Figure 8.
Enhanced GlcNAc exposure on T cell ameliorates EAE during the early disease course. (A) Flow cytometric analysis of WGA binding on living CD4+ T cells in the CNS and spleen of EAE diseased mice in actively immunized C57BL/6 mice normalized to splenic T cells (n = 11 mice from two different experiments). (B–E) EAE was induced in CX3CR1.GFPxRag−/− and CX3CR1.GFPxRag+/− mice (myeloid cells green) via the passive transfer of B6.2D2.RFP.Th17 cells (red). Interactions between myeloid cells and T cells as well as T cell motility parameters were imaged in vivo with intravital two-photon imaging and analyzed during different disease states. For hippocampal slice culture experiments, co-cultures of hippocampal slices from CX3CR1.GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit to identify apoptotic cells, and only living T cells were included for further analysis. (B) Dots represent the percentages of engulfments, temporary contacts and stable contacts among the analyzed T cells in a randomly defined area of the CNS (brainstem) of EAE diseased mice in dependency on disease score. P values indicate whether the slope of linear regression is significantly nonzero (n = 9 mice from two different experiments). (C) Means (± SEM) of percentages of engulfments, temporary contacts, and stable contacts among the detected interactions or the detectable T cell count per region of interest were compared between mice exhibiting a score <2 (n = 3 mice from two different experiments), mice exhibiting a score >2 (n = 6 mice from two different experiments), and hippocampal slice cultures (n = 12 organotypic hippocampal slice cultures from eight different experiments). Statistical analysis was performed using one-sided Mann–Whitney U test. *, P < 0.05; ns, not significant. (D) Mean (± SEM) of T cell motility parameters in the CNS of EAE diseased mice in dependency on disease score. P values indicate whether the slope of linear regression is significantly nonzero (n = 9 mice, 30 T cells per mouse from two different experiments). (E) Normalization of all T cell tracks to a common starting point in a representative experiment of a hippocampal slice co-culture comparing a mouse exhibiting a clinical score of 1.5 and a mouse exhibiting a clinical score of 4.5. Each line represents one individual T cell. Representative of 9 mice from two different experiments. (F–J) EAE was induced actively in C57BL/6 mice and 5 µl PBS or 5 µl GlcNAc-solution (200 mM solved in PBS) were applied intrathecally every second day seven times, either starting at the onset of disease (day 11, F and H) or before the onset of the disease (day 7, G). (F) EAE course in PBS-treated vs. GlcNAc-treated mice (n = 16 PBS-treated mice, 15 GlcNAc-treated mice from 2 different experiments). (G) EAE course in PBS-treated vs. GlcNAc-treated mice. (n = 8 PBS-treated mice, 8 GlcNAc-treated mice from 2 different experiments). (H) Means (± SEM) of percentages of WGA binding T cells in the CNS and spleen of PBS (ctrl) and GlcNAc-treated mice. (I and J) Immunohistochemistry was performed to identify T cells in PBS-treated and GlcNAc-treated mice. (I) Shown are representative images of Iba-1 (green), CD4 (red), and DAPI (blue) in PBS-treated mice (ctrl, n = 13 slices of three different mice from two different experiments) and GlcNAc-treated mice (GlcNAc, n = 18 of three different mice from two different experiments), scale bar = 20 μm, and (J) the quantification of CD4+ T cells. Statistical analysis in F, G, and J was performed using two-sided Student’s t tests comparing the cumulative scores of the distinct groups. Statistical analysis in H was performed using one-way ANOVA with Tukey’s post hoc test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

Enhanced GlcNAc exposure on T cell ameliorates EAE during the early disease course. (A) Flow cytometric analysis of WGA binding on living CD4+ T cells in the CNS and spleen of EAE diseased mice in actively immunized C57BL/6 mice normalized to splenic T cells (n = 11 mice from two different experiments). (B–E) EAE was induced in CX3CR1.GFPxRag−/− and CX3CR1.GFPxRag+/− mice (myeloid cells green) via the passive transfer of B6.2D2.RFP.Th17 cells (red). Interactions between myeloid cells and T cells as well as T cell motility parameters were imaged in vivo with intravital two-photon imaging and analyzed during different disease states. For hippocampal slice culture experiments, co-cultures of hippocampal slices from CX3CR1.GFP pups with pathogenic B6.2D2.CFP.Th17 cells were stained with Image-iT LIVE Red Caspase-3 and -7 Detection Kit to identify apoptotic cells, and only living T cells were included for further analysis. (B) Dots represent the percentages of engulfments, temporary contacts and stable contacts among the analyzed T cells in a randomly defined area of the CNS (brainstem) of EAE diseased mice in dependency on disease score. P values indicate whether the slope of linear regression is significantly nonzero (n = 9 mice from two different experiments). (C) Means (± SEM) of percentages of engulfments, temporary contacts, and stable contacts among the detected interactions or the detectable T cell count per region of interest were compared between mice exhibiting a score <2 (n = 3 mice from two different experiments), mice exhibiting a score >2 (n = 6 mice from two different experiments), and hippocampal slice cultures (n = 12 organotypic hippocampal slice cultures from eight different experiments). Statistical analysis was performed using one-sided Mann–Whitney U test. *, P < 0.05; ns, not significant. (D) Mean (± SEM) of T cell motility parameters in the CNS of EAE diseased mice in dependency on disease score. P values indicate whether the slope of linear regression is significantly nonzero (n = 9 mice, 30 T cells per mouse from two different experiments). (E) Normalization of all T cell tracks to a common starting point in a representative experiment of a hippocampal slice co-culture comparing a mouse exhibiting a clinical score of 1.5 and a mouse exhibiting a clinical score of 4.5. Each line represents one individual T cell. Representative of 9 mice from two different experiments. (F–J) EAE was induced actively in C57BL/6 mice and 5 µl PBS or 5 µl GlcNAc-solution (200 mM solved in PBS) were applied intrathecally every second day seven times, either starting at the onset of disease (day 11, F and H) or before the onset of the disease (day 7, G). (F) EAE course in PBS-treated vs. GlcNAc-treated mice (n = 16 PBS-treated mice, 15 GlcNAc-treated mice from 2 different experiments). (G) EAE course in PBS-treated vs. GlcNAc-treated mice. (n = 8 PBS-treated mice, 8 GlcNAc-treated mice from 2 different experiments). (H) Means (± SEM) of percentages of WGA binding T cells in the CNS and spleen of PBS (ctrl) and GlcNAc-treated mice. (I and J) Immunohistochemistry was performed to identify T cells in PBS-treated and GlcNAc-treated mice. (I) Shown are representative images of Iba-1 (green), CD4 (red), and DAPI (blue) in PBS-treated mice (ctrl, n = 13 slices of three different mice from two different experiments) and GlcNAc-treated mice (GlcNAc, n = 18 of three different mice from two different experiments), scale bar = 20 μm, and (J) the quantification of CD4+ T cells. Statistical analysis in F, G, and J was performed using two-sided Student’s t tests comparing the cumulative scores of the distinct groups. Statistical analysis in H was performed using one-way ANOVA with Tukey’s post hoc test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; ns, not significant.

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