Aguilera-Lizarraga J, Florens MV, Viola MF, et al. Local immune response to food antigens
drives meal-induced abdominal pain. Nature. 2021;590(7844):151-156.
To determine if specific food antigens cause visceral hypersensitivity in irritable bowel syndrome (IBS) patients. Also, to determine correlation between stool bacterial superantigens (SAGS) and IBS symptom severity.
This was a double-blind, placebo-controlled trial of participants with an IBS diagnosis.
This publication included 2 rodent studies that established the likelihood of an infectious influence on immunoglobulin E (IgE) and mast cell–mediated reactions of the viscera. The details of these experiments can be found in the original publication.
The human trial had 4 tiers. All participants were negative for IgE food allergies. All healthy volunteers were without abdominal symptoms, had no gastrointestinal disease or surgery, and were not on gastrointestinal medication. All participants with IBS met Rome III criteria for IBS.
Tier 1—Food antigen injection:
Eight healthy volunteers (8 females, aged 32–54 years) compared to 12 IBS patients meeting the ROME III criteria (8 females, aged 21–39 years).
Tier 2—Trypsin-like activity:
Thirteen healthy volunteers (10 females, aged 23–47 years) compared to 48 IBS patients meeting ROME III criteria (33 females, aged 19–40 years).
Tier 3—Bacterial identification in fecal samples:
Sixty-four healthy volunteers (35 females, aged 32–58 years) compared to 84 IBS patients (66 females, aged 25–50 years).
Tier 4—Biopsy collection for immunofluorescence staining:
Fifteen healthy volunteers (13 females, aged 32–52 years) compared to 22 IBS patients (20 females, aged 27–43 years).
Tier 1: IBS patients and healthy volunteers underwent sigmoidoscopy. During the procedure, food antigens (soy, wheat, gluten, and milk) were placed in the rectal mucosa. Investigators compared the reaction to sodium chloride (NaCl) as a negative control and histamine as a positive control. Sigmoidoscopy was video-recorded, and images of the injection sites were taken prior to injection, immediately after injection, and 12 minutes after injection.
Mucosal edema diameter was measured 12 minutes after injection.
The 95th percentile of the diameter reactions of the negative control (saline) was used as a threshold for determining a positive reaction.
Tier 2: Subjects also underwent biopsy for trypsin-like activity as a surrogate measure of mast cell degranulation and subsequent tryptase activity measurement.
Tier 3: In addition, stool collection was performed using quantitative polymerase chain reaction (qPCR) to identify presence of superantigens. Staphylococcus aureus samples were used as positive controls.
Tier 4: Lastly, subjects had immunofluorescence staining performed after biopsy to determine mast cell volume and the distance of mast cells from nerve fibers.
Primary Outcome Measures
- The presence or absence of colonic mucosal reactions to either soy, wheat, dairy, or gluten in IBS patients vs healthy volunteers (HVs).
- Trypsin-like activity and tryptase activity, which are markers of mast cell activation in IBS patients vs HVs.
- Total levels of IgE+ mast cells in colonic mucosa of IBS patients vs HVs.
- The presence of superantigens (SAGs) in IBS patients compared to HVs.
- Mast cell intensity in IBS patients vs HVs as detected by immunofluorescence.
- All 12 IBS patients showed mucosal reactions to at least 1 of the food antigens tested, whereas only 2 HVs showed single positive reactions to soy and gluten, respectively.
- Important to note is that the immune reaction was local and the local tissue had loss of tolerance vs systemic.
- At baselines, total trypsin-like activity was higher in IBS patients compared to HVs, and tryptase activity was higher after injection of histamine, soy, wheat, and gluten.
- The distance between IgE positive mast cells and nerve fibers was smaller in IBS patients compared to HVs and inversely correlated with the abdominal pain, demonstrating a possible explanation of the mast cell cause of visceral hypersensitivity in IBS.
- 23% of the fecal samples from IBS patients tested positive for S aureus (considered a superantigen) compared to 9% in HVs; 47% of the IBS samples were positive for 1 or more superantigens compared to 17% in HVs.
- Mast cell IgE immunofluorescent intensity was higher in IBS patients and positively correlated with both abdominal pain severity and the mucosal-edema diameter.
Irritable bowel syndrome (IBS) has traditionally been a difficult to manage heterogeneous disorder. Successful IBS care requires patience from all involved and a diligent and comprehensive approach to investigation and treatment—a luxury afforded to few. To date, the role of dietary triggers in IBS has lacked specificity, instead defaulting to generalized dietary recommendations. Understanding how to isolate and individualize dietary impact for those with IBS would certainly lead to improved outcomes, and more satisfied clinicians and patients alike. The recent study by Aguilera-Lizarraga et al on abdominal pain in IBS informs our understanding of how food allergies may exacerbate symptoms of IBS.
