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. 2011 May 31;108(22):9220-5.
doi: 10.1073/pnas.1100667108. Epub 2011 May 16.

Regulation of type 17 helper T-cell function by nitric oxide during inflammation

Wanda Niedbala  1 , Jose C Alves-Filho, Sandra Y Fukada, Silvio Manfredo Vieira, Akio Mitani, Fabiane Sonego, Ananda Mirchandani, Daniele C Nascimento, Fernando Q Cunha, Foo Y Liew
Affiliations

Regulation of type 17 helper T-cell function by nitric oxide during inflammation

Wanda Niedbala et al. Proc Natl Acad Sci U S A. .

Abstract

Type 17 helper T (Th17) cells are implicated in the pathogenesis many of human autoimmune diseases. Development of Th17 can be enhanced by the activation of aryl hydrocarbon receptor (AHR) whose ligands include the environmental pollutant dioxin, potentially linking environmental factors to the increased prevalence of autoimmune disease. We report here that nitric oxide (NO) can suppress the proliferation and function of polarized murine and human Th17 cells. NO also inhibits AHR expression in Th17 cells and the downstream events of AHR activation, including IL-22, IL-23 receptor, and Cyp1a1. Conversely, NO did not affect the polarization of Th17 cells from mice deficient in AHR. Furthermore, mice lacking inducible nitric oxide synthase (Nos2(-/-)) developed more severe experimental autoimmune encephalomyelitis than WT mice, with elevated AHR expression, increased IL-17A, and IL-22 synthesis. NO may therefore represent an important endogenous regulator to prevent overexpansion of Th17 cells and control of autoimmune diseases caused by environmental pollutants.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
NO suppresses the polarization of Th17 cells. Purified CD4+ T cells from BALB/c mice were cultured in round-bottom 96-well plates with mitomycin C-treated antigen-presenting cells as well as anti-CD3, anti-CD28, IL-6, TGF-β, and IL-1 with graded concentration of an NO donor (NOC-18) for up to 120 h. In some cultures, anti–IL-2, anti–IL-4, and anti–IFN-γ antibodies were also added with similar results, except with a higher percentage of IL-17+ cells. (A) IL-17A in the culture supernatant was measured by ELISA, and Il17a expression in the cells was determined by qPCR. Ctrl, control. (B) IL-17– and IL-10–producing cells were determined by intracellular staining (day 4), and cell cycle was estimated by CFSE dilution (day 3) using flow cytometry (FACS). (C) Viability of the cells cultured above was determined by propidium iodide and Annexin V staining and measured by FACS on day 4 of culture. (D) Production of IL-17F was determined by ELISA, and the expression of Il17f mRNA was measured by qPCR. (E) mRNA levels of Il21, Il22, and Il23r in cells were determined by qPCR. (F) Concentrations of IL-2 in the culture supernatant (day 4, 200 μM NOC-18) were determined by ELISA, and the percentage of IL-2+ and IL-10+ cells was assayed by FACS using a cytokine secretion assay. Data are expressed as mean ± SEM (n = 5), representative of three independent experiments. *P < 0.05 compared with control without NOC-18.
Fig. 2.
Fig. 2.
NO suppresses the function of established Th17. CD4+ T cells were polarized for 5 d as in Fig. 1. The cells were then recultured for 3 d with IL-23 (20 ng/mL), anti-CD3 (4 μg/mL), and anti-CD28 (1.5 μg/mL) antibodies in the presence of the NO donor NOC-18 (200 μM) (A) or as indicated. (A) Percentages of IL-17A+ cells and RORγt+ CD4+ T cells in the control and NO-treated cells, together with the isotype control and unactivated cells (Th0), were determined by intracellular staining using FACS, and representative data are shown. Ctrl, control. (B) Quantitation of the FACS data in A. (C) Cell cycle of the established Th17 cells treated with graded concentrations of NOC-18 was determined by CFSE staining. (D) IL-17A, IL-17F, IL-22, and IL-10 concentrations in the culture supernatant were measured by ELISA. Data are mean ± SEM (n = 5), representative of four independent experiments. *P < 0.05 (compared with control without NOC-18).
Fig. 3.
Fig. 3.
NO suppresses Th17 independent of IL-2 or IL-10. CD4+ T cells from WT B6 mice or B6 Il2−/− or Il10−/− mice were purified and polarized to Th17 in the presence of 200 μM NOC-18 for 4 d as in Fig. 1. IL-17A+ and CD4+ T cells were determined by FACS (A). IL-17A concentrations in the culture supernatants of Il2−/− (B) or Il10−/− cells (C) were determined by ELISA. Data are mean ± SEM (n = 5), representative of two independent experiments. *P < 0.05 (compared with control without NOC-18). Ctrl, control.
Fig. 4.
Fig. 4.
NO suppresses the expression of AHR. (A and B) CD4+ T cells from BALB/c mice were cultured (for 3 d, unless indicated otherwise) as in Fig. 1 in the presence of NOC-18 (200 μM, unless indicated otherwise). AHR protein synthesis was analyzed by Western blot in a time- and dose-dependent manner and was also expressed as the ratio to the housekeeping protein β-actin. Ctrl, control; ND, not detectable. Data are mean ± SEM (n = 3). *P < 0.05. The expression of Ahr (C) and Rora and Rorc (D) mRNA was also analyzed by qPCR. Data are mean ± SEM (n = 3), representative of three independent experiments. *P < 0.05 (compared with control without NOC-18). (E and F) CD4+ T cells from BALB/c mice were polarized to Th17 for 3 d as above in the presence of NOC-18 (200 μM) ± FICZ (200 nM). The concentration of IL-17A in the culture supernatant was determined by ELISA (E), and the expression of mRNA of Il17f, Il22, Il23r, and Cyp1a1 was determined by qPCR (F). Data are mean ± SEM (n = 3), representative of three independent experiments. #P < 0.05 compared with control; *P < 0.05 compared with the group with FICZ but without NOC-18. (G) CD4+ T cells from WT B6 mice or B6 Ahr−/− mice were polarized to Th17 for 4 d ± NOC-18 (100 μM) in flat-bottom 96-well plates. IL-17A+ cells were analyzed by intracellular staining, and IL-22 concentration was determined by ELISA. Data are mean ± SEM (n = 3). *P < 0.05; **P < 0.01 compared with WT mice not treated with NOC-18. The difference between NOC-18–treated WT cells and NOC-18–treated Ahr−/− cells was not significant.
Fig. 5.
Fig. 5.
NO suppresses human Th17 cell differentiation. CD4+ T cells were purified from peripheral blood of healthy donors and cultured for 4 d with bead-coated anti-CD3 + anti-CD28, IL-6, TGF-β, IL-1β, IL-21, IL-23, anti–IFN-γ, and anti–IL-4 antibodies in the presence of graded concentrations of NOC-18. (A) Intracellular FACS analysis of IL-17A. Ctrl, control. (B) Concentrations of IL-17A, IL-22, and IL-10 were determined by ELISA. (C) Expression of IL-23R was also determined by FACS. The histogram shows isotype control (gray), Th17 polarized without NOC-18 or FICZ (green), Th17 polarized with 2 μM FICZ (blue), and Th17 polarized with 100 μM FICZ + NOC-18 (red). (D) Percentage of viable cells at the end of culture was analyzed by propidium iodide and Annexin V staining. Data are mean ± SEM (n = 5), representative of three independent experiments. *P < 0.05 (compared with control without NOC-18).
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