Nrf2 deficiency promotes M1 macrophage polarization and inhibits M2 macrophage polarization through autophagy modulation; n = 3 experiments per group. (A,B) Flow cytometry analyses of M1 macrophage polarization in BMDMs. (C,D) Immunofluorescence staining of CD206 and F4/80 in BMDMs. White arrows in the pictures indicate polarized macrophages. (E) Isolated BMDMs were treated with LPS/IFN-γ (15 ng/mL and 50 ng/mL, respectively) for 24 h to induce M1 macrophage polarization, and 0.1 μM RAPA was added simultaneously for autophagy activation. The levels of M1 macrophage markers such as iNOS, IL-6, IL-1β, and TNF-α were measured with qRT-PCR. (F) Isolated BMDMs were treated with IL-4/IL-13 (25 mg/mL) for 24 h to induce M2 macrophage polarization, and 0.1 μM RAPA was added simultaneously for autophagy activation. The levels of M2 macrophage markers such as Arg1, Fizz1, Ym1, and IL-10 were determined by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001.