Intestinal Epithelial Barrier Function Research Articles

Potential Probiotic Weizmannia coagulans WC10 Improved Antibiotic-Associated Diarrhea in Mice by Regulating the Gut Microbiota and Metabolic Homeostasis.

Antibiotic-associated diarrhea (AAD) is a common side effect of long-term and heavy antibiotic therapy. Weizmannia coagulans (W. coagulans) is an ideal probiotic because of its high viability, stability, and numerous health benefits to the host. In this study, the strains were first screened for W. coagulans WC10 (WC10) with a high combined ability based on their biological properties of gastrointestinal tolerance, adhesion, and short-chain fatty acid production ability. The effect of WC10 on mice with AAD was further evaluated. The results showed that WC10 was effective in improving the symptoms of AAD, effectively restoring antibiotic-induced weight loss, and reducing diarrhea status score and fecal water content. In addition, WC10 decreased the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines, alleviated intestinal tissue damage and inflammation, and improved intestinal epithelial barrier function by decreasing serum levels of enterotoxin, DAO, and D-lactic acid, and by increasing the expression of the intestinal mucosal immune factors sIgA and occludin. Importantly, the composition and function of the gut microbiota gradually recovered after WC10 treatment, increasing the number of SCFAs-producing Bifidobacterium and Roseburia. Subsequently, the short-chain fatty acid (SCFA) content was examined and WC10 significantly increased acetate, propionate, and butyrate production. Additionally, metabolomic analysis also showed that WC10 reversed the antibiotic interference with major metabolic pathways. These findings provide a solid scientific basis for the future application of W. coagulans WC10 in the treatment of AAD.

Inhibition of miR-142-3p promotes intestinal epithelial proliferation and barrier function after ischemia/reperfusion injury by targeting FoxM1.

Damage of intestinal barrier function (BF) after ischemia/reperfusion (I/R) injury can induce serious complications and high mortality. MicroRNAs (miRNAs) are involved in intestinal mucosal BF and epithelial proliferation after I/R injury have been reported. We aimed to investigate the role and regulatory mechanism of miR-142-3p (miR-142) in intestinal epithelial proliferation and BF after I/R injury. We detected the proliferation, barrier function and miR-142 expression in clinical ischemic intestinal tissues. Furthermore, we induced an in vivo intestinal I/R injury mouse model and in vitro IEC-6 cells hypoxia/reoxygenation (H/R) injury model. After increasing and decreasing expression of miR-142, we detected the proliferation and barrier function of intestinal epithelial cells after I/R or H/R injury. We found that miR-142 expression was significantly increased in clinical ischemic intestinal mucosa and mouse intestinal mucosa exposed to I/R injury, and there was an inverse relationship between miR-142 and proliferation/BF. Inhibition of miR-142 significant promoted intestinal epithelial proliferation and BF after I/R injury. Furthermore, inhibition of miR-142 improved overall survival rate of mice after I/R injury. MiR-142 directly targeted FoxM1 which was identified by bioinformatics analysis and luciferase activity assay in IEC-6 cells. Inhibition of miR-142 promotes intestinal epithelial proliferation and BF after I/R injury in a FoxM1-mediated manner.

Oral curcumin ameliorates acute murine campylobacteriosis

IntroductionHuman infections with the food-borne enteropathogen Campylobacter jejuni are responsible for increasing incidences of acute campylobacteriosis cases worldwide. Since antibiotic treatment is usually not indicated and the severity of the enteritis directly correlates with the risk of developing serious autoimmune disease later-on, novel antibiotics-independent intervention strategies with non-toxic compounds to ameliorate and even prevent campylobacteriosis are utmost wanted. Given its known pleiotropic health-promoting properties, curcumin constitutes such a promising candidate molecule. In our actual preclinical placebo-controlled intervention trial, we tested the anti-microbial and anti-inflammatory effects of oral curcumin pretreatment during acute experimental campylobacteriosis.MethodsTherefore, secondary abiotic IL-10-/- mice were challenged with synthetic curcumin via the drinking water starting a week prior oral C. jejuni infection. To assess anti-pathogenic, clinical, immune-modulatory, and functional effects of curcumin prophylaxis, gastrointestinal C. jejuni bacteria were cultured, clinical signs and colonic histopathological changes quantitated, pro-inflammatory immune cell responses determined by in situ immunohistochemistry and intestinal, extra-intestinal and systemic pro-inflammatory mediator measurements, and finally, intestinal epithelial barrier function tested by electrophysiological resistance analysis of colonic ex vivo biopsies in the Ussing chamber.Results and discussionWhereas placebo counterparts were suffering from severe enterocolitis characterized by wasting symptoms and bloody diarrhea on day 6 post-infection, curcumin pretreated mice, however, were clinically far less compromised and displayed less severe microscopic inflammatory sequelae such as histopathological changes and epithelial cell apoptosis in the colon. In addition, curcumin pretreatment could mitigate pro-inflammatory innate and adaptive immune responses in the intestinal tract and importantly, rescue colonic epithelial barrier integrity upon C. jejuni infection. Remarkably, the disease-mitigating effects of exogenous curcumin was also observed in organs beyond the infected intestines and strikingly, even systemically given basal hepatic, renal, and serum concentrations of pro-inflammatory mediators measured in curcumin pretreated mice on day 6 post-infection. In conclusion, the anti-Campylobacter and disease-mitigating including anti-inflammatory effects upon oral curcumin application observed here highlight the polyphenolic compound as a promising antibiotics-independent option for the prevention from severe acute campylobacteriosis and its potential post-infectious complications.

