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. 2016 Dec;139(Pt 12):3121-3136.
doi: 10.1093/brain/aww254. Epub 2016 Oct 25.

IL4I1 Augments CNS Remyelination and Axonal Protection by Modulating T Cell Driven Inflammation

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Free PMC article

IL4I1 Augments CNS Remyelination and Axonal Protection by Modulating T Cell Driven Inflammation

Konstantina Psachoulia et al. Brain. .
Free PMC article

Abstract

SEE PLUCHINO AND PERUZZOTTI-JAMETTI DOI101093/AWW266 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Myelin regeneration (remyelination) is a spontaneous process that occurs following central nervous system demyelination. However, for reasons that remain poorly understood, remyelination fails in the progressive phase of multiple sclerosis. Emerging evidence indicates that alternatively activated macrophages in central nervous system lesions are required for oligodendrocyte progenitor differentiation into remyelinating oligodendrocytes. Here, we show that an alternatively activated macrophage secreted enzyme, interleukin-four induced one (IL4I1), is upregulated at the onset of inflammation resolution and remyelination in mouse central nervous system lesions after lysolecithin-induced focal demyelination. Focal demyelination in mice lacking IL4I1 or interleukin 4 receptor alpha (IL4Rα) results in increased proinflammatory macrophage density, remyelination impairment, and axonal injury in central nervous system lesions. Conversely, recombinant IL4I1 administration into central nervous system lesions reduces proinflammatory macrophage density, enhances remyelination, and rescues remyelination impairment in IL4Rα deficient mice. We find that IL4I1 does not directly affect oligodendrocyte differentiation, but modulates inflammation by reducing interferon gamma and IL17 expression in lesioned central nervous system tissues, and in activated T cells from splenocyte cultures. Remarkably, intravenous injection of IL4I1 into mice with experimental autoimmune encephalomyelitis at disease onset significantly reversed disease severity, resulting in recovery from hindlimb paralysis. Analysis of post-mortem tissues reveals reduced axonal dystrophy in spinal cord, and decreased CD4+ T cell populations in spinal cord and spleen tissues. These results indicate that IL4I1 modulates inflammation by regulating T cell expansion, thereby permitting the formation of a favourable environment in the central nervous system tissue for remyelination. Therefore, IL4I1 is a potentially novel therapeutic for promoting central nervous system repair in multiple sclerosis.

Keywords: experimental autoimmune encephalomyelitis (EAE); inflammation; interleukin-4 induced 1 (IL4I1); macrophages; remyelination.