Functional gastrointestinal disorders (FGIDs), including IBS, share a common pathophysiology involving visceral hypersensitivity (VH), abnormal gastrointestinal motility, and gut-brain interactions. More recently research has identified low-grade intestinal inflammation, increased intestinal permeability, immune activation, and disturbances in the microbiome (such as small intestinal bacterial overgrowth) as backbone dysfunctions in FGIDs.1
What distinguishes IBS from other FGIDs are the frequency and timing of abdominal pain. According to the new Rome IV guidelines, abdominal pain at least once per week just before, during, or shortly after a bowel movement is the diagnostic criteria that distinguishes IBS.1 Abdominal pain is 1 of the most debilitating IBS features, and treatments have been developed to address this pain including neuromodulators, acid blockers, low-dose antidepressants, and antispasmodics.2
IBS-related abdominal pain appears to be partly mediated by transient receptor potential vanilloid 1 (TRPV1) channels, according to animal models, though other receptors such as Taste Receptor type 1 (T1R), Taste Receptor type 2 (T2R), G-coupled taste receptors, glutamate-sensing receptors, and acid sensors have been also mentioned in studies. TRPV1 cells are of particular interest related to mast cells.3 In the digestive tract, mast cell populations are in close proximity to gastrointestinal mucosa sensory nerve fibers containing neuropeptides, including visceral afferents expressing TRPV1 receptors. This close spatial association, when coupled with mast cell activation, has been suggested to alter pain perception in response to triggers in FGIDs.4 Of note, TRPV1 binds capsaicin and is responsible for visceral hypersensitivity associated with spicy foods in FGIDs.
Mast cells have an intriguing role in visceral sensitivity. When activated, mast cells release histamine, serotonin, prostaglandins, cytokines, neuronal growth factor, and proteases (ie, tryptase). According to murine models of IBS, histamine, serotonin, and prostaglandins bind with receptors on the afferent neurons and lead to neuronal excitability resulting in visceral hypersensitivity. This binding subsequently triggers to the nerve cells to release more mediators that sprout nerves, recruit fibers to the location, and intensify nerve signaling.4
Most clinicians caring for an IBS patient are faced with the question of “Where do I start?” Address the microbiota/dysbiosis? Or the intestinal permeability? The central nervous system? Or diet? These questions are overwhelming to both clinician and patient alike.
Undoubtedly any clinician will be approached with the question “What about the dietary role in IBS?” While certain dietary approaches have been used with some success to control IBS, such as the low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (low-FODMAP) diet, simple carbohydrate diet (SCD diet), gluten-free/dairy-free diets, and elimination diets, the specificity to the role of food in IBS is lacking.
Food allergies exist in 6% to 8% of children and 1% to 4% of adults, making the detection of true allergies (ie, IgE-mediated) in IBS patients rare.5 Attempts to identify food allergies in IBS patients have not been fruitful. There has been little to no consistency between symptoms and foods being reported on positive skin prick tests or radioallergosorbent tests (RASTs).6
However, many IBS patients feel better when they fast, and many report that certain foods aggravate symptoms. These reactions have been called an atypical food allergy or food intolerance, but classic food allergies in IBS have been disputed.
Atypical food allergy involves both the innate and acquired immune system. However, the reaction is usually delayed. It involves IgE, T lymphocytes, eosinophils, mast cells, and other mucosal cells. The symptoms caused by this reaction can develop up to days after ingestion of the offending food.5 The questions that persist are how do these atypical allergies develop? And, how do we identify them?
The Aguilera-Lizarraga et al paper investigated the mechanism of food-induced abdominal pain in IBS. The authors go into depth to describe the break of tolerance of foods. The first part of their study was preclinical, looking at the events related to infecting rodents with Citrobacter rodentium superantigens simultaneously with food antigen consisting of egg-white albumin (OVA). The comparison/control group of rodents were infused with albumin only. Previous OVA-tolerant mice who were exposed to OVA+SAGs became intolerant to OVA. Yet rodents exposed only to OVA did not develop intolerance. OVA-specific IgE antibodies were detected in the colon of OVA-infected rodents, the site colonized by C rodentium, but not in the small intestine or serum.
This reaction aligns with the superantigen hypothesis of allergy. Superantigens are exotoxins produced by bacteria in order to evade the immune response of the host. SAGs shortcut normal antigen presentation leading to excessive release of inflammatory cytokines and excessive T-cell activation, expansion, and anergy, as well as B-cell activation. Two of the most common superantigens are from Staphylococcus aureus and Streptococcus pyogenes.7
In this setting, a food protein (or friendly microbiota) at the “scene of the crime” can be targeted by the immune system. A victim of friendly fire so to speak. The immune system may now see that molecular pattern of a previously beneficial element as danger. In the Aguilera-Lizzaraga study this involved OVA hanging around the gut at the wrong time (a C rodentium infection).
