Steps in coeliac disease pathogenesis
Our understanding of the key steps underlying the intestinal inflammatory response to coeliac disease has increased dramatically in recent years. These steps include: (i) a (putative) direct response of the epithelium via the innate immune system to toxic proteins in wheat gluten, (ii) modification of wheat gluten proteins by tissue transglutaminase, (iii) role of HLA‐DQ2 in presenting toxic wheat proteins to T cells, and (iv) identification of key toxic protein sequences in wheat (fig 3). These advances have introduced the possibility of novel therapeutics (distinct from the gluten free diet) to treat coeliac disease.
Figure 3Underlying mechanisms in coeliac disease pathogenesis. Wheat gluten is partially digested but key toxic sequences are resistant to intestinal proteases. One gluten peptide (p31‐43/49) may directly induce interleukin 15 (IL‐15) (more ...)
The work of Dicke and Frazer in the 1950s identified the protein component of wheat (gluten) as the toxic fraction. Toxic sequences are now identified in both gliadin and glutenin components as well as similar proteins in rye and barley. Cereals are polyploid in nature which combined with the large allelic variation present in all gluten genes makes even gluten from a single wheat variety a complex mixture. However, the general structure of the protein families and the relevance of the high proline(P) and glutamine(Q) content to human coeliac disease is now understood. Interestingly, the fact that many gliadin peptides possess bulky hydrophobic amino acids followed by proline blocks the activity of intestinal peptidases such as pepsin and chymotrypsin.68,69
Thus gluten peptides able to stimulate T cells (such as a recently described 33mer sequence) are resistant to degradation by all gastric, pancreatic, and intestinal brush border membrane proteases in the human intestine.70
Several recent reports have suggested that one of the first events in coeliac disease pathogenesis may be a direct effect of certain wheat peptides, distinct from those recognised by T cells, on the intestinal epithelium. One such peptide, wheat A‐gliadin p31‐43 (LGQQQPFPPQQPY) or p31‐49 induces changes associated with coeliac disease on intraduodenal administration,71
and in vitro in biopsy based studies.72,73
Changes were observed in patients within four hours, which is surprisingly rapid for a purely T cell mediated response. More recent work demonstrated that this peptide appears to induce interleukin 15, a key cytokine involved in T cell activation, and preincubation with the peptide permits later activation of biopsies by known T cell stimulatory wheat gluten sequences.72
Interleukin 15 also appears to induce expression of a stress molecule, MICA, on enterocytes and upregulates NKG2D receptors on intraepithelial lymphocytes. The interaction between enterocyte MICA and lymphocyte NKG2D results in direct enterocyte killing, and is likely to be one way villous atrophy occurs.73,74
Direct effects of gliadin on enterocytes may also increase intestinal permeability through release of zonulin and effects on intracellular tight junctions.75
Surface tissue transglutaminase may have a role in innate immune signalling by p31‐43 in coeliac disease.76
More precise details of these mechanisms remain to be elucidated. It is not known whether enterocytes, macrophages/dendritic cells, or another cell type in the small intestinal mucosa are directly activated by p31‐43/49, what receptor mechanism is involved, and why these events should be coeliac disease specific.
Involvement of the human leucocyte antigen region (HLA) in coeliac disease was first suggested by serotype based association studies. More detailed analyses at the genetic level implicated HLA‐DQ2.77
Nearly all patients with coeliac disease possess either the HLA‐DQ2 heterodimer (encoded by DQA1*05 and DQB1*02) or HLA‐DQ8 (DQA1*03 and DQB1*0302) compared with approximately one third of Caucasian populations. A European study of 1008 coeliac patients found that 90% possessed genetic variants encoding the HLA‐DQ2 heterodimer (in various forms), 4% with partial HLA‐DQ2 (DQB1*02 without DQA1*05), 2% with partial HLA‐DQ2 (DQA1*05 without DQB1*02), and 6% HLA‐DQ8 without DQ‐278
. Our current understanding of coeliac disease susceptibility implies possession of HLA‐DQ2 is necessary but not sufficient for coeliac disease development (as it is also possessed by 30% of the healthy Caucasian population). Nevertheless, genetic testing for HLA‐DQ is a useful cost effective test to exclude coeliac disease in individuals already following a gluten free diet. Interestingly, HLA‐DQ2 can be encoded in several different ways, leading to variation in the proportion of HLA‐DQ2 molecules present.79
Individuals homozygous for HLA‐DQB1*02 appear to be at highest risk of coeliac disease. The functional role of HLA‐DQ2 in coeliac disease is now clearly defined and explains the genetic association. Cells able to present toxic gluten peptides to T cells do so via HLA‐DQ2. Recent work has even identified the crystal structure and kinetics of dominant wheat gluten peptide binding to HLA‐DQ2 (fig 4).80,81
Figure 4Crystal structure of immunodominant wheat gluten peptide binding to HLA‐DQ2. Putative hydrogen bonding network (red dashes) between DQ2 (α, β chains in green, blue) and α1‐gliadin epitope (amino (more ...)
