophages(? 2 ?M).Itraconazole promotes macrophage phagocytosis and increased the secretion of IL-6 and TNF-? while decreased the secretion of IL-4 and IL-10 in LPS,IL-4,and C.albicans stimulated RAW264.7 cells which means that the macrophage differentiated to classically activated macrophage.Itraconazole not only has antifungal effects,but also has the function of immune activation.The double effects of over antifungal and immune activation promote the removal of fungi.The improvement of M1 polarization and enhancement of phagocytosis of fungi indicated its new application in clinic. |
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