Intestinal barrier dysfunction develops at the onset of experimental autoimmune encephalomyelitis, and can be induced by adoptive transfer of auto-reactive T cells

Abstract

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system with a pathogenesis involving a dysfunctional blood-brain barrier and myelin-specific, autoreactive T cells. Although the commensal microbiota seems to affect its pathogenesis, regulation of the interactions between luminal antigens and mucosal immune elements remains unclear. Herein, we investigated whether the intestinal mucosal barrier is also targeted in this disease. Experimental autoimmune encephalomyelitis (EAE), the prototypic animal model of MS, was induced either by active immunization or by adoptive transfer of autoreactive T cells isolated from these mice. We show increased intestinal permeability, overexpression of the tight junction protein zonulin and alterations in intestinal morphology (increased crypt depth and thickness of the submucosa and muscularis layers). These intestinal manifestations were seen at 7 days (i.e., preceding the onset of neurological symptoms) and at 14 days (i.e., at the stage of paralysis) after immunization. We also demonstrate an increased infiltration of proinflammatory Th1/Th17 cells and a reduced regulatory T cell number in the gut lamina propria, Peyer's patches and mesenteric lymph nodes. Adoptive transfer to healthy mice of encephalitogenic T cells, isolated from EAE-diseased animals, led to intestinal changes similar to those resulting from the immunization procedure. Our findings show that disruption of intestinal homeostasis is an early and immune-mediated event in EAE. We propose that this intestinal dysfunction may act to support disease progression, and thus represent a potential therapeutic target in MS. In particular, an increased understanding of the regulation of tight junctions at the blood-brain barrier and in the intestinal wall may be crucial for design of future innovative therapies.

Conflict of interest statement

Competing Interests: The authors have read the journal's policy and declare that Dr. Shahram Lavasani (SL) is a part time employee and stakeholder of ImmuneBiotech AB. Dr. Anders Bredberg (AB) is a part time employee of ImmuneBiotech AB. Mrs Mehrnaz Nouri (MN), PhD student, is advisor and has received research support from ImmuneBiotech AB. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

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.