Positive Health Online

Introduction

Wheat and other gluten containing grains including rye and barley have become a major part of the human diet ever since they were first cultivated by our ancestors about 10,000 years ago. Little did our ancestors know that consumption of some components of these grains such as gluten and its derivatives would trigger development of a range of clinically significant disorders in at least 1% of the present-day global population. These disorders are known as gluten-related disorders or gluten enteropathies and include coeliac disease (CD),  non-celiac gluten sensitivity (NCGS) and wheat allergy (WA).

Gluten enteropathies are associated with one or more of the following symptoms, anaemia, anxiety, bloating, brain fog, delayed growth in children, depression, diarrhoea, fatigue, headaches, infertility, joint pain, mouth sores, slow weight gain and numbness. As a consequence, it is not possible to distinguish between these different clinical entities on symptom pattern alone. Essentially, symptoms associated with these gluten enteropathies are mostly due to impaired absorption of vital nutrients.

https://commons.wikimedia.org/wiki/File:Fimmu-12-744396-g002.jpg

Different locations of the body that are affected by autoimmune diseases

Picture Credit: Katie Glover, Deepakkumar Mishra, and Thakur Raghu Raj Singh on Wikipedia

About Nutrient Absorption

The luminal or inner wall of the gastrointestinal tract, particularly the small intestine, is lined with a layer of cells called enterocytes.[1] Enterocytes play an important role in absorption of vital nutrients as well as immunological protection of the gastrointestinal tract. These important cells may be damaged by different types of immune mediated inflammatory processes induced by gluten and its derivatives.

Enterocytes are found on the surface of finger-like projections called villi. Spaces between villi are called crypts. This cellular configuration increases the surface area of the gastrointestinal lining to maximise absorption of digested food components.

Following digestion of different food components in the stomach, the digest products pass into the small intestine where nutrients can be absorbed.

The gaps between enterocytes should remain tight so that large partially undigested fragments such as gluten cannot pass through them. Unfortunately, some partial digests of gluten and its derivatives such as gliadin as well as some bacteria, can stimulate the synthesis of abnormally high amounts of a protein called zonulin. Excess zonulin widens the spaces between enterocytes so that undigested gluten fragments can access the sub-enterocyte layer that houses immunocompetent cells.

As described below, the enterocytes have another important property, they also function as antigen presenting cells and interact with immunologically competent cells, T cells, to provide immunological protection for the gastric lining.  

About Gluten

Gluten is a structural protein found in the seeds of cereals rye, wheat and barley.[2] Gluten and its derivatives in these cereals are quite difficult to digest by proteolytic enzymes in the stomach because they contain stretches that are rich in the amino acids proline and glutamine. 

In the small intestine, some gluten is broken down into sub-components such as glutenin and gliadin by the enzyme tissue transglutaminase 2 (tTG), releasing small strings of amino acids in the form of peptides. Some gluten remains partially undigested. These poorly digested particles gradually collect under the enterocyte layer. These particles can then be taken up by local phagocytic dendritic cells which transform some of them into antigens which can be presented to CD4+ helper T cells and subsequently to cytotoxic T cells and antibody producing B cells. These reactions form the basis of the immune mediated inflammatory anti-enterocytic process.

Celiac Disease Pathogenesis from Caio G et al 600x465px.jpg]

 https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-019-1380-z#Fig1

Celiac disease pathogenesis. Partially digested gliadin fragments interact with chemokine receptor 3 on the apical side of epithelium (1) inducing a myeloid differentiation primary response 88-dependent release of zonulin (2). Zonulin interacts with the intestinal epithelium and triggers increased intestinal permeability (3). Functional loss of the gut barrier facilitates gliadin peptide translocation from lumen to the lamina propria (4). Gliadin peptides trigger release of IL-15, keratinocyte growth factor, and IL-8 (5), with consequent recruitment of neutrophils in the lamina propria (6). Simultaneously, alpha-amylase/trypsin inhibitors engage the Toll like receptor 4–MD2–CD14 complex with subsequent up-regulation of maturation markers and release of proinflammatory cytokines (7). Following innate immune-mediated apoptosis of intestinal cells with subsequent release of intracellular tissue transglutaminase, gliadin peptides are partially deamidated (8). Deamidated gliadin is recognized by DQ2/8⁺ antigen presenting cells (9) and then presented to T helper cells (10). T helper cells trigger activation and maturation of B cells, producing IgM, IgG, and IgA antibodies against tissue transglutaminase (11). T helper cells also produce pro-inflammatory cytokines (interferon γ and tumor necrosis factor α) (12), which in turn further increase gut permeability and, together with T killer cells, initiate the enteropathy. Damaged enterocytes express CD71 transporter also on their apical side, resulting in retrotranscytosis of secretory IgA-gliadin complexes (13), thus potentiating gluten trafficking from gut lumen to lamina propria. Ultimately, the interaction between CD4⁺ T cells in the lamina propria with gliadin induces their activation and proliferation, with production of proinflammatory cytokines, metalloproteases, and keratinocyte growth factor by stromal cells, which induces crypt hyperplasia and villous blunting secondary to intestinal epithelial cell death induced by intraepithelial lymphocytes. The hyperplastic crypts (14) are characterized by an expansion of the immature progenitor cells compartment (WNT) and downregulation of the Hedgehog signaling cascade. An increased number of stromal cells known to be part of the intestinal stem cell niche and increased levels of bone morphogenetic protein antagonists, like Gremlin-1 and Gremlin-2, may further contribute to the crypt hyperplasia present in celiac disease

