4.1. Cross-Reactivity between Gluten Proteins
Primarily, gluten is a source of flour and, consequently, bread and flour products. Consumption of gluten-containing food makes such food an immune system target. Digested gluten is a reason for the emergence of different antigens and immunogens. Cross-reactivity implies the reaction between an antibody and an antigen that differs from the immunogen, and it has been shown in glutenin-specific and gliadin-specific T-cells. Such T-cells could respond to gliadin and glutenin and vice versa due to their directivity to repetitive sequence highly homologous in these proteins [69
]. Such cross-reactivity contributes to the development and spread of T-cell response and inflammation. It is also known that CD is characterized not only by inflammation and small intestine tissue remodeling but also by neurologic defects such as axonal neuropathy and cerebellar ataxia [70
]. It has been shown that such neurological implications may occur partly due to the cross-reactivity between antigliadin antibody and synapsin I protein [72
]. This protein is a cytosolic phosphoprotein found in most neurons of the central and peripheral nervous systems. Although gliadins are not homologous to synapsin I, there is a glutamine- and proline-rich-region in C
-terminal sequence of synapsin I, which includes PQP and PQQP motifs similar to those in gliadin.
Evidence of the cross-reactivity of prolamin proteins was also reported in the course of allergy. ω5-gliadin is the major wheat allergen. It was shown that anti-ω5-gliadin antibodies bind to rye γ70-secalin, rye γ35-secalin and barley γ3-hordein [73
]. In about 90% of patients with wheat-dependent exercise-induced anaphylaxis, IgE antibodies against these proteins were found. Rye γ70- and γ35-secalin and barley γ3-hordein cross-react with ω5-gliadin. This probably happens due to the fact that IgE antibodies bind to structurally similar epitopes; found proteins are related to the same evolutionary group of γ-type prolamins.
Even though there is no cross-reactivity between allergens in oat and other gluten species, avenins show immunological cross-reactivity to γ-secalin due to their considerable homology [74
]. First immunogenic peptides in hordein, secalin and avenin were revealed on the basis of T-cell cross-reactivity against wheat gluten proteins [75
]. Epitopes defined in hordein and secalin were recognized by α-gliadin-reactive T-cell lines in vitro while avenin epitopes were not. That explains why rye and barley were considered to be pathogenic for CD patients, whereas oat was included in the “gluten-free” group of food.
Later, it was shown that oat consumption is safe for the majority of CD patients [76
], even for children [77
]. It is well known that the greater the proline residues in storage protein the more pathogenic this protein is for CD patients [78
]. Low proline content may be the reason why oat avenins are less immunogenic compared to wheat prolamins but may still be toxic in large quantities. However, there was no direct (in vivo) evidence of the activation of gluten-reactive T-cells following ingestion of oats. Hardy et al. provided in vivo evidence that ingestion of oats activates avenin-specific T-cells in 10% of CD patients [79
]. Moreover, they showed T-cells to be cross-reactive against hordein and avenin. After oral challenge with barley (and not wheat or rye) the majority of HLA-DQ2.5 CD patients harbor T-cells capable of being activated by avenin peptides ex vivo, but the ingestion of oats itself provides rather weak antigenic stimulation for this population of T-cells. Avenins are probably less stimulatory because they do not contain proteolytically resistant peptides longer that 10 amino acid residues. They have reduced binding stability to HLA-DQ2.5 compared to hordein peptides. This means that avenin-reactive T-cells are activated by the consumption of barley rather than oat.
4.2. Celiac Disease
As described in the introduction, gluten is impregnable by the gastric, pancreatic and intestinal digestive proteases of people carrying HLA-DQ2 or/and DQ8 haplotype. HLA-DQ is a part of the MHC class II antigen-presenting receptor system and distinguishes its own and foreign cells. HLA-DQ protein consists of two subunits, which are encoded by the HLA-DQA1 and HLA-DQB1 genes located on the short arm of the 6 chromosome. Mainly, people with celiac disease have DQ2 or DQ8 isoforms because these receptors bind to gliadin peptides more tightly than other forms.
However, there are multiple DQ2 haplotypes. The most associated with celiac disease (95% of patients) is the two-gene HLA-DQ2 haplotype referred to as DQ2.5. This haplotype is composed of subunits α5
encoded by two adjacent gene alleles DQA1*0501 and DQB1*0201 (Figure 5
). Four percent of CD patients have the DQ2.2 isoform (DQA1*0201:DQB1*0202) and the remaining have DQ8 (encoded by the haplotype DQA1*03:DQB1*0302).
