Figure 6. Blocking PIKFYVE with a small... | Rockefeller University Press

Figure 6.

$Blocking PIKFYVE with a small molecule inhibitor reduces EAE disease and Th17 differentiation in vivo. C57BL/6 mice were immunized with 200 µg of MOG35–55 peptide containing CFA. On day 0 and 2, mice received 200 ng of pertussis toxin. The treatment with apilimod or vehicle was performed daily by oral route (20 mg/kg), starting 1 day before the immunization. (A and B) (A) The clinical score was measured daily and (B) its linear regression was computed using five mice per treatment group. Draining lymph nodes were collected 7 days after EAE induction from apilimod- or vehicle-treated mice and the cells were stimulated with MOG35–55 for 96 h. All cells were stained with viability probe and antibodies for CD3 and CD4. (C and D) A fraction of the cells were also stained with antibodies for (C) IL-17A and Foxp3 or (D) GM-CSF and IFNγ. On day 15 after EAE induction, spinal cords were harvested from apilimod- or vehicle-treated mice. Isolated cells were stimulated, stained with multiple antibody panels, and analyzed by flow cytometry. (E) The level of CD4+ T cell infiltration (CD4+CD45hi cells) was determined. (F) High-dimensional flow cytometry and tSNE analysis measured the infiltration of other immune cell populations into the spinal cord in the apilimod or vehicle treated mice. (G) The abundance of Th17 cells (CD3+CD4+CD45hiIL17+) and Tregs (CD3+CD4+CD45hiFoxP3+) that infiltrated the central nervous system in apilimod- or vehicle-treated mice on day 15 of EAE were measured with flow cytometry. (H) Additionally, the abundance of GM-CSF (CD3+CD4+CD45hiGM-CSF+) and IFNγ (CD3+CD4+CD45hiIFNγ+) that infiltrated the central nervous system was measured on day 15 after EAE induction with flow cytometry. (I) Histological analysis of the leukocyte infiltration and demyelination was performed on spinal cord sections collected on day 30 after EAE induction. The black scale bar represents 100 μM. The data shown depict the mean ± SEM (n = 5 biological replicates). HE, hematoxylin and eosin; LFB, Luxol Fast Blue. P values were calculated by a two-way ANOVA test followed by Sidak test in panels A and B and by Student’s t test in panels C–E, G, and H.$

Blocking PIKFYVE with a small molecule inhibitor reduces EAE disease and Th17 differentiation in vivo. C57BL/6 mice were immunized with 200 µg of MOG_35–55 peptide containing CFA. On day 0 and 2, mice received 200 ng of pertussis toxin. The treatment with apilimod or vehicle was performed daily by oral route (20 mg/kg), starting 1 day before the immunization. (A and B) (A) The clinical score was measured daily and (B) its linear regression was computed using five mice per treatment group. Draining lymph nodes were collected 7 days after EAE induction from apilimod- or vehicle-treated mice and the cells were stimulated with MOG_35–55 for 96 h. All cells were stained with viability probe and antibodies for CD3 and CD4. (C and D) A fraction of the cells were also stained with antibodies for (C) IL-17A and Foxp3 or (D) GM-CSF and IFNγ. On day 15 after EAE induction, spinal cords were harvested from apilimod- or vehicle-treated mice. Isolated cells were stimulated, stained with multiple antibody panels, and analyzed by flow cytometry. (E) The level of CD4⁺ T cell infiltration (CD4⁺CD45^hi cells) was determined. (F) High-dimensional flow cytometry and tSNE analysis measured the infiltration of other immune cell populations into the spinal cord in the apilimod or vehicle treated mice. (G) The abundance of Th17 cells (CD3⁺CD4⁺CD45^hiIL17⁺) and Tregs (CD3⁺CD4⁺CD45^hiFoxP3⁺) that infiltrated the central nervous system in apilimod- or vehicle-treated mice on day 15 of EAE were measured with flow cytometry. (H) Additionally, the abundance of GM-CSF (CD3⁺CD4⁺CD45^hiGM-CSF⁺) and IFNγ (CD3⁺CD4⁺CD45^hiIFNγ⁺) that infiltrated the central nervous system was measured on day 15 after EAE induction with flow cytometry. (I) Histological analysis of the leukocyte infiltration and demyelination was performed on spinal cord sections collected on day 30 after EAE induction. The black scale bar represents 100 μM. The data shown depict the mean ± SEM (n = 5 biological replicates). HE, hematoxylin and eosin; LFB, Luxol Fast Blue. P values were calculated by a two-way ANOVA test followed by Sidak test in panels A and B and by Student’s t test in panels C–E, G, and H.