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.

Yersiniabactin Siderophore of Crohn’s Disease-Associated Adherent-Invasive Escherichia coli Is Involved in Autophagy Activation in Host Cells

Background: Adherent-invasive Escherichia coli (AIEC) have been implicated in the etiology of Crohn’s disease. The AIEC reference strain LF82 possesses a pathogenicity island similar to the high pathogenicity island of Yersinia spp., which encodes the yersiniabactin siderophore required for iron uptake and growth of the bacteria in iron-restricted environment. Here, we investigated the role of yersiniabactin during AIEC infection. Methods: Intestinal epithelial T84 cells and CEABAC10 transgenic mice were infected with LF82 or its mutants deficient in yersiniabactin expression. Autophagy was assessed by Western blot analysis for p62 and LC3-II expression. Results: Loss of yersiniabactin decreased the growth of LF82 in competitive conditions, reducing the ability of LF82 to adhere to and invade T84 cells and to colonize the intestinal tract of CEABAC10 mice. However, yersiniabactin deficiency increased LF82 intracellular replication. Mechanistically, a functional yersiniabactin is necessary for LF82-induced expression of HIF-1α, which is implicated in autophagy activation in infected cells. Conclusion: Our study highlights a novel role for yersiniabactin siderophore in AIEC–host interaction. Indeed, yersiniabactin, which is an advantage for AIEC to growth in a competitive environment, could be a disadvantage for the bacteria as it activates autophagy, a key host defense mechanism, leading to bacterial clearance.

A Network Pharmacology-Based Approach to Investigating the Mechanisms of Fushen Granule Effects on Intestinal Barrier Injury in Chronic Renal Failure

Purpose. Fushen Granule (FSG) is a Chinese medicine prepared by doctors for treating patients with chronic renal failure, which is usually accompanied by gastrointestinal dysfunction. Here, we explore the protective effect of FSG on intestinal barrier injury in chronic renal failure through bioinformatic analysis and experimental verification. Methods. In this study, information on the components and targets of FSG related to CRF is collected to construct and visualize protein-protein interaction networks and drug-compound-target networks using network pharmacological methods. DAVID is used to conduct gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, it is validated by in vitro experiments. In this study, the human intestinal epithelial (T84) cells are used and divided into four groups: control group, model group, FSG low-dose group, and FSG high-dose group. After the experiment, the activity of T84 cells is detected by a MTT assay, and the expressions of tight junction protein ZO-1, claudin-1, nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), malondialdehyde (MDA), and cyclooxygenase-2 (COX-2) are examined by immunofluorescence and/or western blotting. Results. Eighty-six potential chronic renal failure-related targets are identified by FSG; among them, nine core genes are screened. Furthermore, GO enrichment analysis shows that the cancer-related signaling pathway, the PI3K-Akt signaling pathway, the HIF1 signaling pathway, and the TNF signaling pathway may play key roles in the treatment of CRF by FSG. The MTT method showed that FSG is not cytotoxic to uremic toxin-induced injured T84 cells. The results of immunofluorescence and WB indicate that compared with the control group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is decreased and protein expressions level of HO-1, MDA, and COX-2 is increased after urinary toxin treatment. Instead, compared with the model group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is increased and protein expressions level of HO-1, MDA, and COX-2 is decreased after FSG treatment. Conclusion. FSG had a protective effect on urinary toxin-induced intestinal epithelial barrier injury in chronic renal failure, and its mechanism may be related to the upregulation of Nrf2/HO-1 signal transduction and the inhibition of tissue oxidative stress and inflammatory responses. Screening CRF targets and identifying the corresponding FSG components by network pharmacological methods is a practical strategy to explain the mechanism of FSG in improving gastrointestinal dysfunction in CRF.

