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.