Introduction

Functional dyspepsia (FD) is a disorder of gut-brain interaction (DGBI) characterised by relapsing and remitting gastro-duodenal symptoms, in the absence of identifiable pathology via routine clinical screening. An association between FD and wheat-containing foods has been reported in observational studies, and recent work demonstrates increased gut-homing T cells in peripheral blood of FD patients reporting sensitivity to wheat, compared to those without wheat sensitivity and controls. We have also recently shown increased populations of mucosal lymphocytes with a T helper (Th)2- and Th17-like phenotype in FD patients compared to controls, however, a mucosal adaptive lymphocyte response specific to wheat has not been demonstrated.

Aims & Methods

We aimed to examine whether proteins present in wheat could provoke a response from FD duodenal lymphocytes isolated from mucosal biopsies. Lamina propria mononuclear cells (LPMCs) were isolated from duodenal biopsies from 50 FD patients (38 female, 46.8±16.8 years) and 23 controls (11 female, 52.1±13.1 years). LPMCs were exposed to gluten (0.2mg/mL) or gliadin (0.2mg/mL) for 24 hours. Flow cytometry was performed to phenotype lymphocytes using a surface marker panel. Quantitative polymerase chain reaction (qPCR) was used to measure expression of the gliadin-associated T-cell receptor alpha variant (TRAV)26-2.

Results

In response to gliadin (but not gluten) stimulation, the effector Th2-like population was increased in FD LPMCs compared to controls (16.34 ± 13.17 vs 25.83 ± 14.87, p = 0.01) and unstimulated FD LPMCs (19.40 ± 14.35 vs 25.83 ± 14.87, p = 0.04). Duodenal gene expression of TRAV26-2 was decreased in FD compared to controls (0.013 ± 0.015 vs 0.003 ± 0.005, p = 0.04). We identified a positive association between gene expression of this T cell receptor variant and LPMC effector Th17-like cell populations in FD patients (rs = 0.3733, p = 0.04), but not controls (rs = 0.2238, p = 0.485) following exposure to gluten, but not gliadin.

Conclusion

Our findings in this observational study suggest differential T cell responses to gluten and gliadin in a subset of FD patients, and suggest Th2 responses previously reported in FD may occur in response to food components such gliadin. We also show that FD patients have reduced expression of TRAV26-2, previously associated with T cell responses against gliadin in celiac disease. TRAV26-2 expression correlated with Th17-like populations in FD patients, suggesting that specific TCR variants in may be associated with an immune response to the consumption of wheat-containing foods in FD.

Introduction

Functional dyspepsia (FD) is a chronic gastrointestinal disorder characterised by meal-related symptoms, which a subset of patients attribute to the consumption of gluten-containing foods [1]. Recent evidence suggests that subtle duodenal immune activation is a feature of FD, as evidenced by elevated eosinophils [2] and impaired epithelial barrier function [3]. FD patients also exhibit an altered duodenal mucosa-associated microbiota (d-MAM) when compared to controls [4]. Given the microbiome’s involvement in the digestion of gluten, we hypothesised that the d-MAM determines the capacity for gluten to disrupt the epithelium and drive immune responses in FD patients.

Aims & Methods

Duodenal biopsies were collected from 10 FD patients and 7 asymptomatic controls. The d-MAM was cultured from biopsies in a habitat simulating media [5] containing 1% wheat-gluten, for up to 48hrs. Microbial cell pellets were frozen for future microbiome sequencing and the cell-free supernatants, containing d-MAM-digested gluten, were collected. Caco2 cells were challenged with the d-MAM-digested gluten, in a Transwell model of the intestinal epithelium (n=3, 2 technical replicates). Transepithelial electrical resistance (TEER) and protein expression of markers of epithelial integrity were measured in Caco2 cells and compared between treatments. Peripheral blood mononuclear cells (PBMCs) from FD patients (n=20) and controls (n=20) were stimulated with pooled samples of FD or control d-MAM-digested gluten, or left untreated, for 44 hours. For comparison to host enzymatic breakdown of gluten, matched PBMCs were also treated with α-chymotrypsin digested, deaminated gluten (CT gluten). The T cell phenotype and presence of activation-induced markers was assessed in PBMCs by flow cytometry.

