Impact of Dietary Gluten on Regulatory T Cells and Th17 Cells in BALB/c Mice

Dietary gluten influences the development of type 1 diabetes (T1D) and a gluten-free (GF) diet has a protective effect on the development of T1D. Gluten may influence T1D due to its direct effect on intestinal immunity; however, these mechanisms have not been adequately studied. We studied the effect of a GF diet compared to a gluten-containing standard (STD) diet on selected T cell subsets, associated with regulatory functions as well as proinflammatory Th17 cells, in BALB/c mice. Furthermore, we assessed diet-induced changes in the expression of various T cell markers, and determined if changes were confined to intestinal or non-intestinal lymphoid compartments. The gluten-containing STD diet led to a significantly decreased proportion of γδ T cells in all lymphoid compartments studied, although an increase was detected in some γδ T cell subsets (CD8+, CD103+). Further, it decreased the proportion of CD4+CD62L+ T cells in Peyer's patches. Interestingly, no diet-induced changes were found among CD4+Foxp3+ T cells or CD3+CD49b+cells (NKT cells) and CD3−CD49b+ (NK) cells. Mice fed the STD diet showed increased proportions of CD4+CD45RBhigh+ and CD103+ T cells and a lower proportion of CD4+CD45RBlow+ T cells in both mucosal and non-mucosal compartments. The Th17 cell population, associated with the development of autoimmunity, was substantially increased in pancreatic lymph nodes of mice fed the STD diet. Collectively, our data indicate that dietary gluten influences multiple regulatory T cell subsets as well as Th17 cells in mucosal lymphoid tissue while fewer differences were observed in non-mucosal lymphoid compartments

No diet-induced difference was found in the proportion of CD4⁺Foxp3⁺ T cells.

<p>(<b>A</b>) Representative plot of lymphocyte and CD3⁺CD4⁺ gate. (<b>B</b>) Bars represent the proportion of Foxp3⁺ cells (gated on 100% CD4⁺CD3⁺ cells) in various lymphoid compartments. No diet-induced differences was found in the proportion of CD4⁺Foxp3⁺ T cells in all lymphoid compartments tested. Data are presented as mean values ± SEM of two independent experiments with 6 mice in each group. (<b>C</b>) Representative histogram from PLN, showing the proportion of CD4⁺Foxp3⁺T cells in the STD diet mice compared to the GF diet mice. Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN). Black bars: STD diet. Grey bars: GF diet.</p

Increase in CD103⁺ T cells in S and PLN in mice receiving the STD diet.

<p>(<b>A</b>) Bars represent the proportion of CD103⁺ cells, gated on 100% CD3⁺ cells. There was an increased proportion of CD103 expression in S and PLN in BALB/c mice receiving the STD diet. (<b>B</b>) Representative dot plot from PLN showing the differences in CD103⁺ T cells between the two diets. (<b>C</b>) Bars showing the percentages of CD103⁺ cells gated on 100% CD3⁺CD4⁺ lymphocytes. (<b>D</b>) Representative dot plots of CD4⁺CD103⁺ T cells from isolated PLN. Barplots are mean values ± SEM of two independent experiments with 6 mice in each group. Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN). Black bars: STD diet. Grey bars: GF diet. * P<0.05. ** P<0.01.</p

The proportion of CD4⁺T cells expressing CD45RB^high and CD45RB^low was influenced by the diet.

<p>(<b>A</b>) Bars represent percentages of CD45RB^low expressing cells gated on 100% CD3⁺CD4⁺ cells. There was a decreased proportion of CD4⁺ CD45RB^low T cells in S and PP of BALB/c mice receiving the STD diet compared to the GF diet. (<b>B</b>) Percentages of cells expressing CD45RB^high gated on 100% CD3⁺CD4⁺ cells. There was an increased proportion of CD4+CD45RB^high T cells in PP and PLN in mice receiving the STD diet. Data are presented as mean values ± SEM of two independent experiments with 6 mice in each group. (<b>C</b>) Representative dot plot of cells isolated from PLN showing the proportion of CD45RB^low and CD45RB^high CD4⁺ T cells from STD mice vs. GF mice. Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN). Black bars: STD diet. Grey bars: GF diet. * P<0.05. ** P<0.01.</p

No diet-induced difference was found in the proportion of CD3⁺CD49b⁺ (NKT cells) or CD3⁻CD49b⁺ (NK cells).

<p>(<b>A</b>) Bars represent the proportion of CD49b⁺ cells, gated on 100% CD3⁺ cells. No diet induced differences was found. (<b>B</b>) Bars showing the percentages of CD3⁻CD49b⁺ cells, with no significant differences between the two diets. Barplots are mean values ± SEM of two independent experiments with 6 mice in each group. Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN). Black bars: STD diet. Grey bars: GF diet.</p

Fewer CD4⁺ T cells in PP expressed CD62L in mice receiving the STD diet.

<p>(<b>A</b>) Bars represent percentages of CD62L⁺ cells gated on 100% CD3⁺CD4⁺ cells. In BALB/C mice receiving the STD diet, compared to mice receiving the GF diet, there was a decreased proportion of CD4⁺CD62L⁺ T cells in PP. Data are presented as mean values ± SEM of two independent experiments with 6 mice in each group. (<b>B</b>) Representative histograms from PP and PLN showing the differences in CD4⁺CD62L⁺ T cells. Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN).Black bars: STD diet. Grey bars: GF diet. * P<0.05.</p

The STD diet reduced the proportion of γδ T cells.

<p>(<b>A</b>) Bars represent percentages of γδ T cells. The proportion of γδ T cells was reduced in BALB/c mice on the gluten-containing STD diet in all tested lymphoid compartments. (<b>B</b>) Representative dot plots illustrating gating of γδ T cells, and the subsequent analysis of subpopulation of γδ T cells, according to CD8 and CD103 expression. (<b>C</b>) Percentages of γδ T cells expressing CD103. Gluten induced a significant increase in CD103 expression on γδ T cells in MLN and PLN. (<b>D</b>) Percentages of CD8⁺CD103⁺ cells gated on 100% γδ⁺ cells, with an increased proportion in MLN and PLN in mice on the STD compared to the GF diet. (<b>E</b>) Percentages of γδ T cells expressing CD8. The proportion of CD8⁺ γδ T cells was higher in PLN in mice receiving the STD compared to the GF diet. (<b>F</b>) Percentages of CD8⁻CD103⁻ γδ T cells was lower in mice on the STD than on the GF diet, with a significant decrease in MLN and PLN. Barplots are mean values ± SEM of two independent experiments with 6 mice in each group. Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN). Black bars: STD diet. Grey bars: GF diet. * P<0.05. ** P<0.01. *** P<0.001.</p

Summary of changes in T cells (CD3⁺) subsets and cellular markers in BALB/c mice receiving the gluten-containing STD diet compared to the GF diet.

<p>Spleen (S); inguinal lymph nodes (ILN); mesenteric lymph nodes (MLN); Peyer's patches (PP) and pancreatic lymph nodes (PLN). Significant diet-induced changes within specific lymphoid compartment are indicated in each box. ↑: Increased proportion in animals receiving the gluten-containing STD diet. ↓: Decreased proportion in animals receiving the gluten-containing STD diet. I: No diet-induced changes. Boxes with thin line: Subsets of T cells expressing a specific cellular marker e.g. γδ T cells expressing CD103.</p