Figures

Figure 1
Figure 1
Il4i1 is expressed during remyelination in CNS lesions. (A) Graph showing the differential expression pattern of Il4i1 against Mmp12, and Mbp at 5, 14 and 28 dpl. The values were obtained from a previously published rat remyelination transcriptome (Huang et al., 2011). (B) qRT-PCR detection of Il4i1 in lesioned mouse spinal cord at 3, 10 and 20 dpl compared to control non-lesioned tissue (n = 3 per group). (C) In situ hybridization of Il4i1 in a non-lesioned and focally demyelinated mouse spinal cord sections at 10 dpl. Sense control staining shows faint labelling. GM = grey matter; WM = white matter. Scale bar = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA followed by post hoc analysis.
Figure 2
Figure 2
Il4i1 is upregulated in alternatively activated microglia and macrophages through IL-4 receptor signalling. qRT-PCR detection of Il4i1 in untreated, lipopolysaccharide (LPS)- and IL4-treated (A) microglia, (B) BV2 cells, and (C) RAW264.7 cell at 24 h after treatment (n = 3 per group). (D) qRT-PCR for Il4i1 expression in unlesioned wild-type (WT), lesioned wild-type and lesioned II4ra−/− spinal cord at 10 dpl (n = 3 per group). (E) In situ hybridization of Il4i1 in wild-type and II4ra−/− mice at 10 dpl. Spinal cord lesion is encircled. GM = grey matter, WM = white matter. Scale bar = 100 μm. Density of (F) oligodendrocyte precursor cells (PDGFRα+Olig2+) and (G) oligodendrocytes (CC1+Olig2+) from MACS purified primary oligodendrocyte precursor cell cultures at 3 days in vitro after treatment with vehicle (1XPBS) or recombinant IL4I1 for 24 h (n = 5 images per condition). All experimental results were replicated at least twice. **P < 0.01, ***P < 0.001, ****P < 0.0001; ANOVA followed by post hoc analysis.
Figure 3
Figure 3
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.
Figure 4
Figure 4
IL4I1 regulates remyelination and preserves axonal integrity. Quantification of (A) Nkx2.2+Olig2+ and (B) CC1+Olig2+ cells per mm2 in lesions at 5, 10 and 20 dpl in wild-type (WT), Il4i1/ and IL4I1-treated mice. (C) Immunostaining of Olig2 (green), CC1 (red) and DAPI (blue) in lesions at 10 dpl. Lesions are characterized as the cluster of DAPI+ nuclei in the spinal cord ventral funiculous. (D) Electron micrographs of lesion at 10 dpl show reduced remyelinated axons in Il4i1/ lesions, and increased remyelinated axons in IL4I1 treated lesions compared to wild-type. (E) G-ratio analysis of remyelinated axons and corresponding axonal diameter. (F) Scatter plot showing the overall g-ratio in the mouse groups. (G) Electron micrographs of lesions in Il4i1/ mice showing a dystrophic axon and an axon that has undergone Wallerian degeneration. (H) Immunostaining of SMI-32 (green), NF200 (red) and DAPI (blue) in spinal cord lesions at 10 dpl. Axonal dystrophy is detected by SMI-32+NF200+ co-labelling. (I) Graph of relative SMI-32 signal intensity normalized to NF200. For cell counts and fluorescence intensity, n = 3–5 mice per group were used and n = 3 ×10 magnification images per mouse were analysed. Scale bar = 100 μm for immunofluorescence images. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ANOVA followed by post hoc analysis.
Figure 5
Figure 5
IL4I1 injection rescues remyelination impairment in Il4ra/ mice. Quantification of (A) CC1+Olig2+ cells, (B) SMI-32 signal intensity normalized to NF200, and (C) CD11b+iNOS+ macrophages in Il4ra−/− and Il4ra−/− treated mice at 10 dpl. *P < 0.05, **P < 0.01, ***P < 0.001; ANOVA followed by post hoc analysis.
Figure 6
Figure 6
IL4I1 modulates IFN-γ and IL-17 expression in CNS lesions and splenocytes. (A) Normalized expressions of Ifng, Il17 and Il4 in wild-type (WT, n = 4) and IL4I1-treated (n = 6) spinal cord lesions at 10 dpl by qRT-PCR. (B) Normalized expression of Ifng expression in splenocyte cultures treated with PMA+Ionomycin in the presence or absence of recombinant IL4I1 for 24 h (n = 3 per group) followed by qRT-PCR. (C) Cell death assay in splenocyte cultures treated with PMA+Ionomycin with or without recombinant IL4I1 for 24 h, and assessed by LIVE/DEAD® cell stain kit (n = 8 per group).
Figure 7
Figure 7
IL4I1 reverses clinical severity and preserves axons in mice with EAE. (A) Clinical scores of PBS-treated (n = 8) and IL4I1-treated (n = 10) mice for 28 days after EAE induction. Flow cytometry analysis of (B) spinal cord and (C) spleen from untreated and IL4I1 treated mice at 35 days after EAE induction showing the percentage and total number of gated T-bet+CD4+, Rorγt+CD4+ and Gata3+CD4+ cells (samples from n = 2 mice were combined for each group and analysed). (D) Immunostaining of SMI-32 (green), NF200 (red) and DAPI (blue) in spinal cord of untreated and IL4I1 treated mice with EAE at 35 days after EAE induction. Axonal dystrophy is detected by SMI-32+NF200+ co-labelling. (E) SMI-32 signal intensity normalized to NF200 in untreated, and IL4I1-treated mice showing reduced axonal dystrophy in IL4I1 treated mice (n = 3 per group). Scale bar = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; one-way Wilcoxon Rank-Sum Test (EAE), two-tailed Student’s t-test (SMI-32 analysis).

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