Aguilera-Lizarraga et al explain that SAGs trigger T helper 2 (TH2) activation and the upregulation of trypsin (Tpsab1) genes, and genes for inflammatory cytokines interleukin 10 (IL-10), IL-4, and IL-6. Trypsin is a known inducer of tryptase. Tryptase is an enzyme that is released, along with histamine and other chemicals, from mast cells when they are activated as well as in allergic (hypersensitivity) responses.
Aguilera-Lizarraga et al also demonstrated in mice that postinfection exposure to OVA leads to visceral hypersensitivity in the OVA+SAG group. Hence, the model of abdominal pain was substantiated. They proposed that this IgE reaction led to mast cell activation and that the release of histamine and binding of histamine to H1 receptors lead to heightened nervous system signaling.
The role of mast cells has previously been substantiated in IBS patients. Supernatants of mucosal biopsies of patients with IBS contain increased concentrations of histamine, serotonin, trypsin, tryptase, prostaglandin E2, other proteases, and cytokines. In addition, jejunal luminal tryptase release has been shown to be 5 times higher and the expression of both tryptase mRNA and protein enhanced in jejunal tissue in IBS-D, while serum tryptase remained unaltered—again showing local-tissue-level involvement rather than a systemic basis.4
IBS should be considered part immune dysregulation, part micropathology, part microbiota imbalance, part food reactivity, and part gut-brain interaction. Management requires multiple tools.
In the Aguilera-Lizarraga et al study, they proceeded to demonstrate histamine-like reactions to common food antigens in human IBS patients. By injecting antigens of dairy, soy, wheat, and gluten into the colorectal mucosa of IBS patients compared to healthy volunteers, they observed that all 12 of the IBS patients reacted to at least 1 of the antigens, whereas only 2 healthy volunteers reacted to these antigens. Of note, there were similar findings in a study done in 1997 by Bischoff and colleagues8 using a process called colonoscopic allergen provocation (COLAP).
Through stool studies, Aguilera-Lizarraga et al showed that the presences of superantigens (SAGs) was much higher in those with IBS than in healthy volunteers (47% vs 17% respectively). Of interest, 23% of the fecal samples from IBS patients tested positive for S aureus superantigens, compared to 9% in the healthy volunteer group. This implies that superantigens may be involved as a driver in IBS.
Finally, tissue studies in IBS patients compared to healthy volunteers demonstrated significantly increased mast cell activity, increased volume of IgE positive mast cells, and increased proximity of mast cells to afferent nerve cells—meaning mast cell activation is likely an underpinning and the proximity of these mast cells to nerve fibers may be responsible for increased visceral hypersensitivity.
While Aguilera-Lizarraga et al emphasized the colon involvement, a recent study9 used confocal laser endomicroscopy (CLE) to look at the tissue and immune response to different foods in the small intestine of patients with diagnosed IBS. The study clearly showed intolerances and intestinal leakages in IBS patients challenged with wheat, dairy, yeast, and soy compared to healthy volunteers. The study showed disrupted intestinal lining (claudin 2 elevation and epithelial layer leaks) and immune actuation (secreted eosinophilic cationic protein and the histamine marker tryptase). This study was novel in that it captured this immune activity through video.
Both of these studies demonstrate that IBS reactions are happening on a local tissue level and are less likely to be detectable on standard blood tests of any kind. In addition, the studies highlight that immunotherapy and immune modulation may be a key therapeutic strategy for IBS patients. In this regard, Aguilera-Lizarraga et al discuss the use of IgE-inhibiting interventions such as omalizumab, which is a monoclonal antibody; or using spleen tyrosine kinase inhibitors to block mast cell degranulation. Already, mast cell stabilizers are used in IBS such as ketotifen and cromolyn sodium.
For the clinician, this study encourages the exploration of food and microbiota as drivers of IBS symptomology. Quantitative PCR testing of stool is still a relatively new tool available commercially that may identify possible pathobionts that may be involved. Any exposure of the gastrointestinal system to superantigens and possibly mast cell activation through the release of lipopolysaccharide (LPS) may trigger local immune activation and ensuing symptoms in those with IBS. In addition, lactulose hydrogen breath testing can identify patients with SIBO. Microbiota modulation has been shown to help IBS patients especially with issues of stool quality and bloating, making microbiome health a cornerstone of investigation and treatment.10
Food allergy identification, however, still poses tremendous reproducibility and reliability challenges. Until tissue-related studies become validated and routine, we are limited to food trial diets and blood tests. Commercial food reaction testing (eg, IgG, Elisa, Alcat) has been consistently questioned on reliability, utility, and reproducibility.11 IgG panels report several subclasses with 1 being IgG4. The presence of IgG4 is considered a neutralizing antibody that develops in the presences of an IgE. The finding is equivocal, meaning it often indicates tolerance but also can mean ongoing reactivity. So, it is advised that IgE/IgG4 be checked in parallel as IgG4 tends to rise with an anaphylactic IgE response. Of note, one study by Zar et al demonstrated IgG4 directed therapy in IBS patients resulted in improved symptoms.12 Another study by Guo et al13 showed that 12 weeks of an IgG-directed intervention resulted in significantly improved symptoms in 72 IBS-D patients. In Guo’s study, however, there remains a question as to whether the improvement was based on subjects just eating better or due to the specific food elimination.
