The Preliminary Study Of Innate Immune Response Of Candida Albicans On The Monocyte-macrophage Cell Line THP-1 Cells

Chapter I Study on the phagocytosis of the THP-1 monocytesObjective To investigate the phagocytosis on ink and Candida albicans (C. albicans) of the THP-1 monocytes. Methods Propylene glycol monomethyl ether acetate (PMA) was used to induce the THP-1 cells to macrophage. THP-1 cells were incubated with ink and heat-killed C. albicans, and phagocytosis were examined. THP-1 cells pretreated with Cytochalasin-B (Cyt-B) for 30 minutes were incubated with C. albicans to observe the phagocytosis of the THP-1 cells. Results The morphology of THP-1 monocytes become adherent and undergo profound morphological and physiological changes by inducing with PMA. After incubating with ink and heat-killed C. albicans the THP-1 macrophage swallowed lots of ink and C. albicans, and pretreated with Cyt-B inhibited this kind of phenomenon. Conclusions The THP-1 macrophage could swallow ink and C. albicans and actin plays an important role in phagocytosis.Chapter II Study on the effect of Candida albicans on the production of pro-inflammatary factors in human THP-1 monocytes.Objective To investigate the production of TNF-a and IL-6 in human THP-1 monocytes after being challenged by C. albicans. Methods THP-1 cells were challenged with heat-killed C. albicans (105CFU/ml,106CFU/ml) and lipopolysaccharide (100ug/L) in vitro. After culture for different durations, the mRNA expressions of TNF-a and IL-6 were assayed by real time reverse transcription polymerase chain reaction, and the secretion of TNF-a and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). Results With the two-way analysis of variance, the mRNA levels of TNF-a and IL-6 of THP-1 cells treated with different stimulators make differences (F=110.983, P<0.001; F= 294.114, P< 0.001) and C. albicans up-regulated the mRNA levels of TNF-a and IL-6 in a time-dependent (F-701.680, P<0.001; F=1036.557, P<0.001) and dose-dependent manner. Exposure of THP-1 cells to C. albicans at the concentration of 106CFU/ml at the time point of 24h led to increased secretion of TNF-α(6385.70± 533.99 ng/L, P<0.001) and IL-6(924.90±30.13ng/L, P<0.001). Conclusion There was a time- and dose-dependent TNF-α and IL-6 release during the C. albicans infection.Chapter Ⅲ Study on the effect of Candida albicans on the activation of signal pathway in human THP-1 monocytesObjective To investigate the effect of activation of p38MAPK and NF-κB in THP-1 cells after incubation with C. albicans. Methods THP-1 cells were incubated with heat-killed C. albicans. After culture for 30min and 60min, Western blotting was used to analyze p38MAPK and IκBα phosphorylation. NF-κB nuclear translocation was observed by immunofluorescence after THP-1 cells challenged with C. albicans for 16 hours. THP-1 cells pretreated with Dexamethasone were challenged with the same stimulators and were examined the activation of p38MAPK and the mRNA expressions of TNF-α and IL-6.Results THP-1 cells challenged with C. albicans at the time point of 30min and 60min resulted in the activation of p38MAPK and IκBα in a time-dependent manner. C. albicans and LPS induced the NF-κB translocation in contrast with control. THP-1 cells pretreated with 40 μg/L Dexamethasone for 30 minutes challenged with C. albicans didn’t result in the up-regulated actidvation levels of p38MAPK. THP-1 cells pretreated with 40 μg/L Dexamethasone for 30 minutes challenged with C. albicans didn’t result in the up-regulated mRNA levels of TNF-α and IL-6. Conclusions Human THP-1 monocytes may play a role in immune responses against C. albicans through activation of p38MAPK and NF-κB with the increased production of TNF-α and IL-6.Chapter Ⅳ Study on the effect of Candida albicans on the expression of Dectin-1 in human THP-1 monocytesObjective To investigate the effect of C. albicans on the expression of Dectin-1 in human THP-1 monocytes. Methods Flow cytometry was used to assay the expression of Dectin-1 in human THP-1 monocytes after being treated with heat-killed C. albicans and Curdlan(a kind of P-glucan) for different durations. Results After being treated with heat-killed C. albicans and Curdlan for 3 hours, no difference was made on the expression of Dectin-1. After stimulated for 6 hours or longer time, the expression of Dectin-1 was remarkably up-regulated (P<0.05) in a time-dependent manner. Conclusions Candida albicans may induced the up-regulation of the expression of Dectin-1 in human THP-1 monocytes.