OBJECTIVETo investigate the effects of Toll/interleukin 1 receptor domain-containing protein(TcpC)on macrophages and its mechanisms.

METHODSMurine macrophage J774A cells were co-cultured with TcpC producing wild type E. coli strain CFT073 (TcpC(wt)) or tcpc gene-deleted CFT073 mutant (TcpC(mut)) in Transwell system, respectively. Apoptosis of J774A cells co-cultured with TcpC(wt) or TcpC(mut) was analyzed by Annexin/PI double staining. The levels of reactive oxygen species (ROS) in J774A cells were determined by DCFH-DA staining after treatment with TcpC(wt) or TcpC(mut) at 6 h, 12 h,24 h or 36 h. After the ROS was scavenged by N-acetylcysteine (NAC), the changes of J774A cell apoptosis were also examined. The expression of caspase-3 in J774A cells co-cultured with TcpC(wt) or TcpC(mut) in the presence or absence of 0.1 mmol NAC was detected by Western blot.

RESULTSJ774A cells co-cultured with TcpC(wt) for 24 h or 36 h showed significantly increased apoptosis (27.39% ± 4.05% and 28.45% ± 4.55%,respectively) when compared to control group (7.96% ± 1.63% and 10.55% ± 1.44%,P<0.01) or TcpC(mut) group (11.45% ± 2.77% and 19.26%± 2.89%,P<0.01). Levels of ROS in J774A cells treated with TcpC(wt) for 24 h (108.8 ± 9.73) or 36 h (100.3 ± 10.11) were significantly higher than those in control group (56.8 ± 4.11 and 52.8 ± 4.42,P<0.01) or TcpC(mut) (69.7 ± 5.66 and 62.6 ± 4.56, P < 0.01). The pro-apoptotic effects of TcpC(wt) on J774A cells were reversed by 0.1 or 1 mMol NAC treatment. Expression of caspase-3 in J774A cells co-cultured with TcpC(wt) (0.43 ± 0.04) decreased significantly when compared to control group (0.75 ± 0.08,P<0.05) or TcpC(mut) group (0.80 ± 0.12,P<0.05). However,total caspase-3 expression was restored in J774A cells co-cultured with TcpC(wt) in the presence of 0.1 mmol NAC (0.80 ± 0.09).

CONCLUSIONTcpC can promote ROS production in macrophages,hereby inducing macrophage apoptosis.