Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells

Abstract

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.

Fig 1. Cytokine release is dependent on NO^. concentration.

DCs were stimulated for 23 h with different concentrations of DPTA NONOate 10 minutes before maturation with LPS (100ng/ml). Cytokines were measured using Luminex system as described in Material and Methods: (A) IL-10, (B) IL-12p70 and (C) TNF-α. Data are expressed as means ±SEM of 6 independent experiments. *P < 0.05.

Fig 2. NO^. inhibits the release of IL-12 at early time-points.

DCs were stimulated with DPTA NONOATE (0.6mM) 10 min, 1h and 5h before maturation with LPS (100ng/ml). The secretion of cytokines was analyzed using the Luminex system: (A) IL-10 (Due to a high variability of the IL-10 production the values were normalized to the levels of LPS %), (B) IL-12p70 and (C) TNFα. Data are expressed as means ±SEM of 11 independent experiments. *P < 0.05.

Fig 3. NO^. fails to modify the alloantigen T-cell response but enhances the release of IL-17 during a MLR.

DCs were simulated in time-dependent experiments with DPTA NONOATE^. 0.6mM and LPS 100ng/ml. 1×10⁵ responder T-cells used for allogenic MLR assays were added to washed DCs for 6 days. A-E, Bioplex of IL-17, IFN-γ, TNF-α, IL-6, IL-13 in 6-day culture at a DC:T-cell ratio of 1:10. Data are expressed as means ±SEM of 7 independent experiments for IL-17 and 6 independent experiments for IFN-γ, TNF-α, IL-6, IL-13. F, T-cell proliferation was measured by incorporation of thymidine at DC:T-cell ratios of 1:10, 1:50 and 1:100. Data are expressed as means ±SEM from 3 independent experiments done in triplicate. *P < 0.05.

Fig 4. NO^. promotes the release of IL-23, IL-6 and IL-1β during maturation of DCs.

DCs were stimulated with DPTA NONOate (0.6mM) treated DCs 1h or 5h before maturation with LPS (100ng/ml). (A) IL-23 and (B) IL-6 were analyzed in supernatants. (C) IL-1β was analyzed in supernatants, but also in DCs pre-treated with the specific caspase-I inhibitor II Ac-YVAD-CMK (50 M) for 1 h before the DPTA NONOate /LPS stimulation. (D) Cells were harvested and intracellular pro-IL-1β (blotted with a specific pro-IL1β was analyzed by Western blot. Data are expressed as means ±SEM of 6 independent experiments. *P < 0.05 or **P < 0.01.

Fig 5. NO^. modulates the gene expression of IL-12, IL -23, IL-6 and IL-1β during maturation of DCs.

DCs were stimulated with DPTA NONOate (0.6mM) for 30 min before maturation with LPS (100ng/ml). Cells were analyzed at the different time-points indicated. (A) IL-12Bp40, (B) IL-12Ap35, (C) IL-23Ap19, (D) IL-1β and(E) IL-6 levels were assessed by TaqMan real-time reverse transcription—polymerase chain reaction (RT-PCR), using β-actin as endogenous control. Data express the relative gene abundance compared to control conditions, and are shown as mean ±SEM of 4 independent experiments, done in duplicates. *P < 0.05 or **P < 0.01.

Fig 6. IL-1β is required to prime T-cells to produce IL-17 in a mixed lymphocyte reaction condition.

Production of IL-17 by T-cells primed for 6 days with allogenic DCs at a DC:T-cell ratio of 1:10. As described in Materials and Methods, DCs were simulated with DPTA NONOate (0.6mM) 1h before LPS (100ng/ml) stimulation. During the MLR, cells were cultured in the presence of neutralizing antibodies to IL-23, IL-1β and IL-6R. Data are expressed as mean ±SEM of 3 independent experiments. *P < 0.05.

Fig 7. Carboxy-PTIO inhibits the DPTA NONOate induction of IL-1β and IL-23.

DCs were stimulated with Carboxy-PTIO (5μM) 30 min before treatment with DPTA NONOATE (0.6mM in RPMI or electrolyzed water (EW)). After 1h stimulation cells were maturated with LPS (100ng/ml). The secretion of cytokines was analyzed using the Luminex system: (A) IL-1β, (B) IL-23 and (C) IL-12p70. Data are expressed as means ±SEM of 3 independent experiments. *P < 0.05.