#794 LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis

Abstract Background and Aims Long non-coding RNA(LncRNA) has been reported to be associated with intestinal barrier damage. The aim of this study was to explore the mechanism of lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury. Method Human colon cancer cells (Caco-2) were used to establish intestinal epithelial injury models with the urea treatment in different conditions. Cell Counting Kit-8 (CCK-8) and Western blot screening the best concentration and time. The expressions of lncRNA NEAT1 and miR-122-5p were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot and immunofluorescence were used to detect the expression of tight junction proteins Occludin, ZO-1 and Claudin-1. Sodium fluorescein was used to detect the paracellular permeability of intestinal epithelial injury models. The binding of miR-122-5p to lncRNA NEAT1 and Occludin was determined by bioinformatics analysis and dual luciferase reporter assay. Results The best condition for the injury model was urea treatment in 144 mg/dl for 48 hours. With the increase of urea intervention time and concentration, the damage degree of intestinal epithelial cells is aggravated. Based on the qRT-PCR results, lncRNA NEAT1 was significantly down-regulated in the model group. Meanwhile, the tight junction proteins Occludin, ZO-1 and Claudin-1 were significantly reduced. The permeability of sodium fluorescein was significantly increased in the model group. Overexpression of lncRNA NEAT1 can alleviate the above performances. As the target gene of lncRNA NEAT1, miR-122-5p is significantly up-regulated in the model group. The dual luciferase reporter assay proved that miR-122-5p was targets to Occludin. The protective effect of overexpression lncRNA NEAT1 on intestinal epithelial barrier function is reversed by miR-122-5p mimics. Conclusion LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis.

Study on the mechanism of hirudin multi target delaying renal function decline in chronic kidney disease based on the "gut-kidney axis" theory.

The disorder of the "gut-kidney axis" exacerbates renal function decline in chronic kidney disease (CKD), and current CKD therapy is insufficient to address this issue. Hirudin has a palliative effect on the decline of renal function. However, whether hirudin can delay CKD by regulating the "intestinal renal axis" disorder remains unclear. Unilateral ureteral ligation (UUO) induced CKD rat model, and the rats were treated with bifidobacterium and hirudin for 36 days. After 14 and 36 days of modeling, kidney and colon tissues were collected for pathology, western blot (WB) assay, and quantitative real-time PCR (qPCR) detection. Serum samples were collected for renal function testing. Fecal samples were used for 16S rRNA sequencing and research on fecal bacterial transplantation. Lipopolysaccharide combine with adenosine 5'-triphosphate (LPS + ATP)-induced intestinal epithelial cell injury was treated with a nod-like receptor pyrin domain-associated protein 3 (NLRP3) inhibitor and hirudin. Protein expression was detected using WB and qPCR. The kidneys and colons of the CKD rats exhibited varying degrees of lesions. Creatinine (CRE), blood urea nitrogen (BUN), N-acetyl-β-D-glucosidase (NAG) enzyme, and serum uremic toxins were elevated. The expression of claudin-1 and occludin was decreased, NLRP3 inflammatory-related proteins were increased, and the gut microbiota was disrupted. These pathological changes were more pronounced after 36 days of modeling. Meanwhile, high-dose hirudin treatment significantly improved these lesions and restored the intestinal flora to homeostasis in CKD rats. In vitro, hirudin demonstrated comparable effects to NLRP3 inhibitors by upregulating claudin-1 and occludin expression, and downregulating NLRP3 inflammatory-related proteins expression. The dysbiosis of the gut microbiota and impaired intestinal epithelial barrier function in CKD are associated with renal dysfunction in CKD. Hirudin delays the progression of CKD by regulating the disorder of the "gut-kidney axis" and inhibiting the activation of the NLRP3-ASC-caspase-1 pathway.

