Intestinal Epithelial Cell Line Research Articles

IL-22 regulates MASTL expression in intestinal epithelial cells.

Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as a oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic CRC and colitis associated cancer (CAC). CAC is one of the most severe complications of chronic IBD, but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL expression in IBD patients and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from late-stage ulcerative colitis patients compared to controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, facilitating IL-22- mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Further, CAIX stabilizes MASTL by associating with it in response to IL-22 stimulation.

Alleviating Pentatrichomonas hominis-induced damage in IPEC-J2 cells: the beneficial influence of porcine-derived lactobacilli.

Pentatrichomonas hominis is a common intestinal parasitic protozoan that causes abdominal pain and diarrhea, and poses a zoonotic risk. Probiotics, known for enhancing immunity and pathogen resistance, hold promise in combating parasitic infections. This study aimed to evaluate two porcine-derived probiotics, Lactobacillus reuteri LR1 and Lactobacillus plantarum LP1, against P. hominis infections in pigs. Taxonomic identity was confirmed through 16S rRNA gene sequencing, with L. reuteri LR1 belonging to L. reuteri species and L. plantarum LP1 belonging to L. plantarum species. Both probiotics exhibited robust in vitro growth performance. Co-culturing intestinal porcine epithelial cell line (IPEC-J2) with these probiotics significantly improved cell viability compared with the control group. Pre-incubation probiotics significantly enhanced the mRNA expression of anti-oxidative response genes in IPEC-J2 cells compared with the PHGD group, with L. reuteri LR1 and L. plantarum LP1 significantly up-regulating CuZn-SOD、CAT and Mn-SOD genes expression (p < 0.05). The anti-oxidative stress effect of L. reuteri LR1 was significantly better than that of L. plantarum LP1 (p < 0.05). Furthermore, pre-incubation with the probiotics alleviated the P. hominis-induced inflammatory response. L. reuteri LR1 and L. plantarum LP1 significantly down-regulated IL-6、IL-8 and TNF-α gene expression(p < 0.05) compared with the PHGD group. The probiotics also mitigated P. hominis-induced apoptosis. L. reuteri LR1 and L. plantarum LP1 significantly down-regulated Caspase3 and Bax gene expression (p < 0.05), significantly up-regulated Bcl-2 gene expression (p < 0.05) compared with the PHGD group. Among them, L. plantarum LP1 showed better anti-apoptotic effect. These findings highlight the probiotics for mitigating P. hominis infections in pigs. Their ability to enhance anti-oxidative responses, alleviate inflammation, and inhibit apoptosis holds promise for therapeutic applications. Simultaneously, probiotics can actively contribute to inhibiting trichomonal infections, offering a novel approach for preventing and treating diseases such as P. hominis. Further in vivo studies are required to validate these results and explore their potential in animal and human health.

Alternariol Monomethyl-Ether Induces Toxicity via Cell Death and Oxidative Stress in Swine Intestinal Epithelial Cells.

Alternariol monomethyl-ether (AME), together with altenuene and alternariol, belongs to the Alternaria mycotoxins group, which can contaminate different substrates, including cereals. The aim of the present study was to obtain a deeper understanding concerning the effects of AME on pig intestinal health using epithelial intestinal cell lines as the data concerning the possible effects of Alternaria toxins on swine are scarce and insufficient for assessing the risk represented by Alternaria toxins for animal health. Our results have shown a dose-related effect on IPEC-1 cell viability, with an IC50 value of 10.5 μM. Exposure to the toxin induced an increase in total apoptotic cells, suggesting that AME induces programmed cell death through apoptosis based on caspase-3/7 activation in IPEC-1 cells. DNA and protein oxidative damage triggered by AME were associated with an alteration of the antioxidant response, as shown by a decrease in the enzymatic activity of catalase and superoxide dismutase. These effects on the oxidative response can be related to an inhibition of the Akt/Nrf2/HO-1 signaling pathway; however, further studies are needed in order to validate these in vitro data using in vivo trials in swine.

