Figure 6.

E₂ is verified to facilitate trained immunity in primary BMDMs from female and male mice. (A) In vitro trained immunity model for BMDMs. (B) Flow cytometry was used for testing the purity of BMDMs induced by in vitro culture. (C) The mRNA levels of TNFα and IL-6 in male/female BMDMs were detected by qPCR to determine the different intensity of trained immunity between genders. (D) The protein concentrations of TNFα and IL-6 from the supernatant from male/female BMDM cultures were detected by ELISA to determine the different intensity of trained immunity between genders. (E) E₂ activated hallmarks of trained immunity, such as Akt, 4EBP1, and S6 by western blot. (F) E₂ promoted M1 polarization in TI + LPS group from male and female mice. Meanwhile, E₂ maintained the M2 polarization to inhibit the effect of TI (n ≥ 3/group). ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001, paired Student's t-test comparing β-glucan + LPS group and LPS group. *p < 0.05, **p < 0.01, and ***p < 0.001, paired Student's t-test comparing with control group. ∧∧p < 0.01, paired Student's t-test comparing between β-glucan + LPS groups with or without E₂.