by Jianghong Zhong, Tatjana Scholz, Anthony C. Y

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Mannan-induced Nos2 in macrophages enhances IL-17–driven psoriatic arthritis by innate lymphocytes by Jianghong Zhong, Tatjana Scholz, Anthony C. Y. Yau, Simon Guerard, Ulrike Hüffmeier, Harald Burkhardt, and Rikard Holmdahl Science Volume 4(5):eaas9864 May 16, 2018 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 1 Mannan induces the production of NO. Mannan induces the production of NO. (A) The serum NOx status in Ncf1+/+ and Ncf1*/* mice at day 5 after mannan administration (n = 5). Phosphate-buffered saline (PBS) injection was used as negative controls (n = 5). (B) Kinetic effect of l-NAME administration on serum NOx status in Ncf1*/* mice at 2 and 5 days postinjection (DPI) of mannan using PBS as controls. n = 5 mice per group. The black asterisk stands for the significance in comparison at 2 DPI, whereas the red one for 5 DPI. (C) Example of in vivo peroxynitrite imaging at 2 DPI. (D) Statistical analysis of the mean optical radiance at paws per mouse at 2, 5, and 8 DPI. The significance is calculated for each control group in comparison with the l-NAME group. (E) Suppression of mannan-induced Ps-like arthritis by administration of l-NAME. The asterisk stands for the significance comparing l-NAME group with the d-NAME and blank group. For (D) and (E), both Ncf1+/+ and Ncf1*/* mice were divided into three groups (n = 5 per group), that is, l-NAME and d-NAME treatments and nontreated individuals as blank controls. All results are shown as mean ± SEM. Jianghong Zhong et al. Sci Adv 2018;4:eaas9864 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 2 Deficiency of Nos2 alleviates MIP development. Deficiency of Nos2 alleviates MIP development. (A) Levels of serum NOx in naïve Ncf1+/+.Nos2−/− mice and the Ncf1+/+.Nos2+/+ littermates and naïve Ncf1*/*.Nos2−/− mice and their littermates Ncf1*/*.Nos2+/+ mice, compared with serum NOx at 5 DPI. l-NAME was administered daily for 5 days beginning on day 0, and PBS was used as the control (n = 5 mice per group). (B) Comparison of H2O2 generation by fresh bone marrow cells isolated from naïve Ncf1+/+.Nos2−/− mice, naive littermates Ncf1+/+.Nos2+/−, and Ncf1+/+.Nos2+/+ controls under the defined conditions (n = 5 mice per group). (C) Representative tomography of peroxynitrite distribution (red volume) in the paw and the statistical comparison with the volume of peroxynitrite production in mice with genotype of Ncf1+/+.Nos2+/+ (n = 4), Ncf1+/+.Nos2+/− (n = 4), and Ncf1+/+.Nos2−/− (n = 4) at 2 DPI. In (B) and (C), the same marked signs stand for these three kinds of mice groups. (D) Evaluation of the effect of l-NAME in both Ncf1+/+.Nos2−/− and the littermate Ncf1+/+.Nos2+/+ mice after mannan injection (n = 7 mice per group). (E) A study of arthritis was performed in both Ncf1*/*.Nos2−/− mice (n = 6) and the littermate Ncf1*/*.Nos2+/+ mice (n = 10) after mannan injection. All results are shown as mean ± SEM. Jianghong Zhong et al. Sci Adv 2018;4:eaas9864 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 3 NOS2 expression is enhanced upon in vitro activation for patients with PsA. NOS2 expression is enhanced upon in vitro activation for patients with PsA. Following an identical IFN-γ and LPS preactivation procedure, MFIs of NOS2 expression in CD14+ PBMC subpopulation (CD14+), human leukocyte antigen–DR (HLA-DR) and CD11c double-positive cells (TIP DC), and CD11b+ MDSC subpopulation were determined in the cohort of PsA patients (n = 39), compared with those obtained in the HD group (n = 18). All results are shown as mean ± SEM. Jianghong Zhong et al. Sci Adv 2018;4:eaas9864 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 4 NOS2-dependent IL-1α promotes MIP development. NOS2-dependent IL-1α promotes MIP development. (A) The numbers of CD11b+CD45+ live skin cells were calculated for hind paws from both Ncf1+/+ and Ncf1*/* mice with l-NAME or PBS treatment at 5 DPI. n = 6 mice per group including the naïve mice. (B to D) The concentrations of TNF-α, IL-17A, and IL-1α in the supernatant solution of skin cells were quantified after 4 hours of stimulation with PMA/ionomycin. (E) The NOS2-dependent increase of IL-1α was found in the supernatant of skin cells from the mice with MIP disease at day 5, and each group included five mice for the cytokine analysis. (F) Evaluation of the time course was performed in Ncf1*/* mice during IL-1α in vivo neutralization (n = 5 mice per group) after mannan injection. All results are shown as mean ± SEM. Jianghong Zhong et al. Sci Adv 2018;4:eaas9864 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

Fig. 5 Resident IL-17A production requires NO for ILC3 but not γδ T cells. Resident IL-17A production requires NO for ILC3 but not γδ T cells. The Ncf1*/* mice received an arthritogenic dose of mannan at day 0. l-NAME and PBS were injected intraperitoneally daily for 5 days beginning on day 0, and the local skin was excised from hind paws when the mouse was sacrificed using CO2 at day 5. The skin cells were analyzed by flow cytometry for the following cell populations: (i) total number of CD45+ live cells, (ii) CD45+CD3−CD90.2+CD127+RORγt+ live cells (ILC3), and (iii) CD45+βTCR−γδTCR+ live cells (γδ T cells). (A) Schematic illustration of the region of interest (ROI) at the position corresponding to dotted lines for isolating skin samples from the paw. (B) Number of CD45+ live skin cells from the selected ROI in the paw. (C to F) Frequency of IL-17A+ expression was shown within both ILC3 in (C) and (D) and γδ T cells in (E) and (F). Each group included five mice for the intracellular cytokine analysis by flow cytometry. All results are shown as mean ± SEM. Cy7, Cyanine7. Jianghong Zhong et al. Sci Adv 2018;4:eaas9864 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).