Effects of Leptospira interrogans infection on the activities of NADPH oxidase and the levels of ROS in THP-1 and J774A.1 cells

Abstract

Objective To investigate the effects of Leptospira interrogans (L.interrogans) infection on the activities of NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and the levels of reactive oxygen species (ROS) in THP-1 and J774A.1 cells and to understand the bactericidal mechanisms of macrophages in different hosts against L. interrogans. Methods Human mononuclear macrophage cell line (THP-1 cells) and murine mononuclear macrophage cell line (J774A.1 cells) were infected with L. interrogans strain 56601. The activities of NADPH oxidase and the levels of superoxide ion (O2-) were measured with spectrophotography. Changes in the levels of ROS were detected with immunofluorescence assay. Results Compared with the normal cells, the activities of NADPH oxidase in L. interrogans-infected J774A.1 cells changed from 0.619 0 μmol·min-1·mg-1 to 0.305 5 μmol·min-1·mg-1, 6.141 5 μmol·min-1·mg-1, 1.487 1 μmol·min-1·mg-1 and 0.964 6 μmol·min-1·mg-1 after 2, 4, 12 and 24 hours of infection, respectively (P<0.05), while the activities of NADPH oxidase in L. interrogans-infected THP-1 cells were up-regulated from 0.723 5 μmol·min-1·mg-1 to 0.884 2 μmol·min-1·mg-1, 1.897 1 μmol·min-1·mg-1, 1.125 4 μmol·min-1·mg-1 and 0.562 7 μmol·min-1·mg-1, respectively (P<0.05). The levels of O2- in L. interrogans-infected J774A.1 cells at the time points of 2 h, 4 h, 12 h and 24 h after infection increased from 0.189 0 μmol/L to 0.236 3 μmol/L, 0.297 7 μmol/L, 0.324 0 μmol/L and 0.305 7 μmol/L, respectively (P<0.05), while the levels of O2- in L. interrogans-infected THP-1 cells rose from 0.123 7 μmol/L to 0.149 3 μmol/ L, 0.249 0 μmol/ L, 0.270 0 μmol/ L and 0.272 7 μmol/L, respectively (P<0.05). The fluorescence intensity of ROS in THP-1 and J774A.1 cells increased gradually after infection with L. interrogans for 2 h and decreased after reaching the peak at 24 h. Conclusion Both the activities of NADPH oxidase and the levels of O2- in J774A.1 and THP-1 cells were significantly upregulated after infected with L. interrogans, especially in J774A.1 cells. The results of this study provided references for further elucidating the bactericidal mechanisms of macrophages in different hosts against L. interrogans. Key words: Leptospira interrogans; Macrophage; NADPH; ROS; Bactericidal mechanism