A31 COMPLEX ROLE OF DIETARY FIBERS IN IBD: MICROBES MEDIATE FIBER-INDUCED INFLAMMATION

Background Dietary fibers pass through the bowel undigested and are fermented within the intestine by microbes, typically promoting gut health. However, many IBD patients describe experiencing sensitivity to fibers. β-glucan, found on the surface of fungal cells during fungal infection, has been shown to bind to fiber receptors, such as Dectin-1, on host immune cells, resulting in a pro-inflammatory response. These fungal fibres share properties with dietary fibers. Aims As an altered gut microbial composition has been associated with IBD, we hypothesized that the loss of fiber-fermenting microbes populating the gut in IBD could lead to dietary fibers not being efficiently broken down into their beneficial biproducts (e.g. short chain fatty acids; SCFA), resulting in binding of intact fibers to pro-inflammatory host cell receptors. Methods Immune and epithelial cell lines and colonic biopsies cultured ex vivo were incubated with oligofructose or inulin (5g/L), or pre-fermented fibers (24hr anaerobic fermentation). Immune responses were measured by cytokine secretion (ELISA), and expression (qPCR). Barrier integrity was measured by transepithelial resistance (TEER). Food frequency questionnaire (FFQ) data of patient fiber consumption were correlated with gut microbes (shotgun sequencing) and immune responses to fiber in patient biopsies. Results Unfermented oligofructose induced IL-1β secretion in leukocytes (macrophage, T cell, neutrophil) and in colon biopsies from pediatric Crohn disease (CD; n=38) and ulcerative colitis (UC; n=20) patients cultured ex vivo, but not in non-IBD patients (n=21). IL-1β secretion was greater in patients with more severe disease. Pre-fermentation of oligofructose by whole-microbe intestinal washes from non-IBD patients or remission patients reduced secretion of IL-1β, while whole microbe intestinal washes from severe IBD patients were unable to ferment oligofructose or reduce cytokine secretion. Fiber effects on IL-1β secretion in biopsies positively correlated with effects on barrier integrity in T84 cells. Fiber-associated immune responses in patient biopsies cultured ex vivo (ELISA) correlated with fiber avoidance (FFQ) and gut microbiome (sequencing) in matching patient samples. Conclusions Our findings demonstrate that intolerance and avoidance of prebiotic fibers in select IBD patients is associated with the inability to ferment these fibers, leading to pro-inflammatory immune responses and intestinal barrier disruption. This highlights select disease state scenarios, in which administration of fermentable fibers should be avoided and tailored dietary interventions should be considered in IBD patients. Funding Agencies CIHRWeston Foundation

A49 BUTYRATE PRESERVES THE EPITHELIAL MITOCHONDRIAL NETWORK DISRUPTED BY THE CROHN’S DISEASE PATHOBIONT ADHERENT-INVASIVE E. COLI

Background Mitochondria exist in a dynamic network that undergoes continuous cycles of fission and fusion that is tightly controlled. Mitochondrial dysfunction is implicated in several autoimmune and inflammatory diseases. Adherent-invasive E. coli (AIEC) is a pathogenic strain of bacteria associated with Crohn’s disease (CD), that can evoke pro-inflammatory responses. Data from our lab showed that AIEC (strain LF82) infection in gut epithelial cells (T84 human cell line) caused dramatic mitochondrial network fragmentation and loss of mitochondrial membrane potential. Short chain fatty acids (SCFA) produced by commensal bacteria in the gut have a wide range of benefits including enhancing mitochondrial biogenesis. Aims To determine (1) if sodium butyrate (NaB) treatment can protect against mitochondrial dysfunction induced by AIEC (strain LF82) infection in T84 epithelial cells and then (2) to identify the mechanism by which NaB restores mitochondrial functions. Methods We assessed changes in mitochondrial network morphology through confocal microscopy live cell imaging of mitotracker-stained T84 epithelial cells based on unbiased Hoechst-stained nuclei to select the field of view. The effect of NaB on the proliferation of bacteria and their invasion of T84-epithelia was assessed by growth curve analysis and bacterial internalisation assays. Finally, mRNA and protein expression of peroxisome proliferating activator receptor gamma co-activator 1 alpha (PGC1α), a regulator of mitochondrial biogenesis, were assessed by qPCR and Western Blot. Results We confirmed that AIEC infection (strain LF82, 108cfu, 4h) induces massive mitochondrial fragmentation in T84 cells (2x105). We also found that cotreatment of T84 cells with NaB (10mM) and LF82 showed increased percentage of fused mitochondrial networks compared to LF82-treated cells. This result was also seen in cells treated with mitochondrial uncoupler dinitrophenol (DNP; 0.1 mM, 2h) and NaB. Moreover, the protective effect of NaB was not related to inhibition of proliferation of the bacteria as we demonstrated that LF82 growth and its invasive phenotype was not compromised by NaB (3-20mM; 0-24hr). In agreement with the effect of LF82 on mitochondrial functions, LF82 significantly reduced PGC1α mRNA expression in T84 cells, that was prevented by co-treatment with NaB. Conclusions These data suggest the pathogen disruption of the epithelial mitochondrial network is a component of IBD; thus, identifying mitochondrial fission and fusion pathways as novel therapeutic targets to control enteric inflammation. The data underscore the complex interplay between bacteria and the epithelium, such that commensal organisms may preserve the mitochondrial network in the face of challenge from pathogens that seek to disrupt mitochondrial form and function Funding Agencies CIHR

Elevated Extracellular cGMP Produced after Exposure to Enterotoxigenic Escherichia coli Heat-Stable Toxin Induces Epithelial IL-33 Release and Alters Intestinal Immunity

