FECAL LPS CAUSES FODMAP-MEDIATED BARRIER DYSFUNCTION IN IBS-D VIA TLR4-DEPENDENT MAST-CELL ACTIVATION

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.

Background: Irritable bowel syndrome (IBS) is characterized by abdominal pain associated with altered bowel habits including constipation (IBS-C) or diarrhea (IBS-D). The colonic mucosa is richly innervated by extrinsic efferent and afferent nerves and intrinsic enteric neurons that modulate motor, secretory and sensory functions. The complex interplay of these nerves and immune cells are thought to be altered in IBS. Aims: 1. To establish an approach for 3D imaging of innervation in colonic mucosal biopsies and computational quantitation of nerve fibers (NFs), enteric glial cells (EGCs), mast cells (MCs) and the proximity MCs to NFs. 2. To compare these measures between healthy controls (HCs) and IBS patients. 3. To assess their correlations with IBS Symptom Severity Scale (IBS-SSS) and abdominal pain intensity and unpleasantness within 24 h of biopsies. Methods: Sigmoid colonic mucosal biopsies collected from 12 HCs (age: 34±11 yrs, 6 females [F], 6 males [M]) and 15 IBS patients (age: 34±10 yrs; including IBS-C: 5F/2M and IBS-D: 4F/4M) were processed for CLARITY procedure and single or double immunolabeling with 10 marker antibodies (Table). Z-stack confocal images with 150-200 optical sections per sample generated 3D images. Densities of NFs, EGCs and MCs, and the proximity of MCs to pan-NFs (PGP9.5 immunoreactive (ir)), extrinsic and intrinsic primary afferent fibers (SP-ir and Calb-ir, respectively) expressed as % of MCs with shortest distance (≤5.2 µm) to NFs of total MCs (PGP9.5-MC, SP-MC, Calb-MC, respectively) were quantitated using Imaris 9.7. Results: Densities of NFs, EGCs and MCs did not show significant differences between IBS and HCs, but Calb-ir and hpChAT-ir densities tended to be lower in IBS vs HCs (P=0.07, P=0.05 respectively). In IBS, VIP-ir and NPY-ir densities negatively correlated with IBS-SSS (r=-0.67, P=0.013 and r=-0.57, P=0.042 respectively) and NPY with abdominal pain unpleasantness (r=-0.71, P=0.006). Strikingly, SP-MC positively correlated with IBS-SSS (r=0.63, P=0.021; Figure), pain intensity (r=0.61, P=0.026) and unpleasantness (r=0.57, P=0.041). In IBS-D patients only, similar findings were seen showing negative correlations of VIP-ir and NPY-ir densities with IBS-SSS (r=-0.9, P=0.006 and r=-0.57, P-0.042 respectively) and a positive correlation between SP-MC and pain intensity (r=0.8, P=0.03). Conclusions: Novel 3D imaging and computerized quantitation of colonic mucosal NF densities and immune cells revealed differences between IBS and HCs and particularly significant correlations of NF densities and MCs in close proximity to sensory nerves with IBS severity and current abdominal pain scores. These findings support alterations in peripheral neuronal signaling in IBS. These measures may become potential objective markers for IBS symptom severity and therapeutic response (supported by NIH/SPARC and Ironwood).

Introduction: In a mechanistic clinical trial, we have recently shown that a diet low in fermentable oligo-, di-and mono-saccharides (FODMAPs) (LFM) improves colonic barrier function in patients with diarrhea-predominant IBS (IBS-D). However, the underlying mechanisms of FODMAP-mediated barrier dysfunction in IBS-D are poorly understood.

Methods: Fecal supernatants (FS) were obtained from IBS-D patients who responded to 4-week LFM as well as healthy controls (HC). These FS were used to evaluate the effects on barrier function in vivo and in vitro models. For in vitro studies, two-dimensional human colonoids were treated with IBS-D or HC FS. For in vivo studies, IBS-D and HC FS were intracolonically injected into wildtype (WT) mice for 24 hours and barrier function was assessed. The role of fecal Lipopolysaccharide (LPS) in barrier dysfunction was assessed using LPS removal and ultrapure-LPS-RS (potent TLR4 antagonist). To delineate the role of mast cells in barrier function, WT mice were pretreated with cromolyn sodium, a mast cell stabilizer prior to FS administration. Finally, Tlr4 knockout mice (Tlr4^-/-) and mast-cell deficient (MCD) mice with/without mast-cell reconstitution treated with IBS-D and HC FS were also utilized.

Results: Post-LFM, LPS concentration in IBS-D FS was 55% lower than pre-LFM (P=0.0002, n=21 paired samples). Mice injected with pre-LFM IBS-D FS had a 37% decrease in colonic trans-epithelial electrical resistance (TEER) and a 134% increase in plasma 4-kDa fluorescein isothiocyanate (FITC) concentration compared to those injected with HC FS (P<0.001 for both). Mice treated with post-LFM FS had similar TEER and plasma FITC concentrations to those treated with HC FS (P>0.05). Removal of LPS from pre-LFM IBS-D FS or pretreating mice with ultrapure LPS-RS normalized the decrease in TEER and increase in plasma FITC concentration seen with IBS-D FS. Tlr4^-/- mice treated with pre-LFM IBS-D FS had similar colonic TEER and plasma FITC concentrations to Tlr4^-/- mice treated with HC FS (P>0.05, Fig 1). IBS-D FS or LPS alone were unable to induce any changes in TEER in two-dimensional human colonoids compared to HC FS and PBS respectively (P=0.9 for both).
In separate experiments, barrier dysfunction caused by pre-LFM IBS-D FS was also normalized by pretreatment of WT mice with cromolyn sodium (P>0.05). Finally, MCD mice treated with pre-LFM IBS-D FS had similar TEER and plasma FITC concentrations compared to those treated with HC FS. MCD mice reconstituted with WT mast cells have significantly lower TEER and higher plasma FITC levels compared to MCD mice reconstituted with Tlr4^-/- mast cells when they were treated with the same IBS FS (Fig 2).

Conclusion: Fecal LPS causes FODMAP-mediated barrier dysfunction in IBS-D. This is not due to the direct effect of LPS on epithelial cells but due to LPS-mediated tlr4-dependent mast-cell activation.