Study On The Role Of Mannan-binding Lectin In Regulating Candida Albicans-induced DC Maturation And Cytokine Secretion

BackgroundMannan-binding lectin is a plasma protein secreted by liver cells. It belongs to the collectin family members of the c-type lectin super family. Dendritic cell(DC) is the most powerful antigen presenting cell(APC) in vivo, and it is a direct mediator of adaptive immunity response. It has been reported that MBL can induce the differentiation and maturation of DC, and high concentration of MBL can inhibit LPS-induced DC maturation. Until now, however, it has not been reported that Candida albicans(C.albicans) induce DC maturation and MBL whether could regulation such immunological effect. Accordingly, in-depth study of MBL regulates C.albicans induce DC maturation and its regulation mechanism has important significance.ObjectiveAnalyze the role of MBL in C.albicans inducing the differentiation and maturation of DC and the regulation function of the cytokines secretion. And explore its regulation mechanism preliminarily.Methods1. Cell culture:Using Ficoll density gradient centrifugation to isolate human PBMCs, Monocytes(Mo) were isolated by adherent method and cultivated in the serum-free medium containing100μg/L rhIL-4and rhGM-CSF for5days, inducing Mo to differentiate into immature dendritic cells(imDC), then we added different concentrations of MBL and C.albicans and continued to cultivate2to4days, stimulated the maturation of imDC, by human serum albumin(HSA) and TNF-a as contrast. The medium was changed half-quantity at a time every three days, and added the corresponding rhIL-4and rhGM-CSF.2. CD molecule detection:We collected dendritic cells of each culture group, and conducted flow cytometry analysis of DC maturation-related surface CD83, CD86molecules.3. Cytokine detection:We collected the DC supernatants of each culture group, and tested the secretion levels of TNF-α, IL-8, IL-6and IL-1β and other cytokines induced by C.albicans through ELISA.4. Cell binding:We resuspended imDC and C.albicans respectively with binding buffer containing different concentrations of Ca2+, then added the FITC-MBL, stood for30min in37℃photophobic, analyzed the binding of MBL and imDC, MBL and C.albicans by FACS.5. Statistical analysis:the experimental data was analyzed with the SPSS17.0software, the experimental results were expressed by mean±standard deviation (x±s), and the data was statistically analyzed using ANOVA and Independent samples t test, P<0.05indicated statistical difference, P<0.01indicated significant difference.Results1. We observed the situation by200×under the inverted microscope. The cells are irregular in shape and suspension cells increased, which added the rhIL-4and rhGM-CSF to cultivate for3to5days. The cell membrane extended root like protrusions after7days culture cell suspension growth.2. Compared with the blank cells group, the expressions of CD83/CD86of C.albicans stimulation group DC surface were significantly increased, the expressions of CD83/CD86of TNF-α stimulation group DC surface was also increased significantly. There was no significant difference between the two groups, which suggested that C.albicans can induce DC differentiation and maturation. Compared with the C.albicans stimulation group, the expressions of CD83/CD86of C.albicans+MBL stimulation group decreased obviously, yet there was no significant change of C.albicans+HSA stimulation group, which suggested that high concentration of MBL can inhibit C.albicans inducing DC maturation.3. Low concentration MBL (physiological concentration) has no significant effect on C.albicans stimulating DC secretion of IL-8, TNF-α and IL-6, high concentration of MBL (super physiological concentration) has inhibitory effect on the secretion of above cytokines.4. MBL can combine with imDC and C.albicans, and the combination has a Ca2+concentration dependent manner.ConclusionHigh concentration of MBL inhibits C.albicans inducing DC differentiation and maturation, and MBL can inhibit the cytokines secretion of DC stimulated by C. albicans in a concentration dependent manner. And this regulating effect relates to the ligand binding of MBL.