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, 187 (8), 4338-46

Sensitization to Gliadin Induces Moderate Enteropathy and Insulitis in Nonobese diabetic-DQ8 Mice

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Sensitization to Gliadin Induces Moderate Enteropathy and Insulitis in Nonobese diabetic-DQ8 Mice

Heather J Galipeau et al. J Immunol.

Abstract

Celiac disease (CD) is frequently diagnosed in patients with type 1 diabetes (T1D), and T1D patients can exhibit Abs against tissue transglutaminase, the auto-antigen in CD. Thus, gliadin, the trigger in CD, has been suggested to have a role in T1D pathogenesis. The objective of this study was to investigate whether gliadin contributes to enteropathy and insulitis in NOD-DQ8 mice, an animal model that does not spontaneously develop T1D. Gliadin-sensitized NOD-DQ8 mice developed moderate enteropathy, intraepithelial lymphocytosis, and barrier dysfunction, but not insulitis. Administration of anti-CD25 mAbs before gliadin-sensitization induced partial depletion of CD25(+)Foxp3(+) T cells and led to severe insulitis, but did not exacerbate mucosal dysfunction. CD4(+) T cells isolated from pancreatic lymph nodes of mice that developed insulitis showed increased proliferation and proinflammatory cytokines after incubation with gliadin but not with BSA. CD4(+) T cells isolated from nonsensitized controls did not response to gliadin or BSA. In conclusion, gliadin sensitization induced moderate enteropathy in NOD-DQ8 mice. However, insulitis development required gliadin-sensitization and partial systemic depletion of CD25(+)Foxp3(+) T cells. This humanized murine model provides a mechanistic link to explain how the mucosal intolerance to a dietary protein can lead to insulitis in the presence of partial regulatory T cell deficiency.

Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Impaired intestinal barrier function in gliadin-sensitized NOD-DQ8 mice. Sections of small intestine were mounted in Ussing chambers and tissue conductance (mS/cm2) was measured 24 h after the final gavage. Each dot represents an individual mouse; p values were computed using an ANOVA with a post hoc test for multiple comparisons.
FIGURE 2
FIGURE 2
Histopathology of the small intestine showing decreased villus-to-crypt ratio in gliadin-sensitized NOD-DQ8 mice. H&E-stained sections of the proximal small intestine in untreated controls (A) and anti-CD25 mAb-treated (B), gliadin-sensitized (C), anti-CD25 mAb treated plus BSA-sensitized (D), and anti-CD25 mAb treated plus gliadin-sensitized mice (E). Original magnification ×10. F, Quantification of villus/ crypt ratios (n = 8 for each group). Data represented as mean ± SEM; p values were computed using an ANOVA with a post hoc test for multiple comparisons. *p < 0.05 versus control mice, anti-CD25 mAb treated mice, and anti-CD25 + BSA-treated mice.
FIGURE 3
FIGURE 3
Immunohistochemistry showing increased number of CD3+ IELs in gliadin-sensitized mice. CD3+-stained sections of the proximal small intestine in untreated controls (A), anti-CD25 mAb-treated (B), gliadin-sensitized (C), anti-CD25 mAb-treated plus BSA-sensitized (D), and anti-CD25 mAb-treated plus gliadin-sensitized mice (E). Original magnification ×40. Black arrows indicate IELs. F, Quantification of CD3+ cells in villi tips, expressed as IEL per 100 enterocytes (n = 8 for each group). Data are represented as mean ± SEM; p values were computed using an ANOVA with a post hoc test for multiple comparisons. **p < 0.01 versus control mice, anti-CD25 mAb treated mice, and anti-CD25 + BSA-treated mice.
FIGURE 4
FIGURE 4
Anti-gliadin and anti-tTG ELISAs. Serum was collected from NOD-DQ8 mice, and the presence of anti-gliadin IgG Abs (A) and anti-tTG IgA Abs (B) was determined by ELISA. Anti-gliadin and anti-tTG positive reactivity was determined using a positive cutoff value of ≥3 SD above the mean of control mice (dotted line) (40). Each dot represents an individual mouse.
FIGURE 5
FIGURE 5
Gliadin-sensitized NOD-DQ8 mice develop severe insulitis in the presence of an immune dysregulation. Insulitis was determined by evaluating H&E-stained sections of the pancreas for islet infiltration, scoring each islet from grade 0 (no infiltration) to grade 4 (end-stage insulitis with <20% of islet mass remaining) (–40). An average insulitis score was determined for each group of mice: untreated controls (A), anti-CD25 mAb treated mice (B), gliadin-sensitized mice (C), anti-CD25 mAb-treated plus BSA-sensitized mice (D), and anti-CD25 mAb treated plus gliadin-sensitized mice (E). Original magnification ×10. F, Quantification of insulitis scores for multiple mice (n = 9 per group). Data are represented as mean ± SEM; p values were computed using an ANOVA with a post hoc test for multiple comparisons.
FIGURE 6
FIGURE 6
Immunohistochemistry showing the presence of CD3+ lymphocytes in pancreatic islets in NOD-DQ8 mice. Representative CD3+-stained sections of the pancreas in untreated controls (A), anti-CD25 mAb-treated (B), gliadin-sensitized mice (C), anti-CD25 mAb plus BSA-sensitized (D), and anti-CD25 mAb-treated plus gliadin-sensitized mice (E). Original magnification ×20.
FIGURE 7
FIGURE 7
T cells isolated from the PLNs of mice that develop insulitis respond to gliadin stimulation. A, CD4+ T cells were isolated from the PLNs of control and anti-CD25 mAb-treated plus gliadin-sensitized mice and labeled with CFSE. CD4+ T cells were incubated with APCs with PT gliadin (right panels), BSA (center panels), or media alone (left panels) for 4 d. Proliferation was assessed with FACS analysis. Cells were gated on live CD4+ lymphocytes. Gated population represents the percentage of proliferated cells. B, Quantification of proliferation in A for control mice (n = 5) and anti-CD25+ gliadin mice (n = 5). Data are represented as mean ± SEM; p values were computed using an ANOVA for multiple comparisons. **p < 0.01 versus other groups. TNF-α (C), IL-6 (D), and MCP-1 (E) production was assessed in cell culture supernatants from mice that developed insulitis, using a cytometric bead array inflammation kit. The dotted line represents the limit of detection; p values were computed using an unpaired t test.

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