Intestinal Epithelial Cells Research Articles

Phytochemical Constituents of Ocimum gratissimum extract and its effect against gastro-enteric bacteria on the intestinal linings of an albino Wister rats

Enteritis is the inflammation of the small intestines which could lead to diarrhea. Bacterial gastroenteritis is a digestive system problem caused by bacteria. The symptoms include nausea, vomiting, fever, abdominal cramps and diarrhea. These can be treated with antibiotics, but in recent times, especially in economically developing countries like Nigeria, these drugs have become extremely costly resulting to the low purchasing power of the greater populace. Antibiotic resistance due to abuse also necessitated a growing need for alternative and affordable source of anti-bacteria medication. Ocimum gratissimum leaves were collected, selected and pulverized, it was macerated in ethanol for 3days with intermediate agitation, filtered, and concentrated. The extract was subjected to LD50. Phytochemical analysis were done on the plants leaves to check for secondary metabolites. A pilot study of E.coli was carried out with 8 albino rats grouped into four. Different titrations of E.coli were administered intraperitoneal to the rats to determine the minimum amount of E.coli capable of infecting the rats, the stool sample was inoculated in a sterile broth and then cultured in an EMB agar plate selective for E.coli. After 24 hours culture, a 0.8 mL was selected as it showed the least colony. The extract of Ocimum gratissimum was formulated using 0.5%w/v, 1.25%w/v and 0.9% normal saline as control in Group A, B and C respectively. These were used to treat the 20 albino wistar rats grouped A to D (at n = 5). Group D Negative control was not infected with 0.8 mL of E.coli broth. The results showed that Ocimum gratissimum is rich in flavonoids, tannins, saponins, resins, terpenoids, alkaloids, fats and oils. Pictorial results of the histopathology shown that albino rats of group A and C have normal to mild intestinal tract, not erupted or distorted but that it is dose dependent as group B with 1.25%w/v extract restored the intestinal linings of the albino rats to that of the Negative control which was not infected intraperitoneal. Results proved that Ocimum gratissimum has activity against gastroenteritis bacteria E. coli and can serve as an alternative in treatment of bacteria enteritis as it is readily available and affordable.

Protective effect of soluble protein hydrolysate against H2O2‑induced intestinal injury: An interventional study.

The present study aimed to investigate whether soluble protein hydrolysate (SPH) protects against intestinal oxidative stress injury. An in vitro lactate dehydrogenase assay was used to assess the cytotoxicity and protective effect of SPH. For in vivo assessment, friend virus B NIH Jackson mouse pups aged 21 days were administered with 5% w/v soluble protein hydrolysate (SPH) through drinking water for 14 days and then luminally injected with 0.3% or 0.6% H2O2. Thereafter, the fecal samples of mice were collected, and the mice were sacrificed. Intestinal epithelial injury was assessed, and the expressions of 84 oxidative stress‑related genes in intestinal tissues was determined. SPH prophylactically protected against H2O2‑induced oxidative stress injury in human intestinal epithelial cells. An animal model of oxidative stress‑induced intestinal injury was established using 0.3 and 0.6% H2O2. SPH treatment reduced oxidative stress (0.3% H2O2)‑induced gut injury in mice. As no accelerated body growth was observed in SPH‑treated mice, it was hypothesized that the underlying protective mechanism of SPH is not related to nutrient oversupply. Treatment with SPH upregulated five oxidative protective genes that were not consistent between the sexes. Some antioxidative genes, including ferritin heavy polypeptide‑1 (Fth1), heme oxygenase‑1 (Hmox1), NAD(P)H dehydrogenase quinone 1 (Nqo1) and superoxide dismutase 1 (Sod1), were commonly upregulated in both male and female mice. Overall, an antioxidative protective effect was observed following SPH treatment, which may be attributed to the upregulation of genes that protect against oxidative damage. The findings of the present study highlight the promising potential of SPH as a functional food for alleviating intestinal oxidative stress injury.

P-1845. Evaluation of the immunogenicity and protective efficacy of a fusion vaccine based on the class 5a fimbrial tip of Enterotoxigenic Escherichia coli (ETEC) in an Aotus nancymaae CS14+ and CFA/I+ challenge model