Results

Challenge of Caco2 cells with gluten digested by the FD d-MAM resulted in a significant increase in Zonula occludens-1 (p=0.007), and reduction in Myd88 (p=0.03), compared to Caco2 cells challenged with control d-MAM-digested gluten. However, TEER readings were not altered between treatment groups in Caco2 experiments. In PBMC experiments, in response to stimulation with FD or control d-MAM-digested gluten, both FD and control PBMCs exhibited heightened expression of activation markers (CD4⁺OX40⁺CD25⁺), when compared to their respective unstimulated cells (p<0.05). When assessing the T-cell phenotype we observed that FD d-MAM-digested gluten promoted an increase in T-helper (Th) 2 cells in both FD (p=0.007) and control (p=0.0001) PBMCs. Meanwhile, control d-MAM-digested gluten stimulation resulted in elevated Th17.1 cells in FD (p<0.0001) and control (p=0.02) PBMCs, when compared to unstimulated cells. Alternatively, CT gluten stimulation did not alter any of the assessed lymphocyte populations in FD or control PBMCs when compared to unstimulated cells, suggesting host digestion of gluten isn’t sufficient to induce immune responses in FD.

Conclusion

Our findings suggest that the d-MAM of FD patients digest gluten in a way that promotes epithelial barrier remodelling and altered immune responses compared to the d-MAM from asymptomatic individuals. The promotion of a Th2 T-cell phenotype by the FD d-MAM is consistent with reports of increased Th2-associated cells, such as eosinophils, in FD patients [6]. These results highlight the likely role of microbiome-gluten interactions in FD, indicating the potential for novel dietary therapies and microbial approaches to management.

References

1. Potter, M., et al., Incidence and prevalence of self-reported non-coeliac wheat sensitivity and gluten avoidance in Australia. Med J Aust, 2020. 212(3): p. 126-131.
2. Shah, A., et al., Duodenal Eosinophils and Mast Cells in Functional Dyspepsia: A Systematic Review and Meta-Analysis of Case-Control Studies. Clinical Gastroenterology and Hepatology, 2022. 20(10): p. 2229-2242.e29.
3. Puthanmadhom Narayanan, S., et al., Duodenal Mucosal Barrier in Functional Dyspepsia. Clin Gastroenterol Hepatol, 2022. 20(5): p. 1019-1028.e3.
4. Shanahan, E.R., et al., Alterations to the duodenal microbiota are linked to gastric emptying and symptoms in functional dyspepsia. Gut, 2023. 72(5): p. 929-938.
5. Schooth, L., Defining the Role of the Mucosa-Associated Microbiota in Digestive Health and Disease Using Novel ex-vivo Combinations of Microbe Culture with Metagenomics. 2024 [Doctoral thesis, The University of Queensland], UQ eSpace.
6. Burns, G.L., et al., Type 2 and type 17 effector cells are increased in the duodenal mucosa but not peripheral blood of patients with functional dyspepsia. Front Immunol, 2022. 13: p. 1051632.

