Limosilactobacillus reuteri ATCC 6475 metabolites upregulate the serotonin transporter in the intestinal epithelium

The serotonin transporter (SERT) readily takes up serotonin (5-HT), thereby regulating the availability of 5-HT within the intestine. In the absence of SERT, 5-HT remains in the interstitial space and has the potential to aberrantly activate the many 5-HT receptors distributed on the epithelium, immune cells and enteric neurons. Perturbation of SERT is common in many gastrointestinal disorders as well as mouse models of colitis. Select commensal microbes regulate intestinal SERT levels, but the mechanism of this regulation is poorly understood. Additionally, ethanol upregulates SERT in the brain and dendritic cells, but its effects in the intestine have never been examined. We report that the intestinal commensal microbe Limosilactobacillus (previously classified as Lactobacillus) reuteri ATCC PTA 6475 secretes 83.4 mM ethanol. Consistent with the activity of L. reuteri alcohol dehydrogenases, we found that L. reuteri tolerated various levels of ethanol. Application of L. reuteri conditioned media or exogenous ethanol to human colonic T84 cells was found to upregulate SERT at the level of mRNA. A 4-(4-(dimethylamino) phenyl)-1-methylpyridinium (APP+) uptake assay confirmed the functional activity of SERT. These findings were mirrored in mouse colonic organoids, where L. reuteri metabolites and ethanol were found to upregulate SERT at the apical membrane. Finally, in a trinitrobenzene sulphonic acid model of acute colitis, we observed that mice treated with L. reuteri maintained SERT at the colon membrane compared with mice receiving phosphate buffered saline vehicle control. These data suggest that L. reuteri metabolites, including ethanol, can upregulate SERT and may be beneficial for maintaining intestinal homeostasis with respect to serotonin signalling.

Different Mechanism and Efficacy of Dextran-Sodium Sulfate and 2,4,6-Trinitrobenzene Sulphonic Acid in Modeling Ulcerative Colitis in C57BL/6 Mice

Background and Aims: Dextran-sodium sulfate and 2,4,6-trinitrobenzene sulphonic acid are common modeling methods in studying ulcerative colitis. Little attention has been paid to the mechanism differences between the two approaches. Here, we aim to compare the mechanisms and efficacy of these two models and wish to provide fundamental proves for choosing ideal ulcerative colitis models. Methods: Dextran-sodium sulfate and 2,4,6-trinitrobenzene sulphonic acid were applied to induce the colitis in C57BL/6 mice for seven days. Body weight and disease activity index were assessed. Hematology was detected by routine blood test. Histopathology was analyzed by hematoxylin-eosin staining section. Enzyme-linked immunosorbent assay, Western blot and quantitative real-time PCR were used to detect the cytokines protein levels and mRNA levels. Flow cytometry were used to detect the cycles and subsets of splenic cells. Results: Dextran-sodium sulfate induced colitis in C57BL/6 mice showed higher acute immune activities, while 2,4,6-trinitrobenzene sulphonic acid induced colitis showed chronic immune activities with high platelet amounts and activation. Dextran-sodium sulfate is more suitable for modeling acute ulcerative colitis. On the contrary, 2,4,6-trinitrobenzene sulphonic acid is more appropriate for modeling chronic ulcerative colitis. Conclusions: Dextran-sodium sulfate treatment within 7 days in C57BL/6 mice is a suitable experimental model for studying human acute ulcerative colitis with immune response, fecal blood and acute pathogenic damage. Conversely, 2,4,6-trinitrobenzene sulphonic acid treatment within 7 days is more appropriate for studying human chronic ulcerative colitis with hypercoagulable state, IL-2 over-expression state and chronic pathogenic damage.