Abstract Background Enterotoxigenic Escherichia coli (ETEC) colonizes intestinal epithelial cells via colonization factors (CFs), fimbrial, non-fimbrial, and helical adhesins. Once attached, ETEC can release enterotoxins including heat-stable toxin (ST) and/or heat-labile toxin (LT) which cause hypersecretion of fluid in the intestinal lumen resulting in watery diarrhea. Currently, there are no effective countermeasures against ETEC, and antibiotic treatments are losing effectiveness due to increasing antimicrobial resistance. Passive or active immunization against CFs are alluring targets to prevent adhesion, colonization, and ultimately, diarrhea. However, ETEC which infects humans exhibits remarkable heterogeneity and expresses at least 29 immunologically different CFs, making it difficult to create a broadly targeting vaccine that is effective in preventing diarrhea caused by multiple ETEC strains. One third of the CFs that have been described belong to one of three phylogenetic families: CFA/I-like family, which is defined as Class 5 fimbriae divided into three subclasses, 5a, 5b and 5c that are highly related in structure and function. Methods In this work, we evaluated the efficacy of a vaccine based on the CFA/I adhesin-pilin fusion (CfaEB) fused to the pilins of colonization factor CS14 (CsuA2) and CS4 (CsfA) to generate broad cross-protection against members of subclass 5a. We previously established a non-human primate challenge model for CS14+ ETEC (strain WS3294A) and found that a 5x1012 CFU dose was needed to achieve a sufficiently high (70%) attack rate. Therefore, the same challenge dose was used to evaluate the effectiveness of the vaccine candidate CfaEp3 (CfaEB-CsuA2-CsfA) against ETEC H10407 (CFA/I; n=10) and WS3294A (CS14; n=10). Results Animals that received four vaccine doses with dmLT adjuvant, administered intradermally, were protected from challenge with WS3294A (91% efficacy; p=0.05). However, only 20% protection against ETEC H10407 was achieved. Conclusion Although the vaccine candidate did not present cross-protection against members of subclass 5a, the model opens the possibility of future testing of combined vaccines to combat infection with the main pathogenic strains of ETEC encountered by travelers, the military, and civilian populations. Disclosures All Authors: No reported disclosures

E. Coli cytotoxic necrotizing factor-1 promotes colorectal carcinogenesis by causing oxidative stress, DNA damage and intestinal permeability alteration

BackgroundBacterial toxins are emerging as promising hallmarks of colorectal cancer (CRC) pathogenesis. In particular, Cytotoxic Necrotizing Factor 1 (CNF1) from E. coli deserves special consideration due to the significantly higher prevalence of this toxin gene in CRC patients with respect to healthy subjects, and to the numerous tumor-promoting effects that have been ascribed to the toxin in vitro. Despite this evidence, a definitive causal link between CNF1 and CRC was missing. Here we investigated whether CNF1 plays an active role in CRC onset by analyzing pro-carcinogenic key effects specifically induced by the toxin in vitro and in vivo.MethodsViability assays, confocal microscopy of γH2AX and 53BP1 molecules and cytogenetic analysis were carried out to assess CNF1-induced genotoxicity on non-neoplastic intestinal epithelial cells. Caco-2 monolayers and 3D Caco-2 spheroids were used to evaluate permeability alterations specifically induced by CNF1, either in the presence or in the absence of inflammation. In vivo, an inflammatory bowel disease (IBD) model was exploited to evaluate the carcinogenic potential of CNF1. Immunohistochemistry and immunofluorescence stainings of formalin-fixed paraffin-embedded (FFPE) colon tissue were carried out as well as fecal microbiota composition analysis by 16 S rRNA gene sequencing.ResultsCNF1 induces the release of reactive oxidizing species and chromosomal instability in non-neoplastic intestinal epithelial cells. In addition, CNF1 modifies intestinal permeability by directly altering tight junctions’ distribution in 2D Caco-2 monolayers, and by hindering the differentiation of 3D Caco-2 spheroids with an irregular arrangement of these junctions. In vivo, repeated intrarectal administration of CNF1 induces the formation of dysplastic aberrant crypt foci (ACF), and produces the formation of colorectal adenomas in an IBD model. These effects are accompanied by the increased neutrophilic infiltration in colonic tissue, by a mixed pro-inflammatory and anti-inflammatory cytokine milieu, and by the pro-tumoral modulation of the fecal microbiota.ConclusionsTaken together, our results support the hypothesis that the CNF1 toxin from E. coli plays an active role in colorectal carcinogenesis. Altogether, these findings not only add new knowledge to the contribution of bacterial toxins to CRC, but also pave the way to the implementation of current screening programs and preventive strategies.

TccP4: a novel effector identified in the Escherichia albertii strain 1551-2 required for attaching and effacing lesion formation on infected Nck-null cells.

