Altered gene expression in highly purified enterocytes from patients with active coeliac disease

BMC Genomics Altered gene expression in highly purified enterocytes from patients with active coeliac disease Suzanne Bracken 0 Greg Byrne 0 Jacinta Kelly 0 John Jackson 0 Conleth Feighery 0 0 Address: Department of Immunology, St. James's Hospital, Dublin and Trinity College Dublin, Dublin Molecular Medicine Centre , Dublin , Ireland Background: Coeliac disease is a multifactorial inflammatory disorder of the intestine caused by ingestion of gluten in genetically susceptible individuals. Genes within the HLA-DQ locus are considered to contribute some 40% of the genetic influence on this disease. However, information on other disease causing genes is sparse. Since enterocytes are considered to play a central role in coeliac pathology, the aim of this study was to examine gene expression in a highly purified isolate of these cells taken from patients with active disease. Epithelial cells were isolated from duodenal biopsies taken from five coeliac patients with active disease and five non-coeliac control subjects. Contaminating T cells were removed by magnetic sorting. The gene expression profile of the cells was examined using microarray analysis. Validation of significantly altered genes was performed by real-time RT-PCR and immunohistochemistry. Results: Enterocyte suspensions of high purity (98-99%) were isolated from intestinal biopsies. Of the 3,800 genes investigated, 102 genes were found to have significantly altered expression between coeliac disease patients and controls (p < 0.05). Analysis of these altered genes revealed a number of biological processes that are potentially modified in active coeliac disease. These processes include events likely to contibute to coeliac pathology, such as altered cell proliferation, differentiation, survival, structure and transport. Conclusion: This study provides a profile of the molecular changes that occur in the intestinal epithelium of coeliac patients with active disease. Novel candidate genes were revealed which highlight the contribution of the epithelial cell to the pathogenesis of coeliac disease. - 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 genomewide 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 [9-13]. It has been suggested that the direct effect of pro-inflammatory cytokines such as IFN- and TNF- 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 [14-16]. 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- and iNOS [19-21]. 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 9899% 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 (...truncated)