Serine proteases as luminal mediators of intestinal barrier dysfunction and symptom severity in IBS.

OBJECTIVE: The intestinal lumen contains several proteases. Our aim was to determine the role of faecal proteases in mediating barrier dysfunction and symptoms in IBS. DESIGN: 39 patients with IBS and 25 healthy volunteers completed questionnaires, assessments of in vivo permeability, ex vivo colonic barrier function in Ussing chambers, tight junction (TJ) proteins, ultrastructural morphology and 16 s sequencing of faecal microbiota rRNA. A casein-based assay was used to measure proteolytic activity (PA) in faecal supernatants (FSNs). Colonic barrier function was determined in mice (ex-germ free) humanised with microbial communities associated with different human PA states. RESULTS: Patients with IBS had higher faecal PA than healthy volunteers. 8/20 postinfection IBS (PI-IBS) and 3/19 constipation- predominant IBS had high PA (>95th percentile). High-PA patients had more and looser bowel movements, greater symptom severity and higher in vivo and ex vivo colonic permeability. High-PA FSNs increased paracellular permeability, decreased occludin and increased phosphorylated myosin light chain (pMLC) expression. Serine but not cysteine protease inhibitor significantly blocked high-PA FSN effects on barrier. The effects on barrier were diminished by pharmacological or siRNA inhibition of protease activated receptor-2 (PAR-2). Patients with high-PA IBS had lower occludin expression, wider TJs on biopsies and reduced microbial diversity than patients with low PA. Mice humanised with high-PA IBS microbiota had greater in vivo permeability than those with low-PA microbiota. CONCLUSION: A subset of patients with IBS, especially in PI-IBS, has substantially high faecal PA, greater symptoms, impaired barrier and reduced microbial diversity. Commensal microbiota affects luminal PA that can influence host barrier function.

Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

Intestinal barrier function and microbiota are integrally related and play critical roles in maintenance of host physiology. Sex is a key biologic variable for several disorders. Our aim was to determine sex-based differences in response to perturbation and subsequent recovery of intestinal barrier function and microbiota in healthy humans. Twenty-three volunteers underwent duodenal biopsies, mucosal impedance, and in vivo permeability measurement. Permeability testing was repeated after administration of indomethacin, then 4 to 6 wk after its discontinuation. Duodenal and fecal microbiota composition was determined using 16S rRNA amplicon sequencing. Healthy women had lower intestinal permeability and higher duodenal and fecal microbial diversity than healthy men. Intestinal permeability increases after indomethacin administration in both sexes. However, only women demonstrated decreased fecal microbial diversity, including an increase in Prevotella abundance, after indomethacin administration. Duodenal microbiota composition did not show sex-specific changes. The increase in permeability and microbiota changes normalized after discontinuation of indomethacin. In summary, women have lower intestinal permeability and higher microbial diversity. Intestinal permeability is sensitive to perturbation but recovers to baseline. Gut microbiota in women is sensitive to perturbation but appears to be more stable in men. Sex-based differences in intestinal barrier function and microbiome should be considered in future studies.-Edogawa, S., Peters, S. A., Jenkins, G. D., Gurunathan, S. V., Sundt, W. J., Johnson, S., Lennon, R. J., Dyer, R. B., Camilleri, M., Kashyap, P. C., Farrugia, G., Chen, J., Singh, R. J., Grover, M. Sex differences in NSAID-induced perturbation of human intestinal barrier function and microbiota.

Constipation-Predominant Irritable Bowel Syndrome Females Have Normal Colonic Barrier and Secretory Function.

OBJECTIVES: The objective of this study was to determine whether constipation-predominant irritable bowel syndrome (IBS-C) is associated with changes in intestinal barrier and secretory function. METHODS: A total of 19 IBS-C patients and 18 healthy volunteers (all females) underwent saccharide excretion assay (0.1 g 13C mannitol and 1 g lactulose), measurements of duodenal and colonic mucosal barrier (transmucosal resistance (TMR), macromolecular and Escherichia coli Bio-Particle translocation), mucosal secretion (basal and acetylcholine (Ach)-evoked short-circuit current (Isc)), in vivo duodenal mucosal impedance, circulating endotoxins, and colonic tight junction gene expression. RESULTS: There were no differences in the in vivo measurements of barrier function between IBS-C patients and healthy controls: cumulative excretion of 13C mannitol (0-2 h mean (s.e.m.); IBS-C: 12.1 (0.9) mg vs. healthy: 13.2 (0.8) mg) and lactulose (8-24 h; IBS-C: 0.9 (0.5) mg vs. healthy: 0.5 (0.2) mg); duodenal impedance IBS-C: 729 (65) Ω vs. healthy: 706 (43) Ω; plasma mean endotoxin activity level IBS-C: 0.36 (0.03) vs. healthy: 0.35 (0.02); and in colonic mRNA expression of occludin, zonula occludens (ZO) 1-3, and claudins 1-12 and 14-19. The ex vivo findings were consistent, with no group differences: duodenal TMR (IBS-C: 28.2 (1.9) Ω cm2 vs. healthy: 29.8 (1.9) Ω cm2) and colonic TMR (IBS-C: 19.1 (1.1) Ω cm2 vs. healthy: 17.6 (1.7) Ω cm2); fluorescein isothiocyanate (FITC)-dextran (4 kDa) and E. coli Bio-Particle flux. Colonic basal Isc was similar, but duodenal basal Isc was lower in IBS-C (43.5 (4.5) µA cm-2) vs. healthy (56.9 (4.9) µA cm-2), P=0.05. Ach-evoked ΔIsc was similar. CONCLUSIONS: Females with IBS-C have normal colonic barrier and secretory function. Basal duodenal secretion is decreased in IBS-C.