Escherichia albertii is a pathogen that causes sporadic cases and outbreaks of diarrhea. The main virulence feature of this bacterium is the attaching and effacing (AE) lesion formation on infected intestinal epithelial cells, which is characterized by the formation of pedestal-like structures that are rich in F-actin. The Brazilian E. albertii 1551-2 strain can recruit F-actin using both the Nck-dependent and the Nck-independent pathways, the latter of which uses an adaptor protein named Tir-cytoskeleton coupling protein (TccP/EspFU). Genome analyses of the 1551-2 strain unveiled the existence of a gene encoding a putative novel TccP subtype in addition to a gene encoding for the TccP3 subtype. Amino-acid sequence comparison with known TccP subtypes (TccP/EspFU, TccP2, and TccP3) confirmed that the protein represents a novel TccP subtype-named here TccP4. Lack of TccP4 led to an approximately 96% reduction in the ability of the tccP3 deletion mutant of strain 1551-2 to induce the F-actin-rich pedestals formation in the infected Nck-null mouse embryonic fibroblasts (MEF) cells. The tccP4 gene was distributed widely in E. albertii, including the strains first separated from other E. albertii strains, suggesting that this gene was acquired at a very early stage during the diversification of E. albertii. The highly variable genetic organization of the tccP4-containing regions and the presence of various mobile genetic elements in this region may explain the lack of tccP4 in E. albertii strains belonging to various lineages.IMPORTANCEE. albertii, one of the new members of the genus Escherichia, is a diarrheagenic pathogen. The main characteristic of its pathogenicity is the formation of attaching and effacing (AE) lesions on the surface of infected epithelial cells. Here we identified a novel subtype of the TccP type 3 secretion system (T3SS) effector family (termed TccP4), which is required for the recruitment of F-actin during the AE lesion formation in infected host cells by the E. albertii 1551-2 strain. We also revealed that TccP4 is unique to E. albertii and widely distributed in this species, suggesting that the tccP4 gene was acquired at a very early stage during the diversification process of E. albertii. These findings expand our understanding of the function and diversity of this important T3SS effector family.

Intestinal Epithelial Ptpn23 Is Essential For Gut Barrier Integrity And Prevention Of Fatal Bacterial Translocation.

Protein tyrosine phosphatase non-receptor type 23 (PTPN23) regulates the internalization of growth factor receptors such as the epithelial growth factor receptor (EGFR). Given the crucial function of such receptors in intestinal epithelial cells (IECs), we assessed the involvement of PTPN23 in intestinal homeostasis and epithelial proliferation. We generated mouse models with constitutive (PTPN23fl/flVilCre+/-) or inducible (PTPN23fl/flVilCreERT+/-) deletion of PTPN23 in IEC. To elucidate the functional consequences of PTPN23 deletion in IEC, we performed barrier function studies, flow cytometry, RNAseq and in vivo experiments applying EGFR inhibition, antibiotic treatment, or co-housing approaches to further delineate the observed phenotype. Deletion of PTPN23 in IECs resulted in a severe early-onset phenotype in both models. Mice were characterized by elongated colon, epithelial hyperproliferation, splenomegaly and diarrhea leading to the death of the mice within 3 weeks of PTNP23 deletion. Compromised gut barrier integrity resulted in enhanced bacterial translocation accompanied by reduced IgA transcytosis in PTPN23fl/flVilCreERT+/- compared to wild-type mice. Although EGFR surface expression was increased upon PTPN23-deletion, inhibition of EGFR signaling did not prevent disease. In contrast, and in accordance with defective bacterial handling, antibiotic treatment, but not co-housing, fully rescued the phenotype. The absence of PTPN23 in IECs leads to lethal dysregulation of intestinal homeostasis, triggered by bacterial infiltration due to defects in the intestinal epithelial barrier and impaired IgA transcytosis. Thus, we identify PTPN23 as a novel key player in preserving intestinal epithelial homeostasis, ultimately preventing bacterial overgrowth and excessive immune activation in the intestine.

Antioxidative Function of Zinc and Its Protection Against the Onset and Progression of Kidney Disease Due to Cadmium

Chronic kidney disease (CKD) is now the world’s top seventh cause of death from a non-communicable disease, and its incidence is projected to increase further as its major risk factors, including obesity, diabetes, hypertension, and non-alcoholic fatty liver disease (NAFLD), continue to rise. Current evidence has linked the increased prevalence of CKD, diabetes, hypertension, and NAFLD to chronic exposure to the metal pollutant cadmium (Cd). Exposure to Cd is widespread because diet is the main exposure route for most people. Notably, however, the health risk of dietary Cd exposure is underappreciated, and the existing tolerable exposure guidelines for Cd do not afford health protection. New health-protective exposure guidelines are needed. From one’s diet, Cd is absorbed by the intestinal epithelium from where it passes through the liver and accumulates within the kidney tubular epithelial cells. Here, it is bound to metallothionine (MT), and as it is gradually released, it induces tubular damage, tubulointerstitial inflammation and fibrosis, and nephron destruction. The present review provides an update on our knowledge of the exposure levels of Cd that are found to be associated with CKD, NAFLD, and mortality from cardiovascular disease. It discusses the co-existence of hypertension and CKD in people environmentally exposed to Cd. It highlights nuclear and mitochondrial targeting and zinc deficiency as the universal cytotoxic mechanisms of Cd. Special emphasis is placed on the novel antioxidative function of zinc involving de novo heme biosynthesis and the induced expression of heme oxygenase-1 (HO-1). Other exogenous biomolecules with promising anti-Cd toxicity are highlighted.