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is a major diarrheal pathogen in children in low- to middle-income countries. Previous studies identified heat-stable enterotoxin (ST)-producing ETEC as a prevalent diarrheal pathogen in children younger than 5 years. While many studies have evaluated the interaction of ETEC heat-labile enterotoxin (LT) with host epithelium and immunity, few investigations have attempted similar studies with ST. To further understand ST pathogenesis, we examined the impact of ST on cGMP localization, epithelial cell cytokine production, and antibody development following immunization. In addition to robust intracellular cGMP in T84 cells in the presence of phosphodiesterase inhibitors (PDEis) that prevent the breakdown of cyclic nucleotides, we found that prolonged ST intoxication induced extracellular cGMP accumulation in the presence or absence of PDEis. Further, ST intoxication induced luminal cGMP in vivo in mice, suggesting that secreted cGMP may have other cellular functions. Using transcriptome sequencing (RNA-seq) and quantitative PCR (qPCR), we demonstrated that ST intoxication, or treatment with the clinically used ST mimic linaclotide, altered inflammatory cytokine gene expression, including the interleukin 1 (IL-1) family member IL-33, which could also be induced by cell-permeative 8-Br-cGMP. Finally, when present during immunization, ST suppressed induction of antibodies to specific antigens. In conclusion, our studies indicate that ST modulates epithelial cell physiology and the interplay between the epithelial and immune compartments.

Doxorubicin increases permeability of murine small intestinal epithelium and cultured T84 monolayers

Enteric bacteria and/or their products are necessary for doxorubicin (DXR)-induced small intestine mucosal damage. While DXR does not induce gross loss of epithelium, others have shown elevated serum endotoxin after DXR administration. However, the mechanism of movement is unknown. We hypothesized that DXR treatment resulted in increased paracellular translocation of bacteria or bacterial products through the small intestinal epithelium. We measured permeability after DXR administration using transepithelial resistance and macromolecular flux and assessed tight junctional gene expression and protein localization both in vitro using T84 cells and ex vivo using murine jejunum. DXR treatment increased flux of 4 kDa dextrans in mouse jejenum, but increased flux of 4, 10 and 20 kDa dextrans in T84 cells. Following DXR, we observed increased permeability, both in vitro and ex vivo, independent of bacteria. DXR induced increased expression of Cldn2 and Cldn4 in murine small intestine but increased only CLDN2 expression in T84 cells. DXR treatment induced disorganization of tight junctional proteins. We conclude that DXR increases paracellular transit of small macromolecules, including bacterial products, through the epithelium, by altering expression of tight junctional components and dynamic loosening of cellular tight junctions.

Development of a Cell-Based Assay for Identifying KCa3.1 Inhibitors Using Intestinal Epithelial Cell Lines

Inhibition of the KCa3.1 potassium channel has therapeutic potential in a variety of human diseases, including inflammation-associated disorders and cancers. However, KCa3.1 inhibitors with high therapeutic promise are currently not available. This study aimed to establish a screening assay for identifying inhibitors of KCa3.1 in native cells and from library compounds derived from natural products in Thailand. The screening platform was successfully developed based on a thallium flux assay in intestinal epithelial (T84) cells with a Z′ factor of 0.52. The screening of 1352 compounds and functional validation using electrophysiological analyses identified 8 compounds as novel KCa3.1 inhibitors with IC50 values ranging from 0.14 to 6.57 µM. These results indicate that the assay developed is of excellent quality for high-throughput screening and capable of identifying KCa3.1 inhibitors. This assay may be useful in identifying novel KCa3.1 inhibitors that may have therapeutic potential for inflammation-associated disorders and cancers.

How the colonic environment influences Enterohaemorrhagic E. coli outer membrane vesicle production, and the interaction between outer membrane vesicles with human host cells

Enterohaemorrhagic E. coli (EHEC) may instigate bloody diarrhoea and haemolytic uraemic syndrome (HUS) due to Shiga toxin (Stx) production. Stx has been detected within outer membrane vesicles (OMVs), which are membrane-derived nanosized proteoliposomes. During colonisation, EHEC encounters many environmental surroundings such as the presence of bile salts and carbon dioxide (CO2). Here, the influence of different intestinal cues on EHEC OMV production was studied. OMV yield was quantified by densitometric analysis of outer membrane proteins F/C and A, following OMV protein separation by SDS-PAGE. Compared to cultures in Luria broth, higher OMV yields were attained following culture in human cell growth medium and simulated colonic environmental medium, with further increases in the presence of bile salts. Interestingly, lower yields were attained in the presence of T84 cells and CO2. The interaction between OMVs and different human cells was also examined by fluorescence microscopy. Here, OMVs incubated with cells showed internalisation by semi confluent but not fully confluent T84 cell monolayers. OMVs were internalised into the lysosomes in confluent Vero and Caco-2 cells, with Stx being transported to the Golgi and then the Endoplasmic reticulum. OMVs were detected within polarised Caco-2 cells, with no impact on the transepithelial electrical resistance by 24 hours. These results suggest that the colonic environmental factors influences OMV production in vivo. Additionally, results highlight the discrepancies which arise when using different cells lines to examine the intestine. Nevertheless, coupled with Stx, OMVs may serve as tools of EHEC which are involved in HUS development.