Study of a potentially fatal food-triggered disease has uncovered a process that may contribute to many autoimmune disorders.
The history of gluten
The advancement of agriculture 10,000 years ago came at a dear price: the emergence of an illness now known as coeliac disease (CD), which is triggered by ingesting a protein in wheat called gluten or eating similar proteins in rye and barley.
Gluten and its relatives had previously been absent from the human diet. But once grains began fuelling the growth of stable communities, the proteins undoubtedly began killing people (often children) whose bodies reacted abnormally to them.
Identification of gluten as the trigger occurred after World War II, when Dutch paediatrician Willem-Karel Dicke noticed that a war-related shortage of bread in the Netherlands led to a significant drop in the death rate among children affected by CD; from greater than 35% to essentially zero. He also reported that once wheat was again available after the conflict, the mortality rate soared to previous levels.
Turning to the biological effects of gluten, early investigators learned that repeated exposure in CD patients causes the villi (fingerlike structures in the small intestine) to become chronically inflamed and damaged, so that they are unable to carry out their normal function of breaking food down and shunting nutrients across the intestinal wall to the bloodstream (for delivery throughout the body).
Fortunately, if the disease is diagnosed early enough, and patients stay on a gluten-free diet, the architecture of the small intestine almost always returns to normal, or close to it, and gastrointestinal symptoms disappear.
Fuller details of the many mechanisms through which gluten affects immune activity are still being studied, but one insight in particular has already proved useful in the clinic: a hallmark of the aberrant immune response to gluten is the production of antibody molecules targeted to an enzyme called tissue transglutaminase. This enzyme leaks out of damaged cells in inflamed areas of the small intestine and attempts to help heal the surrounding tissue.
Discovering that these antibodies are so common in CD added a new tool for diagnosing the disorder and also allowed my team and other researchers to assess the incidence of the disease in a new way – by screening people for the presence of this antibody in their blood. Before then, doctors had only nonspecific tests and thus the most reliable way to diagnose the disease was to review the patient’s symptoms, confirm the intestinal inflammation by taking a biopsy of the gut and assess whether a gluten-free diet relieved symptoms. (Screening for antibodies against gluten is not decisive, because they can also occur in people who do not have CD.)
It is also now clear that CD often manifests in a previously unappreciated spectrum of symptoms driven by local disruptions of nutrient absorption from the intestine. Disruption of iron absorption, for example, can cause anaemia and poor folate uptake can lead to a variety of neurological problems. By robbing the body of particular nutrients, CD can thus produce such symptoms as osteoporosis, joint pain, chronic fatigue, short stature, skin lesions, epilepsy, dementia, schizophrenia and seizure.
The gluten-immune dysfunction connection
CD provides an enormously valuable model for understanding autoimmune disorders because it is the only example where the addition or removal of a simple environmental component, gluten, can turn the disease process on and off. (Although environmental factors are suspected of playing a role in other autoimmune diseases, none have been positively identified.)
Gluten has a peculiar structure: it is unusually rich in the amino acids glutamine and proline. This property renders part of the molecule impervious to our protein-chopping machinery, leaving small protein fragments, or peptides, intact. Even so, in healthy people, most of these peptides are kept within the gastrointestinal tract and are simply excreted before the immune system even notices them. And any gluten that sneaks across the gastrointestinal lining is usually too minimal to excite a significant response from a normally functioning immune system.
CD patients, on the other hand, have inherited a mix of genes that contribute to a heightened immune sensitivity to gluten. For example, certain gene variants encoding proteins known as histocompatibility leukocyte antigens (HLAs) play a role. In those with CD, 95% possess either the DQ2 or the DQ8 HLA gene, whereas just 30-40% of the general population have one of those versions. This finding, and others, suggest that HLA DQ2 and DQ8 are not the sole cause of immune hyperactivity but that the disease, nonetheless, is nearly impossible to establish without one of them. The reason these HLAs are key becomes obvious from studies of the function of the proteins they specify.
The HLA DQ2 and DQ8 proteins are made by antigen-presenting cells. These immune sentinels gobble up foreign organisms and proteins, chop them, fit selected protein fragments into grooves on HLA molecules and display the resulting HLA-protein complexes on the cell surface for perusal by immune system cells called helper T lymphocytes. T cells that can recognise and bind to the displayed complexes then call in reinforcements.
