Journal: The FASEB Journal

Article Title: FOXP3⁺ Macrophage‐Derived Amphiregulin Promotes White Matter Repair of Experimental Autoimmune Encephalomyelitis

doi: 10.1096/fj.202500075rr

Figure Lengend Snippet: FIGURE 4 | AREG secreted by FOXP3+ macrophages promotes remyelination of OPCs. (A) Venn diagram on ligands produced by macrophages for OPCs differentiation among NicheNet, genes exhibiting FOXP3 binding as determined by CUT&RUN, and the differentially upregulated genes in FOXP3+ BMDM from the bulk RNA-seq. (B) Representative sections labeled with IBA1 (red) and AREG (green) of WT EAE mice and CKO EAE mice at the chronic phase were displayed. N = 5, compared with the WT control group, by one-way ANOVA (mean ± SD). Scale bar = 20 μm. (C) EAE peak phase spinal cord slices were displayed with IBA1 (green), AREG (red) and FOXP3 (blue). Chi-square and Fisher exact test were calculated for correlation between FOXP3 expression and AREG expression in macrophages. (D) Expression of AREG in FOXP3+ macrophages by flow cytometry. N = 5, compared with FOXP3− cells, by Student's t test (mean ± standard deviation). (E) Culture supernatants of WT and Foxp3-KO BMDM (± myelin pre-treated) were collected and assessed AREG concentration with ELISA. Experiments repeated 3 times, compared with WT BMDM, by one-way ANOVA (mean ± SD). (F) Expression of Areg in WT and Foxp3-KO BMDM (± myelin treated) by qPCR. Experiments repeated 3 times, by Student's t test (mean ± SD). (G) IGV visual analysis of the FOXP3 binding peak in the Areg promoter region from CUT&RUN. The arrows refer to the poten- tial binding site of FOXP3. (H) Quantitative analysis using anti-FOXP3 antibody and qPCR. Experiments were repeated 3 times, by Student's t test (mean ± SD). **p < 0.01, ***p < 0.001.

Article Snippet: The following primary antibodies were used: rabbit anti- Olig2 (Abcam, ab109186, 1:300), mouse anti- CC1 (Abcam, ab16794, 1:1000), rabbit anti- MBP (Proteintech, 10458- 1- AP, 1:500), rabbit anti- NG2 (Abcam, ab129051, 1:500), rabbit anti- dMBP (Millipore, ab5864, 1:2000), chicken antiNeurofilament H (Biolegend, 822601, 1:400), mouse anti- Ki67 (Abcam, ab279653, 1:500), mouse polyclonal anti- FOXP3 (Invitrogen, 14- 5773, 1:500), rabbit monoclonal anti- FOXP3 (Invitrogen, 700914, 1:500), rabbit anti- LC3A/B (Cell signaling technology, 12 741T, 1:1000), rabbit anti- Iba1 (Wako, 019- 19741, 1:1000), goat anti- AREG (R&D Systems, AF989, 1:500), and rabbit polyclonal anti- TMEM119 (catalog PA5- 119617, Invitrogen, 1:500).

Techniques: Produced, Binding Assay, RNA Sequencing, Labeling, Control, Expressing, Flow Cytometry, Standard Deviation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: FOXP3⁺ Macrophage‐Derived Amphiregulin Promotes White Matter Repair of Experimental Autoimmune Encephalomyelitis

