High frequency but impaired function of Tregs in active CD patients

Abstract

Objectives: Coeliac disease (CD) is an immune-mediated systemic disorder elicited by gluten and characterized by a small intestinal enteropathy. Besides active CD, we recognize an early stage of CD characterized by a normal small intestinal mucosa and positive CD serology. The aim of the study was to investigate the immunoregulatory mechanisms acting in the gut of active and potential CD patients. We evaluated the expression of IFN-γ and IL-10 in CD4+ T cells and the frequency of Foxp3+ regulatory T cells (Tregs) in duodenal biopsies. Finally, we investigated the functional properties of intestinal Foxp3+ Tregs from both groups of patients and the effects of IL-15 on their suppressive activity. Methods: CD4+ T cells were isolated from duodenal biopsies of 11 controls, 13 active CD and 15 potential CD patients (7 Marsh 0 (M0) and 8 Marsh 1 (M1)). IFN-γ and IL-10 expression were assessed by flow cytometry. The frequency of CD4+ CD25+ Foxp3+ Tregs frequency and function were investigated. Intestinal Tregs suppressive function was assessed on autologous peripheral blood responder CD4+ CD25− T cells (Tresp), in the presence of a polyclonal stimulus and with or without IL-15. Results: CD4+ IFN-γ+ T cell frequency was higher in duodenal biopsies of active (mean ± sd: 12.9 ± 3.2) and M1 potential CD (17.8 ± 5.9) than controls (4.9 ± 0.7; p < 0.05 and p < 0.05 respectively). On the contrary the highest number of CD4+ IL10+ T cells was observed in M0 potential CD patients (17 ± 5.8) compared to active (6.7 ± 1.7, p < 0.05) and control patients (6.1 ± 0.6; p < 0.05). The percentage of CD4+ CD25+ Foxp3+ T cells was significantly increased in active (14.1 ± 2.7%) and potential CD (9.9 ± 3.2%) if compared to controls (6.5 ± 3.1%; p < 0.01 and p < 0.05 respectively). In particular, in potential M1 CD it was significantly higher (10.9 ± 3.1%; p < 0.05). Intestinal Tregs from both active and potential CD exerted significant suppressive effects on Tresp cells (p < 0.05) in in vitro suppressive assay. Interestingly IL-15 was able to counteract their suppressive activity in active CD but not in potential CD. Conclusions: IFN-γ expression in CD4+ T cells correlates with CD stage, as it is increased in M1 potential and active patients; on the contrary in early CD (M0) a high frequency of IL-10 producing CD4+ T cells was found. This might prevent the progression towards mucosal damage down-regulating IFN-γ production and promoting Foxp3+ Tregs. These cells are significantly increased in the small intestinal mucosa of CD patients and are functionally active; however their suppressive activity is impaired by IL-15 only in active CD patients, suggesting a relation between high IL-15 expression, an impaired Tregs function and villous atrophy in active CD.