Comparison of a brand-name drug and its generic drugs - Impact of anti-human immunodeficiency virus drug exposure on the intestinal epithelial barrier

The use of unapproved anti-human immunodeficiency virus (HIV) drugs for its treatment and prevention has recently increased in Japan. Our research group investigated generic drugs of the Truvada combination tablet (TVD) that have not been approved in Japan, and reported differences in the pharmaceutical properties of their formulations. We also demonstrated that the pharmaceutical excipients in generic drugs may differ from those in TVD, and showed that some pharmaceutical excipients may affect epithelial cell barrier function. The present study investigated differences in the effects of TVD and its generic drugs (Generic A and Generic B) on epithelial cell barrier function. No significant differences were observed in epithelial cell barrier function following exposure to TVD, Generic A, and Generic B. However, significant increases in the mRNA levels of ABCB1 and Occludin, which regulate the epithelial cell barrier, were detected following exposure to Generic B. Therefore, the administration of Generic B for a longer duration or at a higher concentration may lead to changes in epithelial barrier function. Since the treatment and prevention of HIV requires the long-term (chronic) administration of drugs, the drugs used may change, such as from a brand-name drug to a generic drug and also from one generic drug to another. The present results suggest that the effects of brand-name and generic drugs, such as on intestinal epithelial barrier function, may differ with changes in the drugs being administered.

Intestinal Epithelial Cell-specific Knockout of METTL3 Aggravates Intestinal Inflammation in CLP Mice by Weakening the Intestinal Barrier.

Many studies have demonstrated that the expression of methyltransferase- like 3 (METTL3) is altered in various inflammatory diseases. Its specific mechanistic role in the intestinal inflammatory response during sepsis remains limited and requires further investigation. Explore the potential mechanism of METTL3 in the intestinal inflammatory response during sepsis. Immunohistochemical analysis was utilized to detect the expression of METTL3 in the necrotic intestine of patients with intestinal necrosis and the small intestine of cecal ligation and puncture (CLP) mice. Mice were subjected to the CLP and Sham surgeries, intestine tissue was harvested and performed HE staining, and ELISA to examine intestinal inflammatory responses, while TUNEL staining was applied to detect intestinal cell apoptosis. Additionally, ELISA was used to detect diamine oxidase (DAO) and intestinal fatty acid binding protein (I-FABP) levels in intestinal tissue. Immunohistochemistry and RT-qPCR were also employed to examine the mRNA and protein expression levels of Zona Occludens 1 (ZO-1) and Claudin-1. Finally, transcriptomic sequencing was performed on the small intestine tissues of METTL3 Knock-out (KO) and Wild-type (WT) mice in response to sepsis. METTL3 exhibited lower expression level in the necrotic intestine of patients and the small intestine of CLP mice. Loss of METTL3 in CLP mice triggered significantly higher expression of TNF-α and IL-18, down-regulated expression of ZO-1 and claudin-1, and decreased expression of DAO and I-FABP in the intestinal tissue. KEGG enrichment analysis showed that the differential genes were significantly enriched in immune-related pathways. This study reveals a novel mechanism responsible for exacerbated intestinal inflammation orchestrated by METTL3. Particularly, METTL3 null mice displayed decreased ZO- 1 and Claudin-1 expression, which largely hampered intestinal epithelial barrier function, resulting in bacterial and toxin translocation and intestinal immune activation and inflammation against sepsis.

Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions.

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB116-132) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier.

The Role of Ferroptosis in Inflammatory Bowel Disease: Mechanisms and Therapeutic Implications

Inflammatory Bowel Disease (IBD), encompassing Crohn's disease and ulcerative colitis, is characterized by chronic inflammation of the gastrointestinal tract, with an increasing incidence worldwide. Recent advancements in cellular biology have identified ferroptosis, a form of programmed cell death driven by iron-dependent lipid peroxidation, as a critical player in the pathology of IBD. This article reviews the current understanding of ferroptosis and its distinctive mechanisms, including the role of GPx4, Nrf2-HO-1 pathways, and iron metabolism in the context of IBD. It also examines the dual nature of iron in intestinal health and disease, contributing to both physiological functions and pathological processes through oxidative stress and inflammation. The implications of ferroptosis in the intestinal epithelial cell death, barrier function, and immune response are discussed, highlighting its potential as a novel therapeutic target. Despite the promising insights, the article underscores the need for further research to elucidate the complex mechanisms of ferroptosis in IBD and to translate these findings into effective therapeutic strategies. The emerging evidence positions ferroptosis at the crossroads of metabolic, inflammatory, and cell death pathways, offering a unique perspective on the interplay between nutrition, genetics, and immunity in intestinal health and disease.