PSII-6 The interactive effects of deoxynivalenol and lipopolysaccharides on gut integrity and inflammation in IPEC-J2 cells

Abstract The objective of this research is to investigate the interactive effects of deoxynivalenol (DON) and lipopolysaccharides (LPS) on gut integrity and inflammation using porcine intestinal epithelial cell line (IPEC-J2). Three experiments were conducted in this study: Exp 1: cells were treated with control, DON (0.5 ppm) or LPS (10 ug/mL) individually and combination of DON and LPS for 24 h, then measure the Lactate dehydrogenase (LDH) release and interleukin 8 (IL8) production in medium and measure the transepithelial electric resistance (TEER) and FITC-dextran passage rate at the end of each experiment. 2) Treat the cells with control or DON for 24 h, then split the two groups for control or LPS treatment for additional 24 h; same measurements were taken as in experiment 1; 3) Treat the cells with control or LPS for 24 h, then split the two groups for control or DON treatment for additional 24 h; same measurements were taken as in previous experiments. In each experiment, six replications were done. Data were analyzed for the effect of DON, LPS and their interaction. Treatments did not affect the LDH in all experiments except for the high LDH release in the treatment of LPS followed by DON in experiment 3. In experiment 1, DON, LPS or combination of DON and LPS significantly reduced the TEER value by 15.1%, 21.7% and 23.9% respectively with only DON has effect on FITC-dextran passage rate (P &amp;lt; 0.001). LPS significantly increased IL8 secretion in the medium (P &amp;lt; 0.001). In experiment 2, DON or LPS decreased TEER value whereas the cells treated with DON before were more susceptible to LPS (23.6% more reduction compared without DON treatment before). Similarly, the FITC-dextran passage rate was also increased by LPS in the cells exposed to DON before (P &amp;lt; 0.05). In addition, LPS significantly increased IL8 secretion no matter the cells was exposed to DON before. In experiment 3, DON or LPS decreased TEER value (P &amp;lt; 0.05) and cells treated with LPS before were more susceptible to DON (11.8% more reduction compared with no LPS treatment before). However, only DON increased FITC-dextran passage rate. The secretion of IL8 increased by DON or LPS with greatest secretion when cells treated with DON which exposed to LPS before (P &amp;lt; 0.05). In conclusion, the results suggested that cells were more susceptible to LPS when the cells were exposed to DON before or vice versa, and the synergetic effect is limited when LPS and DON were added to cells simultaneously.

The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into four treatment groups: (1) basal diet without infection (NC), (2) basal diet with E. maxima infection (PC), (3) carnosine at 10.0 mg/kg feed with PC (HCS), and (4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on day 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1β gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1β, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection.

Single-cell transcriptomics predicts colony stimulating factor-1 regulation of age-dependent intestinal epithelial restitution