The function of the HLA class II molecule—to present short peptides to T cells—implied that specific dietary wheat, rye, and barley peptides might be presented via HLA‐DQ2 in coeliac disease. Although intestinal T cells specific for wheat gluten were first identified in 1993,82
it was only with the discovery that intestinal T cells specifically recognise deamidated gluten peptides that more precise identification was possible.83
HLA‐DQ2 preferentially binds peptides with negatively charged amino acids present in key positions. Tissue transglutaminase (the target of the antiendomysial autoantibody response in coeliac disease) plays a key role in conversion of specific glutamine residues to glutamate in the intestinal mucosa, generating negatively charged amino acids better able to bind HLA‐DQ2.83
In the intestine, tissue transglutaminase is found just below the epithelium and in the brush border. Deamidation of glutamine residues within a protein sequence is biochemically predictable.84
It remains unclear however why antitissue transglutaminase antibodies are such specific and sensitive indicators of coeliac disease. Such antibodies may recognise transglutaminase‐wheat peptide crosslinked complexes85
although whether these have a primary role in disease pathogenesis or are a bystander secondary phenomenon is uncertain.
Identification of the key dietary wheat, rye, and barley peptides presented by T cells is critical for immunologically based therapies aimed at inducing tolerance and attempts at genetic modification of grains to render them non‐toxic. Initial presentation of dietary antigen to naïve T cells occurs in the mesenteric lymph nodes; these cells recirculate in peripheral blood via the thoracic duct, and home back to the intestine via specific cell adhesion molecules (fig 3). The majority of studies have focused on intestinal T cell lines and clones generated in vitro from small bowel biopsy samples. These methods have identified a large number of different HLA‐DQ2 restricted peptides from wheat α/β, γ, and ω gliadins and low molecular weight glutenins,68,86,87,88
sequences from rye and barley homologous to wheat peptides,89
and a sequence from oat avenins.89,90
However, whether such intestinal biopsy derived T cells generated artificially over many weeks are truly representative of responses in vivo has been unclear. Furthermore, consistency of responses across coeliac individuals has been variable. Arentz‐Hansen et al
found intestinal T cells responsive to one of two overlapping peptides (QLQPFPQPELPY, PQPELPYPQPELPY) in all 17 HLA‐DQ2 coeliac subjects.87
In contrast, another study found poor consistency among 20 coeliac subjects.86
Methodological differences are likely to explain these variations but raise concerns as to the relevance of identified peptides to human disease in vivo.
Key recent advances and unanswered questions in mechanisms of coeliac disease pathogenesis
- HLA‐DQ2 presents T cell stimulatory wheat gluten peptides.
- Tissue transglutaminase modifies gluten peptides to generate more potent T cell stimulatory sequences.
- Immunodominant T cell stimulatory gluten peptides defined.
- Gluten peptides may also activate the innate immune system.
- Resistance of toxic gluten peptides to degradation in the intestine.
- Mechanisms underlying innate immune system activation by gliadin peptides.
- Does the autoantibody response to tissue transglutaminase have a pathogenic role?
- How gluten peptides enter the intestinal mucosa, and mechanisms of presentation to T cells.
- Identification of further genetic (and environmental) factors predisposing to coeliac disease.
- Factors regulating oral tolerance and suppression of immune responses to gluten.
- Can a good animal model of coeliac disease be generated, particularly to study novel therapeutics?
An alternative strategy has been developed by Anderson et al
, and uses peripheral blood from coeliac subjects drawn after oral gluten challenge.91
In individuals following a gluten free diet for a minimum of two weeks, peripheral blood T cell responses can be reliably detected within a window of three days to two weeks after in vivo oral bread challenge.92
That these T cells are HLA‐DQ2 restricted and express gut homing molecules strongly suggests the assay is detecting freshly activated cells recirculating via the thoracic duct (fig 3) and might provide a more accurate picture of the in vivo response to wheat gluten. This method has been used with a library of peptides to “map” toxic wheat sequences,91
and due to the high throughput nature offers the possibility of exhaustive screening of all grains for potentially toxic peptide sequences in coeliac disease.93
Interestingly, results from both peripheral blood and intestinal T cell clone methods identified the same key sequence (PQPELPY) later shown to directly bind to HLA‐DQ2 (fig 4).80,87,91
Responses to this sequence were found in the peripheral blood of 50/57 tested coeliac subjects (but not healthy controls) after in vivo gluten challenge and represented 50% of the total wheat gliadin response.92
Other work has shown that in vivo intraduodenal administration of a peptide containing this sequence can exacerbate coeliac disease.94
Further assessment of other wheat, rye, and barley sequences will be necessary but it would appear that a few key “dominant” peptide epitopes critical for coeliac disease development can be identified with promise for immunotherapies and modified grains.