Disclosure

JP, EH, CN, SS, SR, MS, KH, GB, and KD have no disclosures to report.
NT: Financial support from: BluMaiden (microbiome advisory board) (2021), Comvita Mānuka Honey (2021) (digestive health), Biocodex (functional dyspepsia tool), Microba (microbiome advisory board) outside the submitted work. In addition, Dr. Talley has a patent Nepean Dyspepsia Index (NDI) 1998, Biomarkers of IBS licensed, a patent Licensing Questionnaires Talley Bowel Disease Questionnaire licensed to Mayo/Talley, a patent issued, “Diagnostic marker for functional gastrointestinal disorders” Australian Patent Application WO2022256861A1via the University of Newcastle and UniQuest (University of Queensland) and ”Methods and compositions for treating age-related neurodegenerative disease associated with dysbiosis” US Application No. 63/537,725. Dr. Talley is supported by funding from the National Health and Medical Research Council (NHMRC) to the Centre for Research Excellence in Digestive Health and he holds an NHMRC Investigator grant.
SK: Grants from National Health and Medical Research Council (Ideas Grant and Centre for Research Excellence), grants from Viscera Labs (Research contract), grants from Microba Life Science (Research contract), personal fees from Gossamer Bio, personal fees from Anatara Lifescience, personal fees from Immuron, personal fees from Microba Life Science.

Introduction

Disruptions in tight junction (TJ) protein expression leading to duodenal epithelial barrier impairment may contribute to increased intestinal permeability, potentially playing a role in functional dyspepsia (FD) pathophysiology. Currently published studies evaluated the role of several TJ proteins in FD patients with inconsistent results. Therefore, we conducted this systematic review and meta-analysis to evaluate the duodenal mucosal expression of several TJ proteins in FD.

Aims & Methods

We performed a systematic electronic search on PubMed, EMBASE, and Scopus using predefined keywords. Diagnosis of FD by ROME III or ROME IV criteria was considered acceptable. Full articles satisfying our inclusion and exclusion criteria were included. The principal summary outcome was the mean difference (MD) of several TJ proteins in FD patients and control subjects. The study aimed to find if patients diagnosed with functional dyspepsia have lower gene expression of intestinal TJ proteins compared to healthy subjects.

Results

A total of 8 and 5 studies were included in our qualitative and quantitative synthesis, respectively, with a total population of 666 participants, out of which 420 were FD patients. No significant differences were observed between FD patients and controls in the expression of claudin-1 (-0.102 [CI 95% -0.303,0.099]), claudin-2 (0.161 [CI 95% -0.134,0.456)], claudin-3 (0.278 (CI 95% -0.280,0.837]), claudin-4 (0.045 [CI 95% -0.264,0.354]), ZO-1 (-0.221[CI 95% -0.683, 0.241]), ZO-2 (-0.070 [CI 95% -0.147,0.007]), ZO-3(-0.129 [CI 95% -0.376, 0.118]), B-catenin (-0.135 [CI 95% -0.484,0.214]), E-cadherin (-0.083 [CI 95% -0.229,0.063]), and occludin (-0.158 [CI 95% -0.409, 0.093]).

Conclusion

The expressions of all evaluated proteins including claudin-1, claudin-2, claudin-3, claudin-4, ZO-1, ZO-2, ZO-3, B-catenin, E-cadherin, and occludin did not significantly differ between FD patients and controls. However, due to the limited number of included studies, results should be interpreted with caution.

Introduction

Functional Dyspepsia (FD), a common disorder of gut-brain interaction [1], is associated with alterations in gastric accommodation, gastric emptying, gastric sensitivity, gastroduodenal permeability, and the gut microbiome. Resilience, the ability to maintain function under personal or physical stress, may play a role in the development of FD, although no studies have evaluated this theory. We hypothesize that FD patients have lower resilience than the general population, which may lead to a new therapeutic target.

Aims & Methods

This prospective questionnaire study compared patients with FD (Rome IV criteria) with internal medicine clinic patients without dyspepsia at a tertiary referral center. Participants completed the FD symptom severity score [2], Brief Resilience Scale (BRS) [3], Connor-Davidson Resilience Scale (CDRS) [4], hospital anxiety and depression scale (HADS) [5], early childhood adverse life event questionnaire [6], and perceived stress score [7]. Questionnaires were distributed to clinic patients meeting inclusion/exclusion criteria.

