T.marneffei strain used in our study was isolated from skin lesion of patient with Talaromycosis, cultured at 25℃ and 37℃ for 7 days for morphological identification(Figure 1). The DNA was extracted for PCR amplification and agarose gel electrophoresis, the primer sequence is shown in Table 1,and the amplified products were sequenced and analyzed.
A model of vitro T.marneffei infection with M1 macrophages was constructed in our research (Figure 2).THP-1 cells were subcultured in Roswell Park Memorial Institute medium(RPMI 1640,Gibco)containing 10% of heat inactivated fetal bovine serum (Gibco)and 1% penicillin-streptomycin solution(100U/ml penicillin G,0.1mg/ml streptomycin sulfate, Solarbio, #P1400). THP-1 cells were differentiated into macrophages by incubating with 50ng/μl PMA(Phorbol 12-myristate 13-acetate,Meilunbio,16561-29-8) for 48 hours in Dulbecco's Modified Eagle Medium(DMEM, Gibco) which contained 10% of heat inactivated fetal bovine serum and 1% penicillin-streptomycin solution. Macrophages polarized to M1 macrophages under the stimulation of 100 ng/μl LPS (Meilunbio,MB5198) and 20ng/μl IFN-γ(Novoprotein,C04) for 24 hours. At this time, T.marneffei infected M1 macrophages at a multiplicity of infection (MOI) of 5 at 37℃ for 24, 48 and 72 hours respectively,the cells and supernatant were collected for subsequent experiments. M1 macrophages in DMEM without T.marneffei were used as control cells.
To verify the first hypothesis, T.marneffei were grinded in liquid nitrogen, and the proteins were released in the cleavage of yeast buster proteins extraction reagent (Merck,71186-3CN)for western blot. Yeasts cultured on BHI medium at 37℃ for 7 days were used for immunoelectron microscopy(IEM).
To verify the other hypothesis, the expression of CD86 on yeast cells was detected by IEM and IHC, the changes of CD86 expression in co-culture system were detected by WB,IF and ELISA.
Cellular proteins were lysed by RIPA(Cell Signaling,#9086) on ice and boiled for 5 minutes before adding sodium dodecyl sulfate(SDS) buffer. Proteins were isolated on SDS-PAGE gel and transferred to PVDF membrane for non-specific antibody sealing with 5% skim milk. Antibody labeling was performed in PVDF membrane in turn. Primary antibody (Rabbit anti-CD86 antibody from Abcam, ab53004),β-actin antibody(Rabbit polyclonal to beta actin from Abcam, ab8227), second antibody(Goat Anti-Rabbit IgG H+L (HRP) from Engibody,AT0097). Finally the protein bands were photographed by chemiluminescence and calculated quantitatively by Image J.
Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Blocking non-specific antibody with PBS which contained 0.1% Triton-X 100 and 1% FBS at room temperature for 20 minutes. For labeling, cells were incubated overnight at 4℃ with the primary antibody(Rabbit anti-CD86 antibody from Abcam, ab53004). The negative control group was incubated with PBS instead of primary antibody. After three times washing with PBS, cells were incubated for 1 hour avoiding light at room temperature with second antibody(Goat anti-Rabbit IgG (H+L),Alexa Fluor 488 AffiniPure from YASEN,33106ES60). The images were then observed and collected under the confocal microscope, the fluorescence intensities were calculated quantitatively by Image J.
Enzyme-linked Immunosorbent Assay(ELISA)
The content determination of CD86 in macrophages and supernatant were assayed by using an double-antibody sandwich ELISA kit (Bioss,bsk00643).
Immune Electron Microscopy (IEM)
Cells were fixed overnight with a phosphate buffer (PH=7.2) of 4% paraformaldehyde and 1% glutaraldehyde, and dehydrated with gradually high concentration of ethanol. Cells were embedded with Lowicrys K4M resin and polymerized under UVA at -20℃. The cells were ultra thin sectioned to a thickness of 50-70nm,incubated with the primary antibody(Mouse anti-CD86/B7-2 from NOVUS,NBP2-25208) and second antibody(6nm Colloidal Gold AffiniPure Goat Anti-Mouse IgG (H+L) from Jackson Immuno Research, RRID:AB 2338729) on a nickel net. The images were observed and collected under transmission electron microscope after conventional staining.
Cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Blocking non-specific antibody with PBS which contained 0.1% Triton-X 100 and 1% FBS at room temperature for 20 minutes. For labeling, cells were incubated overnight at 4℃ with the primary antibody(Rabbit anti-CD86 antibody from Abcam, ab53004) and for 1 hour at room temperature with second antibody(Goat Anti-Rabbit IgG HRP from Abcam,ab6721).Finally the target protein appears brown under the reaction with 3,3’-diaminobenzidine(DAB). The negative control group was incubated with PBS instead of primary antibody. The images were then observed and collected under light microscope.
Statistical software SPSS 22.0 was used for analysis, counting data were represented by chi-square test, measurement data were represented by mean± standard deviation (X±SD), comparison between groups was performed by independent sample T test, and P < 0.05 was considered statistically significant.
Ethics Statement: The authors confirm that the ethical policies of the journal, as noted on the journal’s author guidelines page, have been adhered to. No ethical approval was required as this is an original article with no original research data.