Probiotic Consortia Protect the Intestine Against Radiation Injury by Improving Intestinal Epithelial Homeostasis

PurposeRadiation-induced intestinal injuries (RIII) commonly occur during abdomin-pelvic cancer radiotherapy; however, no effective prophylactic or therapeutic agents are available to manage RIII currently. This study aimed to clarify the potential of probiotic consortium supplementation in alleviating RIII. Materials and MethodsMale C57BL/6J mice were orally administered a probiotic mixture comprising Bifidobacterium longum BL21, Lactobacillus paracasei LC86, and Lactobacillus plantarum Lp90 for 30 days before exposure to 13 Gy of whole abdominal irradiation (WAI). The survival rates, clinical scores, and histological changes in the intestines of mice were assessed. The impacts of probiotic consortium treatment on intestinal stem cells (ISCs) proliferation, differentiation, and epithelial barrier function, oxidative stress, and inflammatory cytokines were evaluated. A comprehensive examination of the gut microbiota composition was conducted through 16S rRNA sequencing, while changes in metabolites were identified using liquid chromatography-mass spectrometry. ResultsThe probiotic consortium alleviated RIII, as reflected by increased survival rates, improved clinical scores, and mitigated mucosal injury. The probiotic consortium treatment exhibited enhanced therapeutic effects at the histological level when compared to individual probiotic strains, although there was no corresponding improvement in survival rates and colon length. Moreover, probiotic consortium stimulated ISCs proliferation and differentiation, enhanced the integrity of the intestinal epithelial barrier, and regulated redox imbalance and inflammatory responses in irradiated mice. Notably, the treatment induced a restructuring of gut microbiota composition, particularly enriching short-chain fatty acid-producing bacteria. Metabolomic analysis revealed distinctive metabolic changes associated with probiotic consortium, including elevated levels of anti-inflammatory and anti-radiation metabolites. ConclusionsThe probiotic consortium attenuated RIII by modulating the gut microbiota and metabolites, improving inflammatory symptoms, and regulating oxidative stress. These findings provide new insights into the maintenance of intestinal health with the probiotic consortium supplementation and will facilitate the development of probiotic-based therapeutic strategies for RIII in clinical practice.

Lactate Protects Intestinal Epithelial Barrier Function from Dextran Sulfate Sodium-Induced Damage by GPR81 Signaling.

The dysregulation of the intestinal epithelial barrier significantly contributes to the inflammatory progression of ulcerative colitis. Recent studies have indicated that lactate, produced by gut bacteria or derived from fermented foods, plays a key role in modulating inflammation via G-protein-coupled receptor 81 (GPR81). In this study, we aimed to investigate the potential role of GPR81 in the progression of colitis and to assess the impact of lactate/GPR81 signaling on intestinal epithelial barrier function. Our findings demonstrated a downregulation of GPR81 protein expression in patients with colitis. Functional verification experiments showed that Gpr81-deficient mice exhibited more severe damage to the intestinal epithelial barrier and increased susceptibility to DSS-induced colitis, characterized by exacerbated oxidative stress, elevated inflammatory cytokine secretion, and impaired expression of tight-junction proteins. Mechanistically, we found that lactate could suppress TNF-α-induced MMP-9 expression and prevent the disruption of tight-junction proteins by inhibiting NF-κB activation through GPR81 in vitro. Furthermore, our study showed that dietary lactate could preserve intestinal epithelial barrier function against DSS-induced damage in a GPR81-dependent manner in vivo. Collectively, these results underscore the crucial involvement of the lactate/GPR81 signaling pathway in maintaining intestinal epithelial barrier function, providing a potential therapeutic strategy for ulcerative colitis.

Campylobacter jejuni Surface-Bound Protease HtrA, but Not the Secreted Protease nor Protease in Shed Membrane Vesicles, Disrupts Epithelial Cell-to-Cell Junctions.

