[Polarization of human acute monocytic leukemia THP-1 cells-derived macprophages induced by proteins ].

OBJECTIVE

To investigate the polarization of human acute monocytic leukemia THP-1 cells-derived macrophages induced by proteins , so as to provide insights into the elucidation of the mechanisms underlying host immune responses to hookworm infections.

METHODS

The culture of was established and maintained in the laboratory, and the third- (L3) and fifth-stage larvae (L5) were collected under a sterile condition for preparation of L3 and L5 proteins. The culture of THP-1 cells was established, stimulated with 500 ng/mL PMA to yield M0 macrophages that were adherent to the plate wall. The LPS + IFN-γ group, IL-4 + IL-13 group, L3 protein group and L5 protein group were given stimulation with 500 ng/mL LPS plus 100 ng/mL IFN-γ, IL-4 and IL-13 (both 100 ng/mL), L3 protein (5 mg/mL) and L5 protein (5 mg/mL), respectively, while the negative control group was given no stimulation. The cell morphology was observed using microscopy, the expression of M1/M2 macrophages-specific genes was quantified using a quantitative real-time PCR (qPCR) assay, and the surface markers of M1/M2 macrophages were detected using flow cytometry, while the levels of cytokines secreted by M1/M2 macrophages were measured using enzyme-linked immunosorbent assay (ELISA) following stimulations, so as to examine the polarization of THP-1-derived macrophages induced by proteins .

RESULTS

Following stimulation with PMA, THP-1 cells appeared wall-adherent M0 macrophages, and polarized to typical M1 macrophages following stimulation with LPS + IFN-γ, and typical M2 macrophages following stimulation with IL-4 + IL-13, IL-3 protein or L5 protein. There was a significant difference in the proportion of M1 macrophages among the negative control group, the LPS + IFN-γ group, the IL-4 + IL-13 group, the L3 protein group and the L5 protein group ( = 3 721.00, < 0.001), with the highest proportion detected in the LPS + IFN-γ group, and there was also a significant difference in the proportion of M2 macrophages among groups ( = 105.43, < 0.001). There were significant differences among groups in terms of the expression of ( = 191.95, < 0.001), ( = 129.95, < 0.001), ( = 82.89, < 0.001), ( = 11.30, < 0.001), ( = 9.51, < 0.001) and genes ( = 12.35, < 0.001). In addition, there were significant differences in the proportion of positive CD86 and CD206 expression among groups ( = 24 004.33 and 832.50, < 0.001). Higher IL-1β and TNF-α levels were measured in the LPS + IFN-γ group than in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group ( < 0.001), and greater TGF-β1 and IL-10 levels were seen in the IL-4 + IL-13 group, the L3 protein group and the L5 protein group than in the negative control group and the LPS + IFN-γ group ( < 0.05).

CONCLUSIONS

Both L3 and L5 proteins of may induce the polarization of THP-1-derived macrophages to M2 type .