Journal: Journal of Neuroinflammation
Article Title: Myeloid ZNRF1 suppresses autoimmune demyelination and neuroinflammation by regulating MHC-II-mediated T cell activation
doi: 10.1186/s12974-025-03550-z
Figure Lengend Snippet: ZNRF1 deficiency in myeloid cells promotes EAE pathogenesis and neuroinflammation in mice. Age-matched female Znrf1 F/F and Znrf1 Δmye mice were immunized with 200 μg of MOG 35-55 peptide emulsified in CFA, followed by i.p. injection of 200 ng PTX to induce EAE. a Clinical scores of EAE progression were monitored and recorded daily (N = 42). b Body weights of immunized mice were measured and normalized to their respective weights at day 8 post-immunization. c At day 18 post-EAE induction, spinal cords from female Znrf1 F/F and Znrf1 Δmye mice were harvested to prepare single-cell suspensions. Cells were stained with antibodies against CD45, CD4, CD8, and CD11b, followed by flow cytometry analysis to quantify leucocytes (CD45 + ), Th cells (CD45 + CD4 + ), cytotoxic T cells (CD45 + CD8 + ), and myeloid cells (CD45 + CD11b + ) ( N = 6). d Flow cytometry analysis of Th1 (CD4 + IFNγ + ) and Th17 (CD4 + IL-17A + ) cells in the spinal cords of Znrf1 F/F ( N = 5) and Znrf1 Δmye mice ( N = 5) at day 18 post-EAE induction. e – g Spinal cord tissue sections from Znrf1 F/F and Znrf1 Δmye mice were collected and quantified at day 18 post-EAE induction. Tissue sections were subjected to H&E and LFB staining ( e ), and IHC staining using antibodies against CD3 (T cells) and CD68 (macrophages) ( f ) or IFNγ (Th1 cells) and IL-17A (Th1 cells) ( g ). Scale bars: 200 μm (whole spinal cord sections) and 100 μm (enlarged region #1 and #2). h - i Whole-mount imaging of the anterior ( h ) and posterior ( i ) spinal cord was performed in Znrf1 F/F and Znrf1 Δmye mice after EAE induction. At day 18 post-EAE induction, Znrf1 F/F and Znrf1 Δmye mice were perfused with wheat germ agglutinin (WGA) to label blood vessels (red). Spinal cords were collected and immunostained with antibodies against CD3 (green) to detect CD3 + T cells, lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1) (magenta) for lymph vessels, and DAPI (white) for nuclei. Tissue clearing was performed as described in the METHODS section. Confocal images from whole-mount spinal cords and enlarged regions are shown. Scale bars: 2000 μm (whole-mount anterior spinal cord for Znrf1 F/F and Znrf1 Δmye mice and whole-mount posterior spinal cord for Znrf1 Δmye mice), 1000 μm (whole-mount posterior spinal cord for Znrf1 F/F mice), 200 μm (enlarged region #1), 100 μm (enlarged region #2 and #3). ( a , b ) Data are presented as mean ± SD. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, determined by the Wilcoxon matched-pairs signed-rank test. ( c and d ) Representative flow cytometry plots and quantified numbers of infiltrating immune cells in the CNS are shown. Data are presented as mean ± SD. ns, not significant. * P < 0.05, ** P < 0.01, *** P < 0.001, determined by the unpaired Student’s t-test. ( e – g ) Representative spinal cord sections and quantification of infiltrating immune cells or demyelination in Znrf1 F/F and Znrf1 Δmye mice. Data are presented as mean ± SD. ns, not significant. * P < 0.05, ** P < 0.01, determined by the unpaired Student’s t-test
Article Snippet: The slides were incubated with anti-CD3 antibody (#790–4341; clone, 2GV6; Ventana Medical Systems, Tucson, Arizona), anti-CD68 antibody (#ab125212; polyclonal; Abcam, Cambridge, UK), anti-IFNγ antibody (#BS-0480R; polyclonal, Bioss, Boston, USA), and anti-IL-17A antibody (#BS-1183R; polyclonal, Bioss, Boston, USA) at a 1:100 dilution for 120 min using the automated Ventana Benchmark XT system (Ventana Medical Systems).
Techniques: Injection, Staining, Flow Cytometry, Immunohistochemistry, Imaging
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