Fundamental functions of the intestinal epithelium include the digestion of food, absorption of nutrients, and its ability to act as the first barrier against intruding microbes. Campylobacter jejuni is a major zoonotic pathogen accounting for a substantial portion of bacterial foodborne illnesses. The germ colonizes the intestines of birds and is mainly transmitted to humans through the consumption of contaminated poultry meat. In the human gastrointestinal tract, the bacterium triggers campylobacteriosis that can progress to serious secondary disorders, including reactive arthritis, inflammatory bowel disease and Guillain-Barré syndrome. We recently discovered that C. jejuni serine protease HtrA disrupts intestinal epithelial barrier functions via cleavage of the tight and adherens junction components occludin, claudin-8 and E-cadherin. However, it is unknown whether epithelial damage is mediated by the secreted soluble enzyme, by HtrA contained in shed outer-membrane vesicles (OMVs) or by another mechanism that has yet to be identified. In the present study, we investigated whether soluble recombinant HtrA and/or purified OMVs induce junctional damage to polarized intestinal epithelial cells compared to live C. jejuni bacteria. By using electron and confocal immunofluorescence microscopy, we show that HtrA-expressing C. jejuni bacteria trigger efficient junctional cell damage, but not soluble purified HtrA or HtrA-containing OMVs, not even at high concentrations far exceeding physiological levels. Instead, we found that only bacteria with active protein biosynthesis effectively cleave junctional proteins, which is followed by paracellular transmigration of C. jejuni through the epithelial cell layer. These findings shed new light on the pathogenic activities of HtrA and virulence strategies of C. jejuni.

INFLAMMATION-TRIGGERED CLAUDIN-23 DOWNREGULATION IMPACTS INTESTINAL EPITHELIAL BARRIER FUNCTION

Abstract Intestinal epithelium maintains gut homeostasis by protecting the underlying tissue compartments from harmful contents. Although it is suggested that epithelial barrier function compromise contributes to the pathogenesis of inflammatory bowel disease (IBD), the underlying mechanisms are not well understood. Claudins (CLDN) are tight junctions (TJ) proteins that determine epithelial barrier properties during homeostasis and whose expression is altered in IBD, thereby contributing to a leaky barrier. We recently identified the expression of an atypical CLDN family member, CLDN23, in the regulation of homeostatic intestinal epithelial barrier function. Analysis of the human colonic crypt-luminal axis revealed increased CLDN23 expression in differentiated intestinal epithelial cells (IEC) facing the lumen compared to the proliferative crypt-base IECs. Complementary cell biologic approaches using IEC-specific Cldn23 knockout mice (Cldn23ERΔIEC) and human colon cell lines revealed that CLDN23 strengthens epithelial barrier function. Mechanistically, CLDN23 promoted redistribution of barrier-forming CLDN3 and CLDN4 from the lateral membrane to the TJ through cis- and trans-interactions, resulting in narrowed CLDN3 and CLDN4 pore diameters. Since inflammation compromises the epithelial barrier and contributes to disease pathogenesis in IBD, we first examined CLDN23 expression in colonic mucosal tissue from IBD individuals. Immunofluorescence and RNA scope labeling revealed that CLDN23 expression was significantly decreased in colonic epithelial cells of IBD individuals. Furthermore, reduced CLDN23 expression was also detected in primary colonic epithelial cells (colonoids) from mice treated with pro-inflammatory cytokines TNFα and IFNγ compared to untreated colonoids. Barrier function assays of control Cldn23f/f and Cldn23ERΔIEC mice-derived colonoids treated with TNFα and IFNγ identified decreased transepithelial electrical resistance in colonoids lacking CLDN23 and treated with these pro-inflammatory cytokines. These findings suggest a protective role for CLDN23 in regulating barrier function under homeostatic and inflammatory conditions.

P172 Spatial Transcriptomics Characterization of S1P Pathway Genes in Ulcerative Colitis and Crohn’s Disease Colon Tissues

