Abstract: Objective To analyze the functions of mouse dendritic cells infected with Cryptosporidium parvum in the presence of Toll-like receptor 4 (TLR4). Methods Cryptosporidium cysts were purified and excysted, then labeled by 5'(6)-carboxyldiacetic acid fluorescein succinimidyl ester. Its species was identified by PCR. Mouse bone marrow dendritic cells were prepared and cultured. On day 7 of culture, 1 ml of dendritic cell culture was transferred to a 6-well plate, and 1 ml of labeled excysted Cryptosporidium parvum was added (2 × 10⁵/well), together with 0.5 ml of TLR4 antibody (at a final concentration of 1 μg/ml) (assigned into the TLR4 group) or 0.5 ml RPMI 1640 (infection group). Cells in the control group were incubated with 1.5 ml RPMI 1640 only. Each group had 3 replicates. After 2-hour incubation, flow cytometry was performed to detect the relative expression level of CD11c. Flowjo software was used to compare the maturation state of dendritic cells. After 24 h of infection, adhesion of Cryptosporidium sporozoites to dendritic cells was observed under a fluorescence microscope. The difference in the expression level of CD11c was analyzed with chi-square test. Results PCR amplification on the purified Cryptosporidium generated a band of 830 bp, which was identified to be Cryptosporidium parvum. Flow cytometry showed that the relative expression level of CD11c on dendritic cells was 67.67 ± 1.80 in the TLR4 antibody group and 83.37 ± 3.73 in the infection group, which were significantly different (P < 0.05), and both had significant difference with the control group (7.06 ± 0.02) (P < 0.05). Flowjo software analysis showed that the peak of CD11c expression in the TLR4 antibody group was lower than that in the infection group, implying a lower maturity of dendritic cells in the TLR4 antibody group than in the infection group. Fluorescence microscopy showed adhesion of Cryptosporidium sporozoites to dendritic cells in the TLR4 group and the infection group. Conclusion TLR4 can induce maturation of dendritic cells after infection with Cryptosporidium parvum.

Key words: Cryptosporidium parvum, Dendritic cell, TLR4, Antibody, Degree of ripeness, Adhesion