Figure 1.
Increased plasma levels of intestinal permeability markers during EAE progression.
Sodium fluorescein, Na-F (MW 376 Da) (A), FITC-BSA (MW 66 KDa) (B) in unimmunized animals (control), EAE mice at day 7 (EAE7) and day 14 (EAE14) following immunization, and mice at day 14 after immunization with CFA without MOG followed by administration of pertussis toxin (CFA). Marker levels were measured in plasma samples taken at 1 h after gavage with a saline solution containing the indicated marker for Na-F and 2 h for FITC-BSA. The results are expressed as mean ±SD, (n = 7–10). * represents a p-value≤0.05, ** a p-value≤0.01.
Figure 2.
Altered intestinal architecture in the small intestine.
H&E-stained sections from duodenum, jejunum and ileum were isolated from control, EAE7 and EAE14 animals (A). The sections were examined for crypt depth, defined as the length from crypt base to villus-crypt junction (B), villi length (C), submucosa (D) and muscularis thickness (E). Arrows demonstrate approximate measurements for villus length (1) crypt depth (2) submucosa thickness (3) and muscularis thickness (4) (A, original magnification ×40, insets ×100). Each bar represents mean ±SD of 7–9 analyzed sections per animal, (n = 5). **represents a p-value≤0.01 and *** a p-value≤0.001.
Figure 3.
Increased zonulin expression in the small intestine.
Immunohistochemical staining of zonulin in sections from duodenum, jejunum and ileum, from healthy controls, EAE7 and EAE14 mice (A). Arrows show zonulin both in enterocytes and lamina propria on top of the villi. Semi-quantitative analysis of zonulin staining (B). Staining intensity was expressed as positive pixels/mm2 and converted as ratio to the mean values from relevant sections in the healthy control animals. Data shown are mean ±SD from 4–6 animals for each group. ** represents a p-value≤0.01 and *** a p-value≤0.001 between control and EAE7 or EAE14 animals.
Figure 4.
Increased peripheral and intestinal inflammation.
Intestinal lamina propria (LP), Peyer's patches (PP), mesenteric lymph nodes (MLN) and spleen were isolated from unimmunized controls and EAE7 and EAE14 mice. Single cell suspensions were prepared and analyzed by flow cytometry for presence of T cells expressing IFN-γ, IL-17, or Foxp3. The lymphocytes were gated and cells were analyzed for expression of the indicated cytokines. The dot plots (A, C, and F) show the percentage of double positive cells (upper right quadrants) out of total T cells and the data are representative of one of three independent experiments. The histograms (B, D, and G) show results from all performed experiments, mean ±SD (n = 5). The frequency of CD4+IFN-γ+T cells (A) and relative percentage (B). The frequency of CD3+IL-17+ T cells (C; upper right), CD3−IL-17+ cells (C; upper left) and relative percentage of CD3+IL-17+ T cells (D). A histogram shows IL-17 expression by gated γδ T cells (E). The frequency of CD4+ Foxp3+ Tregs, dot plots from one representative experiment (F) and total results (G). * represents a p-value≤0.05, ** a p-value≤0.01 and *** a p-value≤0.001 in comparison with the controls.
Figure 5.
Increased IL-6 and TNF-α expression in intestinal macrophages and dendritic cells.
Intestinal lamina propria (LP), Peyer's patches (PP) and mesenteric lymph nodes (MLN) isolated from unimmunized controls, and EAE7 and EAE14 mice were analyzed by flow cytometry for presence of F4/80+ macrophages and CD11c+ DC expressing IL-6 (A) and TNF-α (B). Data are representative of one of three independent experiments.
Figure 6.
Increased intestinal permeability and altered intestinal morphology after adoptive transfer of encephalitogenic T cells.
Na-F (A) , FITC-BSA (B) in plasma from mice receiving un-stimulated lymph node cells (Control), MOG-reactive T cells (adoptively transferred EAE) and OVA-reactive T cells, (n = 3–5). Mice were gavaged with a marker molecules as described in Figure 1. H&E-sections from duodenum, jejunum and ileum isolated from animals receiving MOG-reactive T cells (EAETransfer) (C). The sections were examined for crypt depth (D), villus length (E), submucosa (F) and muscularis thickness (G). Arrows demonstrate approximate measurements for villus length (1) crypt depth (2) submucosa thickness (3), muscularis thickness (4) and highlight the differences between the groups (C, original magnification ×40, insets ×100). Each bar represents mean ±SD of 7–9 analyzed sections per animal, (n = 5). Immunohistochemical analysis of zonulin expression from mice receiving MOG-reactive and OVA-reactive T cells (H). Arrows show zonulin both in enterocytes and lamina propria on top of the villi. Semi-quantitative analysis of zonulin staining (I). Staining intensity was expressed as positive pixels/mm2 and converted as ratio to the mean values from relevant sections in the control animals. Data shown are mean ±SD from 3–5 animals for each group. ** represents a p-value≤0.01 and *** a p-value≤0.001.