Uptake of iron from ferrous fumarate can be mediated by clathrin-dependent endocytosis in Hutu-80 cells

Iron uptake in the intestinal epithelium is associated with transport of ferrous iron via the DMT1 transporter (SLC11a2; NRAMP2). In later years, uptake of iron from complex sources, such as nanoparticles, has been found to be mediated through endocytosis. Here we propose that iron from the simple salt ferrous fumarate, a common iron supplement, can be absorbed by clathrin-mediated endocytosis. We used siRNA to silence DMT1 transporter expression, pharmacological inhibition of endocytosis, and Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to show that iron uptake from ferrous fumarate can be mediated by both transport via DMT1 and by clathrin-dependent endocytosis in Hutu-80 cells. Iron uptake (ferritin L) from ferrous fumarate (0.5 mM, 24 h) in DMT1 silenced cells was significantly decreased (60% ± 11%) in comparison to iron controls while a 1-h dose of ferrous fumarate (0.5 mM) significantly decreased ferritin L formation in the presence of the clathrin inhibitor chlorpromazine (61% ± 10%, in post-confluent cells and 37% ± 9% in non-confluent cells). A pilot showed a similar trend for Ferritin (H) levels (confluent cells) and for total cellular iron load (non-confluent cells). ToF-SIMS analysis revealed diminished membrane-associated iron load in endocytosis-inhibited ferrous fumarate treated cells. The reported results support a clathrin-mediated endocytosis mechanism for uptake of iron from ferrous fumarate in addition to iron uptake by DMT1. More studies are needed to understand what determines which uptake mechanism are employed and to which extent.

Single-cell analyses of intestinal epithelium reveal the dysregulation of gut immune microenvironment in systemic lupus erythematosus

BackgroundThe small intestine harbors a rich array of intestinal intraepithelial lymphocytes (IELs) that interact with structural cells to collectively sustain gut immune homeostasis. Dysregulation of gut immune homeostasis was implicated in the pathogenesis of multiple autoimmune diseases, however, whether this homeostasis is disrupted in a lupus autoimmune background remains unclear.MethodsWe performed single-cell RNA sequencing (scRNA-seq) analyses to elucidate immune and structural milieu in the intestinal epithelium of MRL/Lpr lupus mice (Lpr mice) and MRL/Mpj control mice (Mpj mice). Comprehensive analyses including unsupervised clustering, trajectories, and cellular communication were performed. The primary findings from scRNA-seq were further validated by quantitative polymerase chain reaction (qPCR), flow cytometry, and in vivo experiments including selenium supplementation.ResultsWe observed a significant reduction in CD8αα + IELs, accompanied by a marked increase in CD8αβ + IELs in Lpr mice. Additionally, subsets of CD8 + IELs exhibiting significantly enhanced effector functions were found to be markedly enriched in Lpr mice. Intercellular communication patterns within intestinal epithelial immune and structural cells were found to be specifically altered in Lpr mice. Moreover, scRNA-seq revealed significantly decreased intestinal TCRγδ T cells (γδT) associated with reduced aryl-hydrocarbon receptor repressor (AHRR) expression and subsequent oxidative stress and ferroptosis in Lpr mice. Antioxidant selenium effectively reversed the loss of γδT in Lpr mice, improved the gut barrier, and alleviated lupus symptoms.ConclusionsOur high-resolution single-cell atlas enhances the understanding of the immune and structural milieu of intestinal epithelium in lupus and provides new insights into lupus pathogenesis mediated by intestinal immune dysregulation.

Hatching of whipworm eggs induced by bacterial contact is serine-protease dependent.

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage, and visualised the effects of structural changes in the polar plugs, leading to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing 'hatch-ready' L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes.

Morin Reactivates Nrf2 by Targeting Inhibition of Keap1 to Alleviate Deoxynivalenol-Induced Intestinal Oxidative Damage

As a prevalent mycotoxin found in cereal foods and feed, deoxynivalenol (DON) disrupts the orderly regeneration of intestinal epithelial tissue by interfering with the intracellular antioxidant defense system. However, the potential of mulberry leaf-derived Morin, a natural flavonoid active substance with clearing reactive oxygen species (ROS), to mitigate DON-induced intestinal oxidative damage remains unclear. Our investigation demonstrates that Morin effectively reverses the decline in growth performance and repairs damaged jejunal structures and barrier function under DON exposure. Furthermore, the proliferation and differentiation of intestinal stem cells (ISCs) is enhanced significantly after Morin intervention. Importantly, Morin increases the levels of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) in the serum and jejunal tissue, while reducing the accumulation of ROS and malondialdehyde (MDA). Molecular interaction analysis further confirms that Morin targets inhibition of Keap1 to activate the Nrf2-mediated antioxidant system. In summary, our results suggest that Morin alleviates the oxidative damage induced by DON by regulating the Keap1/Nrf2 pathway, thereby restoring the proliferation and differentiation activity of ISC, which provides new insights into Morin mitigating DON damage.

