Friday, May 13, 2011

Hold the Carrots! Vitamin A May Exacerbate Celiac Disease Symptoms-New study reveals how retinoic acid and IL-15 cooperatively promote gut inflammation

Public Interest Note:
            Chances are either you or someone you know has to be very cautious about the types of food that is eaten.  Besides the familiar culprits of Salmonella spp. or E. coli-contaminated foods, millions of Americans must be particularly aware of the foods they consume to prevent violent immune reactions.  It is estimated that millions of Americans are allergic to certain foods1, 10% of the public have food intolerances2, and millions suffer from autoimmune diseases triggered by food (3 million alone have Celiac Disease3).  In the past few years, popular media outlets have increased their coverage regarding food allergies.  This trend is most likely in response to the 2010 study published in The Journal of the American Medical Association (JAMA) established that only 8% of children and 5% of adults actually suffer from food allergies4.  These data came as a shock to the public since it seemed that if anyone off the street could guess the incidence rate of food allergies in America, the majority of people would probably guess closer to 25-50%.  The issue of what an allergy is therefore appears to be a fabulous example of how wide the knowledge gap is between scientists and the public (and even some medical doctors).  Clearly, there is a lot of confusion out there about how the human body reacts to certain foods.  Part of that confusion is because we don’t fully understand the mechanisms that lead or cause such reactions to food.  Of particular interest to scientists is to determine what factors cause our immune system to react to food that should be harmless and why some people are more susceptible to these diseases than others.

Tolerating Gluten: The Immunology Behind The Disease
Celiac Disease is an AUTOIMMUNE DISORDER, which occurs when there is a break in oral tolerance and the immune system overreacts to gluten by producing highly inflammatory cytokines and inhibiting regulatory T cell function-which results in tissue destruction of the intestines. DePaolo, et al. show new data describing how retinoic acid exacerbates inflammation in the gut due to the high concentration of IL-15 in the guts of Celiac Disease Patients. Figure from
            Since many people are mistaken that if someone has a reaction to food it must be an allergy, let’s be clear: Celiac Disease IS NOT A FOOD ALLERGY.  To be classified as an allergy, individuals produce antibodies much like the ones that are made to fight infections, except food allergy-associated antibodies (IgE) are specific for certain ingredients in food (not pathogens).  Circulating IgE antibodies activate mast cells-which release lots of histamine and inflammatory mediators.  People with Celiac Disease do NOT produce IgE against gluten, but rather suffer from severe inflammation in their intestines in response to gluten that ultimately leads to an autoimmune reaction that destroys the small intestinal tissue.  Autoimmunity occurs when a person’s own body triggers an immune attack on itself.  At this point you might (and should) be thinking: Why would your body react so viciously to something as harmless as wheat?  Doesn’t your immune system know any better? 
The answer to these questions is multi-faceted and still rigorously evaluated by immunologists.  Although there are many factors that contribute to a spectrum of autoimmune disorders, the common theme is that they all cause harm because the body has lost the ability to tolerate itself.  Our bodies have evolved to utilize a number of mechanisms to prevent our own immune cells from leading a crusade against our own tissues.  Peripheral immune tolerance is the ability of our immune cells being able to become active, inflammatory reactors to pathogens while at the same time ignoring our own proteins or harmless foods we eat.  Tolerance can be achieved by deleting auto-reactive immune cells and possessing powerful regulatory cells to inhibit inflammation, both of which help keep our immune systems functioning properly.  So when there is a breach of tolerance, we have a problem.  That problem is that our bodies become deregulated and can’t distinguish threatening agents from harmless ones. 
Think about the last breakfast you ate.  Perhaps there was some fruit, toast, and eggs.  With each bite you take, you increase the number of foreign substances entering your body.  Therefore your guts are one of the most, if not the most, infiltrated organs housing thousands of bits of interlopers for brief moments at a time.  Each of these food particulates is seen by your body’s immune system as foreign, as a potential threat to our health, and yet most of us don’t hurl in pain every time we sit down for a meal. Our intestines are full of anti-inflammatory immune cells called T regulatory cells (Tregs).  They are related to the reactive, inflammatory-prone T cells discussed above except Tregs do not promote inflammation.  In fact, Tregs do quite the opposite.  Even when reactive T cells recognize a foreign food protein and begin to initiate an inflammatory response in our intestines (which happens every time we eat), the Tregs quiet those reactive T cells by producing anti-inflammatory molecules allowing your gut immune cells to essentially ignore the harmless food.  This complex regulated system is called oral tolerance.    For this reason, Tregs are essential to the maintenance of a healthy, tolerant gut.  It is pretty amazing that our bodies have evolved such sophisticated mechanisms to keep us healthy and balanced.  Unfortunately, this level of immunological sophistication fails some of us. 