Abstract Background Inflammatory bowel diseases (IBD), such as ulcerative colitis (UC) and Crohn’s disease (CD) are chronic inflammatory diseases of the gastrointestinal tract, related to the confluence of environmental and immunological factors in genetically predisposed individuals. Activated T and B cells migrate from the lymphoid tissue into the circulation through the interaction of Sphingosine-1-phosphate (S1P) and the S1P1 receptor (S1P1), which can perpetuate inflammation and lead to tissue damage. Targeting of cellular migratory pathways has emerged as a novel therapeutic paradigm in IBD. The aim of this study is to characterize the S1P pathway-related genes and signaling pathways driving the pathogenesis of UC and CD. Methods Spatial transcriptomics analysis was performed in health volunteer (HV, n=2), UC (n=4) and CD (n=2) FFPE colon tissues using the GeoMX platform and NanoString whole transcriptome atlas (WTA) assay. The WTA panel was run on 8 samples stained with antibodies to S1P1, pan Cytokeratin, and CD45. Six regions of interests (ROIs) were selected in each sample based on the staining and each ROI were split into two areas (CD45+ or CD45-). Differential gene expression and enriched pathways analysis including S1P pathway genes were performed in CD45+ and CD45- segments in HV, UC and CD colon tissues. Results A total of 6085 and 6054 differentially expressed genes were identified from the CD45+ segments in UC and CD respectively, compared to HV colon (P=0.05), including the genes that are involved in T cell activation (e.g., HLA-DPA1, CCDC13), immune cell migration (e.g., ADAM15, CCL5) and tissue damage (e.g., FABP6, PDK4). Interestingly, significant differences were identified in UC and CD in S1P pathway gene expression. Specifically, SMPD1, a gene involved in generating ceramide and regulating inflammatory responses, was the most significantly up-regulated gene from both CD45+ and CD45- compartments in UC compared to HV colon. While CERS2 and CERS6, genes related to S1P metabolism, were significantly down-regulated genes in CD45+ and CD45- compartment in UC respectively, as compared to HV colon. Additionally, RTN4 (participates in the intestinal epithelial barrier function) and PLPP3 (dephosphorylates extracellular S1P to sphingosine) were most significantly down-regulated genes from CD45+ segments in both UC and CD compared to HV colon. Conclusion In summary, our study successfully validated the GeoMx transcriptome analysis approach in FFPE colon tissue and assessed the S1P pathway gene signature difference in HV, UC and CD colon tissues supporting the pathway’s role in IBD. Results could also facilitate the identification of potential S1P pathway UC and CD disease biomarkers.

P095 Accelerated degradation of gut barrier-protecting 3-aminobenzoic acid exacerbate colitis in Inflammatory Bowel Disease

Abstract Background Disruption of intestinal epithelial cell (IEC) barrier function is crucial for the development of inflammatory bowel disease (IBD). Decreased diversity and compositional changes in intestinal bacteria, namely dysbiosis, impair IEC barrier function in IBD, but the underlying mechanism is not fully understood. This study aims to identify the impact of dysbiosis-driven changes in intestinal bacterial metabolites on IEC permeability in patients with IBD. Methods Caco2 cells were cultured on the trans-well inserts and stimulated with a library of intestinal bacterial metabolites. Proportional changes of trans-epithelial electrical resistance (TEER) were calculated at 48 hours after stimulation (ΔTEER%) and the permeation of 70-kDa FITC-dextran was evaluated using a Qubit fluorometer. Fecal samples were collected from 10 IBD patients with active colonic inflammation (5 ulcerative colitis and 5 Crohn’s disease) and 5 healthy controls (non-IBD, NIBD). Fecal bacteria-derived metabolites were quantified by LC-MS/MS and compared between IBD patients and NIBD controls. 16SrRNA-based metagenomic analysis was performed on the same fecal samples using QIIME2 followed by a PICRUSt2-based functional analysis of microbial communities. Anti-inflammatory effect of 3-aminobenzoic acid (3-ABA) was examined in C57BL/6 mice using a dextran sodium sulfate (DSS)-induced colitis model. Results Among 119 intestinal bacterial metabolites, 4-ABA decreased epithelial permeability the most with the highest ΔTEER% value (control vs. 4-ABA, 1.8 vs. 30.9 %, p<0.05). 3-ABA, one of three structural isomers of ABA, also decreased epithelial permeability in a dose dependent manner with higher ΔTEER% value than 4-ABA and reduced permeation of FITC-dextran (control vs. 3-ABA, 8.3 vs 3.4 nM, p<0.05). Fecal level of 3-ABA was significantly lower in IBD patients than in NIBD controls (7.0 vs. 505.0 μM). Functional prediction of intestinal microbiota demonstrated that IBD patients were significantly more enriched with the bacteria involved in the degradation of ABA than NIBD controls. Rectal administration of 3-ABA significantly improved body-weight loss, shortening of colon length, histologic scores, and the expression of inflammatory cytokines in mice with DSS-induced colitis (control vs. 3-ABA; body-weight loss,15.33 vs. 18.91 g; colon length, 6.11 vs. 7.35 cm; histologic score, 2.14 vs 0.60). Conclusion Accelerated degradation of luminal 3-ABA by intestinal bacteria may disrupt intestinal epithelial barrier function and exacerbate colitis in patients with IBD.

A novel Trichinella spiralis serine proteinase disrupted gut epithelial barrier and mediated larval invasion through binding to RACK1 and activating MAPK/ERK1/2 pathway.

