Fig 1.
NQO1 promotes Th17 differentiation in vitro.
Isolated CD4+ CD62Lhi naïve T cells were stimulated with anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) antibodies and cultured with Th17(β) (stimulated with IL-6 +TGFβ) and Th17(23) (stimulated with IL-1β +IL-6 +IL-23) skewing conditions for 3 days. (A) Real-time PCR analysis of Nqo1 gene expression in WT (n = 3). (B) IL-17A and IL-22 production in the cell culture supernatant was measured by ELISA. (n = 5) (C) On days 3, the differentiated cells were stimulated with PMA, Ionomycin, and BFA for 4 h. The plot indicates frequencies of CD4+ IL-17A+ T cells (n = 3). (D) Histogram showing internal staining of CD4+ RORγt+ expression (n = 3). Each graph indicates the mean ± SE, One way ANOVA.
Fig 2.
NQO1 promotes Th17 differentiation by inhibiting IL-10 production.
(A, C) Purified naïve CD4+ T cells from WT, Nqo1 KO, Il-10 KO, or Nqo1/Il-10 double knock-out were stimulated with anti-CD3/CD28 antibody in each differentiation conditions for 3 days. Cytokines secreted into the cell culture supernatant were measured by ELISA (n = 4–8). (B) Naïve CD4+ T cells from Il-10 venus reporter mice, and Nqo1 KO/Il-10 venus reporter mice were differentiated into Th17 (β) or Th17 (23) cells (n = 3). The ratio of IL-10 expressing CD4+ cells cultured under each condition for 3 days was determined. Data are representative of three independent experiments. Graphs indicate the mean ± SE, One way ANOVA.
Fig 3.
Nqo1 deficiency ameliorates EAE severity depend on IL-10.
(A, B) The graph shows clinical scores and induction ratio for EAE in WT, Nqo1 KO mice after immunization with a MOG-CFA mixture and p-toxin. Mice were sacrificed under anesthesia at end of experiment (n = 4). (C) Absolute numbers of CNS infiltrating cells were analyzed. Total CD4+ cells are presented in left panel and IL-17 expression CD4+ cells are shown in right panel (n = 4). (D) Splenocytes from immunized mice were re-stimulated with 50 μM MOG peptide for 72 h. Frequencies of CD4+ IL-17+, IFNγ+ cells (%) were measured by FACS (n = 4). (E, F) The graph indicates clinical scores for adoptive transfer EAE in recipient mice (n = 5). Mice were sacrificed under anesthesia at 23 days (end of experiment) or at the humane endpoints. Data are representative of three independent experiments. Graphs indicate the mean ± SE. (A-C) Unpaired student T-test, (D-F) One way ANOVA.
Fig 4.
NQO1 inhibits IL-10 production by regulating ROS.
(A-C) Naïve CD4+ T cells from Nqo1 KO and WT mice were differentiated into Th0, Th17(β), and Th17(23) cells with the indicated cytokines for 1 day. Cells were loaded with the ROS indicator, DCFDA, DHE, or mtROS detecting reagent and the fluorescence intensities were detected by flow cytometry (n = 3). (D) IL-10 secretion in the cell culture supernatant with or without NAC (5 mM) as measured by ELISA (n = 4). (D) The expressions of a lot of antioxidative enzymes were measured by RT-PCR (n = 4). These graphs indicate the mean ± SE, One way ANOVA.
Fig 5.
NQO1 inhibits IL-10 production by controlling c-maf upregulation by ROS in Th17(β) cells.
(A) Isolated naïve CD4+ T cells from Nqo1KO or WT mice stimulated with Th0, Th17(β), and Th17(23) skewing cytokine conditions for 3 days. The expression of c-maf was determined by RT-PCR (n = 5). (B) Naïve CD4+ T cells from CD4cre/+ or c-mafFlox/Flox, CD4cre/+ mice were differentiated into Th0, Th17(β), or Th17(23) cells for 3 days. Secreted IL-10 in the cell culture supernatant was measured by ELISA (n = 4). (C) Naïve CD4+ T cells from CD4cre/+ or Nqo1 KO, CD4cre/+ or c-mafFlox/Flox, CD4cre/+ or Nqo1 KO, c-mafFlox/Flox, CD4cre/+ mice were cultured in Th17(β) skewing conditions for 3 days. The secretion of IL-10 in the supernatant was measured by ELISA (n = 5). (D) c-maf gene expression with or without the antioxidant NAC (5 mM) was measured by RT-PCR (n = 4). Graphs indicate the mean ± SE, One way ANOVA.