Figure 4. Spinal cord myeloid cells express IL-3Rɑ and aggravate EAE by instigating immune cell recruitment to the CNS.

(A) Representative immunofluorescent images of IL-3Rɑ (CD123) and CD11b on spinal cord sections of 4 WT EAE mice at disease peak. Scale bar is 100 μm.

(B) Flow plots showing the cell gating strategy for leukocytes and non-leukocytes in the spinal cords of WT EAE mice at peak disease.

(C) Flow cytometric analysis and quantification of IL-3Rɑ expression by spinal cord cells at peak disease (n=9 mice).

(D) qPCR analysis of Il3rɑ expression by myeloid cells sorted from the spinal cord at disease peak (n=4-5 mice).

(E) Mean clinical score and corresponding AUC analysis of WT and Il3rɑ^−/− mice during EAE (n=8-12 mice/group; two-way ANOVA and Mann–Whitney U-test).

(F) Representative histological sections of demyelination in diseased spinal cords from 5 WT and 5 Il3rɑ^−/− mice stained for myelin by Luxol fast blue and counterstained with Cresyl Echt Violet Solution at disease peak. Scale bars represent 500 μm for overview images and 100 μm for the inset images.

(G) Quantification of demyelination area in diseased spinal cords from WT and Il3rɑ^−/− mice (n=5 mice/group; two-way ANOVA and Mann–Whitney U-test).

(H) Flow cytometric analysis of spinal cord CD45⁺ leukocyte subsets in WT and Il3rɑ^−/− mice at peak disease (n=5-6 mice/group; Mann–Whitney U-tests).

(I) Transcript expression analysis in myeloid cells sorted from the spinal cord of WT EAE mice at the peak of disease and stimulated with rIL-3 (n=5 group; Mann–Whitney U-tests).

(J) Measurement of CCL2 in the media of monocyte cultures exposed to rIL-3 (n=5 group; Mann–Whitney U-test).

(K) Enumeration of immune cells in the brain of mice 24 hours after stereotactic injection of 0, 1, 10, or 100 ng of rIL-3 (n=3-8 mice/group; one-way ANOVA).

Mean±s.e.m., *p<0.05, **p<0.01, ***p<0.001.

WT, wildtype; EAE, experimental autoimmune encephalomyelitis; MFI, mean fluorescence intensity; rIL-3, recombinant interleukin-3.

See also Figure S4.