Figure 5.

TNF-α-induced ROS production is responsible for hematopoietic suppression by LPS. (A) ROS scavenger NAC or ablation of LPS-induced TNF-α rescues progenitor growth. WT, Fancc−/− mice or their littermates deficient for Tnfa were injected with PBS or a single dose of LPS (1 mg/kg). Mice were injected with N-acetyl-L-cysteine (NAC; 100 mg/kg) 30 min before and after LPS injection. 24 h after LPS injection, BM cells were isolated and subjected to clonogenic assay. Data shown represent the number (mean ± SD) of total number of colonies from three independent experiments. (B) Anti-oxidant NAC or ablation of LPS-induced TNF-α restores HSC self-renewal ability. 2 × 10⁶ Fancc−/− BM mononuclear cells isolated from the mice described in (A) were transplanted, along with 1 × 10⁶ competitor cells from B6.BoyJ mice (CD45.1⁺), into lethally irradiated recipient mice and long-term engraftment was evaluated at 16 weeks post-transplantation. Data represent mean ± SD of three independent experiments with 3 recipients per group for each experiment. (C) ROS production. WT, Fancc−/− mice or their littermates deficient for Tnfa were injected with PBS or a single dose of LPS (1 mg/kg). Mice were injected with 20 μg of a TNF-α neutralizing antibody 30 min after LPS injection. 24 h after LPS injection, BM cells were isolated and labeled with CM-H2DCFDA followed by flow cytometry.