In patients with CD, tissue transglutaminase released by intestinal epithelial cells attaches to undigested gluten and modifies the peptides in a way that enables them to bind extremely strongly to DQ2 and DQ8 proteins. In consequence, when antigen-presenting cells under intestinal epithelial cells take up the complexes of tissue transglutaminase and gluten, the cells join the gluten to the HLAs and dispatch them to the cell surface, where they activate T cells, inducing the T cells to release cytokines and chemokines (chemicals that stimulate further immune activity). These chemicals and enhancement of immune defences would be valuable in the face of a microbial attack, but in this instance they do no good and harm the intestinal cells responsible for absorbing nutrients.
CD patients also tend to have other genetic predispositions, such as a propensity for over- producing the immune stimulant IL-15 and for harbouring hyperactive immune cells that prime the immune system to attack the gut in response to gluten.
What role might antibodies to tissue transglutaminase play in this pathological response to gluten? The answer is still incomplete, but scientists have some idea of what could happen. When intestinal epithelial cells release tissue transglutaminase, B cells of the immune system ingest it — alone or complexed to gluten. They then release antibodies targeted to the enzyme. If the antibodies are home to tissue transglutaminase sitting on or near intestinal epithelial cells, the antibodies might damage the cells directly or elicit other destructive processes. But no one yet knows whether they in fact, cause such harm.
Bringing validity to the leaky gut theory
In the past nine years my colleagues and I have learned that unusual intestinal permeability also appears to participate in CD and other autoimmune diseases. Indeed, a growing body of evidence suggests that virtually the same trio of factors underpins most, and perhaps all, autoimmune diseases: an environmental substance that is presented to the body, a genetically based tendency of the immune system to overreact to the substance and an unusually permeable gut.
It is fair to say that the theory that leaky gut contributes to CD and autoimmunity in general was initially greeted with great skepticism, partly because of the way scientists thought of the intestines.
In the late 1980s I was working on a vaccine for cholera. At that time, the cholera toxin was believed to be the sole cause of the devastating diarrhoea characteristic of that infection. To test this hypothesis, my team deleted the gene encoding the cholera toxin from the bacterium Vibrio cholerae.
But when we administered our attenuated bacteria to volunteers, the vaccine provoked enough diarrhoea to bar its use. I felt completely disheartened. Years of hard work were literally down the toilet and we were faced with two unattractive options: giving up and moving on to another research project or persevering and trying to understand what went wrong. Some intuition that there was more to this story prompted us to choose the latter path, and this decision led us to discover a new toxin that caused diarrhoea by a previously undescribed mechanism. It changed the permeability of the small intestine by disassembling those supposedly inert tight junctions, an effect that allowed fluid to seep from tissues into the gut. This “grout” was interesting after all.
This is how we came to discover Zonulin. This discovery led us to propose that it is the enhanced intestinal permeability in CD patients that allows gluten, the environmental factor, to seep out of the gut and to interact freely with genetically sensitised elements of the immune system. That understanding, in turn, suggests that removing any one factor of the autoimmunity-causing trinity – the environmental trigger, the heightened immunity or the intestinal permeability – should be enough to stop the disease process.
Given the apparently shared underpinning of autoimmune disorders in general, researchers who investigate those conditions are eager to learn whether some therapeutic strategies for CD might also ease other autoimmune conditions that currently lack good treatments.
The Microbiome: The Future?
People with CD are born with a genetic susceptibility to it. So why do some individuals show no evidence of the disorder until late in life?
Microbes, collectively known as the microbiome, may differ from person to person and from one population to another, even varying in the same individual as life progresses. Apparently they can also influence which genes in their hosts are active at any given time. Hence, a person whose immune system has managed to tolerate gluten for many years might suddenly lose tolerance if the microbiome changes in a way that causes formerly quiet susceptibility genes to become active. If this idea is correct, CD and indeed, other auto-immune conditions might one day be prevented or treated by ingestion of selected helpful microbes or probiotics.
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