| Objective To Analyze the effect of Penicillium marneffei (PM) secondary metabolites on morphology, activity and polarization of mouse macrophage cell line RAW264.7, and to explore the role of PM secondary metabolites in the pathogen resistant to host immune defense.Methods The culture filtrates was isolated from yeast culture of PM shaking cultivated in DMEM at 37℃, then RAW264.7 were cultured in DMEM with PM culture filtrates on different concentration. (1) Morphology of macrophages were observed by light microscopy; (2) Phagocytic activity of macrophages were observed by periodic acid-schiff stain; (3) The effect of PM culture filtrates on toxicity and proliferation of macrophages were assayed by WST-8; (4) The effect of PM culture filtrates on apoptosis and necrosis of macrophages were analyzed by Annexin V-FITC/PI flow cytometry. (5) The effect of PM culture filtrates on cytokines (TNF-α, IL-1β,IL-12, IL-10) secretion in macrophage were detected by ELISA; (6) The effect of PM culture filtrates on macrophage CD 16/32 and CD206 expressions were analyzed by immunofluorescence.Result (1) RAW264.7 were cultured in DMEM with PM culture filtrates appeared as growth density decrease, cell enlarged, cell swollen and rounded, even a few macrophages appeared cytoplasmic vacuoles and cell disintegration. (2) Macrophages were cultured with PM culture filtrates for 24 hours on the concentration varied 10% to 50%, then the phagocytic rate and phagocytic index were calculated respectively in the treated groups and the control. The average phagocytic rate and phagocytic index in treated groups were 83.687 ± 6.279 and 5.969 ±1.170 respectively after 24 h, and they were 78.402 ± 5.522 and 6.602 ± 0.784 respectively after 48 h. They were significant differently in comparison with the control (P<0.05). (3) The OD values were assayed by WST-8 test. The average of OD values were 0.996 ±0.152 and 0.564 ± 0.269 respectively after 24 h and 48 h in treated group, which have significant decreased in comparison with the control group (24 h P<0.05,48 h P<0.001). At 24 h, the average of survival rate(%), apoptosis rate(%) and necrosis rate(%) of macrophages in the treated group were 76.41±10.99,9.13 ±4.10 and 11.03 ± 8.34 respectively. At 48 h, they were 58.91 ± 21.00,13.18 ± 5.51 and 18.14 ± 12.73 respectively. In comparison with the control, survival rate significantly decreased (P<0.05), apoptosis rate significantly increased (P<0.05) and necrosis rate has no statistical significance (P>0.05). (5) The concentrations (pg/ml) of TNF-α, IL-1β, IL-12 and IL-10 in the treated group at 24 h were as followed:1807.300 ± 193.759,4.769 ± 3.736,3.180 ± 1.141 and 35.431 ± 16.005 respectively. In comparison with the control, TNF-a, IL-10 and IL-12 significantly increased (P <0.05), and concentrations of IL-1β significantly decreased (P<0.01). At 48 h, the concentrations of TNF-α, IL-1β,IL-12 and IL-10 followed as 1491.307±695.238,7.153±4.128,4.084±1.631 and 20.878±11.696 respectively. In comparison with the control, TNF-a, IL-10 and IL-12 significantly increased (P<0.01), IL-1β significantly decreased (P<0.01), and IL-12 have no statistical significance (P>0.05). (6) Macrophages that cultured in 20% PM culture filtrates expressed CD206, but not CD16/32. On the other hand, macrophages that cultured with PM conidia expressed CD206.Conclusion (1) The secondary metabolites of PM culture filtrates can activate macrophages to express CD206 and to secrete TNF-a and IL-10. (2) The secondary metabolites of PM culture filtrates activate macrophages in the early stage(24 h～48 h) may be associated with CD206 expression and TNF-a secretion, which increase the chemotaxis of macrophages to engulf more PM cells. However, the secondary metabolites of PM culture filtrates may have toxic effect on macrophages and induce apoptosis and necrosis of macrophages. |
PDF Full Text Request