After the gluten enters into the digestive system, prolamin proteins are not fully hydrolyzed by proteases, which results in the emergence of gluten peptides. They are deamidated by tTG enhancing their affinity to MHC II molecules. Deamidated peptide is then recognized by DQ molecule on the surface of a dendritic cell and is presented to T cells inducing immune response. It is interesting that both DQ2 and DQ8 lack canonical aspartic acid residue at DQβ57. It results in the compensation of this negative charge by negatively charged residues either in the T cell receptor or in the deamidated peptide. Absence of aspartic acid residue leads to cross-reactive and stronger responses by T cells [80
Nowadays, it is clear that distinct gluten peptides are involved in celiac disease in a different manner. Peptides are divided into two groups: toxic and immunogenic. Toxic peptides are capable of inducing mucosal damage when administered in vivo on the intestine, whereas peptide is considered to be immunogenic if it is able to specifically stimulate HLA-DQ-restricted T cell lines isolated from peripheral blood of CD patients [82
]. Peptides differ from each other in the degree of the immunogenicity. It is remarkable that some are immunodominant, meaning that they evoke strong T cell response in almost every CD patient, whereas the immunogenic do not. Immunogenicity is enhanced after tTG deamidation procedure. It is worth mentioning that tTG is more likely activated after inflammation, but it is still not clear whether the deamidation of peptides is by tTG initiates inflammation or vice versa [82
All gluten proteins (gliadin and glutenin from wheat, hordein from barley, secalin from rye and avenin from oat) possess their own sets of toxic and immunogenic peptides (or epitopes) with distinct immunogenicities. However, gliadin peptides are known to be the most toxic and numerous, specifically derived from α- and γ-gliadin: the strongest and most common adaptive response to gluten is directed toward an α2-gliadin fragment of 33 amino acids in length [8
]. Digestion of gliadin results in the emergence of two pieces: 25-mer (p31-55, it can be degraded to smaller peptides) and 33-mer (p57-89). Peptide p31-43 of α2-gliadin may directly induce interleukin-15 production from enterocytes and dendritic cells. Peptide p57-89 is deamidated by tTG and presented to T cells by HLA-DQ molecules. Glutenin peptides are also involved in T cell response [83
]. Peptides may enter the cell by endocytosis, with their entrance into the cells requiring 37 °C temperature and Ca2+
in the media [84
]. It has been shown that these peptides possess structural configuration characterized by a left-handed polyproline II helical conformation that is preferred by MHC class II ligands [85
(1) Properties of 33-mer peptide from α-gliadin
The α-gliadin 33-mer is one of the digestion-resistant gluten peptides that is highly reactive to isolated celiac T cells and is the main immunodominant toxic peptide in celiac patients. It is located in the N
-terminal repetitive region of α-gliadin and contains six overlapping copies of three different DQ2-restricted epitopes (Figure 6
Using RNA-amplicon sequencing (NGS) technology it was shown that α-gliadins can be separated into six types and only one type contains all the immunogenic peptides and epitopes, whereas the other five types do not contain all the epitopes disabling 33-mer peptide formation [30
]. Thus, distinct types of α-gliadins differ mainly in the number of repeat blocks consisting in interspersed motifs PFPPQQ and PYPQPQ.
Six epitopes of type 1 α-gliadin are DQ2.5-glia-α1a/b and DQ2.5-glia-α2 (Figure 6
). There is also a partial overlap with 33-mer DQ2.5-glia-α3 epitope associated only with type 1 of gliadins. 33-mer is able to self-assemble in a concentration-dependent manner through structural transition [32
]. It obtains polyproline II structure based on type II beta-turn with increase of peptide concentration.
33-mer reaches lamina propria and after deamidation plays a central role in the pathogenic cascade of celiac disease by activating the adaptive immune response. 33-mer enters the cell by intracellular pathway, excluding paracellular entrance. Gliadin-derived peptides can also be transcytosed from the apical of the intestinal epithelium to the basolateral side along with transferrin and IgA, avoiding entrance to the late endocytic compartment [87
In vivo experiments revealed that 33-mer gliadin-derived peptide is undigested by enzymes of the intestinal brush border. Moreover, in a monkey model of gluten sensitivity, 33-mer peptide can be detected in the serum when the disease starts, indicating that this peptide can trespass the mucosa intact in vivo [88
Then, 33-mer is deamidated by tTG present in the intestinal brush border and presented by dendritic cell to T cell (in mesenteric lymph nodes). T cells reach peripheral blood through the thoracic duct and product interferon-γ resulting in intestine epithelial cytotoxicity, while another peptide p31-43 has been reported to induce the innate immune response necessary to initiate the T-cell adaptive response through production of interleukin-15.