doi: 10.1096/fj.202500075rr

Figure Lengend Snippet: FIGURE 4 | AREG secreted by FOXP3+ macrophages promotes remyelination of OPCs. (A) Venn diagram on ligands produced by macrophages for OPCs differentiation among NicheNet, genes exhibiting FOXP3 binding as determined by CUT&RUN, and the differentially upregulated genes in FOXP3+ BMDM from the bulk RNA-seq. (B) Representative sections labeled with IBA1 (red) and AREG (green) of WT EAE mice and CKO EAE mice at the chronic phase were displayed. N = 5, compared with the WT control group, by one-way ANOVA (mean ± SD). Scale bar = 20 μm. (C) EAE peak phase spinal cord slices were displayed with IBA1 (green), AREG (red) and FOXP3 (blue). Chi-square and Fisher exact test were calculated for correlation between FOXP3 expression and AREG expression in macrophages. (D) Expression of AREG in FOXP3+ macrophages by flow cytometry. N = 5, compared with FOXP3− cells, by Student's t test (mean ± standard deviation). (E) Culture supernatants of WT and Foxp3-KO BMDM (± myelin pre-treated) were collected and assessed AREG concentration with ELISA. Experiments repeated 3 times, compared with WT BMDM, by one-way ANOVA (mean ± SD). (F) Expression of Areg in WT and Foxp3-KO BMDM (± myelin treated) by qPCR. Experiments repeated 3 times, by Student's t test (mean ± SD). (G) IGV visual analysis of the FOXP3 binding peak in the Areg promoter region from CUT&RUN. The arrows refer to the poten- tial binding site of FOXP3. (H) Quantitative analysis using anti-FOXP3 antibody and qPCR. Experiments were repeated 3 times, by Student's t test (mean ± SD). **p < 0.01, ***p < 0.001.

Article Snippet: Once clinical symptoms develop in mice, WT EAE mice were treated with intrathecal injection (i.t.) of anti- AREG (2 μg per mouse, Clone #206220, Cat# AF989, R&D Systems), control IgG (R&D Systems) every other day for 5 days.

Techniques: Produced, Binding Assay, RNA Sequencing, Labeling, Control, Expressing, Flow Cytometry, Standard Deviation, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: FOXP3⁺ Macrophage‐Derived Amphiregulin Promotes White Matter Repair of Experimental Autoimmune Encephalomyelitis

doi: 10.1096/fj.202500075rr

Figure Lengend Snippet: FIGURE 6 | The secretion of AREG by FOXP3+ macrophages facilitates white matter repair in the EAE. (A–D) WT C57BL/6 female mice were induced EAE, and intrathecal injected (i.t.) with anti-AREG (2 μg per mouse) or the same volume of control IgG every other day for 5 days once EAE symptoms developed. Mice were sacrificed at 28 dpi, and spinal cords were collected for staining and TEM analysis. (E–H) Foxp3 CKO mice (Lyz2Cre-ERT2foxp3fl/fl) were induced EAE, and i.t. with rmAREG (0.2 μg per mouse) or the same volume of PBS for five consecutive days once EAE symptoms developed. Mice were sacrificed at 28 dpi, and spinal cords were collected for staining and TEM analysis. (A, E) Experimental proce- dures displayed above. EAE scores displayed below. N = 6 per group. Data were shown as mean score. Data were analyzed with two-way ANOVA (mean ± standard error), compared with the WT group or CKO group. (B, F) Immunostaining of MBP (red). The white dashed lines outlined the de- myelination lesions. N = 5, compared with the WT group or CKO group, by Student's t test (mean ± SD). Scale bar = 50 μm. (C, G) The demyelination lesions were displayed by LFB staining. N = 5, compared with the WT group or CKO group, by Student's t test (mean ± SD). Scale bar = 100 μm. (D, H) Left: Representative TEM images of WT and CKO mice at 28 dpi. Scale bar = 2.5 μm. Left of the Middle: Quantification of the number of myelinated axons per 1000 μm2, N = 3, compared with the WT group, by Student's t test (mean ± SD). Right of the Middle: Scatter plots of g-ratio as a function of axon diameter. N = 3 per group. Right: The g-ratio of the small-sized (diameter < 0.4 μm), middle-sized (diameter = 0.4–0.8 μm) and large (diame- ter > 0.8 μm) axonal fibers were compared. N = 3, versus WT group or CKO group, by Student's t test (mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Once clinical symptoms develop in mice, WT EAE mice were treated with intrathecal injection (i.t.) of anti- AREG (2 μg per mouse, Clone #206220, Cat# AF989, R&D Systems), control IgG (R&D Systems) every other day for 5 days.

Techniques: Injection, Control, Staining, Immunostaining