Intestinal diseases associated with ischemic injury, which damages the principal barrier against noxious luminal contents, result in unacceptably poor outcomes in newborns. Ischemia-induced loss of the intestinal epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age-dependency of intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier versus older patients. While this age-dependence in repair has not been demonstrated in traditional rodent models, we have developed a highly translational pig model of intestinal ischemic injury and repair that does reflect this difference. We have shown that, while juvenile (weaned) pigs recover rapidly after ischemic intestinal injury, barrier repair is markedly underdeveloped in neonatal (nursing) pigs due to complete failure of epithelial restitution. Importantly, we found that restitution in neonates can be rescued by the direct application of homogenized mucosa from ischemia-injured small intestine from juvenile pigs. The mechanisms that allow for this restitution and rescue remain to be defined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells collected from recovering ischemic jejunum of juvenile pigs were processed for single cell RNA sequencing, unbiased clustering and upstream regulator analysis. A porcine intestinal epithelial cell line (IPEC-J2) and banked tissues from prior rescue experiments were qualitatively and functionally assessed for activity of predicted upstream regulators. Single cell transcriptomics in recovering juvenile epithelium revealed a subcluster of absorptive enterocytes that express several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation by many potential molecules, including colony stimulating factor-1 (CSF-1) which is known to induce cell migration in non-intestinal epithelial tissues. To begin validating this prediction, we demonstrated that CSF-1 was enriched in the ischemic juvenile mucosa which rescues neonatal restitution and documented expression of the CSF-1 receptor (CSF1R) in both neonatal and juvenile epithelium, indicating that these cells are equipped to respond to CSF-1. CSF-1 and CSF1R co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued (but not control) neonates. Further, the CSF1R inhibitor BLZ945 reduced restitution in scratch wounded IPEC-J2 cells. Single cell transcriptomics have the power to inform potential novel therapeutic targets, such as CSF-1, to improve mucosal recovery in neonates with intestinal failure in this unique and powerful pig model. NIH K01 OD028207, NIH R01 HD095876, NIH U01 TR002953, NIH T32 OD011130, NIH P30 DK034987, USDA NIFA 1007263 and 07985. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cell-Free Culture Supernatant of Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 Reduces the Pathogenicity of NetB-Positive Clostridium perfringens in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77-85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity.

Gentamicin Susceptibility and Comparison of Adhesion and Invasion of Caco-2 and HD11 Cell Lines by Salmonella enterica Serotypes

Foodborne Salmonella serovars are important facultative intracellular pathogens that cause gastroenteritis in humans. Four strains from three of the more predominant Salmonella serovars in poultry were studied: Typhimurium, Enteritidis, and Heidelberg. Gentamicin susceptibility was determined using an agar disc diffusion test and minimum inhibitory concentration (MIC) assays for S. Typhimurium ATCC 14028 and S. Heidelberg ARI-14. Both strains were susceptible to gentamicin in disc diffusion. The MIC of gentamicin was approximately 125 mg/ml for all strains tested. These strains’ adhesion and invasion abilities were determined with two different cell lines, a human intestinal epithelial cell line (Caco-2) as well as a chicken macrophage cell line (HD11). Attachment percentages for each Salmonella strain were greater than the strain’s ability to invade cells. Similar attachment percentages to Caco-2 cells were observed for S. Typhimurium and S. Heidelberg. Attachment percentages were lower in HD11 cells than in Caco-2 cells, although Salmonella exhibited higher apparent HD11 invasion, likely from HD11 phagocytosis. Salmonella Enteritidis showed lower rates of adhesion and invasion in HD11 cells compared to Salmonella Typhimurium. Developing a better understanding of Salmonella virulence mechanisms is critical to reducing Salmonella infections.

Selection of Fermentation Supernatant from Probiotic Strains Exhibiting Intestinal Epithelial Barrier Protective Ability and Evaluation of Their Effects on Colitis Mouse and Weaned Piglet Models.

The intestinal epithelial barrier can prevent the invasion of pathogenic microorganisms and food antigens to maintain a consistent intestinal homeostasis. However, an imbalance in this barrier can result in various diseases, such as inflammatory bowel disease, malnutrition, and metabolic disease. Thus, the aim of this study was to select probiotic strains with epithelial barrier-enhancing ability in cell-based model and further investigate them for their improving effects on colitis mouse and weaned piglet models. The results showed that selected specific cell-free fermentation supernatants (CFSs) from Ligilactobacillus salivarius P1, Lactobacillus gasseri P12, and Limosilactobacillus reuteri G7 promoted intestinal epithelial cell growth and proliferation, strengthening the intestinal barrier in an intestinal epithelial cell line Caco-2 model. Further, the administration of CFSs of L. salivarius P1, L. gasseri P12, and L. reuteri G7 were found to ameliorate DSS-induced colitis in mice. Additionally, spray-dried powders of CFS from the three strains were examined in a weaned piglet model, only CFS powder of L. reuteri G7 could ameliorate the feed/gain ratio and serum levels of D-lactate and endotoxin. In conclusion, a new potential probiotic strain, L. reuteri G7, was selected and showed ameliorating effects in both colitis mouse and weaned piglet models.