Gut epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism by which larval penetration of gut epithelium is not completely elucidated. Previous studies showed that proteases secreted by T. spiralis intestinal infective larvae (IIL) degraded tight junctions (TJs) proteins of gut epithelium and mediated larval invasion. A new T. spiralis serine proteinase (TsSPc) was identified in the IIL surface proteins and ES proteins, rTsSPc bound to the intestinal epithelial cell (IECs) and promoted larval invasion of IECs. The aim of this study was to characterize the interacted proteins of TsSPc and IECs, and to investigate the molecular mechanisms of TsSPc mediating larval invasion of gut mucosa. IIFT results showed natural TsSPc was detected in infected murine intestine at 6, 12 hours post infection (hpi) and 3 dpi. The results of GST pull-down, mass spectrometry (MS) and Co-IP indicated that rTsSPc bound and interacted specifically with receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells. rTsSPc did not directly hydrolyze the TJs proteins. qPCR and Western blot showed that rTsSPc up-regulated RACK1 expression, activated MAPK/ERK1/2 pathway, reduced the expression levels of gut TJs (occludin and claudin-1) and adherent protein E-cad, increased the paracellular permeability and damaged the integrity of intestinal epithelial barrier. Moreover, the RACK1 inhibitor HO and ERK1/2 pathway inhibitor PD98059 abolished the rTsSPc activating ERK1/2 pathway, they also inhibited and abrogated the rTsSPc down-regulating expression of occludin, claudin-1 and E-cad in Caco-2 monolayer and infected murine intestine, impeded larval invasion and improved intestinal epithelial integrity and barrier function, reduced intestinal worm burdens and alleviated intestinal inflammation. rTsSPc bound to RACK1 receptor in gut epithelium, activated MAPK/ERK1/2 pathway, decreased the expression of gut epithelial TJs proteins and disrupted the epithelial integrity, consequently mediated T. spiralis larval invasion of gut epithelium. The results are valuable to understand T. spiralis invasion mechanism, and TsSPc might be regarded as a vaccine target against T. spiralis invasion and infection.

Characterizing core microbiota and regulatory functions of the pig gut microbiome.

Domestic pigs (Sus scrofa) are the leading terrestrial animals used for meat production. The gut microbiota significantly affect host nutrition, metabolism, and immunity. Hence, characterization of the gut microbial structure and function will improve our understanding of gut microbial resources and the mechanisms underlying host-microbe interactions. Here, we investigated the gut microbiomes of seven pig breeds using metagenomics and 16S rRNA gene amplicon sequencing. We established an expanded gut microbial reference catalog comprising 17 020 160 genes and identified 4910 metagenome-assembled genomes. We also analyzed the gut resistome to provide an overview of the profiles of the antimicrobial resistance genes in pigs. By analyzing the relative abundances of microbes, we identified three core-predominant gut microbes (Phascolarctobacterium succinatutens, Prevotella copri, and Oscillibacter valericigenes) in pigs used in this study. Oral administration of the three core-predominant gut microbes significantly increased the organ indexes (including the heart, spleen, and thymus), but decreased the gastrointestinal lengths in germ-free mice. The three core microbes significantly enhanced intestinal epithelial barrier function and altered the intestinal mucosal morphology, as was evident from the increase in crypt depths in the duodenum and ileum. Furthermore, the three core microbes significantly affected several metabolic pathways (such as "steroid hormone biosynthesis," "primary bile acid biosynthesis," "phenylalanine, tyrosine and tryptophan biosynthesis," and "phenylalanine metabolism") in germ-free mice. These findings provide a panoramic view of the pig gut microbiome and insights into the functional contributions of the core-predominant gut microbes to the host.

Induced acute hyperglycemia modifies the barrier function of the intestinal epithelium by tissue inflammation and tight junction disruption resulting in hydroelectrolytic secretion in an animal model.

Diabetic-metabolic syndrome (MetS-D) has a high prevalence worldwide, in which an association with the rupture of the intestinal epithelium barrier function (IEBF) has been pointed out, but the functional and morphological properties are still not well understood. This study aimed to evaluate the impact of acute hyperglycemia diabetes on intestinal tight junction proteins, metabolic failure, intestinal ion and water transports, and IEBF parameters. Diabetes was induced in male Rattus norvegicus (200-310 g) with 0.5 mL of streptozotocin (70 mg/kg). Glycemic and clinical parameters were evaluated every 7 days, and intestinal parameters were evaluated on the 14th day. The MetS-D animals showed a clinical pattern of hyperglycemia, with increases in the area of villi and crypts, lactulose:mannitol ratio, myeloperoxidase (MPO) activity, and intestinal tissue concentrations of malondialdehyde (MDA), but showed a reduction in reduced glutathione (GSH) when these parameters were compared to the control. The MetS-D group had increased secretion of Na+, K+, Cl-, and water compared to the control group in ileal tissue. Furthermore, we observed a reduction in mRNA transcript of claudin-2, claudin-15, and NHE3 and increases of SGLT-1 and ZO-1 in the MetS-D group. These results showed that MetS-D triggered intestinal tissue inflammation, oxidative stress, complex alterations in gene regulatory protein transcriptions of intestinal transporters and tight junctions, damaging the IEBF and causing hydroelectrolyte secretion.