Recently, measuring the complement subtype (C3d) along with IgG4 levels has been made available commercially. C3d/immunoglobulin IgG measures both the innate and adaptive responses of the immune system. If C3d antigen is elevated as it pertains to a certain food, there is a likelihood that the innate immune system is activated to exposures to the food antigen. This biomarker should be further evaluated and validated. It is quite intriguing as C3d is known as a driver for mast cell activation, and more studies are eagerly awaited.14
Other intriguing tests are the lymphocyte response assay (LRA), the Antigen Leukocyte Cellular Antibody Test (ALCAT), measuring leukocyte activation, and the similar chemical mediator release testing. The latter 2 measure various mediators related to mast cell release that may be provided by a food antigen. This appears highly relevant to the scenario in the IBS pain patient. Our late and cherished colleague Ather Ali, ND, and his group at Yale studied leukocyte activation. Ali’s group conducted a parallel-group, double-blind, randomized, controlled trial of 58 adults with IBS and measured food reactions using the leukocyte activation test.15 Subjects followed an elimination diet based on the test result for some time. The intervention group had significantly greater increases in mean global improvement score after 4 weeks. The intervention group also had significantly greater reductions in the IBS Symptom Severity Scale at 4 weeks and 8 weeks. There were no significant differences between intervention and comparison groups in mean adverse reactions or quality-of-life scores.
LRA testing looks at delayed TYPE III and TYPE IV allergic reactions along with IgG, IgM, and IgA reactions, perhaps being the most complete look at the atypical food allergy scenario. To date, no studies have been published using LRA to identify food-related triggers in IBS.
Testing can be expensive and impractical in a clinical setting, leaving clinicians no recourse but to implement subjective therapeutic diet interventions. The low FODMAP diet removes an extensive variety of foods, and has been reported to improve symptoms of IBS. A study of 20 patients with diarrhea-predominant IBS (IBS-D) or IBS-M reported that the low FODMAP diet decreased serum levels of proinflammatory IL-6 and IL-8. But the downside is that without FODMAP foods, there may be a reduction in diversification of the gut microbiota. In the same study, there was a decrease in levels of fecal bacteria (Actinobacteria, Bifidobacterium, and Faecalibacterium prausnitzii) and total short chain fatty acids (including n-butyric acid) compared with baseline. Low FODMAP diets appear to reduce symptoms for many people with IBS, but the long-term effects of simplifying the flora is a concern.16 Recently low-FODMAP diets have included a reintroduction phase of the diet in an attempt to remedy this concern and allow patients to resume a more normalized diet long term.17
The gold standard continues to be a strict elimination diet, but compliance is limited to a select few patients who have the time, resources, and the determination required to complete this inconvenient intervention. Some providers, including myself, often do a modified version of an elimination diet with a smaller list (ie, wheat, yeast, dairy, and soy) as a starting point.
Regardless of which food interventions are pursued as part of a treatment plan, Aguilera-Lizarraga et al substantiated that approaches to IBS must include mast-cell-stabilizing strategies. Many classic naturopathic treatments (eg, quercetin) have been employed routinely in supporting those with IBS, but more sophisticated immune-modulating herbs like Scutellaria baicalensis should be considered as well.18 This study also suggests that knowing the contributions of the patient’s atypical food intolerances and possible microbiota (SAGs) is likely key to understanding one’s unique needs regarding treatment.
IBS does not typically exist as a singular entity in those suffering from it, although most studies do not include symptoms outside of the ROME criteria. Most often, IBS is a long-lasting condition and there are concomitant conditions in a clinical setting. Perhaps simple approaches for the recently diagnosed IBS patient may be successful, but many long-term sufferers will require additional considerations and complex strategies to find relief.
IBS should be considered part immune dysregulation, part micropathology, part microbiota imbalance, part food reactivity, and part gut-brain interaction. Management requires multiple tools. Addressing food allergy and histaminic reactions, while also assessing and addressing gut pathobionts, should yield some clinical benefit in a certain percentage of IBS patients.