Credit: Caio, G et al.  Celiac disease: a comprehensive current review. BMC Med. 17: 142-155. 2019. https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-019-1380-z#Fig1 

Coeliac Disease

CD is one of the most undiagnosed forms of ill-health. It is considered an autoimmune disease because of the formation of specific gluten induced autoantibodies which cause inflammation of the lining of the gastrointestinal tract. It is also associated with other autoimmune diseases such as type1 diabetes, thyroid autoimmunity and Sjögren’s syndrome. As in many other autoimmune disorders it is more common in females and is associated with a particular HLA antigen.[3,4]

For many decades, the pathogenesis of CD was poorly understood. In recent years, since identification of the specificity of the causative autoantibody, understanding of CD has greatly advanced.

In the past it was considered that CD was an uncommon gastrointestinal disorder mainly affecting children who appeared to suffer from malnutrition. Common symptoms included growth retardation and diarrhoea. Now, partly because of the development of more specific and sensitive diagnostic tests, it has been found that at least 1% or more of the population of all ages suffer from this disorder. It is thought however that many more suffer from undiagnosed CD.

Those who suffer from untreated CD may exhibit a combination of one or more of a range of symptoms as indicated above. In the long term though, untreated CD may lead to the development of more serious complications such as osteoporosis, reproductive disorders, some cancers, brain disorders and dermatitis herpetiformis, a disorder characterised by an unpleasant skin rash in about 20% of cases. Clearly, because there is such a wide range of symptoms associated with CD, it is difficult to diagnose this condition on symptom profile alone. As indicated, this is partly one of the reasons why many people may suffer from undiagnosed CD.

Aetiology of CD

Damage to the absorptive enterocyte layer is bought about by initiation of the adaptive immune system, the innate immune system and an apoptotic process. Images which illustrate the immunological processes that underpin the pathogenesis of CD are shown in Refs. [3] and [4].

From an immunological standpoint, treatment of gluten derivatives by tGT releases peptides which have a strong affinity for MHC class 2 molecules, in particular HLA DQ2 and/or DQ8. (That is why there is a strong association between CD and HLA DQ2 and DQ8). These peptides become bound to CD4+ T helper cells, the cells which support the formation of antibodies by B cells.

Peptide presentation by HLA-DQ2/DQ8 protein subunits on the surface of dendritic cells to gluten-specific CD4+ helper T cells induces two levels of immune response, enterocyte damage by CD8+ cytotoxic T cells and activation of specific B cells which produce antibodies against them. Because the enzyme tGT is bound to gluten derived fragments including gliadin, some B cells see this complex as an immunogen and consequently produce antibodies against tGT as well as parts of gliadin. In fact, tGT is recognised as the primary disease specific autoantigen in CD.

Significance of Different Antibody Types

There are a number of different antibody types. Two classes of antibody are significant with respect to the pathogenesis of CD namely IgG ( and its sub-classes) and IgA antibodies.

It is important to understand the significance of antibody types and subclasses because they have a major influence on CD pathogenicity and diagnosis.

Igg Sub-Classes

There are four different IgG sub-classes, IgG1, 2, 3 and 4. IgG1 and IgG3 subclasses, when bound to their immunological targets activate and fix the complement system very efficiently.

