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Multicenter Study
. 2015 Sep;149(3):681-91.e10.
doi: 10.1053/j.gastro.2015.05.013. Epub 2015 May 19.

Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease

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Free PMC article
Multicenter Study

Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease

Mala Setty et al. Gastroenterology. .
Free PMC article

Abstract

Background & aims: The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy.

Methods: We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy.

Results: IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors.

Conclusions: A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.

Keywords: Cytotoxic Intraepithelial Lymphocytes; Heat Shock Protein; Interleukin-15; Natural Killer Receptors.

Figures

Figure 1
Figure 1. Potential CD patients both fail to upregulate activating and downregulate inhibitory NK receptors to the same extent as active CD patients
Representative flow cytometry analysis of NK receptors on freshly isolated TCR-αβ+CD8+ intra-epithelial cytotoxic T lymphocytes (IE-CTL) in control, At Risk, and active CD patient groups. (A) At Risk TG2neg subjects conserve similar levels of inhibitory NKG2A receptors expression as compared to controls. In contrast, At Risk TG2pos (potential CD) patients (p=0.0181) and active CD patients (p<0.0001) have lower proportions of IE-CTL with inhibitory NKG2A receptors as compared to At Risk TG2neg and controls (p<0.0001). (B) At Risk TG2neg (p=0.0156) and active CD patients (p=0.0002), but not At Risk TG2pos patients (p=0.38), showed an increased proportion of IE-CTL with activating CD94 receptors as compared to controls and to At Risk TG2pos patients (p=0.0072 and p=0.0005 respectively). (C) At Risk TG2neg (p=0.0015) and active CD patients (p<0.0001) showed an increased proportion of IE-CTL with activating NKG2D receptors as compared to controls and to At Risk TG2pos (p=0.033 and p=0.0044 respectively). Percentage of positive cells for each patients’ group is shown in A and B, mean fluorescence intensity (MFI) of NKG2D is shown in C. *p<0.05, **p<0.01, ***p<0.001, **** p<0.0001 (Mann Whitney test).
Figure 2
Figure 2. At Risk TG2neg family members but not At Risk TG2pos patients express innate epithelial stress markers and upregulate activating NK receptors
(A) Hsp27, Hsp70 and IL-15 expression was assessed in intestinal epithelial cells (IEC) by immunohistochemistry (IHC). The distribution of the number of positive innate stress markers for each group is represented on a Box plot (left) and the relative proportion of controls, At Risk TG2neg, At risk TG2pos and GFD CD patients within the groups of individuals presenting 0, 1, 2 or 3 positive innate markers is shown (right). IEC from controls and At Risk TG2pos subjects show a significantly lower number of innate immune markers as compared to IEC from At Risk TG2neg and GFD subjects. Pairwise Wilcoxon rank tests were performed between patient groups: control vs. At risk TG2pos p=0.41, vs. At Risk TG2neg p=0.002, vs. GFD p=0.0037; At risk TG2pos vs. At Risk TG2neg p=0.0005, vs. GFD p=0.0017; At Risk TG2neg vs. GFD p=0.4. *p<0.05, **p<0.01, ***p<0.001. (B) Representative IHC images of Hsp27, Hsp70 and IL-15 staining are shown for each category. (C) At Risk TG2pos potential CD subjects who have high levels of IL-15 expression (IL-15pos) in IEC tend to have lower levels of inhibitory NKG2A receptors (left panel) (p=0.03) but have also low levels of the activating NKG2D (right panel), while At Risk TG2neg who have high levels of IL-15 expression show an increase in IE-CTL with high NKG2D (right panel). However, these individuals retain high levels of inhibitory NKG2A receptors (left panel). *p<0.05. **p<0.01, ***p<0.001(Mann Whitney test).
Figure 3
Figure 3. At Risk TG2neg family members but not At Risk TG2pos subjects display ultrastructural alterations of intestinal epithelial cells
Ultrastructural examination of small intestinal absorptive enterocytes from 5 control subjects, 5 At Risk TG2pos and 4 TG2neg subjects, 5 active CD and 3 GFD CD patients was performed by transmission electron microscopy (EM). (A) Representative images from two patients in each patient group are shown. Below each image the semi-quantitative EM score (1 to 4) and the expression of innate epithelial stress markers are indicated. (B) The semi-quantitative EM scoring for all subjects examined within each group was significantly different for all groups compared to controls (vs. TG2neg p=0.05, vs. Active CD p=0.029 vs. GFD p=0.023) except for potential CD patients (At Risk TG2pos subjects). [a] Out of the two potential patients showing an EM score of 3, one has a family history of CD and the other had an associated autoimmune condition (type-1 diabetes). (C) A significant increase of interquartile range (IQR) of MV length was found in At Risk TG2neg patients relative to controls (p=0.0039). [b] The two patients with the highest IQR had a family history of CD, partially overlapping with the IQR of At Risk TG2neg individuals.
Figure 4
Figure 4. Innate epithelial stress and adaptive immune responses are both required for IE-CTL to acquire a “fully active” killer phenotype and induce villous atrophy
Proposed “two-hit” model of CD pathogenesis. The adaptive immune response in CD is characterized by anti-TG2 antibodies (1) that are induced in patients with adaptive anti-gluten immunity (2). Epithelial stress is characterized by the expression of innate epithelial stress markers (i.e. Hsp27, Hsp70 and IL-15) and the presence of ultrastructural alterations in the small intestinal enterocytes (3). Each of these responses is necessary but not sufficient to induce full activation of IE-CTL, characterized by high levels of activating and low levels of inhibitory NK receptors (4), and epithelial destruction resulting in villous atrophy (5). At Risk TG2neg subjects show signs of intestinal epithelial stress, but lack the adaptive immune response. Although they upregulate activating NK receptors, the expression of high levels of inhibitory NK receptors on IE-CTL prevents extensive IEC killing. In contrast, Potential CD patients show the typical adaptive immune response of CD, but lack the epithelial stress response. Hence, they fail to upregulate activating NK receptors and often maintain inhibitory NK receptors to some extent on IE-CTL. Only active CD patients, who have anti-gluten specific adaptive immunity and innate epithelial alterations develop villous atrophy as a result of their IE-CTL acquiring a fully activated NK phenotype characterized by the loss of inhibitory NK receptors and upregulation of activating NK receptors.

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