Cellular uptake and transport mechanism of lycopene-loaded nanomicelles formed by amphiphilic peptide self-assembly in the intestinal epithelium.

This study aimed to elucidate the transport mechanism of lycopene-loaded nanomicelles to improve intestinal absorption of lycopene. The interactive mechanism between lycopene and nanomicelles was investigated through isothermal titration calorimetry (ITC). The cytotoxicity, cellular uptake, endocytosis, and intracellular transport pathways of lycopene-loaded nanomicelles were investigated using the Caco-2 cell model. The ITC results demonstrated that nanomicelles/lycopene binding was an entropy-driven spontaneous exothermic reaction, and hydrophobic interactions were the main driving force. Lycopene-loaded nanomicelles were not cytotoxic, and uptake of lycopene by Caco-2 cells increased 2.20-fold after nanoencapsulation. The results of intracellular transport of lycopene-loaded nanomicelles indicated that the endoplasmic reticulum, Golgi apparatus, and lysosomes play key roles in this process. The intracellular transport results showed that the endoplasmic reticulum, Golgi apparatus, and lysosomes were important organelles for intracellular transport of lycopene-loaded nanomicelles. Nanomicelles effectively enhance the cellular uptake of lycopene and are promising carriers for its delivery. This study contributes to the understanding of the transport mechanism of nanomicelles through intestinal epithelial cells. © 2025 Society of Chemical Industry.

Disrupting of IGF2BP3-stabilized CLDN11 mRNA by TNF-α increases intestinal permeability in obesity-related severe acute pancreatitis

BackgroundObesity is a significant risk factor for severe acute pancreatitis (SAP) and is typically associated with increased intestinal permeability. Understanding the role of specific molecules can help reduce the risk of developing SAP. Claudin 11 (CLDN11), a member of the Claudin family, regulates the permeability of various internal barriers. However, the role and mechanism of CLDN11 in the intestinal permeability of obesity-related SAP remain unclear.MethodsWe evaluated intestinal permeability and the expression of CLDN11 in experimental obesity-related SAP. A recombinant adeno-associated virus carrying CLDN11 was used to treat experimental obesity-related SAP. The interaction between CLDN11 mRNA and insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) protein was predicted through bioinformatics analysis and validated by RNA immunoprecipitation and RNA pull-down assay. Additionally, tumor necrosis factor-α (TNF-α) treatment in Caco-2 cells was conducted, and the IGF2BP3/CLDN11 axis was detected. Moreover, we conducted anti-TNFα therapy and evaluated intestinal permeability and pancreatic inflammation in experimental obesity-related SAP.ResultsDownregulation of CLDN11 was observed in the intestinal epithelial cells of experimental obesity-related SAP. When the expression of CLDN11 in intestinal epithelial cells of experimental obesity-related SAP was increased exogenously, intestinal epithelial permeability and pancreatic inflammation were relieved. Overexpression of CLDN11 reduced the paracellular permeability of Caco-2 monolayer cells, while knockdown of CLDN11 increased it. IGF2BP3 bound to and regulated the stability of CLDN11 mRNA. TNF-α treatment downregulated IGF2BP3 and CLDN11 in vitro. Anti-TNFα therapy reduced intestinal permeability, alleviated pancreatitis, and improved the expression of IGF2BP3 and CLDN11 in intestinal epithelial cells in experimental obesity-related SAP.ConclusionCLDN11 regulates intestinal permeability in obesity-related SAP. Mechanistically, an increase in TNF-α impaired the stability of IGF2BP3-dependent CLDN11 mRNA in obesity-related SAP.