It has been shown that 33-mer of α-gliadin is very similar to protein Prn of B. pertussis
, which causes pertussis. These results show that neither pertussis immunization nor disease induces production of antibodies reactive against the peptide, and thus it is unlikely that either pertussis immunization or disease contributes to CD pathogenesis on the basis of cross-reactive antibodies [89
(2) Repertoire of gluten peptides active in CD
It has been established that deamidated forms of gluten peptides are more toxic than their amidated forms. tTG preferably deamidates sites QXP (X—any amino acid residue), which are abundant in immunodominant peptides. Interestingly, both DQ2 and DQ8 molecules lack the aspartic acid residue at β57 position present in other DQ molecules. DQ2 and DQ8 molecules possess positively charged pockets containing five anchor positions and a least three of them (P4, P6, and P7) prefer to bind negatively charged amino acids [90
]. Crystallographic structure of DQ2 complexes with immunodominant epitope revealed that glutamic acid residue fits in the P4, P6 and P7 anchor positions and proline residue—in the P1, P3, P6 and P8 positions [91
]. Analogous report regarding DQ8 complexes revealed only two glutamic acid-preferred positions (P1 and P9). This explains the lower number of gluten peptides active in DQ8 individuals.
Long-term T cell lines (TCL) or T cell clones (TCC) raised against gluten are used to identifying gluten immunogenic peptides. Anderson et al. established an approach that detects gluten-specific T cells in the peripheral blood (peripheral blood mononuclear cells, PBMC) after 3 days of consumption of gluten-containing food by an interferon-γ EliSpot assay [92
]. A comprehensive, quantitative mapping of T cell epitopes was used to screen all the unique 20-mer sequences of gliadins, glutenins, hordeins, and secalins. Independently, this screening and EliSpot assay provided the set of immunodominant epitopes from wheat, barley, and rye (Table 3
]. Almost all DQ2 immunogenic peptides of α-gliadins map the N
-terminal 57–89 region (33-mer). DQ2-restricted ω-gliadin peptides strictly related to α-gliadin 17-mer: it contains two overlapping copies of 9-mer epitopes. Immunogenic peptides of γ-gliadins are spread along all the sequences. Similarly, few DQ2-restricted sequences from secalin and hordein proteins were reported to stimulate intestinal CD4+ T cell lines or clones [75
Only 3 DQ8-restricted epitopes were identified using T cell lines or T cell clones: two for α-gliadin, γ-gliadin and one for glutenin [95
]. Additional peptides were discovered as a result of the work by Tye-Din et al. [93
]. Furthermore, it was shown that HLA-DQ8-associated CD appears not to be exclusively dependent on deamidation by tTG [95
Avenins differ from other groups of prolamins due to their low content of proline and glutamine residues. Nevertheless, a few gliadin-like and glutenin-like avenin-derived peptides were identified [96
]. Avenin peptides were divided into three groups: low-stimulatory short peptides (six residues), stimulatory (27 and 10 residues) and peptides with upregulated stimulatory capacity (10 and 14 residues). Larger peptides (27 residues) are commensurable in size with 33-mer and induce response of dendritic cells in not only CD patients but also in control healthy patients. Whereas peptides with the appropriate size and disposition of amino acids residues (10 and 14 residues) are likely to go through a differential endocytic pathway.
Thus, immunogenic sequences were identified in all gluten proteins of Triticeae and oat. These studies revealed that amongst all the DQ2-restricted peptides of wheat, barley, rye and oat prolamins, there is a hierarchy of T cell recognition depending on the specific cereal ingested. Furthermore, an evident redundancy in DQ2-restricted peptide recognition occurs, i.e., activated by a dominant peptide T cells are capable of recognizing and responding to a large number of related gluten sequences and vice versa. However, there is no clear difference in the immunogenicity strength between DQ8-restricted peptides. Altogether, DQ8-type of HLA molecules are less strongly associated with celiac disease, compared to DQ2. Oat peptides possess the lowest immunogenic activity though avenin peptides are capable of inducing T cell response.
