Tissue-infiltrating lymphocytes analysis reveals large modifications of the duodenal "immunological niche" in coeliac disease after gluten-free diet

Abstract

Objectives: The role of T lymphocytes in the pathogenesis of Celiac disease (CD) is well established. However, the mechanisms of T-cell involvement remain elusive. Little is known on the distribution of T subpopulations: T-regulatory (Treg), Th17, CD103, and CD62L cells at disease onset and after gluten-free diet (GFD). We investigated the involvement of several T subpopulations in the pathogenesis of CD.

Methods: We studied T cells both in the peripheral blood (PB) and the tissue-infiltrating lymphocytes (TILs) from the mucosa of 14 CD patients at presentation and after a GFD, vs. 12 controls.

Results: Our results extend the involvement of Treg, Th1, and Th17 cells in active CD inflammation both in the PB and at the TILs. At baseline, Tregs, Th1, and Th17 cells are significantly higher in active CD patients in TILs and PB. They decreased after diet. Moreover, CD62L+ TILs were increased at diagnosis as compared with GFD patients.

Conclusions: Our data show significant modifications of the above-mentioned subpopulations both in the PB and TILs. The increase of suppressive Tregs in active CD both in the PB and TILs is intriguing. T lymphocytes are known to have a crucial role in the pathogenesis of CD. We have shown that gluten trigger results in systemic recruitment of T lymphocytes, the unbalance between pro-inflammatory and anti-inflammatory populations and the increase of CD62L+ T cells in TILs. Our results delineate a more complete picture of T-cell subsets in active vs. GFD disease. Our data of T-cell subpopulations, combined with known data on cytokine production, support the concept that duodenal micro-environment acts as an immunological niche and this recognition may have an important role in the diagnosis, prognosis and therapeutical approach of CD.

Figure 1

T-bet expression in PBMC and TIL from circulating T cells and B cells. Mean percentage of CD4+ T-bet (a), CD8+ T-bet (b) T cells, and CD19+ T-bet B cells (c) in peripheral blood and in duodenal mucosa of healthy subjects and celiac disease. Box plots express the first (Q1) and third (Q3) quartiles within a given data set by the upper and lower horizontal lines in a rectangular box, in which there is a horizontal line showing the median. The whiskers extend upwards and downwards to the highest or lowest observation within the upper (Q3+1.5 × interquartile range) and lower (Q1–1.5 × interquartile range) limits. P-values indicate statistical significances (<0.05) between the different groups. GFD, gluten-free diet; MFI, mean fluorescence intensity.

Figure 2

Immunohistochemistry for FoxP3 and IL-17 of gut mucosa in CD patients. Immunohistochemistry for FoxP3 and IL-17 proteins in a representative case of celiac disease (a for FoxP3 and c for IL-17) and in normal mucosa (b for FoxP3 and d for IL-17) (Avidin–Biotin–Peroxidase complex method in paraffin section lightly counterstained with ematoxylin. Original magnification, × 100 (a,b and d); × 200 (c).

Figure 3

Tregs and Th17 expression in PBMC and TIL from CD patients before and after GFD. PMBC Tregs (CD3+/CD4+/CD25+/CD127−) (a) are augmented in CD patients and decrease after GFD, returning to similar value of control patients. TIL Tregs (b) are significantly augmented in CD patients and significantly decrease after GFD. PMBC Th17 cells (c) are significantly augmented in CD patients and significantly decrease after GFD. TIL Th17 cells (d) are augmented in CD patients and significantly decrease after GFD. ^#P<0.05 post-GFD vs. pre-GFD levels. GFD, gluten-free diet; PBMC, peripheral blood mononuclear cell; TIL, tissue-infiltrating lymphocytes; Treg, T-regulatory.