Ag85B regulates myeloid dendritic cell maturation and suppresses expression of TSLPR and OX40L mediated by TSLP in vitro

AIM: To investigate the maturation of mice immature myeloid dendritic cells(mDCs) induced by antigen(Ag)85B of mycobacterium tuberculosis, and the expression of TSLPR and OX40L mediated by TSLP in vitro.METHODS: Recombinant mouse GM-CSF(rmGM-CSF) and rmIL-4 were used to induce bone marrow precursor cells of C57BL/6 mice to differentiate into immature mDCs in vitro. mDCs were identified followed by purification using CD11c binding magnetic beads. The morphological characteristic of mDCs was observed under inverted phase-contrast microscope and scanning electron microscope. The surface phenotypes of mDCs were determined by flow cytometry. To obtain the optimal concentrations of Ag85B and TSLP, immature mDCs were cultured with different concentrations of Ag85B or TSLP at 0(control group), 50, 100 and 200 μg/L for 24 h, and the expression of cell surface molecules CD80, CD86, TSLPR and OX40L was detected by flow cytometry. In addition, the expression of TSLPR and OX40L in Ag85B and TSLP-co-stimulated mDCs was determined by flow cytometry.RESULTS: After 7 d of culture in vitro, the cells showed irregular dendritic protrusions under the inverted-phase contrast microscope, and had wrinkles and dendritic splits under scanning electron microscope, conformed to the morphological characteristics of immature mDCs. The mDCs cells expressed higher level of specific marker CD11c, lower level of co-stimulatory molecules CD80 and CD86, which conformed to the phenotype of immature mDCs. The CD80⁺ and CD86⁺ cell ratios of mDCs displayed significant increases in 50, 100 and 200 μg/L Ag85B or TSLP groups compared with control group(P<0.05). The ratios of TSLPR⁺ and OX40L⁺ cells did not differ among different concentrations of Ag85B groups. The ratios of TSLPR⁺ and OX40L⁺ cells were significantly increased in 100 μg/L and 200 μg/L TSLP groups compared with control group and 50 μg/L TSLP group(P<0.05). Under the circumstance of optimal Ag85B or TSLP treatment concentration at 200 μg/L, there was significantly decreased in TSLPR and OX40L cell ratio of mDCs in Ag85B group or Ag85B combined with TSLP group when compared with TSLP group(P<0.05), and no significant difference among Ag85B group, Ag85B combined with TSLP group and control group was observed.CONCLUSION: Ag85B enhances mDCs maturation by up-regulating the expression of co-stimulatory molecules CD80 and CD86, and inhibit the expression of pro-inflammatory specific molecules TSLPR and OX40L on TSLP-activated mDCs, indicating that Ag85B modifies the development of asthmatic airway inflammation through the pathway of TSLP-activated mDCs.