The complement system comprises a series of enzymes which interact sequentially to promote a profound inflammatory response and attract phagocytic cells. It also kills cells to which the terminal complement component C9 is bound by inflicting a lethal molecular hole in them. On the other hand, IgG2 and IgG4 sub-classes activate the complement system very poorly, if at all. Interestingly, it is now becoming clear that CD is associated mostly with anti-gluten/tTG antibodies that are of the IgG1 and 3 sub-classes.[5] As discussed below, it is recognized that the less severe form of enteritis, NCGS, is associated with the formation of IgG2 and 4 types of autoantibodies. Thus, determination of the type of IgG autoantibody may be helpful in distinguishing between the clinical entities CD and NCGS.

Interestingly, antibodies of the IgG sub-classes IgG1 and IgG3 are able to cross the placenta to offer immunological protection for the unborn child. CD mothers with IgG1 and/or IgG3 anti-tGT antibodies may harm their children before and after birth because their anti-tGT autoantibody passes into the baby’s circulation during pregnancy.

IgA Antibody

As well as the production of IgG antibodies, the enterocytic layer houses B cells that can synthesise anti -tGT antibodies of the IgA type. IgA antibodies can exist in a dimeric form which enables them to pass through to the outside of the enterocytic layer further hindering the absorption of vital nutrients.

Following both an antibody and cell mediated immunological attack on enterocytes and exposure to gluten derivatives, a localised apoptotic process is initiated which also contributes to the death of enterocytes. Apoptosis is a term used to describe a specific biological process called programmed cell death in which cells spontaneously self-destruct. Cellular destruction is brought about by expression of a protein called p53. This protein instructs cells to kill themselves by destroying their own DNA.

Diagnosis of CD

To ensure the accuracy of any diagnostic test for CD it is important that the subject has consumed gluten prior to the test because diagnostic antibodies rapidly disappear on a gluten free diet.

Histological Examination

The results of histological and immunohistochemical tests on a biopsy specimen are highly diagnostic for CD. The results of these tests reveal the extent of enterocytic damage and immunological involvement and whether the damage is due to T cell cytotoxicity or antibody mediated enterocytic destruction.

Confirmatory Serological Tests

Detection of various antibodies in the blood can be helpful in the diagnosis of CD. Detection of anti-tGT antibody of the IgA type is highly specific and sensitive for the diagnosis of CD. Unfortunately, about 5% of CD patients are IgA deficient. In these cases, suitable diagnostic tests should include identification of IgG antibodies against tTG as well as detection of IgG antibodies directed against deaminated gliadin peptide (DGP) and epithelial membrane antigen (EMA). Detection of IgG antibody against EMA is highly specific for CD.[3]

Exclusion Tests

A diagnosis of CD can be excluded by genetic typing for the HLA class 2 antigens DQ2 or DQ8 because around 95 % of CD sufferers are positive for one or both of these two HLA antigens. Lack of the HLA antigens DQ2 or DQ8 excludes a diagnosis of CD. HLA typing for these antigens cannot be used to confirm a diagnosis of CD because around 30% of the population are positive for one or both of these HLA antigens.

Other Considerations

It is clear that gluten is the predominant trigger for the onset of CD, however, recent findings have raised the possibility that changes in the composition of the gut microbiome including bacteria, yeasts and viruses may also play a role in increasing susceptibility to development of CD. Alterations in the composition of the gut microbiome may upregulate the expression of zonulin. As stated above, excess zonulin widens the spaces between enterocytes giving  particulate matter access to the sub-enterocytic regions.

About Non-Coeliac Gluten Sensitivity [NCGS]

For many years, it had been debated whether NCGS was in fact a distinct disease entity. Now, in light of the results of many in-depth investigations, it is accepted that NCGS is a recognized disease entity.[6] It is defined as a syndrome characterised by development of intestinal and extra-intestinal symptoms following the ingestion of gluten-containing foods, particularly wheat, in subjects that are not affected by either CD or WA. The exclusion of CD and WA for the diagnosis of NCGS remains a key step due to the lack of specific or reliable biomarkers for NCGS. Importantly, NCGS cases do not develop he same degree of pathological intestinal damage or sensitization to wheat proteins as in CD and WA, respectively.

Some form of intestinal barrier dysfunction has been suggested to play a role in the pathogenesis of NCGS because serum levels of intestinal fatty acid-binding protein (FABP2), a biomarker used for evidence of intestinal injury, are raised in those suffering NCGS.

It is thought that NCGS occurs more frequently than CD, perhaps even  more than 10% but precise estimations are not possible because of a lack of definitive diagnostic biomarkers.

Gastrointestinal and/or extra intestinal symptoms can be triggered in NCGS susceptible cases by ingestion of gluten. The most common gastrointestinal symptoms are bloating, abdominal pain, diarrhoea, nausea and reflux. The extra intestinal manifestations are widely heterogeneous and include headache, general tiredness, blurred mind, fibromyalgia, lack of well-being, dermatitis, joint pain and depression. Consequently, it is not possible to distinguish between CD and NCDS on symptomology alone.

