Altered gene expression in highly purified enterocytes from patients with active coeliac disease.(Research article)

Authors: Suzanne Bracken [1]; Greg Byrne [1]; Jacinta Kelly [1]; John Jackson [1]; Conleth Feighery (corresponding author) [1]

Background

Coeliac disease is a permanent intolerance to dietary prolamins from wheat, barley and rye. Ingestion of these proteins in susceptible individuals gives rise to an inflammatory lesion in the small intestine characterised by crypt hyperplasia and villous atrophy [1]. While progress has been made in understanding the mechanisms by which prolamins activate the immune system, the molecular events that ultimately lead to the intestinal lesion are, as yet, ill defined.

Coeliac disease has a strong HLA association with approximately 95% of coeliac patients expressing the HLA-DQ2 molecule [2]. A large population-based study showed the disease concordance rate between monozygotic twins to be 75% [3]. This rate is considerably higher than that for other multifactorial diseases such as Crohn's disease [4] or insulin dependent diabetes mellitus [5]. However, in the study by Greco

et al , the concordance rate for coeliac disease in HLA-matched dizygotic twins was found to be only 11% [3]. Thus, while the evidence points to a very strong HLA genetic contribution to coeliac disease, other non-HLA-linked genes must play a role.

Additional linkage studies have been performed in coeliac disease in an attempt to identify susceptibility loci other than the 6p21 HLA locus. Evidence has been found for linkage with the non-HLA loci 2q33, 5q31-33 and 19p13 [6] and candidate gene association studies within these loci have concentrated on genes known to be immunologically relevant to disease pathogenesis. Recent genome-wide association studies have identified a region harbouring IL-2 and IL-21 as a further potential genetic susceptibility region predisposing to celiac disease [7, 8]. However, so far no gene has been conclusively proven to confer a risk of coeliac disease. Hence, a hypothesis-free approach to selecting genes for study, as employed here, may be useful.

Much research in coeliac disease has focused on the role of T-cells and the pro-inflammatory cytokines they produce [9, 10, 11, 12, 13]. It has been suggested that the direct effect of pro-inflammatory cytokines such as IFN-[gamma] and TNF-[alpha] may contribute to the characteristic coeliac lesion [9]. Members of the metalloproteinase (MMP) family have been implicated in coeliac disease pathology. These enzymes are capable of tissue remodelling by degradation of proteins in the extracellular matrix and basement membrane. Several studies have demonstrated elevated levels of MMP expression in the coeliac lesion [14, 15, 16].

It has been proposed that dysregulated differentiation of epithelial cells in the small intestine may also play a role in the generation of the coeliac lesion. Diosdado

et al have suggested that stem cells in the villous crypt proliferate, but do not receive the signal to differentiate leading to the development of undifferentiated, hyperplastic crypts and subsequently, villous atrophy [17]. It has recently been reported that gliadin can directly cause up-regulation of several epithelial cell surface molecules such as HLA-DR, ICAM-1 and MICA [18]. Furthermore, other studies have reported increased expression of several cytokines in the epithelium of patients with active celiac disease including IL-15, MIF, TNF-[alpha] and iNOS [19, 20, 21]. Thus, the intestinal enterocyte is emerging as a potential contributor to coeliac disease pathogenesis and must be studied further.

The purpose of this study was to examine the role of the epithelial cell in coeliac disease, employing a gene microarray based technique. This allowed for the analysis of the simultaneous expression of thousands of gene transcripts, in a hypothesis-free manner [22]. Epithelial cells were isolated from biopsies taken from coeliac patients with active disease and compared to controls, thereby examining the gluten-induced inflammatory environment of the coeliac lesion. In the study, 102 genes were found to have significantly altered expression. Further studies using RT-PCR and immunohistochemistry were used to validate altered expression of gap junction protein alpha 4 and small proline rich protein 3.

Results

Microarray analysis of coeliac duodenal epithelial cells

DTT/EDTA treatment was employed to strip the epithelial layer from patient intestinal biopsies. Magenetic cell sorting was then used to deplete CD3

+ cells, and enterocyte suspensions with purities of 98-99% were routinely obtained (Figure 1). Using Atlas Glass Human 3.8I oligonucleotide micoarrays (BD Biosciences Clontech, UK), we analysed the gene expression profile of a homogeneous population of duodenal epithelial cells taken from patients with active coeliac disease, in comparison to control patients. Of the 3,800 genes present on the array (all of which have been previously annotated), 3549 had sufficient data across the five experiments for comparison. Many of these genes showed fold-change ratios with little or no deviation from 1. Thus, to focus on only differentially expressed genes, the list was filtered on a fold-change of 1.25-fold. A fold-change of 1.25 has been described in previously published microarray experiments [23, 24] and has been shown to indicate reliable differences in gene expression [23]. This filtering yielded a list of 1,256 genes with which to perform analysis. Of these 1,256 genes, 827 were up-regulated and 429 were down-regulated. A p-value of 0.05 was used as a cut-off to distinguish significantly expressed genes, which yielded a gene list of 102 (68 up-regulated and 34 down-regulated) genes which are presented in Table 1. These 102 genes were grouped according to functional categories, including protein transport, ion transport, proliferation, differentiation, anti-apoptosis/survival, structural, adhesion, metabolism, transcription, protein biosynthesis, signal transduction, cell cycle and DNA repair, and immune response and inflammation (categories of genes and up/down-regulation are summarised in Figure 2 and Table 2 respectively).Figure 1: Flow cytometric analysis of purified epithelial cells . A: Size versus granularity plot. B and C correspond to the population of cells within gate 1; and demonstrate control antibody and BerEP-4 expression, respectively. D and E correspond to the population of cells within gate 2; and demonstrate control antibody and CD3 expression, respectively. [figure omitted]Figure 2: 102 differentially expressed genes organised by category. [figure omitted]Table 1 caption: Differentially expressed genes. [table omitted]Table 2 caption: Summary of microarray results by gene category. [table omitted]

Verification of Selected Genes by Real-time PCR

In order to corroborate the microarray gene expression results genes were selected for validation by real-time RT-PCR using the same patient samples. Miron

et al have demonstrated that the popular strategy of selecting genes with the greatest fold-increase generally fails as a global validation strategy while a random selection of genes for validation (10-25 genes) is a more robust technique [25]. We therefore used random-stratified sampling as a gene selection method. Ten genes representing a range of p-values were randomly selected for quantitative RT-PCR analysis.

The RT-PCR expression values were calculated from a standard curve and the mean of the values calculated for the coeliac samples was divided by the mean for the control samples to give a ratio. The RT-PCR ratios were then compared to the microarray ratios (Table 3). Eight of the ten genes showed up-regulation of expression in coeliac disease with both microarray and real-time RT-PCR analysis. One gene showed up-regulation of expression in coeliac disease with microarray analysis and non-differential expression with real-time RT-PCR. One gene showed down-regulation of expression in coeliac disease with microarray analysis and up-regulation of expression with real-time RT-PCR analysis. Thus, 80% of the genes tested in this study were found to be validated, which compares well to a reported average RT-PCR confirmation rate of 70% [26].

Table 3 caption: Comparison of fold-changes obtained for genes by microarray and by TaqMan RT-PCR [table omitted]

Immunohistochemical Analysis

Two of the genes validated by real-time RT-PCR, were selected for …