Intracellular iron increases IL-6 and TNF-α expression and secretion in BMDMs. (A-D) BMDMs were isolated from Fpn1^flox/flox and Fpn1^LysM/LysM mice and incubated with 100 ng/mL LPS for the times indicated. Relative IL-6 (A) and TNF-α (B) mRNA levels, and IL-6 (C) and TNF-α (D) protein concentrations in the cell culture medium. (E-F) Relative IL-6 (E) and TNF-α mRNA (F) levels in BMDMs from wild-type mice. Cells were incubated overnight with FAC (100μM) or DFO (50μM) and then treated with 100 ng/mL LPS for the times indicated. (G-H) BMDMs from Fpn1^flox/flox and Fpn1^LysM/LysM mice were incubated overnight with FAC (100μM) or DFO (50μM) and then treated with 100 ng/mL LPS for the times indicated. IL-6 (G) and TNF-α (H) protein concentration in the cell culture medium. Data are representative of 3 independent experiments with similar results. For quantitative RT-PCR analysis, results were normalized to the internal control β-actin and presented as relative expression level calculated by the 2^ΔΔC_t method. Data are mean ± SEM. *P < .05.