Concentration of IL-17A (A), IL-17F (B) CXCL5 (C), CXCL8 (D), IL-5 (E), Eotaxin I (F), IL-12p40 (G) and IL-12p70 (H) IFN-β (I), IP10 (J) in serum of six patients with Neuromyelitis optica (NMO) with spinal cord lesions greater than three vertebral segments (MR>3seg) and 10 patients with NMO with spinal cord lesions less than three vertebral segments (MR<3seg). Cytokine concentrations were measured by Luminex multiplex assay and calculated from a standard linear regression of known quantities of each cytokine. Neutrophil elastase (K) was measured in plasma from six NMO patients and seven healthy volunteers by ELISA. Data are represented as mean ± SEM and p-values from two-tailed Student’s t-tests are shown.

To induce Th1 and Th17 experimental autoimmune encephalomyelitis (EAE), donor mice were sacrificed 10 days after MOG immunization (see methods) and spleens and draining lymph nodes were isolated and restimulated with 20 µg/ml MOG_35–55 in the presence of IL-12 (Th1) or IL-23 (Th17) for 3 days and transferred to healthy recipient mice to induce EAE. (A) Quantification of levels of IL-17, CXCLI, IL-5, IFN-γ and IL-2 in spleen culture supernatants of Th17 and Th1 EAE animals at peak of disease after 3 days of restimulation with MOG_35–55. Cytokine secretion was measured by ELISA. Results are shown as mean ± SEM of three animals per group. IFN-γ:*p < 0.04; IL-17: *p < 0.023 **p < 0.005; CXCLI:*p < 0.039 **p < 0.008; IL-5: *p < 0.026.
(B) Flow cytometry analysis of IFN-γ and IL-l7-secreting CD4+cells from central nervous system (CNS) of Th17 and Th1-induced EAE mice at peak of disease. *p < 0.05.
(C–E) Flow cytometry analysis of MHCII⁺ and GR1⁺ cells of CD11b⁺ cells from CNS of Th17 and Th1-induced EAE mice at peak of disease (C). Quantification of GR1^hiMHCII^lo (D) and GR1^loMHCII^hi (E) cells of CD11b⁺ cells from CNS. Data represent individual values of 4–6 animals, mean and SEM.*p < 0.03,**p < 0.002.
(F, G) Representative microphotographs of CNS infiltrates of Th17 (F) and Th1 (G) -induced EAE mice isolated by cytospin. Cells were stained by Wright–Giemsa. Arrows show neutrophilic polymorphonuclear cells.
(H, I) Histology of inflammatory infiltrates in the optic tract of Th17 (H) and Th1 (I)-induced EAE stained with H&E at the peak of disease. Scale bars: 100 µm.
(J) Quantification of lesion volume in optic tract of Th17 and Th1-induced EAE presented as percentage of lesion volume of the optic tract.

(A) Representative immunofluorescence stainings of neutrophil elastase (red), histone (green) and DNA (blue) in human neutrophil cultures stimulated with media only, C5a, IFN-β and C5a + IFN-β for 4h. 20×. (B) Quantification of mean number of neutrophil extracellular traps (NETs) in neutrophil cultures of four healthy controls stimulated with media only, C5a, IFN-β and C5a + IFN-β. p-value from a two-tailed Student’s t-test is shown.

(A, B) Clinical scores from mice with passive experimental autoimmune encephalomyelitis (EAE) induced by adoptive transfer of Th17 (A) or Th1 (B) cells. Mice were treated with the indicated dose of Sivelestat or PBS i.p. every day from day 6 to 16 after transfer. Arrows depict initiation and termination of treatment. Results represent mean clinical score of 5–14 mice per group. Error bars represent means ± SEM. *p < 0.05, ** p < 0.01, # p < 0.005. (C–F) Histology of spinal cord and brain sections from Th17- or Th1-induced EAE mice treated with Sivelestat or PBS. Sections of spinal cord and brain were obtained 9 days after transfer and stained with H&E (C) and Luxol fast blue (F). Scale bars: 100 µm (C), 200 µm (F). Quantification of number of inflammatory foci in spinal cord (D) and brain (E). Data represent mean ± SEM of three mice per group.*p < 0.03, **p < 0.009. (G–I) Concentration of IL-17 (G), IL-2 (H) and IL-5 (I) from supernatants of MOG_35–55-stimulated spleen cells taken from mice 9 days after transfer of encephalitogenic Th17 or Th1 cells. Mice were treated with Silvelestat or PBS starting at day 6. Data represent mean ± SEM of three mice per group. *p < 0.05.