Tea Polyphenols Relieve the Fluoride-Induced Oxidative Stress in the Intestinal Porcine Epithelial Cell Model

Prolonged excessive intake of fluoride (F) can result in fluorosis, leading to a range of tissue oxidative damages. Therefore, mitigating the oxidative stress induced by fluorosis has become a significant research concern. Consequently, how to relieve oxidative stress caused by fluorosis is an urgent matter. In the present study, intestinal porcine epithelial (IPEC-J2) cells were chosen to explore the underlying mechanism of tea polyphenols (TPs) on F-induced oxidative stress. The results show that the cytotoxicity of IPEC-J2 cells induced by F presented a dose-dependent manner according to cell viability. Additionally, F treatment inhibited the activity of T-SOD, CAT, and GSH-Px as well as their transcription levels, increased the reactive oxygen (ROS) formation and cell damage rates, and then promoted cell apoptosis through the results of TUNEL and mitochondrial membrane potential detection when compared with the IPEC-J2 cells from the control group. As the main antioxidant ingredient in tea, TPs alleviated F-induced cell oxidation and apoptosis via blocking F-induced ROS generation and LDH’s release, as well as promoting the transcription of tight junction (TJ) proteins and the activities of antioxidant enzymes in IPEC-J2 cells. These results provide a new treatment strategy for F-induced intestinal oxidative impairment.

Rhanterium Epapposum Essential Oil and Its Primary Compounds Control Infection, Inflammation, and Serum Electrolyte Imbalance in Mice with Giardiasis.

The present experimental study seeks to evaluate the in vitro and in vivo effects, as well as the potential mechanisms of action, of Rhanterium epapposum essential oil (REE) and its main constituents against Giardia lamblia infection. The analysis of REE was performed using the Gas Chromatography-Mass Spectrometry (GC-MS) detector. The in vitro effects of REE and its main constituents on viability of G. lamblia cysts and the human intestinal epithelial cell line, as well as their effects on plasma membrane permeability, the reactive oxygen species (ROS) generation, and trophozoite adherence were assessed by MTT assay. Effects of oral administration of REE and its main constituents at doses 10 and 20mg/kg on the number of Giardia cysts, the serum level of electrolytes of sodium (Na+) and potassium (K+), as well as the gene expression of inflammatory genes of TNF-α, IL-1β, IL-10, NF-κB p65, and Toll-like receptor 4 (TLR4)) were assessed. The 50% inhibitory concentrations (IC50) value of REE, MC, CP, LN and MTZ against G. lamblia cysts was 26.7, 32.1, 35.3, 45.1, and 42.1µg/mL, respectively; indicated that REE, MC, and CP displayed high antigiardial effects in comparison with MTZ. The obtained 50% cytotoxic concentration (CC50) value of REE, MC, CP, LN, and MTZ on HIEC-6 cells was 279.1, 363.4, 394.2, 478.3, and 422.1µg/mL, respectively. REE and its main compounds significantly (p < 0.001) increased the plasma membrane permeability and ROS generation in Giardia trophozoites, while, reduced the Giardia trophozoites adherence. In vivo, REE, MC, CP, and LN mainly at the dose of 20mg/kg considerably reduced the number and viability of Giardia cysts, modulated the serum level of electrolytes Na and K in the infected mice compared with the mice treated with MTZ (P < 0.01); whereas inhibited the inflammation cytokines in the infected mice compared with the mice treated with MTZ (p < 0.001). The current investigation showed that R. epapposum essential oil and its main compounds controlled the Giardia infection in mice by inhibiting inflammation, and serum electrolyte imbalance. After confirmation in further studies, these compounds may be considered for development as a novel therapeutic option for the treatment of giardiasis.

Inflammation-Induced Th17 Cells Synergize with the Inflammation-Trained Microbiota to Mediate Host Resiliency Against Intestinal Injury.

Inflammation can generate pathogenic Th17 cells and cause an inflammatory dysbiosis. In the context of inflammatory bowel disease (IBD), these inflammatory Th17 cells and dysbiotic microbiota may perpetuate injury to intestinal epithelial cells. However, many models of IBD like T-cell transfer colitis and IL-10-/- mice rely on the absence of regulatory pathways, so it is difficult to tell if inflammation can also induce protective Th17 cells. We subjected C57BL6, RAG1-/-, or JH-/- mice to systemic or gastrointestinal (GI) Citrobacter rodentium (Cr). Mice were then subjected to 2.5% dextran sodium sulfate (DSS) to cause epithelial injury. Fecal microbiota transfer was performed by bedding transfer and co-housing. Flow cytometry, qPCR, and histology were used to assess mucosal and systemic immune responses, cytokines, and tissue inflammation. 16s sequencing was used to assess gut bacterial taxonomy. Transient inflammation with GI but not systemic Cr was protective against subsequent intestinal injury. This was replicated with sequential DSS collectively indicating that transient inflammation provides tissue-specific protection. Inflammatory Th17 cells that have a tissue-resident memory (TRM) signature expanded in the intestine. Experiments with reconstituted RAG1-/-, JH-/- mice, and cell trafficking inhibitors showed that inflammation-induced Th17 cells were required for protection. Fecal microbiota transfer showed that the inflammation-trained microbiota was necessary for protection, likely by maintaining protective Th17 cells in situ. Inflammation can generate protective Th17 cells that synergize with the inflammation-trained microbiota to provide host resiliency against subsequent injury, indicating that inflammation-induced Th17 TRM T cells are heterogenous and contain protective subsets.

