Legends
FIGURE 1. Effects of β-damascone on DC-mediated activation of T cells.
A. Structure of β-damascone.
B. The concentrations of IL-2 in the culture media of OT-II splenocytes. A total of 1.0 x 105 cells obtained from the whole spleen of OT-II were maintained in the presence or absence of 500 ng OVA peptide and the indicated concentration of β-damascone (β-D) in 200 µL culture media for 72 h.
C . Cell viabilities of DCs and T cells in the presence of β-damascone. BMDCs and naïve CD4+ T cells were preincubated with the indicated concentrations of β-damascone for 24 h. Cell viability was judged with DAPI staining.
D. Division of OT-II CD4+ T cells cocultured with OVA-pulsed BMDCs. CFSE-labeled OT-II CD4+ T cells (5.0 x 104) were cocultured with or without C57BL/6 BMDCs (1.0 x 104), which were pretreated with 500 ng OVA peptide, in the presence or absence of the indicated concentrations of β-damascone in 200 µL of culture media for 72 h.
E. Division of CD4+ T cells via stimulation with anti-CD3 and anti-CD28 Abs. CD4+ T cells, which were isolated from the spleen of C57BL/6 mice, were labeled with CFSE, and then incubated with plate coated Abs as described in the Materials and Methods.
F. Effects of β-damascone on the frequencies of Th1 and Th2 cells developed under polarization conditions in a DC-dependent manner. OT-II naïve CD4+ T cells were cocultured with OVA-pulsed C57BL/6 BMDCs under Th1 (top) or Th2 (bottom) polarization conditions in the presence or absence of β-damascone (30 µM) for 72 h as described in the Materials and Methods section using a gating strategy shown in Supplementary Figure S2A .
G . Frequencies of Th1 and Th2 cells induced under polarization conditions in an APC-independent manner. C57BL/6 naïve T cells were stimulated with anti-CD3 and anti-CD28 Abs under Th1 (top) or Th2 (bottom) polarization conditions.
The Tukey-Kramer test was used (B , D , E ,F and G ). *; p < 0.05, **; p< 0/01, n.s.; not significant.
FIGURE 2. β-Damascone suppressed LPS-induced activation of DCs.
A . Messenger RNA expression levels in BMDCs.
B. Concentrations of cytokines in the culture media of BMDCs.
C. Cell surface expression levels of MHC class II and CD86 on BMDCs.
D. A Western blot profile of phosphorylated IκB.
BMDCs pretreated with 50 µM β-damascone for 24 h were stimulated with 100 ng/mL LPS. The cells were harvested at 3 h and 24 h after LPS stimulation to determine mRNA levels (A ) and cell surface protein expression levels (C ), respectively, and culture media were collected at 24 h after stimulation to measure the concentration of cytokines (B ). For Western blot analysis, cells at 1 h after LPS stimulation were lysed, and aliquots containing a total of 10 µg protein were added to each lane (D ). The data represent the mean ± SEM of three independent experiments (A , B , andC ) performed in triplicate samples (A and B ). A gating strategy of flowcytometric analysis of BMDCs is shown inSupplementary Figure S2B .
The Tukey-Kramer test (A , and B ) or Dunnett’s test (C ) was used. *; p < 0.05, **; p< 0/01.
FIGURE 3. Suppressive effects of β-damascone on the stimulation of DCs by various TLR ligands.
The effects of β-damascone (50 µM) on mRNA expression levels (A , B , and C ) and protein release of cytokines (D , E , F ) in BMDCs stimulated with polyI:C (A and D ), CpG (B and E ), or R848 (C and F ). The data represent the mean ± SEM of three independent experiments performed in triplicate samples.
The Tukey-Kramer test was used. *; p < 0.05, **;p < 0/01.
FIGURE 4. β-Damascone activated the NRF2 signaling in DCs, and NRF2 deficiency reduced the suppressive effects of β-damascone.
A. A Western blot profile showing NRF2 protein levels in BMDCs following treatment with β-damascone. BMDCs were incubated in the presence of 50 µM β-damascone for the indicated hours.
B. The mRNA levels of Hmox1 in BMDCs. BMDCs cultured in the presence or absence of 50 µM β-damascone for 24 h were incubated with or without 100 µg/mL LPS for an additional 3 h.
C. The mRNA levels of Hmox1 in BMDCs generated from NRF2-deficient mice (Nrf2 -/-) and control mice (Nrf2+ /-).
D. Th1 development induced by coculture withNrf2 -/- BMDCs or control BMDCs.
E. LPS-induced cytokine production fromNrf2 -/- BMDCs and control BMDCs.
The data represent the mean ± SEM of three independent experiments performed in triplicate samples. The Tukey-Kramer test was used. *;p < 0.05, **; p < 0/01, n.s.; not significant.
FIGURE 5. Oral administration of β-damascone ameliorated the pathology of CHS.
A. Schematic of the oral administration schedule of β-damascone in the DNFB-induced CHS model. The indicated amounts of β-damascone in 200 µl saline were orally administered every day. p.o.; per os.
B. Ear swelling of CHS mice at the indicated time points.
(Ear swelling) = (ear thickness after challenge) – (ear thickness before the first challenge)
Data were pooled from 2 independent experiments. n = 9-10.