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Updated: May 6, 2026
Author Spotlight: THP-1 Macrophage Response to LPS/ATP — Unveiling the Pyroptosis, Apoptosis, and Necroptosis Spectrum
Published on: May 3, 2024
[TcpC induces apoptosis of macrophages through promoting ROS production]
Da-Yong Zhang1, Yi-Qian Lin, Fei He
1Department of Medicine, Zhejiang University City College School of Medicine, Hangzhou 310015, China.
Objective:
To investigate the effects of Toll/interleukin 1 receptor domain-containing protein(TcpC)on macrophages and its mechanisms.
Methods:
Murine 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.
Results:
J774A 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).
Conclusion:
TcpC can promote ROS production in macrophages,hereby inducing macrophage apoptosis.
Insights
Toll/interleukin 1 receptor domain-containing protein (TcpC) from E. coli induces macrophage apoptosis by increasing reactive oxygen species (ROS) production. Scavenging ROS with N-acetylcysteine reversed these pro-apoptotic effects, highlighting ROS as a key mediator.
Area of Science:
- Immunology and Microbiology
- Cellular and Molecular Biology
Background:
- Toll/interleukin 1 receptor domain-containing protein (TcpC) is a virulence factor produced by certain E. coli strains.
- The precise mechanisms by which TcpC influences host immune cells, particularly macrophages, remain incompletely understood.
Purpose of the Study:
- To elucidate the effects of TcpC on macrophage apoptosis.
- To investigate the underlying molecular mechanisms, focusing on reactive oxygen species (ROS) generation.
Main Methods:
- Murine macrophage J774A cells were co-cultured with wild-type E. coli producing TcpC (TcpC(wt)) or a TcpC-deficient mutant (TcpC(mut)).
- Apoptosis was assessed using Annexin/PI staining, and ROS levels were measured via DCFH-DA staining.
- The role of ROS was further examined by scavenging with N-acetylcysteine (NAC), and caspase-3 expression was analyzed by Western blot.
Main Results:
- TcpC(wt) significantly increased J774A cell apoptosis and ROS production compared to control and TcpC(mut) groups.
- The pro-apoptotic effects induced by TcpC were reversed upon ROS scavenging with NAC.
- TcpC(wt) treatment led to decreased caspase-3 expression, which was restored by NAC, suggesting ROS-mediated apoptosis.
Conclusions:
- TcpC promotes ROS production in macrophages.
- This ROS generation is a key mechanism by which TcpC induces macrophage apoptosis.
- Targeting ROS may offer a strategy to mitigate TcpC-induced immune cell damage.
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