Celiac Disease: Current Treatment Options & Why This Research Paper Matters
       Celiac Disease patients are genetically susceptible individuals who develop inflammatory T cell reactions in their gut in response to gluten.  Currently, the only treatment is to live a life completely void of gluten (found in wheat, barley), which is not always easy to do.  It has been recently shown that retinoic acid (RA) can increase the number of Tregs in the gut under homeostatic conditions.  The benefits of retinoic acid has been well acknowledged and widely regarded by doctors everywhere for its great anti-inflammatory ability.  RA and vitamin A is important for maintaining vision and enhancing skin health because of its anti-inflammatory properties (which is why they are often found in anti-aging crèmes and acne medications).  Because vitamin A is naturally abundant in many foods (sweet potatoes, carrots, melons), many doctors and scientists have speculated its use as a treatment for individuals suffering from inflammatory gut diseases such as Celiac Disease5.
Since this discovery, some researchers and doctors have speculated the use of RA as a possible treatment for the disease.  However, as DePaolo, et al. illustrates in their recent Nature paper, RA has a completely different function in a highly inflammatory gut like that seen in Celiac Disease patients and thereby warns against the use of RA as a treatment option for this autoimmune disease.

What the &*%$#! Does the Title Mean?!

1)             An adjuvant is something that boosts the inflammatory response, without an adjuvant T cells activity is too weak to cause much of an inflammatory effect.  (Think of anergy!  The reason why immune cells become anergic is because they are sub-optimally activated.  This happens because the signal they receive is very weak alone, unless that signal is coupled with another which together transforms the quiet immune cell to an active, powerful member of the immune system!) 
2)             IL-15 is a cytokine, a small molecule released by an immune cell called dendritic cells (DCs), which help activate T cells.  It has been previously demonstrated that individuals with Celiac Disease have a lot more IL-15 than healthy people in their gut6.  The reason for this remains unknown.
3)             An antigen is what the immune system recognizes which activates the inflammation.  Antigens are proteins that must be processed into smaller pieces called peptides by DCs (dendritic cells are special kind of immune cell that belongs to a very select group of cells appropriately named: Professional Antigen Presenting Cells). The processed antigen is then presented by the APC to activate T cells.

Compile this information together and we can infer that: IL-15 acts and RA act cooperatively to induce a powerful inflammatory immune response against antigens that you eat.  

Ready for an adventure? Read on for a guided-tour through the scientific data!
This study was thoroughly designed beginning with the first figure in the paper, which establishes a link between the high levels of IL-15 in the gut and the breach in oral tolerance in Celiac Disease patients.  To do this, the researchers isolated DCs and T cells from mice and mixed them together in a culture dish.  To this cell culture, they then added TGF-beta (an essential cytokine for Treg development).  When they add TGF-beta and RA, the number of Tregs doubles, so clearly RA can substantially promote the development of Tregs. The purpose of these first experiments is to serve as controls, which provides interesting data, but this information is already known and published previously7.  So, why is DePaolo and colleagues’ study published in Nature?  Because what they show next is what happens when IL-15 is added to the mix-something no one had demonstrated before.   Turns out that IL-15 dramatically reduces the number of Tregs in half, even in the presence of TGF-beta!  What was more amazing was when they added IL-15, TGF-beta and RA to the DC/T cell mixture.  The number of Tregs developed was even more dramatically reduced!  Clearly, RA contributes to two completely different outcomes depending on the existence of IL-15.  Merely making this connection leaves many questions still unanswered including: Can this effect happen outside of the culture dish and inside a living organism? What’s the mechanism of Tregs inhibition? and How is all of this connected to Celiac Disease?
Let’s begin with the first question: Does IL-15 act synergistically with RA to inhibit Tregs development in vivo?  To examine the relevance of their experiments with the DC/T cell culture, the scientists fed mice the protein, chicken ovalbumin (OVA), a commonly used and well-accepted model antigen to study oral tolerance in mice.   As we would expect, in a wild-type, healthy mouse, Tregs develop normally in the mesenteric lymph node (MLN-the lymphoid organ in the gut that houses the DCs and T cells).  But when they feed OVA to mice that were genetically engineered to overexpress IL-15 in the MLN, number of Tregs found is reduced in half compared to wild-type mice!
The experiment was then repeated by feeding mice with OVA in the presence of RA and again the mice that had lots of IL-15 present in their gut developed significantly fewer Tregs than the non-genetically manipulated mice.  So it seems that the scientist’s in vitro findings were vindicated in vivo, but they still haven’t demonstrated clearly how this is all happening.