Increased Mucosal Thrombin is Associated with Crohn’s Disease and Causes Inflammatory Damage through Protease-activated Receptors Activation

Background and Aims Thrombin levels in the colon of Crohn’s disease patients have recently been found to be elevated 100-fold compared with healthy controls. Our aim was to determine whether and how dysregulated thrombin activity could contribute to local tissue malfunctions associated with Crohn’s disease. Methods Thrombin activity was studied in tissues from Crohn’s disease patients and healthy controls. Intracolonic administration of thrombin to wild-type or protease-activated receptor-deficient mice was used to assess the effects and mechanisms of local thrombin upregulation. Colitis was induced in rats and mice by the intracolonic administration of trinitrobenzene sulphonic acid. Results Active forms of thrombin were increased in Crohn’s disease patient tissues. Elevated thrombin expression and activity were associated with intestinal epithelial cells. Increased thrombin activity and expression were also a feature of experimental colitis in rats. Colonic exposure to doses of active thrombin comparable to what is found in inflammatory bowel disease tissues caused mucosal damage and tissue dysfunctions in mice, through a mechanism involving both protease-activated receptors -1 and -4. Intracolonic administration of the thrombin inhibitor dabigatran, as well as inhibition of protease-activated receptor-1, prevented trinitrobenzene sulphonic acid-induced colitis in rodent models. Conclusions Our data demonstrated that increased local thrombin activity, as it occurs in the colon of patients with inflammatory bowel disease, causes mucosal damage and inflammation. Colonic thrombin and protease-activated receptor-1 appear as possible mechanisms involved in mucosal damage and loss of function and therefore represent potential therapeutic targets for treating inflammatory bowel disease.

Transmural impedance detects graded changes of inflammation in experimental colitis

Ulcerative colitis is a chronic disease in which the mucosa of the colon or rectum becomes inflamed. An objective biomarker of inflammation will provide quantitative measures to support qualitative assessment during an endoscopic examination. Previous studies show that transmural electrical impedance is a quantifiable biomarker of inflammation. Here, we hypothesize that impedance detects spatially restricted areas of inflammation, thereby allowing the distinction between regions that differ in their severity of inflammation. A platinum ball electrode was placed into minimally inflamed (i.e. normal) or 2,4,6-trinitrobenzene sulphonic acid (TNBS)-inflamed colonic regions of rats and impedance measurements obtained by passing current between the intraluminal and subcutaneous return electrode. Histology of the colon was correlated with impedance measurements. The impedance of minimally inflamed (normal) tissue was 1.5–1.9 kΩ. Following TNBS injection, impedance significantly decreased within the inflammatory penumbra ( p < 0.05), and decreased more in the inflammatory epicentre ( p = 0.02). Histological damage correlated with impedance values ( p < 0.05). Thus, impedance values of 1.5–1.9, 1.3–1.4 and 0.9–1.1 kΩ corresponded to minimally inflamed, mildly inflamed and moderately inflamed tissue, respectively. In conclusion, transmural impedance is an objective, spatially localized biomarker of mucosal integrity, and distinguishes between severities of intestinal inflammation.

Nod2 Protects the Gut From Experimental Colitis Spreading to Small Intestine

Background and Aims Nucleotide oligomerization domain 2 [NOD2] mutations are key risk factors for Crohn’s disease [CD]. NOD2 contributes to intestinal homeostasis by regulating innate and adaptive immunity together with intestinal epithelial function. However, the exact roles of NOD2 in CD and other NOD2-associated disorders remain poorly known. Methods We initially observed that NOD2 expression was increased in epithelial cells away from inflamed areas in CD patients. To explore this finding, Nod2 mRNA expression, inflammation, and cytokines expression were examined in the small bowel of wild-type [WT], Nod2 knockout and Nod2 mutant mice after rectal instillation of 2,4,6-trinitrobenzene sulphonic acid [TNBS]. Results In WT mice, Nod2 upregulation upstream to rectal injury was associated with pro-inflammatory cytokine expression but no overt histological inflammatory lesions. Conversely, in Nod2-deficient mice the inflammation spread from colitis to ileum and duodenum. Conclusions Nod2 protects the gut from colitis spreading to small intestine.