Results

38 FD (mean age 43 years old, 87% female) and 31 control (mean age 57 years old, 39% female) patients completed the survey. FD patients exhibited lower resilience on the CDRS (96.83 vs. 90.26, P<0.04, CI -12.93 to -0.21) but not on the BRS (3.018 vs. 3.022, P<0.96, CI -0.15 to 0.13). FD patients and controls scored no different on the HADS anxiety index (17.05 vs. 18.38, P<0.064, CI -2.74 to 0.08) and depression scores (P<0.14, CI -0.21 to 1.5). The FD group reported "high" perceived psychological stress while the control group reported a “moderate” level (P<0.004, CI 2.002 to 9.827). In childhood, FD patients had significantly more adverse childhood events (29.3% vs. 15%, P<0.004, CI 0.05 to 0.25). FD patients were more likely to have been exposed to a household member suffering from a psychiatric illness compared to control patients (47% vs. 20%, P<0.049, CI 0.001 to 0.5). FD patients were also more likely to report psychological abuse in childhood (53% vs. 17%, P<0.03, CI 0.034 to 0.67). No other significant differences were found in types of early childhood trauma including physical abuse, familial substance abuse, witnessed maternal violence, or criminal behavior by a household member.

Conclusion

This novel, prospective questionnaire pilot study shows that FD patients have lower resilience compared to the general outpatient population on the CDRS. The long-term symptoms of FD may further decrease resilience. While anxiety and psychological stress are not strongly associated with FD, the significant exposure to childhood household members with a psychiatric diagnosis and significantly higher prevalence of self reported psychological abuse may explain the higher prevalence of antidepressant use in this population, especially considering the significantly higher level of self perceived psychological stress [8]. Otherwise, early childhood trauma appears similar between FD patients and patients without FD and is thus not likely to influence resilience markers. Understanding the contribution of resilience to development of FD may lead to therapies enhancing resilience, improving symptoms, and patient well-being.

References

1. El-Serag HB, Talley NJ. Systemic review: The prevalence and clinical course of functional dyspepsia. Aliment Pharmacol Ther. 2004;19:643-654.
2. Taylor F, Higgins S, Carson RT, et al. Development of a symptom-focused patient-reported outcome measure for functional dyspepsia: the functional dyspepsia symptom diary (FDSD). Am J Gastroenterol. 2018 Jan,113(1):39-48.
3. Smith BW, Dalen J, Wiggins K, et al. The brief resilience scale: assessing the ability to bounce back. Int J Behav Med. 2008;15(3):194-200
4. Connor KM & Davidson JR. 2003. Development of a new resilience scale: The Connor-Davidson Resilience Scale (CD-RISC). Depress. Anxiety, 18; 76-82
5. Zigmond AS & Snaith RP. 1983. The hospital anxiety and depression scale. Acta Psychiatrica Scandinavica, 67: 361-370
6. Felitti VJ, Andra RF, Nordenburg D, et al. Relationship of childhood abuse and household dysfunction to many of the causes of death in adults. The adverse childhood experiences (ACE) study. Am J Prev Med. 1998 May;14(4):245-58
7. Cohen S, Kamarck T, Mermelstein R. A global measure of perceived stress. J Health Soc Behav. 1983 Dec;24(4):385-96.
8. Esterita T, Dewi S, Suryatenggara FG, Glenardi G, et al. Association of Functional Dyspepsia with Depression and Anxiety: A Systematic Review. J Gastrointestin Liver Dis. 2021 Jun 18;30(2):259-266.

Introduction

The etiology of gastroesophageal reflux disease (GERD) is unknown but reflux-induced esophageal injury as a result of cytokine-mediated inflammation has been implicated (1). There is emerging evidence duodenal immune activation may be associated with an increased risk of GERD (2). Celiac disease is strongly associated with GERD which improves upon removal of gluten (3). The duodenum is the first major part of the gut to process food and swallowed aerosol antigens, and is a master controller of upper gut motility. The role of duodenal inflammation in GERD, if any, remains understudied. Assessing T-helper (Th) subsets using chemokine staining has been previously documented and allows the use of archived gut samples (4). We aimed to identify if there is a duodenal Th signal in GERD.