It is known the inflammatory cytokines, IL-12 and IFNgamma, is very important in inhibiting Treg development.  It is also known that DCs can produce a lot of IL-12 in response to inflammatory stimuli8.  DePaolo, et al contributed new data to show IL-15 and RA can also induce IL-12 production from DCs.  They repeated their OVA-feeding experiments in mice that are unable to produce IL-12.  In these IL-12-deficient mice, IL-15 and RA do not affect Treg development in the gut!  Next, they assessed IL-12 levels in the MLN of mice fed OVA, substantially more IL-12 was found in their MLN of IL-15 overexpressing mice than wild-type mice and the amount of IL-12 is even more elevated in mice that were fed OVA and RA. This inflammatory effect can be inhibited when they block either RA or IL-12 signaling.  To construct a more complete signaling mechanism, they do a series of important molecular biology experiments to figure out which transcription factor is regulating IL-12 production from DCs in the presence of IL-15 and RA.  In three figures, the research group convincingly illustrates the mechanism in which DCs respond to IL-15 and RA by producing high levels of IL-12 (via JNK-mediated transcription), which is then secreted and acts on nearby T cells to inhibit their development into Tregs. 
Image depicting the role of MHC molecule, HLA-DQ2 (or 8) contributes to inflammation in the gut via the production of IFNgamma and TNFalpha, which when goes unregulated, leads to tissue destruction in the intestines. Image from
Whew!  This is a lot of data so far and we are almost at the end of the road-but there’s one more figure that really makes this paper outstanding.  The final set of experiments published depicts the novelty of this research.  These scientists, for the first time, develop a physiologically relevant mouse model to study Celiac Disease.  It is already understood that people can be genetically susceptible to developing Celiac Disease, if they express a certain major histocompatability complex (MHC) on their APCs.  The same cells responsible for processing foreign proteins, presents these peptides to T cells via MHC.  It has been known that the expression of MHC molecule HLA-DQ8 (or HLA-DQ2) is associated with celiac disease development because both molecules selectively bind gluten peptides tightly9 .  As with common genetic screening test, just because you express a certain molecule or gene, does not always result in disease development.  In fact, 40% of people express either celiac-disease-associated MHC, but only 2% of these individuals develop Celiac Disease10.  So clearly, HLA-DQ8/-DQ2 is not the only thing contributing to the onset of disease in these patients.  DePaolo and colleagues decided to breed their IL-15 overexpressing mice with mice that only express HLA-DQ8 to test the hypothesis that gluten is fed to mice that express HLA-DQ8, the DCs in the gut will process and present gluten peptides via HLA-DQ8 to activate T cells, but because these mice express lots of IL-15 in the gut too, the T cells will not develop into Tregs.  So if T cells get activated, but do not turn into regulatory cells-what happens?  When they did this experiment, they observed a quite remarkable effect: the gut T cells turned into inflammatory, IFNgamma-producing effector T cells in the mice that expressed both HLA-DQ8 and high IL-15.  The mice that only expressed HLA-DQ8 (low IL-15 levels) did not develop any inflammatory T cells.  This effect, consistent with their previous figures, was even more dramatically induced in the presence of RA.  Lastly, they correlate these findings with active Celiac Disease patients.
In conclusion, what this extraordinary research team provided us with a physiological-relevant mouse model to further study the mechanism of Celiac Disease development in mice, offered insight into the importance and mechanism of not only oral tolerance, but also how gut immunity may be affected in related gut inflammatory conditions such as inflammatory bowel disease.  Furthermore, this research highlights the important role of tissue environment in determining RA function.  Before this publication, everywhere people looked-skin, eyes, etc. RA and vitamin A were beneficial agents that promoted anti-inflammation.  However, in the gut-where IL-15 can reach high levels- RA has a completely different role as an adjuvant that promotes severe inflammation and effector T cell activation.  Finally and importantly, this collaborative research effort revealed a promising, new target (IL-15) for the treatment of a disease that has no cure. DePaolo RW, Abadie V, Tang F, Fehlner-Peach H, Hall JA, Wang W, Marietta EV, Kasarda DD, Waldmann TA, Murray JA, Semrad C, Kupfer SS, Belkaid Y, Guandalini S, & Jabri B (2011). Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens. Nature, 471 (7337), 220-4 PMID: 21307853

References and Further Reading:
1: “Alergy Facts and Figures”. Asthma and Allergy Foundation of America.
3: Adams, Jefferson. “Is Celiac Disease America’s Most Under-Diagnosed Health Problem? Celiac Disease and Gluten-free Diet Information Since 1995. (2009).
4: Schneider Chafen, Jennifer J., et al. “Diagnosing and Managing Common Food Allergies: A Systematic Review”. The Journal of the American Medical Association. 303(18): 1848-1856. 2010.
5: Mora, J. Rodrigo, et al. “Vitamin effects on the immune system: vitamins A and D take center stage”. Nature Reviews Immunology.  8, 685-698 (2008).
7: Mucida, D., et al. “Reciprocal Th17 and Regulatory T cell Differentiation Mediated by Retinoic Acid. Science. 317:5855, 256-260. (2007)
8: “Caretto, D., et al. “Cutting edge: the Th1 response inhibits the generation of peripheral regulatory T cells.” J. Immunology. 184, 30-34 (2010.)
9: Lundin, KEA. “HLA-DQ8 as an Ir gene in celiac disease”.Gut. 52(1):7-8 (2003).


  1. This was very helpful; thank you!

  2. Thanks Shardule for reading Escaping Anergy! If you don't mind, could you tell me what was particularly helpful in this post? Always looking for ways to improve the posts, so if there was something explained particularly well or if it's just the topic of diet or celiac disease that was interesting, I'd love to know! Thanks again and please continue to check out Escaping Anergy for more posts about how your diet influences your immune system (and other amazing immunology discoveries!!)