4. Discussion
In the current study, we identified β-damascone from approximately 150 compounds as a novel immunomodulator through 2-step screening monitoring of DC-mediated immunoresponses. By conducting in vivo experiments using a mouse model, we demonstrated that oral intake of β-damascone, which activates the NRF2 pathway, ameliorated CHS inflammation. NRF2 plays a role as a master regulator of antioxidant and anti-electrophilic responses by transactivating the genes encoding antioxidant enzymes and drug metabolizing enzymes. In a previous study using a 2,4-dinitrochlorobenzene (DNCB)-induced CHS model, NRF2-deficient mice exhibited more severe inflammation accompanied by increased chemokine production and neutrophil recruitment in the skin than control mice12. This observation that NRF2 deficiency deteriorates CHS may be coincident with our results, indicating the important roles of NRF2 in DC-mediated immune responses. In addition to CHS, various immunorelated inflammatory diseases and autoimmune diseases, including colitis and psoriasis, are reported to be exacerbated in NRF2 knockout mice 13-16. Although the effects of NRF2 deficiency are not restricted to DC function, because NRF2 is ubiquitously expressed, these studies of NRF2-deficient mice support the possibility that β-damascone could be useful for the prevention and/or treatment of inflammatory diseases and autoimmune diseases. We found that β-damascone significantly suppressed TLR7-mediated gene expression and protein production of IL-6, IL-12p40, and TNF-α in BMDCs (Figure 3C and3F ). Because a topical application of imiquimod17,18, a ligand of TLR7 and TLR8, causes psoriasis-like pathology in mouse skin through activation of dermal DCs and Langerhans cells 19, we expect that β-damascone ameliorates psoriasis, even from the point of view of modification of DC function. We will evaluate the effects of β-damascone on various immunorelated diseases, such as psoriasis, inflammatory bowel disease, and multiple sclerosis using a mouse model in the near future.
Our results using Nrf2 -/- DCs indicated that the suppressive effects of β-damascone on IL-12 production in DCs and Th1 development activity of DCs are dependent on NRF2. In contrast, the suppressive effects of β-damascone on the production of TNF-α and IL-6 were still observed in Nrf2 -/- DCs, suggesting that β-damascone modulates DC function through additional target(s) other than NRF2. It has been reported that the induction of the proinflammatory cytokine genes, Il1a , Il1b , and Il6is inhibited by NRF2, which binds to these genes and subsequently inhibits the recruitment of RNA polymerase II to target genes in macrophages 20. However, under our experimental conditions, we could not find a significant contribution of NRF2 in the suppression of the production of IL-6 and TNF-α in DCs. Further detailed analysis to clarify other target(s) of β-damascone could lead to the development of novel anti-inflammatory immunomodulators.
In the current study, we identified β-damascone as the most effective compound from an aroma library and confirmed that oral administration of β-damascone ameliorated CHS. Although we obtained β-damascone as a natural compound candidate of immunomodulator in the current study, there is room for improvement; briefly, β-damascone could be developed to a drag carrying stronger activity and lesser toxicity by chemical structural modification. Further research using an organic chemical approach considering β-damascone as a lead compound should be conducted to develop a novel immunomodulator.