Aims & Methods

Two retrospective case control studies. Study 1; Duodenal biopsies of 3 groups: 1) GERD (n=22), defined as abnormal acid exposure time on 24-pH monitoring, with or without positive symptom association probability (GERD-SAP+ and GERD-SAP-). 2) Healthy controls (n=17): no symptoms, endoscopic or histological abnormalities. 3) Disease controls: Eosinophilic duodenitis (EoD): (n=8). Blinded histological review of duodenal samples to assess cellular and architectural changes. Dual Immunohistochemistry (IHC) was optimized to identify CD4-Th subsets using the chemokine staining patterns: Th1(CD4+CCR4-CCR5+CCR6-), Th2 (CD4+CCR4+CCR5-CCR6-) and Th17(CD4+CCR4+CCR5-CCR6+). Immunofluorescence confirmed co-staining. Dual IHC and blinded quantitative intensity measuring (H-score) were performed using HALO software. Clinical and histological data were collected. One-way Anova was used to compare signal intensity to controls. A positive signal is defined as a statistically significant higher co-staining of CD4-chemokine stain compared to controls. Study 2: Same methods used to assess esophageal T-helper signal in reflux esophagitis (LA Grade B and higher) (RE) (n=16) compared to eosinophilic esophagitis (EoE) (n=31), lymphocytic esophagitis (LE) (n=12) and healthy controls (n=11).

Results

Considering duodenal histopathology (study 1), no differences were found in any of the mean inflammatory cell counts and no architectural abnormalities comparing GERD to healthy controls. Considering duodenal IHC, the CD4 and CD8 intensities were not significantly higher in GERD compared to controls (p=0.96 and 0.96, respectively). GERD- SAP+ demonstrated a significantly higher Th2 intensity than controls (p=0.01), with no difference in Th1(p=0.55) and Th17 intensities (p= 0.27). A significantly higher Th17 intensity was found in EoD (p=0.006). Duodenal Th2 intensity was significantly higher in GERD with a history of atopy (p=0.035), upright reflux (p=0.037), low esophageal contraction amplitude (p=0.0025) and failed peristalsis (p=0.048). Th1 and Th17 intensities were not higher in any of the GERD subgroups compared to controls. Considering esophageal IHC (study 2), CD8 intensity was significantly elevated in RE (p=0.002), but the CD4 intensity was not (p=0.059). RE had a higher Th2 intensity with no difference in other Th markers (p<0.0001). Th-17 intensity was higher in EoE and LE (p=0.0017 and p<0.0001).

Conclusion

A significantly increased duodenal Th-2 signal is found in established GERD when present with a positive symptom association probability, while an increased esophageal Th-2 signal is found in documented reflux esophagitis. The role of immune activation in GERD deserves further investigation.

References

1. Souza RF, Huo X, Mittal V, et al. Gastroesophageal reflux might cause esophagitis through a cytokine-mediated mechanism rather than caustic acid injury.
2. Ronkainen J, Aro P, Walker MM, et al.. Duodenal eosinophilia is associated with functional dyspepsia and new onset gastro-oesophageal reflux disease. Aliment Pharmacol Ther. 2019 Jul;50(1):24-32.
3. Irani MZ, Eslick GD, Burns GL, et al. Coeliac disease is a strong risk factor for Gastro-oesophageal reflux disease while a gluten free diet is protective: a systematic review and meta-analysis. EClinicalMedicine. 2024;71
4.Oki M, Ohtani H, Kinouchi Y, et al. Accumulation of CCR5+ T cells around RANTES+ granulomas in Crohn's disease: a pivotal site of Th1-shifted immune response? Laboratory investigation. 2005;85(1):137-45.

