THE LUMINAL MICROBIOME REGULATES GLUTAMINE SYNTHETASE LEVELS IN A SUBSET OF PATIENTS WITH DIARRHEA-PREDOMINANT IRRITABLE BOWEL SYNDROME LEADING TO BARRIER DYSFUNCTION

Objectives
Ageing seems to have a beneficial effect on irritable bowel syndrome (IBS) (Sperber et al. Gastroenterology 2021), and the odds of developing IBS are lower when age is above 50 years (Lovell & Ford CGH 2012). Older age may also affect symptom reporting and quality of life. In this study, we aimed to characterize IBS patients of different age, by comparing patient reported outcomes and measures of gut physiology.

Methods
IBS patients (n=1677; 74% females; Rome II–IV) that had completed questionnaires and gut physiology testing in studies of pathophysiologic mechanisms were included. Patient reported outcomes were gastrointestinal (GI) symptom severity (GSRS-IBS), psychological distress (HADS), somatic symptom severity (PHQ-12), GI-specific anxiety (VSI), and quality of life (IBS-QOL). Subsets of the patients underwent the following gut physiology tests: transit time by radiopaque markers and/or the wireless motility capsule, a rectal sensitivity test (barostat), anorectal manometry, and a lactulose/mannitol urinary excretion test for small bowel permeability.
Linear regression analyses (controlled for sex) were done to assess if age was associated with the dependent variables. IBS patients were stratified into age groups (‘younger’ 18–29 years; ‘intermediate’ 30–49 years; ‘older’ ≥50 years) and groups were compared by ANOVA.

Results
Most of the patient reported outcomes and measures of gut physiology were associated with age (Table 1). Younger patients had more severe GI symptoms compared to both patients of intermediate and older age, except for diarrhea (younger vs. intermediate) and constipation (younger vs. older). No differences in severity of GI symptoms were observed between patients of intermediate and older age. Younger age was associated with higher levels of general and GI-specific anxiety, and more severe somatic symptoms, and older age was associated with better QOL (Table 2). Older patients had longer oroanal transit times and colonic transit times compared to younger IBS patients, as well as a higher proportion with delayed transit time. Rectal urgency, discomfort, and pain thresholds were lower (=more sensitive) in patients of younger/intermediate age compared to older patients. Anorectal function differed between the groups with lower resting anal sphincter pressures in older patients compared to the other age groups. Small bowel permeability did not differ between the age groups (Table 2).

Conclusion
The overall symptom burden seems to decrease in IBS with increasing age, which may partly be related to age-related changes in GI sensorimotor function. Age is an important factor to take into consideration in the management of patients with IBS, as well as in pathophysiological research.

Introduction: Glutamine synthetase (GLUL) catalyzes the conversion of glutamate to glutamine. GLUL has been reported as a target of microRNA-29a and increased microRNA-29a along with decreased GLUL has been reported in patients with diarrhea-predominant IBS (D-IBS) with increased intestinal permeability. However, it is unclear if decreased GLUL is the cause of increased intestinal permeability in a subset of IBS-D patients and there is no data on the regulation of GLUL in IBS-D.
Methods: GLUL mRNA levels were quantified using colon biopsies obtained from 28 treatment naïve IBS-D patients. IBS-D patients in the lowest quartile were considered “low-GLUL” while those in the highest were considered “normal-GLUL” IBS-D patients. Fecal Supernatant (FS) from low-GLUL and normal-GLUL IBS-D patients along with healthy controls (HC) were intracolonically administered to wild-type mice for 24 hours. In vivo and ex vivo barrier function was assessed along with mucosal GLUL, microRNA-29a, and glutamine levels. To assess the rescue effects of glutamine in barrier dysfunction caused by IBS-D FS, glutamine in drinking water (10mg/mL) was provided ad libitum for 72 hours prior to FS administration. Finally, the role of luminal lipopolysaccharide (LPS) in GLUL regulation was studied using pharmacological inhibition and genetic knockout models.
Results: Mice treated with low-GLUL IBS-D FS had lower colonic mucosal levels of GLUL mRNA, microRNA-29a, and glutamine compared to mice treated with normal-GLUL IBS-D and HC FS. This was associated with lower colonic trans-epithelial electrical resistance (TEER), the higher plasma concentration of 4-kDa fluorescein isothiocyanate (FITC) following intraoral gavage, and lower colonic Claudin-1 and ZO-1 mRNA levels in the low-GLUL IBS-D FS treated mice compared to the other two groups. Glutamine rescue completely reversed the effect of low GLUL IBS-D FS on colonic barrier function but did not change the GLUL and microRNA-29a levels. Pre-treatment of mice with ultrapure LPS-RS (Tlr4 antagonist) or treatment of low GLUL IBS-D FS with LPS removal assay reversed the effect of FS on microRNA-29a, GLUL mRNA, and mucosal glutamine levels. This was accompanied by the normalization of TEER, 4KDa-FITC leakage, and tight junction mRNA levels. Intracolonic administration of LPS alone (E. coli O111:B4) increased microRNA-29a, and reduced GLUL mRNA and glutamine levels. Finally, GLUL mRNA, microRNA-29a levels, and glutamine levels were similar between Tlr4-/- mice treated with low GLUL IBS-D and HC FS. There was no evidence of barrier dysfunction in Tlr4^-/- mice treated with low GLUL IBS-D FS.
Conclusion: Luminal LPS regulates colonic miRNA-29a and GLUL levels in a subset of patients with IBS-D. Low colonic GLUL levels cause low mucosal glutamine and barrier dysfunction in rodent models, which is reversed with glutamine supplementation.