Macleaya cordata Extract Inhibits LPS-Induced IPEC-J2 Inflammation Through TLR4-Mediated NF-κB and MAPK Signaling Pathways

Objectives: The Macleaya cordata has long been utilized as a traditional medicinal remedy for treating inflammation in certain countries, and it is also being considered as a potential alternative to antibiotic growth promoters in livestock breeding. However, the molecular mechanisms underlying its anti-inflammatory characteristics have not been thoroughly investigated. In this study, we assessed the anti-inflammatory effects of the Macleaya cordata extract (MCE) and elucidated the molecular mechanism in the porcine intestinal epithelial cell line J2 (IPEC-J2) stimulated by lipopolysaccharide (LPS). Methods: The IPEC-J2 cells were pretreated with various concentrations of MCE (50 ng/mL, 100 ng/mL, and 150 ng/mL) prior to stimulation with LPS to induce of inflammation. Subsequently, the secretion levels and mRNA expression of the inflammatory cytokines IL-6, TNF-α, IFN-γ, and IL-10 were assessed. The impact of MCE on the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways was investigated. Results: The results demonstrated that MCE significantly suppressed the production and mRNA expression levels of pro-inflammatory cytokines in LPS-induced IPEC-J2 cells, while promoting the expression of anti-inflammatory cytokines. Moreover, it down-regulated the phosphorylation of NF-κB (p65) and attenuated the expression of phosphorylated JNK, ERK, and p38 MAPKs in a concentration-dependent manner. Furthermore, MCE downregulated the expression of TLR4 and MyD88, which serve as crucial upstream signaling molecules. The findings of this study suggest that the anti-inflammatory effect of MCE is attributed to its ability to regulate inflammatory cytokines and mediators by inhibiting the TLR4-mediated NF-κB and MAPK signaling pathways. Conclusions: Therefore, MCE holds promise as a potential therapeutic and preventive agent for managing inflammatory reactions and diseases in livestock and poultry breeding, warranting further investigation.

Innovative probiotic Escherichia coli nissle 1917-derived outer membrane vesicles coupled microspheres demonstrate anti-inflammatory and barrier-enhancing properties in colitis

Occurrence of inflammatory bowel disease (IBD) is increasing worldwide and has become a global health threat. Nonetheless, the effective prevention and available treatment interventions of IBD are limited. Bacterial outer membrane vesicles (OMVs) derived from probiotic bacteria such as Escherichia coli Nissle 1917 (EcN) exhibit promising anti-inflammatory, anti-oxidant, immunomodulatory, and gut microbiota modulatory functions. However, the application of OMVs in vivo encounters potential challenges due to poor tolerance against gastric juice. A delivery system of probiotic-EcN-derived OMVs was established by aldehyde-coupling to silica microspheres (SAP@OMVs) in this study. These SAP@OMVs microspheres were nontoxic to murine macrophage RAW 264.7 and human intestinal epithelial cell lines Caco-2. In the dextran sulfate sodium (DSS)-induced acute colitis models, the application of SAP@OMVs significantly improved mouse survival and alleviated the harmful effects of DSS by maintaining colon length, reducing colon injury, decreasing expression of inflammatory factors such as TNF-α and IL-1β, and increasing expression of the tight junction proteins gene zonula occludens-1 (ZO-1). The SAP@OMVs restored gut microbiota with increasing abundance of the Lactobacillus. In conclusion, the novel vehicle of OMVs demonstrated anti-inflammatory effects, representing a crucial strategy for clinical translation of OMVs as a potential therapeutic intervention for IBD.