Trichinella spiralis excretory/secretory proteins mediated larval invasion via inducing gut epithelial apoptosis and barrier disruption.

Intestinal larva invasion is a crucial step of Trichinella spiralis infection. Intestinal infective larvae (IIL) and their excretory/secretory proteins (ESP) interact with gut epithelium, which often results in gut epithelium barrier injuries. Previous studies showed when T. spiralis invaded intestinal epithelium cells, the IIL ESP disrupted the tight junctions (TJs) of Caco-2 monolayer, but the mechanism is not clear. The IIL ESP might cause gut epithelial apoptosis, weaken the gut barrier and aid the larval invasion. The aim of this study was to investigate whether T. spiralis IIL ESP participate in enterocyte apoptosis and disrupt gut epithelial barrier to promote the larval invasion. Cell viability was assessed by CCK-8 assay and the results showed that 200 μg/ml of IIL ESP incubated with Caco-2 cells for 18 h inhibited the Caco-2 cell viability. The results of trans-epithelial electrical resistance (TEER) and FITC-dextran showed that IIL ESP decreased the TEER, increased FITC-dextran flux in Caco-2 monolayer. qPCR, Western blot and immunofluorescence test (IFT) showed that IIL ESP decreased the mRNA and protein expression of TJs (ZO-1, E-cad, Occludin and Claudin-1). The IIL ESP-induced Caco-2 cell apoptosis was observed by DAPI, Hoechst 33358, TUNEL and Annexin V/PI staining. Besides, flow cytometry revealed an increasing apoptosis rate in Caco-2 cells after the IIL ESP treatment. qPCR and Western blot analysis indicated that IIL ESP activated caspases (Caspase 3, Caspase 9 and Caspase 8), up-regulated the pro-apoptotic factors (Bax and Cytochrome c) and down-regulated the anti-apoptosis molecule Bcl-2. Interestingly, pretreatment of Caco-2 cells with apoptosis inhibitor Z-VAD-FMK abrogated and recovered the barrier function of Caco-2 monolayer destroyed by IIL ESP. Furthermore, the Z-VAD-FMK pretreatment also impeded the in vitro larva invasion of Caco-2 monolayer. T. spiralis IIL ESP induced gut epithelial apoptosis, reduced the TJs expression, damaged gut epithelial integrity and barrier function, and promoted larval invasion. These findings provided a basis of further understanding the interaction mechanism between T. spiralis and host gut epithelium, and they were valuable to the development new prevention and therapeutic strategy of early T. spiralis infection.

DOP075 Tissue fibrosis modulates interleukin (IL)-13 signaling in intestinal epithelial cells

Abstract Background Inflammatory Bowel Diseases (IBDs) are characterized by chronic inflammation of the intestinal tissue and persistent disruption of the intestinal epithelial barrier. A common and dangerous complication of chronic inflammation during IBD is fibrosis, a mechanical stiffening of the intestinal tissue [1,2]. Previous work suggests that immune cell activity, for example Interleukin (IL)-13-mediated macrophage activation, can be mechanosensitive [3,4]. However, it is not currently well understood how fibrosis affects the inflammatory response in intestinal epithelial cells. Methods To address how tissue stiffness impacts intestinal inflammation, we studied the effects of inflammatory cytokine signaling in intestinal epithelial cells using a recently developed intestinal organoid monolayer model. We cultured intestinal organoid monolayers on soft (0.5kPa), intermediate (5kPa) or stiff (15kPa) hydrogels to mimic physiological or fibrotic conditions and studied the cellular response to IL-13, an inflammatory cytokine implicated in IBD and loss of barrier integrity. To investigate the impacts of substrate stiffness and IL-13 signaling on epithelial cell function, we combined high-resolution live microscopy, quantitative image analysis using AI-based models and biophysical measurements. Results We found that substrate mechanosensing and IL-13 signaling interact synergistically to modulate intestinal epithelial stem cell differentiation, leading to a dramatic increase in the frequency of secretory cells. Furthermore, organoids cultured on stiff substrates no longer respond to IL-13 treatment, suggesting that fibrotic conditions may promote an inflamed-like state even in the absence of inflammatory cytokines. To further investigate cross-talk between substrate mechanics and IL-13 signalling, we inhibited downstream IL-13 signalling and cell contractility. We found that both the stiffness- and IL-13-induced phenotypes could be rescued by either inhibiting myosin-2 activity or blocking downstream IL-13 signalling, further indicating a co-dependence and synergy of substrate stiffness and IL-13. Further experiments implicate the relocalization of myosin-2 to the cell cortex and increased nuclear accumulation of the mechanosensitive protein YAP in mediating this response. Experiments using mouse models of colitis further support a role for YAP in this process. Conclusion Taken together, this work reveals how tissue mechanics modulate the inflammatory response in the intestinal epithelium. Future translational work based on these findings will inspire new approaches to mitigate the effects of fibrosis during IBD.

