3.4 Involvement of the NRF2 pathway in the suppressive effects of β-damascone on DCs
Some substances derived from plants, especially phytochemicals (e.g., sulforaphane), often activate the NRF2 pathway by directly acting on Keap1, resulting in the induction of antioxidant and anti-inflammatory responses 11. To clarify whether β-damascone activates the NRF2 pathway in DCs, we determined the NRF2 protein and Hmox1mRNA levels in DB-treated DCs. As shown in Figure 4A , a Western blot analysis revealed that the amount of NRF2 protein was increased in DCs in the presence of β-damascone and peaked at 1 h after the addition of DB to the culture medium. The mRNA levels of Hmox1 , which is a target gene of NRF2, were markedly upregulated by β-damascone much higher than those induced by LPS (Figure 4B ). These results showing the increase in NRF2 protein and Hmox1 mRNA levels in β-damascone-treated DCs suggest the possibility that β-damascone activates the NRF2 pathway in DCs. Then, we investigated the roles of NRF2 in DB-mediated modification of DC function by usingNrf2-/- DCs. First, it was confirmed that the β-damascone-induced increase in Hmox1 mRNA levels in DCs was almost abolished by NRF2 deficiency (Figure 4C ). When OVA-pulsed Nrf2-/- BMDCs were cocultured with OT-II CD4+ T cells under a Th1-polalizing conditions, the suppression of Th1 development by β-damascone was not observed, whereas control (Nrf2+/- ) DC-dependent Th1 development was significantly suppressed in the presence of β-damascone (Figure 4D ), as was the case for WT BMDCs (Figure 1E ). Furthermore, IL-12p40 release from LPS-stimulated DCs tended to increase by Nrf2 deficiency in DCs, and the suppressive effect of β-damascone on IL-12p40 production was markedly reduced inNrf2-/- BMDCs compared with that inNrf2+/- BMDCs (Figure 4E ). These results demonstrate that DC functions, including Th1 induction and IL-12 production, were suppressed by β-damascone depending on activation of the NRF2 pathway in DCs.