D-Mannose promotes recovery from experimental colitis by inducing AMPK phosphorylation to stimulate epithelial repair.

Delayed mucosal healing and impaired intestinal epithelial barrier function have been implicated in the pathogenesis of ulcerative colitis (UC). Accordingly, restoration of epithelial barrier function as a means to reshape mucosal homeostasis represents an important strategy for use in the treatment of UC. In this study, we examined the role and mechanisms of D-mannose in the recovery of colitis as assessed in both animal and cell models. We found that D-mannose ameliorated inflammation, promoted mucosal healing in the colon and therefore was able to induce the recovery of UC. Furthermore, D-mannose increased the expression of tight junction (TJ) proteins and reduced the intestinal permeability during the recovery of colitis. Moreover, D-mannose inhibited M1 macrophage polarization and promoted M2 macrophage polarization via inducing AMPK phosphorylation while reducing mTOR phosphorylation in both models. In addition, increased TJ protein expression and decreased paracellular permeability were observed in NCM460 cells when incubated with the supernatants of D-mannose-treated RAW264.7 cells, suggesting that M1/M2 polarization induced by D-mannose modulates the expression of TJ proteins. Further study showed that D-mannose significantly upregulated the expression of TJ proteins in DSS-treated NCM460 cells by inducing AMPK phosphorylation, indicating a direct protective effect on epithelial cells. Finally, the protective effects of D-mannose were significantly abrogated by the presence of compound C, an AMPK inhibitor. Taken together, our data indicate that D-mannose can alleviate inflammation and foster epithelial restitution in UC recovery by inducing the TJ protein expression, which are achieved by inducing AMPK phosphorylation in the epithelium and/or macrophages.

SIRT1 Stabilizes β-TrCP1 to Inhibit Snail1 Expression in Maintaining Intestinal Epithelial Integrity to Alleviate Colitis

Dysfunction of the intestinal epithelial barrier comprising the junctional complex of tight junctions and adherent junctions leads to increased intestinal permeability, which is a major cause of uncontrolled inflammation related to inflammatory bowel disease (IBD). The NAD+-dependent deacetylase SIRT1 is implicated in inflammation and the pathologic process of IBD. We aimed to elucidate the protective role and underlying mechanism of SIRT1 in cell-cell junction and intestinal epithelial integrity. The correlation of SIRT1 expression and human IBD was analyzed by GEO or immunohistochemical analyses. BK5.mSIRT1 transgenic mice and wild-type mice were given dextran sodium sulfate (DSS) and the manifestation of colitis-related phenotypes was analyzed. Intestinal permeability was measured by FITC-dextran and cytokines expression was analyzed by quantitative polymerase chain reaction. The expression of the cell junction-related proteins in DSS-treated or SIRT1-knockdown Caco2 or HCT116 cells was analyzed by Western blotting. The effects of nicotinamide mononucleotide in DSS-induced mice colitis were investigated. Correlations ofthe SIRT1-β-TrCP1-Snail1-Occludin/Claudin-1/E-cadherin pathway with human IBD samples were analyzed. Reduced SIRT1 expression is associated with human IBD specimens. SIRT1 transgenic mice exhibit much-reduced manifestations of DSS-induced colitis. The activation of SIRT1 by nicotinamide mononucleotide bolsters intestinal epithelial barrier function and ameliorates DSS-induced colitis in mice. Mechanistically, DSS downregulates SiRT1 expression, leading to destabilization of β-TrCP1 and upregulation of Snail1, accompanied by reduced expression of E-cadherin, Occludin, and Claudin-1, consequently resulting in increased epithelial permeability and inflammation. The deregulated SIRT1-β-TrCP1-Snail1-Occludin/Claudin-1/E-cadherin pathway correlates with human IBD. SIRT1 is pivotal in maintaining the intestinal epithelial barrier integrity via modulation of the β-TrCP1-Snail1-E-cadhein/Occludin/Claudin-1 pathway.