Antioxidative enzyme NAD(P)H quinone oxidoreductase 1 (NQO1) modulates the differentiation of Th17 cells by regulating ROS levels

Abstract

NAD(P)H quinone oxidoreductase 1 (NQO1) is a flavoprotein that catalyzes two-electron reduction of quinone to hydroquinone by using nicotinamide adenine dinucleotide (NADPH), and functions as a scavenger for reactive oxygen species (ROS). The function of NQO1 in the immune response is not well known. In the present study, we demonstrated that Nqo1-deficient T cells exhibited reduced induction of T helper 17 cells (Th17) in vitro during Th17(23)- and Th17(β)- skewing conditions. Nqo1-deficient mice showed ameliorated symptoms in a Th17-dependent autoimmune Experimental autoimmune encephalomyelitis (EAE) model. Impaired Th17-differentiation was caused by overproduction of the immunosuppressive cytokine, IL-10. Increased IL-10 production in Nqo1-deficient Th17 cells was associated with elevated intracellular Reactive oxygen species (ROS) levels. Furthermore, overproduction of IL-10 in Th17 (β) cells was responsible for the ROS-dependent increase of c-avian musculoaponeurotic fibrosarcoma (c-maf) expression, despite the lack of dependency of c-maf in Th17(23) cells. Taken together, the results reveal a novel role of NQO1 in promoting Th17 development through the suppression of ROS mediated IL-10 production.

Fig 1. NQO1 promotes Th17 differentiation in vitro.

Isolated CD4⁺ CD62L^hi 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 CD4^cre/+ or c-maf^Flox/Flox, CD4^cre/+ 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 CD4^cre/+ or Nqo1 KO, CD4^cre/+ or c-maf^Flox/Flox, CD4^cre/+ or Nqo1 KO, c-maf^Flox/Flox, CD4^cre/+ 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.