Genetic typing for HLA DQ2 and/or DQ8 is not helpful in excluding or confirming the diagnosis of NCGS because only about 50% of NCGS patients are positive for one or both of these CD associated HLA antigens.

In NCGS, in contrast to CD, IgA autoantibody against tTG is absent. Also, CD4+ T helper cells are not increased in in the gastrointestinal tract of NCGS patients. There is evidence however that NCGS is driven by a low level autoimmune process because it occurs more commonly in younger females and is associated with other autoimmune diseases particularly autoimmune thyroid disease.  Antibody against nuclear components may also be present in many NCGS patients. Antibodies against nuclear components are a common feature of many autoimmune diseases.

https://commons.wikimedia.org/wiki/File:Gliadin-immuno-innate.png

Gluten Immunochemistry
This image shows the alpha-2 gliadin 33mer that contains 6 T-cell epitopes, when deamidated.
The bottom part shows the innate epitopes on alpha-9 gliadin

Picture Credit: Pdeitiker on Wikipedia

Interestingly, IgG antibodies of different sub-classes allow partial distinction between CD and NCGS patients and provide a possible explanation for the difference in disease severity between CD and NCDS. In the blood, as stated, anti-gliadin antibodies of the IgG sub-classes IgG1 and 3 have been found to be associated with CD. By contrast, anti-gliadin antibodies of the IgG2 and 4 sub-classes are more strongly associated with NCDS.[5] This is an important observation with respect to the different disease manifestations between CD and NCDS because, as indicated above, IgG1 and 3 antibodies are able to activate the complement system more effectively than IgG2 and 4 sub-classes. These findings are also consistent with a mild form of autoimmunity in some NCGS cases.

Histological examination of biopsy specimens from NCGS patients do not reveal evidence of major enterocytic damage as in CD.

Other Differences

It is thought that some wheat components other than gluten derived gliadins and glutenins, such as fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) may be involved in development of NCGS.  (FODMAPs are short-chain carbohydrates that are poorly absorbed in the small intestine and subsequently fermented in the colon.) FODMAPs and amylase and trypsin inhibitors (ATIs), may also act as triggers of some of the extra-intestinal clinical manifestations in NCGS cases because NCGS patients have reported remission of symptoms after following a low-FODMAP diet. (ATIs are a group of low-molecular weight proteins that are highly resistant to gastrointestinal proteases and can be found in the endosperm of plant seeds, where they act as natural pesticide).

Although the role of ATIs in NCGS remains uncertain, it is considered  that they may induce inflammation by activating elements of the innate immune system such as complement. Much more work is needed to fully understand the pathogenesis of NCGS.

Diagnosis of NCGS

There is a lack of specific biomarkers for the diagnosis of NCGS and  consequently, its diagnosis is mostly based on the exclusion of CD and WA. Distinction between these different disorders may be determined by  the clinical assessment of the patient while undergoing a double- or single-blind placebo-controlled gluten challenge. This is a complex time consuming process summarised by Cardenas-Torres and colleagues.[6] Basically, following a challenge with either gluten or its derivatives, clinical manifestations would be expected  within days in subjects suffering from NCGS. By contrast, clinical signs would be expected to be seen in a matter of weeks in subjects with CD.

Optimizing The Microbiome

Studies have shown that onset of CD in genetically predisposed individuals is influenced by factors such as caesarean birth, formula feeding and drug exposure. They have also established that intestinal infections also contribute to the risk of development of  CD. These findings implicate the composition of the gut  microbiome in susceptibility to development of CD.[7] (The microbiome refers to the composition of different microbes such as bacteria, fungi and viruses that occupy a particular anatomical site, in this case, the gastrointestinal tract.)

With respect to CD, elevated levels of several genera of undesirable bacteria including bacteroides, Escherichia, and prevotella have been found. By contrast, beneficial bacteria such as lactobacilli and bifidobacteria have been found to be less abundant in the gut microbiome of CD patients.[7]

Thus far it is unclear to what extent alterations in the composition of the microbiome influence the development of NCGS.

Microbiome and CD

Even though the prescribed way to improve the clinical symptoms of CD is to follow a gluten free diet, such a diet alone is not able to fully recover the optimal composition of the gut microbiome. Consequently, it is concluded that enrichment of helpful probiotics such as different strains of lactobacillus and bifidobacterium in the gut in addition to a gluten free diet may be needed to fully restore optimal health of the gastrointestinal tract in CD patients.