Intestinal barrier dysfunction in irritable bowel syndrome: a systematic review.

BACKGROUND AND AIM: Irritable bowel syndrome (IBS) is a complex and heterogeneous disorder. Sensory, motor and barrier dysfunctions are the key physiological endophenotypes of IBS. Our aim is to review studies evaluating barrier dysfunction in adults and children with IBS, as well as to link those changes with IBS symptomatology and quality of life. METHODS: A comprehensive and systematic review of multiple databases was performed up to March 2020 to identify studies comparing intestinal permeability in IBS patients with healthy controls. Both in vivo and in vitro studies were considered. RESULTS: We identified 66 studies, of which 27 used intestinal probes to quantify barrier function. The prevalence of barrier dysfunction differed between PI-IBS (17-50%), IBS-D (37-62%) and IBS-C (4-25%). At a group level, permeability was increased compared with healthy controls in IBS-D (9/13 studies) and PI-IBS (4/4 studies), but only a minority of IBS-C (2/7 studies) and not in the only IBS-M study. All four studies in children with IBS demonstrated loss of barrier function. A heterogeneous set of tight junction genes were found to be altered in small and large intestines of adults with IBS, but these have not been evaluated in children. Positive associations were identified between barrier dysfunction and bowel disturbances (6/9 studies), abdominal pain (9/13 studies), overall symptom severity (1/6 studies), depression and anxiety (1/1 study) and quality of life (1/4 studies). Fecal slurry or supernatants of IBS patients were found to induce barrier disruption in animal models (5/6 studies). CONCLUSIONS: Barrier dysfunction is present in a significant proportion of adult and all pediatric IBS studies, especially in the IBS-D and PI-IBS subtype. The majority of studies indicated a positive association between loss of barrier function and symptoms such as abdominal pain and changes in the bowel function.

Duodenal mucosal secretory disturbances in functional dyspepsia.

BACKGROUND: There is increased recognition of duodenal disturbances (inflammation, altered mucosal protein expression, and chemosensitivity) in functional dyspepsia (FD). Besides sensorimotor functions, enteric submucosal neurons also regulate epithelial ion transport. We hypothesized that duodenal mucosal ion transport and expression of associated genes are altered in FD. METHODS: Duodenal mucosal ion transport (basal and acetylcholine- and glucose-evoked changes in short-circuit current [Isc]) and expression of associated genes and regulatory miRNAs were evaluated in 40 FD patients and 24 healthy controls. RESULTS: Basal Isc (FD: 88.2 [52.6] µA/cm2 vs healthy: 20.3 [50.2] µA/cm2 ; P ≤ .0001), acetylcholine-evoked Isc (FD: Emax 50.4 [35.8] µA/cm2 vs healthy: 16.6 [15] µA/cm2 ; P ≤ .001), and glucose-evoked Isc responses (FD: Emax 69.8 [42.1] µA/cm2 vs healthy: 40.3 [24.6] µA/cm2 ; P = .02) were greater in FD than in controls. The Emax for glucose was greater in FD patients on selective serotonin reuptake inhibitors. In FD, the mRNA expression of SLC4A7 and SLC4A4, which transport bicarbonate into cells at the basolateral surface, and the apical anion exchanger SLC26A3 were reduced (false discovery rate <0.05), the serotonin receptor HTR4 was increased, and the serotonin transporter SLC6A4 was decreased. Selected miRNAs (hsa-miR-590-3p, hsa-miR-32-5p) that target genes associated with ionic transport were upregulated in FD. CONCLUSIONS: Compared to controls, FD patients had greater baseline and agonist-evoked duodenal mucosal secretory responses. These findings may be explained by reduced gene expression, which would be anticipated to reduce luminal bicarbonate secretion. The upregulated miRNAs may partly explain the downregulation of these genes in FD.

Small intestinal microbial dysbiosis underlies symptoms associated with functional gastrointestinal disorders.

Small intestinal bacterial overgrowth (SIBO) has been implicated in symptoms associated with functional gastrointestinal disorders (FGIDs), though mechanisms remain poorly defined and treatment involves non-specific antibiotics. Here we show that SIBO based on duodenal aspirate culture reflects an overgrowth of anaerobes, does not correspond with patient symptoms, and may be a result of dietary preferences. Small intestinal microbial composition, on the other hand, is significantly altered in symptomatic patients and does not correspond with aspirate culture results. In a pilot interventional study we found that switching from a high fiber diet to a low fiber, high simple sugar diet triggered FGID-related symptoms and decreased small intestinal microbial diversity while increasing small intestinal permeability. Our findings demonstrate that characterizing small intestinal microbiomes in patients with gastrointestinal symptoms may allow a more targeted antibacterial or a diet-based approach to treatment.