Gingipain-carrying outer membrane vesicles from Porphyromonas gingivalis cause barrier dysfunction of Caco-2 cells by releasing gingipain into the cytosol

Ingestion of Porphyromonas gingivalis, a periodontal pathogen, disrupts the intestinal barrier in mice. However, the involvement of outer membrane vesicles (OMVs) secreted from P. gingivalis in the destruction of the intestinal barrier remains unclear. In this study, we tested the hypothesis that OMVs carrying gingipains, the major cysteine proteases produced by P. gingivalis, affects the intestinal barrier function. OMVs increased the permeability of the Caco-2 cell monolayer, a human intestinal epithelial cell line, accompanied by degradation of the tight junction protein occludin. In contrast, OMVs prepared from mutant strains devoid of gingipains failed to induce intestinal barrier dysfunction or occludin degradation in Caco-2 cells. A close histological examination revealed the intracellular localization of gingipain-carrying OMVs. Gingipain activity was detected in the cytosolic fraction of Caco-2 cells after incubation with OMVs. These results suggest that gingipains were internalized into intestinal cells through OMVs and transported into the cytosol, where they then directly degraded occludin from the cytosolic side. Thus, P. gingivalis OMVs might destroy the intestinal barrier and induce systemic inflammation via OMV itself or intestinal substances leaked into blood vessels, causing various diseases.

Intestinal epithelial cells of Japanese flounder (Paralichthys olivaceus) as an in vitro model for studying intestine immune function based on transcriptome analysis

Japanese flounder (Paralichthys olivaceus) is an economically crucial marine species, but diseases like hemorrhagic septicemia caused by Edwardsiella tarda have resulted in significant economic losses. E. tarda infects various hosts, and its pathogenicity in fish is not fully understood. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and are representative of typical PAMP molecules that cause activation of the immune system. The PoIEC cell line is a newly established intestinal epithelial cell line from P. olivaceus. In order to investigate whether it can be used as an in vitro model for studying the pathogenesis of E. tarda and LPS stimulation, we conducted RNA-seq experiments for the PoIECs model of E. tarda infection and LPS stimulation. In this study, transcriptome sequencing was carried out in the PoIEC cell line after treatment with LPS and E. tarda. A total of 62.52G of high-quality data from transcriptome sequencing results were obtained in nine libraries, of which an average of 87.96% data could be aligned to the P. olivaceus genome. Data analysis showed that 283 and 414 differentially expressed genes (DEGs) in the LPS versus Control (LPS-vs-Con) and E. tarda versus Control groups (Et-vs-Con), respectively, of which 60 DEGs were shared in two comparation groups. The GO terms were predominantly enriched in the extracellular space, inflammatory response, and cytokine activity in the LPS-vs-Con group, whereas GO terms were predominantly enriched in nucleus and positive regulation of transcription by RNA polymerase II in the Et-vs-Con group. KEGG analysis revealed that three immune-related pathways were co-enriched in both comparison groups, including the Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway, and Cytokine-cytokine receptor interaction. Five genes were randomly screened to confirm the validity and accuracy of the transcriptome data. These results suggest that PoIEC cell line can be an ideal in vitro model for studies of marine fish gut immunity and pathogenesis of Edwardsiellosis.

Orally Administrated Glutamate Restored EAAT1 and 3 Expression Levels Suppressed in 5-Fluorouracil-damaged Intestinal Epithelial Cells.

5-Fluorouracil (5-FU) treatment induces intestinal mucositis, with diarrhea as the primary symptom. Mucositis significantly reduces patients' quality of life (QOL). Amino acids such as glutamate are beneficial for treating gastrointestinal disorders; however, the underlying mechanism remains unclear. Therefore, this study aimed to clarify the role of excitatory amino acid transporters (EAATs) in 5-FU-induced intestinal injury. The rat intestinal epithelial cell line (IEC-6) was used to evaluate whether the EAAT inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (L-trans-PDC) affects 5-FU-induced cytotoxicity. Mice with 5-FU-induced mucositis were used to determine the effects of glutamate on EAATs expression levels. Treatment with L-trans-PDC suppressed IEC-6 cell growth. It also exacerbated the 5-FU-induced cell growth suppression and increased inflammatory cytokine expression. In addition, mice treated with 5-FU+Glutamate showed higher EAAT1,3 expression than 5-FU only-treated mice. Decreased EAAT levels worsen intestinal cell damage caused by 5-FU, suppress cell growth, and induce inflammation. This study contributes to the understanding EAAT and its relationship with intestinal mucositis, which can aid in the development of novel preventive strategies for cancer chemotherapy.

