The Effect Of Talaromyces Marneffei Infection On CD86 Expression In THP-1 Cells

Objective: To investigate the effect of Talaromyces Marneffei(T.marneffei)on CD86 expression in THP-1 cells after infection and discuss the potential mechanisms.Methods:1?The fungal strains were isolated from the skin tissue of patients with skin and soft tissue infections,and the isolates were respectively cultured at27? and 37? for morphological identification;2 ?DNA of the isolate was extracted,PCR amplification and gel electrophoresis were performed,PCR amplified products were sequenced and aligned on NCBI;3?Immunoelectron microscopy and immunohistochemistry were used to detect the CD86 expression on T.marneffei;4?Human mononuclear cells THP-1 were differentiated to THP-1-M? macrophages with PMA(Phorbol 12-Myristate 13-Acetate),then THP-1-M? macrophages were induced differentiating to M1 macrophages by LPS(Lipopolysaccharides)and IFN-?(Interferon-?).Two types of cells were cultured:isolated M1 macrophages and M1 macrophages with T.marneffei.The relative expression of CD86 in both cultures was detected by western blot at 24,48 and72 h;5?The fluorescence intensity of CD86 in both cultures was detected by immunofluorescence at 24,48 and 72 hours;6 ?The expression of CD86 in macrophages and its presence in supernatant in both cultures was determined by Enzyme-linked immunosorbent assay at 24,48,and 72 h;7?The expression of CD86 on T.marneffei in the co-culture group was detected by Immuno-electron microscope;8?The expression of CD86 on T.marneffei in the co-culture group was detected by immunohistochemistry.Results:1?The tissue isolates were hyphe form at 27 ?,which appeared as baskets under the microscope.They were yeast form at 37 ? which is characterized by short rod structure under the microscope;2?Sequence alignment of PCR amplified products showed that the similarity of DNA between the isolates and Talaromyces marneffei was 99%;3?CD86 on T.marneffei was not detected by either immunoelectron microscopy or immunohistochemistry;4?Western blot showed that the expression of CD86 decreased with time in the culture contained T.marneffei,the relative expression of CD86 was significantly lower in the co-culture group than that in the group of isolated M1 macrophages at 72 h,P<0.05;The relative expression of CD206 in the co-culture group at 48 h was higher than other time,and it was almost negative at 72h;5 ?Immunofluorescence showed that the expression of CD86 decreased with time in the culture contained T.marneffei,the fluorescence intensity of CD86 was significantly lower in the co-culture group than that in the group of isolated M1 macrophages at 72 h,P<0.05;6?Enzyme-linked immunoassay showed that the expression of CD86 in macrophages decreased with time in the culture contained T.marneffei,but CD86 content in supernatant increased.The expression of CD86 protein in macrophages were significantly lower in the co-culture group than that of the group of M1 macrophages at 72 h,P < 0.05,but the content of CD86 in supernatant was significantly higher in the co-culture group than that of the group of M1 macrophages at 72 h,P < 0.01;7?Immunoelectron microscopy showed that specific colloidal gold particles on T.marneffei after co-culturing with macrophages;8?Immunohistochemistry showed that T.marneffei were CD86 positive after co-culturing with macrophages.Conclusion: 1?After T.marneffei infection,CD86 expression on THP-1decreased,and with the progression of infection,insufficient polarization of M1 macrophages gradually appeared;2 ? T.marneffei may adsorb or uptake CD86 in supernatant produced by macrophages during contact with THP-1 cells,thus leading to the consumption of CD86 in macrophages.