Disclosure

Nicholas J. Talley: Disclosures: GutSee Ltd (consulting microbiome, 2025), Brown University, Agency for Health Care Research and Quality (fiber and laxation) (2024), Rome Foundation (member gastroduodenal committee) (present) Biocodex (FD diagnostic tool) (present) Microba (consulting microbiome - commencing 2025) Comvita Manuka Honey (FD trial consulting) (2025), BluMaiden outside the submitted work. In addition, Dr. Talley has a patent Nepean Dyspepsia Index (NDI) 1998, a patent Licensing Questionnaires Talley Bowel Disease Questionnaire licensed to Mayo/Talley, “Diagnostic marker for functional gastrointestinal disorders” Australian Provisional Patent Application
2021901692, ”Methods and compositions for treating age-related neurodegenerative disease associated with dysbiosis” US Patent Application No. 63/537,725. Boards: The Gut Foundation Board (2025-present), Asia Pacific Association of Medical Editors (APAME) Board (President 2023-2025), Doctors for the Environment (Chair 2024-present), Luminal Faculty GESA (Chair) (2023-2025) Editorial: UptoDate (Section Editor), Mayo Clinic Proceeding Associate Editor (2023-present)
Financial support: Dr. Talley is supported by funding from the National Health and Medical Research Council (NHMRC) to the Centre for Research Excellence in Digestive Health and he holds an NHMRC Investigator grant.

Introduction

Approximately 50% of patients with disorders of gut-brain interaction (DGBI) report intolerance of wheat products [2]. While dietary gluten restrictions would be a feasible approach to managing gastrointestinal symptoms, in DGBI patients with gluten-related symptoms, there are challenges in relation to compliance and even adverse long-term effects of a gluten and wheat-free diet.

Aims & Methods

This study aims to test the feasibility of a double-blind gluten exposure test and determine the rate of patients with DGBI who respond with a change of symptom severity after exposure to gluten.
From a cohort of 70 consecutive patients referred to our Integrated Care Clinic for patients with complex DGBI, 35 agreed to participate in a double-blind gluten exposure test. This test was aligned with the gluten exposure test outlined in the Salerno criteria [1]. After exclusion of coeliac disease (endoscopy and serology), a seven-week gluten-free run-in, patients received one week of encapsulated gluten (8 g per day) or identical placebo. Each week with daily gluten exposure or placebo exposure was separated by one week of washout. Gluten and placebo were administered in a double-blind randomised fashion. The Structured Assessment of Gastrointestinal Symptoms (SAGIS) score was used at baseline before commencing the gluten exposure test and at the end of every week to assess changes of gastrointestinal symptoms.

Results

34 patients completed the challenge test, of whom seven initially had self-reported symptoms associated with gluten or wheat consumption. Only one of these seven patients responded to gluten exposure with a >25% increase of the SAGIS score. 16 out of 34 patients (47 %, 95% CI 30-65) reported improved gastrointestinal symptoms while on a gluten-free diet. Overall, 2 of the 34 patients (5.9%, 95% CI 0.7-20.0) showed a deterioration of symptoms during the gluten challenge week, while 8 patients (23.5%, 95% CI 10.8-41.2) reported a reduction of the symptoms while exposed to placebo. No effects of gluten exposure on extraintestinal symptoms were observed.

Conclusion

A standardised, double-blind gluten exposure test is suitable in the routine clinical setting to rule out gluten intolerance in patients with self-reported gluten sensitivity. Overall, gluten exposure is rarely associated with an increase of gastrointestinal symptoms in patients with DGBI. Self-reported non-celiac wheat sensitivity is a poor predictor for developing symptoms during double-blind gluten exposure.

References

[1] Catassi C, Elli L, Bonaz B, et al. Diagnosis of Non-Celiac Gluten Sensitivity (NCGS): The Salerno Experts' Criteria. Nutrients 2015;7:4966-77.
[2] Shah A, Talley NJ, Kang S, et al. Self-reported non-celiac wheat sensitivity in patients with chronic unexpalined (functional) gastrointestinal symptoms: gut function, immune activation and duodenal mucosa associated microbiome. Gastroenterology 2020;158:S-488