Some foods that may contain healthy probiotics include  yogurt, cottage cheese, tempeh, sauerkraut and miso soup.

About Wheat Allergy

 In contrast to CD and NCGS, WA has a different pathogenetic background. It is not an autoimmune disease. Its pathogenesis involves an inflammatory process brought about by either an IgE mediated allergic reaction directed against a number of wheat proteins such as albumin, globulins, gliadin or glutenin, or induction of a gastrointestinal eosinophilia, an influx of eosinophils.[8]

Those who suffer from WA share many of the symptoms experienced by subjects suffering from CD and NCGS.

WA occurs mainly in children. It usually resolves spontaneously as they reach puberty. There is a genetic basis to WA because it occurs more frequently in families in which other family members suffer from some other form of allergy  such as asthma.

Allergy involves the production of antibodies of the IgE type.  IgE antibodies are unusual in that they can bind to the surface of mast cells. Mast cells are found throughout the body, especially surrounding mucosal tissues such as the  gastrointestinal tract. When an antigen binds to an IgE antibody bound to  a mast cell, the mast cell bursts open releasing potent inflammatory mediators such as histamine, heparin, a variety of cytokines, chondroitin sulphate, and neutral proteases. IgE mediated reactions are immediate and can have extremely serious consequences such as anaphylaxis.

Diagnosis of Wheat Allergy [WA]

As in other forms of enteritis, damage to the gastrointestinal tract can be identified by histological examination of biopsy tissue.  Histological examination would also identify an abnormal influx of eosinophils. Eosinophils are a type of white cell involved in allergies and parasite infections.

IgE mediated WA may be diagnosed by identification of raised levels of IgE in the blood as well as detection of specific IgE antibodies directed against some of the wheat proteins

Skin prick tests may be helpful in the diagnosis of WA. Skin tests involve pricking the skin beneath a drop of a solution of the suspected wheat allergen. A positive result is recognized by the development of an inflammatory wheal around the skin prick site within minutes.

Dietary wheat component challenges may be used in  a diagnostic setting. However, these challenges should only be performed under strict medical supervision since they are likely to produce serious anaphylactic responses.

Conclusions

It is clear that gluten as well as other wheat derived proteins and probably FODMAPs  play a significant role in development of CD, NCGS and WA.

It is recommended that those with a family history of autoimmunity (and are DQ2 or DQ8 positive) reduce gluten intake as much as possible to reduce the possibility of development of CD. Once CD or NCGS has developed, it is essential that a gluten free diet is maintained as well as  normalization of the gut microbiome.  A FODMAP restricted diet may also be helpful.  Normalization of the gut microbiome has many other health benefits.

A gluten and FODMAP free diet is also helpful in relieving symptoms of irritable bowel syndrome.

Because different communities may have different dietary customs, it is recommended that concerned subjects seek advice from an authoritative local dietician to formulate appropriate dietary modifications taking into account local customary and environmental  dietary conditions .

Finally, because of the remarkable range of intestinal and extra-intestinal symptoms associated with CD, it would be helpful to refer to the results of IgG and IgA anti-tGT tests to exclude a form of CD as the cause of any unwanted symptoms. Alternatively, it may be useful to note symptom changes following a gluten free diet for a period of a few weeks. 

References

1. Snoeck V et al. The role of enterocytes in the intestinal barrier function and antigen uptake. Microbes Infect. 7(7-8): 997-1004. 2005.
2. Shewry P R et al. The structure and properties of gluten: an elastic protein from wheat grain. Phil Trans R Soc Lond. B357: 33–142. 2002.
3. Caja S et al. Antibodies in celiac disease: implications beyond diagnostics. Cell Mol Immunol. 8(2):103-109. 2011.
4. Caio, G et al. Celiac disease: a comprehensive current review. BMC Med. 17: 142-155. 2019.
5. Uhde et al. Subclass profile of IgG antibody response to gluten differentiates non-celiac gluten sensitivity from celiac disease. Gastroenterology. 159(5): 1965-196. 2020.
6. Cárdenas-Torres F et al. Non-Celiac Gluten Sensitivity: An Update. Medicina (Kaunas). 57(6): 526-538. 2021.
7. Lupu VV et al. Advances in understanding the human gut microbiota and its implication in pediatric celiac disease-a narrative review. Nutrients. 15(11): 2499-2516. 2023.
8. Cianferoni A. Wheat allergy: diagnosis and management. J Asthma Allergy.9:13-25.2016.