DOP127 A novel epithelial junction gene associated with very early onset IBD: a mechanistic study using patient organoids and transgenic mouse models

Abstract Background Very early onset inflammatory bowel disease (VEO-IBD) is often associated with a severe disease course and unique characteristics including underlying monogenic etiologies in some cases. In this study, we mechanistically defined the function of CGNL1, an epithelial junctional protein gene whose pathogenic variants may contribute to VEO-IBD. Methods Whole exome sequencing (WES) was performed on the affected patient and all first-degree relatives. Intestinal epithelial organoids (IEOs) were generated from the patient’s mucosal biopsies and were characterized by microscopy, whole-mount immunostaining, transcriptomics and transmission electron microscopy (TEM). In addition, we generated a new Cgnl1-/- mice for dextran-sodium sulfate (DSS)-induced colitis and infection by Citrobacter rodentium which directly compromises intestinal epithelium. Integrated analysis was performed to determine CGNL1 expression in single-cell datasets. Results In this patient with VEO-IBD who required early total colectomy with pouch formation, coexistent primary sclerosing cholangitis (PSC) and celiac disease, WES identified two rare, novel, pathogenic variants in the CGNL1 gene. Both pathogenic variants were also present in the patient’s younger sister, who has a similar history including fulminant UC and PSC-autoimmune hepatitis overlap syndrome. Furthermore, multiple relatives of the patient suffer from IBD and other inflammatory conditions including celiac disease, eosinophilic esophagitis, and type-1 diabetes. The RNA and protein levels of CGNL1 were diminished in the patient’s intestinal epithelium compared to that of healthy individuals and other IBD patients. The patient’s IEOs from pouch body and pre-pouch ileum formed smaller 3D spheroids with altered cell composition and significantly impaired barrier function compared to those from normal controls (Figure 1). TEM demonstrated disrupted tight junctions and adherens junctions in the patient’s IEOs. Similarly, impaired junctional complexes in intestinal epithelial cells and elevated intestinal permeability were present in Cgnl1-/- mice, which also showed increased susceptibility to DSS-induced colitis (Figure 2) and C. rodentium infection. Single-cell analysis revealed diminished expression of CGNL1 in intestinal and hepatic epithelial cells from patients with IBD or PSC, respectively. Conclusion The pathogenic CGNL1 variants may contribute to VEO-IBD and other inflammatory GI disease by disrupting gut barrier integrity. This study expands our understanding of epithelial barrier impairment in the pathogenesis of VEO-IBD and suggests the intestinal epithelium as a therapeutic target for some IBD patients.

P0013 Protein-Energy Malnutrition Induces Microbiome-Dependent Long-Lasting Changes in Intestinal Epithelial Composition

Abstract Background Protein-energy malnutrition (PEM) in childhood is associated with increased susceptibility to infections, endocrine disorders, metabolic syndrome and organ dysfunction later in life1-4. However, the mechanisms behind these long-term effects are poorly understood. While the gut microbiome plays an important role in the aetiology of PEM, little is known about the long-term epithelial changes caused by the microbiome5-7. The aim of this project was to analyse the effects of PEM on the intestinal epithelium in the presence and absence of the microbiome. Methods Conventionally maintained (CONV-R) and germ-free (GF) mice were exposed to isocaloric PEM (0.7% protein) for 4 weeks, followed by a 6-week recovery period (20% protein). Multi-omics was used to analyse the effects of PEM. Changes in the composition of the intestinal epithelium were validated by immunostaining. An organoid differentiation model was used to analyse the hypotheses formulated under ex vivo PEM conditions. Results PEM caused a long-lasting reduction in body weight in CONV-R mice and extensive changes in the intestinal epithelium, including a significant depletion of Paneth cells, compared to GF mice. Furthermore, PEM disrupted microbiota composition and function, particularly affecting amino acid and lipid metabolism. Using a multi-omics approach, we identified the microbiota-derived PPAR-GDF15 axis as a critical mediator of PEM-induced Paneth cell dysfunction. These findings were validated using an ex vivo intestinal organoid model of PEM, which revealed that Paneth cell depletion requires not only the stress-induced cytokine GDF15 as a mediator of PEM, but also a secondary microbiota-dependent hit including lipid metabolites such as 9-HODE and 12,13-diHOME, which act as PPAR agonists. Conclusion PEM induces long-term microbiome-dependent changes in the intestinal epithelium. This malnutrition-microbiome-host axis may be a promising target for therapeutic intervention to counteract the persistent effects of malnutrition.