Research ArticleIMMUNOLOGY

Mannan-induced Nos2 in macrophages enhances IL-17–driven psoriatic arthritis by innate lymphocytes

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Science Advances  16 May 2018:
Vol. 4, no. 5, eaas9864
DOI: 10.1126/sciadv.aas9864
  • Fig. 1 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.

  • Fig. 2 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.

  • Fig. 3 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.

  • Fig. 4 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.

  • Fig. 5 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+CD3CD90.2+CD127+RORγt+ live cells (ILC3), and (iii) CD45+βTCRγδTCR+ 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.

  • Table 1 Optical parameters of tissues at 535 nm with unit of mm−1.
    BoneSkinSubcutaneous tissue
    Absorption coefficient0.010.710.10
    Reduced scattering coefficient2.184.181.73

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaas9864/DC1

    fig. S1. Representative flow cytometry gating scheme for NOS2 expression analysis in human IFN-γ/LPS–activated MDSCs and in vitro–differentiated TIP DCs.

    fig. S2. Representative flow cytometry gating scheme for skin ILC3 analysis at the paw.

    fig. S3. Representative flow cytometry gating scheme for skin γδ T cells analysis at the paw.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Representative flow cytometry gating scheme for NOS2 expression analysis in human IFN-γ/LPS–activated MDSCs and in vitro–differentiated TIP DCs.
    • fig. S2. Representative flow cytometry gating scheme for skin ILC3 analysis at the paw.
    • fig. S3. Representative flow cytometry gating scheme for skin γδ T cells analysis at the paw.

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