4.3. Wheat Allergy
Wheat flour triggers IgE-mediated food allergy and is one of the top eight food allergens. Wheat allergy commonly develops in childhood [97
]. When an allergen specifically binds to IgE antibodies, it induces the activation of mast cells and basophils. In the case of wheat, it is believed that allergy occurs due to a breach in oral tolerance and as a consequence of Th2-biased immune dysregulation that induces sensitization and B-cell-specific allergen IgE production [98
]. Gluten proteins causing allergy include some types of ω-gliadin as well as non-gluten protein of wheat such as profilin, serpin, α-purothionin, etc. Non-gluten flour proteins and some γ-, α/β-gliadins can cause an occupational respiratory allergy such as baker’s asthma, which appears after the inhalation of flour by millers or bakers. ω5-gliadins trigger another type of allergy—food allergy—referred to as wheat-dependent exercise-induced anaphylaxis (WDEIA), which develops after the ingestion of wheat followed by intense physical exercise.
Recently, it was shown that γ-, α/β-, ω5-, ω1,2-gliadins contain IgE-binding epitopes as well as HMW and LMW subunits of glutenin [99
] (Table 4
). Nevertheless, major allergenic protein of wheat is ω5-gliadin possessing N
-terminal sequence SRLL, which can be crucial for allergy pathogenesis, and repetitive region consists almost entirely of peptides FPQQQ and QQIPQQ [14
γ-gliadin, α/β-gliadin and ω1,2-gliadin are causative allergens in both WDEIA patients and those with baker's asthma [100
]. Epitopes QQPFP and PQQPF of gliadin are also involved in atopic dermatitis-related wheat allergy [102
] as well as QQQPP motif in LMW-GS [103
Nowadays, 3D structure of known IgE allergenic epitopes helps to elucidate its conformation and to produce recombinant allergens for further research.
4.4. Non-Celiac Gluten Sensitivity (NCGS)
The first reported NCGS cases were described as longstanding and previously unresolved history of abdominal pain, discomfort, bloating, altered bowel habit and fatigue with exclusion of celiac disease. NCGS is more frequently diagnosed in adults rather than in children [104
]. In most cases, NCGS reveals itself a few hours after gluten digestion [104
]. Similarly to CD patients, patients with NCGS suffer from nutritional deficiencies, coexisting autoimmunity, and a decreased bone mineral density compared with the general population. The prevalence of HLA-DQ2 and/or HLA-DQ8 genotypes is ~50% in NCGS comparable to the general population [105
], but there is no anti-tTG2 antibodies identified. Gluten only triggered an innate immune response in NCGS and provoked an additional adaptive immune response with increased expression of IL-6, IL-21, IL-17 and IFN-γ [106
]. However, gastrointestinal symptoms other than intestinal permeability and adaptive immune responses are not involved in the process. In NCGS, gliadins do not induce mucosal inflammation in vitro or the activation of basophils as seen in CD [107
It has been suggested that in NCGS gluten-related peptides enter the systemic circulation and cause extraintestinal manifestations such as ataxia, neuropathy and encephalopathy [108
]. Moreover, it has been proposed that gluten causes depression, anxiety, autism and schizophrenia in patients with NCGS [109
], and also reported that psychosis might be a manifestation of NCGS [110
Nowadays, gluten-related disorders have often been recognized as commonly mimicking irritable bowel syndrome (IBS) because of the similar symptoms such as abdominal pain, bloating, bowel habit abnormalities (either diarrhea or constipation) [111
]. Indeed, both can coexist independently without necessarily sharing a common pathophysiological basis. Furthermore, the microbiome may also play a role in the pathogenesis of NCGS [112
]. Gut microbiota composition and metabolomic profiles may influence the loss of gluten tolerance and subsequent onset of gluten intolerance in genetically-susceptible individuals [113
]. Gut microbiota could become a target for further therapy [114
Recently, a standardizing protocol was reported for the diagnostic confirmation of NCGS [3
]. It implies assessment of the clinical response to GFD and consequent effect of the gluten challenge. It is important that patients are on a normal, gluten-containing diet for proper evaluation, which is not always possible. For these two assessment steps, a modified version of the Gastrointestinal Symptom Rating Scale (GSRS) is used. The GSRS protocol is based on reviews and the clinical experience and allows for evaluation of gastrointestinal and extra-intestinal symptoms. Patients name one to three symptoms and the Numerical Rating Scale (NRS) measures the severity score from 1 to 10. However, there are still difficulties in diagnosing and managing NCGS. Even though the precise mechanism and biochemical markers for the NCGS disease have still not identified, this protocol could be used to establish the prevalence of this condition.