Nickel Sulfate Promotes IL-17A Producing CD4+ T Cells by an IL-23-Dependent Mechanism Regulated by TLR4 and Jak-STAT Pathways  Rami Bechara, Diane Antonios, Hayat Azouri, Marc Pallardy  Journal of Investigative Dermatology  Volume 137, Issue 10, Pages 2140-2148 (October 2017) DOI: 10.1016/j.jid.2017.05.025 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Effect of metallic haptens on the production of IL-23 and IL-12 by human MoDCs. Immature DCs were treated or not with 500 μM of NiSO4, NiCl2, CoCl2, or 5 μM of K2Cr2O7 or 25 ng/ml of LPS for the indicated time. ELISA was performed to measure (a) IL-23, (b) IL-12p40, and (c) IL-12p70 production after 24 hours. Values are presented as mean ± SEM of four independent experiments. (d) Kinetics of IL-23 production by NiSO4-activated MoDCs. Values are presented as mean ± SEM of two independent experiments. (e) il-23p19, (f) il-12p40, and (g) il-12p35 mRNA levels were quantified by qRT-PCR. Results are expressed as fold factor compared with untreated samples and corrected by the expression of β-actin and gapdh. Values are presented as mean ± SEM of four independent experiments. *P ≤ 0.05, Mann-Whitney. CoCl2 , cobalt chloride; K2Cr2O7, potassium dichromate; LPS, lipopolysaccharide; MoDC, monocyte-derived dendritic cell; NiCl2, nickel chloride; NiSO4, nickel sulfate; qRT-PCR, quantitative real-time reverse transcriptase-PCR; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 2140-2148DOI: (10.1016/j.jid.2017.05.025) Copyright © 2017 The Authors Terms and Conditions

Figure 2 NiSO4-activated MoDCs promote IL-17A-producing-CD4+ T cells through IL-23 production. (a–d) Frequency of IL-17A and/or IFN-γ-positive CD4+ T cells cultured for 9 days with NiSO4-activated MoDCs or control. Cytokine content was measured by intracellular staining after 5 hours of PMA/ionomycine activation and after gating on viable CD4+ T cells. (e–h) Anti-IL-23p19 neutralizing Ab or control Ab were added to NiSO4-activated MoDCs 1 hour before the coculture, for 9 days, with allogeneic CD4+ T cells. Cytokine content was measured by intracellular staining after 5 hours of PMA/ionomycine activation and after gating on viable CD4+ T cells. One representative experiment is shown in (i). Each circle represents a donor for a total of six independent experiments with six different donors. #P ≤ 0.05, Wilcoxon signed rank test. Ab, antibody; MoDC, monocyte-derived dendritic cell; NiSO4, nickel sulfate; PMA, phorbol 12-myristate 13-acetate. Journal of Investigative Dermatology 2017 137, 2140-2148DOI: (10.1016/j.jid.2017.05.025) Copyright © 2017 The Authors Terms and Conditions

Figure 3 IFN-γ regulates IL-23 production by NiSO4 in human MoDCs. DCs were treated or not with NiSO4, in addition or not of IFN-γ (1,000 U/ml) for the indicated time. ELISA was performed to measure (a) IL-23, (b) IL-12p40, and (c) IL-12p70 production after 24 hours. Values are presented as means ± SEM of four independent experiments (a–c). (d) Kinetics of IL-23 production by NiSO4 or NiSO4- and IFN-γ-activated DCs. Values are presented as mean ± SEM of two independent experiments. (e) Molarity ratio of IL-23 to IL-12p70 for each condition of stimulation based on the molecular weight of IL-23 and IL-12p70 and their respective concentrations obtained by ELISA at 24 hours after stimulation. (f) il-23p19, (g) il-12p40, and (h) il-12p35 mRNA levels were quantified by qRT-PCR. Values are presented as mean ± SEM of four independent experiments *P ≤ 0.05, Mann-Whitney. MoDC, monocyte-derived dendritic cell; NiSO4, nickel sulfate; qRT-PCR, quantitative real-time reverse transcriptase-PCR; SEM, standard error of the mean. Journal of Investigative Dermatology 2017 137, 2140-2148DOI: (10.1016/j.jid.2017.05.025) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Production of IL-23 and IL-12 in NiSO4-activated MoDCs is TLR4, p38MAPK, and NF-κB dependent. At day 5, immature DCs were pretreated or not (control) for (a–c) 1 hour with an anti-TLR4 or isotype control antibodies (1, 5, and 10 μg/ml) or (d–f) 30 minutes with the inhibitor of the p38MAPK pathway (SB203580, 20 μM) or for 1 hour with either DMSO or with the inhibitor of the NF-κB pathway (BAY 11-7085, 3 μM). Cells were then treated or not with NiSO4 (500 μM) for 24 hours. ELISA was performed after 24 hours of stimulation. Values are presented as mean ± SEM of four independent experiments. *P ≤ 0.05, Mann-Whitney. MAPK, mitogen-activated protein kinase; MoDCs, monocyte-derived dendritic cells; NiSO4, nickel sulfate; SEM, standard error of the mean; TLR, toll-like receptor. Journal of Investigative Dermatology 2017 137, 2140-2148DOI: (10.1016/j.jid.2017.05.025) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Inhibition of the Jak-STAT pathway enhances the IL-23/IL-12p70 ratio in NiSO4 or NiSO4 and IFN-γ-activated MoDCs. At day 5, immature DCs were pretreated with either DMSO or with the inhibitor of the Jak-STAT pathway (Jak Inhibitor I, 0.5 μM) and subsequently treated or not with NiSO4 (500 μM) (a–c) in the presence or not of IFN-γ (1,000 U/ml) (d–f) for 24 hours. Cell supernatants were assayed by ELISA. Results are expressed in pg/ml. Values are presented as mean ± SEM of four independent experiments. *P ≤ 0.05, Mann-Whitney. (g) Molarity ratio of IL-23 to IL-12p70 for each condition of stimulation based on the molecular weight of IL-23 and IL-12p70 and their respective concentrations obtained by ELISA after 24 hours of stimulation. MoDC, monocyte-derived dendritic cell; NiSO4, nickel sulfate; SEM, standard error of the mean; STAT, signal transducer and activator of transcription. Journal of Investigative Dermatology 2017 137, 2140-2148DOI: (10.1016/j.jid.2017.05.025) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Inhibition of the Jak-STAT pathway in NiSO4-treated MoDCs modulates the Th1/Th17 balance. (a–d) Frequency of IL-17A and/or IFN-γ-positive CD4+ T cells cultured for 9 days with MoDCs treated or not with NiSO4. In some conditions, MoDCs were pretreated with Jak Inhibitor I (0.5 μM) for 2 hours and then activated or not with NiSO4. Cytokine content was measured by intracellular staining after 5 hours of PMA/ionomycine activation and after gating on viable CD4+ T cells. One representative experiment is shown in (e). Each circle represents a donor for a total of six independent experiments with six different donors. #P ≤ 0.05, Wilcoxon signed rank test. MoDC, monocyte-derived dendritic cell; NiSO4, nickel sulfate; PMA, phorbol 12-myristate 13-acetate; STAT, signal transducer and activator of transcription; Th, T helper. Journal of Investigative Dermatology 2017 137, 2140-2148DOI: (10.1016/j.jid.2017.05.025) Copyright © 2017 The Authors Terms and Conditions