aww254-F3: IL4I1 modulates inflammation in CNS lesions. Quantification of macrophage subpopulations in lesions of wild-type, Il4i1−/− and IL4I1 treated mice at 5, 10 and 20 dpl. (A) CD11b+ iNOS+ cell quantification. (B) Immunostaining of iNOS (green), CD11b (red) and DAPI (blue) at 10 dpl. (C) CD11b+Ym1+ cell quantification. (D) Immunostaining of Ym1 (green), CD11b (red) and DAPI (blue) at 10 dpl. (E) Quantification of the ratio of iNOS+/Ym1+ cells in lesions. (F) Flow cytometry analysis of iNOS+CD11b+ cells in lesioned spinal cord of wild-type (WT) and IL4I1-treated mice at 10 dpl. For cell counts, n = 3–5 mice per group were used and n = 3 × 10 magnification images per mouse were analysed. Scale bar = 100 μm.

Mentions: To determine if IL4I1 modulates inflammation during CNS remyelination, focal demyelination was performed on Il4i1 knockout (Il4i1−/−) mice. These mice do not display obvious developmental or behavioural abnormalities (according to MMRRC repository). The absence of Il4i1 expression in CNS lesion was confirmed by qRT-PCR and in situ hybridization (Supplementary Fig. 2A and B). To examine the distribution of proinflammatory macrophage subpopulations in lesions, immunostaining analysis for CD11b and iNOS co-labelling, corresponding to CAM, was performed on wild-type and Il4i1−/− mice at 5, 10 and 20 dpl. In wild-type lesions, we found that the relative density of CD11b+iNOS+ macrophages was high at 5 dpl, and reduced at 10 and 20 dpl (Fig. 3A and B). In Il4i1−/− lesions, as in the wild-type, we observed a relatively high density of CD11b+iNOS+ macrophages at 5 dpl. However, this density remained significantly elevated across all three post-lesion time points, unlike their wild-type counterparts (Fig. 3A and B). To assess the distribution of other proinflammatory cell populations, CNS lesions at 10 dpl were co-immunostained of tenascin-C and GFAP to identify reactive astrocytes, or intracellular fibronectin (IST-9) to identify meningeal fibroblasts. We found that mice deficient in Il4i1 displayed enhanced tenascin-C+ and IST-9+ staining in lesions, suggesting increased gliosis (Supplementary Fig. 3). These results suggest that inflammation remains unresolved in the absence of Il4i1 expression.Figure 3