Trypanosoma cruzi interaction with host tissues modulate the composition of large extracellular vesicles

Trypanosoma cruzi is the protozoan that causes Chagas disease (CD), an endemic parasitosis in Latin America distributed around the globe. If CD is not treated in acute phase, the parasite remains silent for years in the host's tissues in a chronic form, which may progress to cardiac, digestive or neurological manifestations. Recently, studies indicated that the gastrointestinal tract represents an important reservoir for T. cruzi in the chronic phase. During interaction T. cruzi and host cells release extracellular vesicles (EVs) that modulates the immune system and infection, but the dynamics of secretion of host and parasite molecules through these EVs is not understood. Now, we used two cell lines: mouse myoblast cell line C2C12, and human intestinal epithelial cell line Caco-2to simulate the environments found by the parasite in the host. We isolated large EVs (LEVs) from the interaction of T. cruzi CL Brener and Dm28c/C2C12 and Caco-2 cells upon 2 and 24 h of infection. Our data showed that at two hours there is a strong cellular response mediated by EVs, both in the number, variety and enrichment/targeting of proteins found in LEVs for diverse functions. Qualitative and quantitative analysis showed that proteins exported in LEVs of C2C12 and Caco-2 have different patterns. We found a predominance of host proteins at early infection. The parasite-host cell interaction induces a switch in the functionality of proteins carried by LEVs and a heterogeneous response depending on the tissues analyzed. Protein–protein interaction analysis showed that cytoplasmic and mitochondrial homologues of the same parasite protein, tryparedoxin peroxidase, were differentially packaged in LEVs, also impacting the interacting molecule of this protein in the host. These data provide new evidence that the interaction with T. cruzi leads to a rapid tissue response through the release of LEVs, reflecting the enrichment of some proteins that could modulate the infection environment.

Bacillus subtilis Induces Human Beta Defensin-2 Through its Lipoproteins in Human Intestinal Epithelial Cells.

Human intestinal epithelial cells (IECs) play an important role in maintaining gut homeostasis by producing antimicrobial peptides (AMPs). Bacillus subtilis, a commensal bacterium, is considered a probiotic. Although its protective effects on intestinal health are widely reported, the key component of B. subtilis responsible for its beneficial effects remains elusive. In this study, we tried to identify the key molecules responsible for B. subtilis-induced AMPs and their molecular mechanisms in a human IEC line, Caco-2. B. subtilis increased human beta defensin (HBD)-2 mRNA expression in a dose- and time-dependent manner. Among the B. subtilis microbe-associated molecular patterns, lipoprotein (LPP) substantially increased the mRNA expression and protein production of HBD-2, whereas lipoteichoic acid and peptidoglycan did not show such effects. Those results were confirmed in primary human IECs. In addition, both LPP recognition and HBD-2 secretion mainly took place on the apical side of fully differentiated and polarized Caco-2 cells through Toll-like receptor 2-mediated JNK/p38MAP kinase/AP-1 and NF-κB pathways. HBD-2 efficiently inhibited the growth of the intestinal pathogens Staphylococcus aureus and Bacillus cereus. Furthermore, LPPs pre-incubated with lipase or proteinase K decreased LPP-induced HBD-2 expression, suggesting that the lipid and protein moieties of LPP are crucial for HBD-2 expression. Q Exactive Plus mass spectrometry identified 35 B. subtilis LPP candidates within the LPP preparation, and most of them were ABC transporters. Taken together, these results suggest that B. subtilis promotes HBD-2 secretion in human IECs mainly with its LPPs, which might enhance the protection from intestinal pathogens.

Olive Mill Waste-Water Extract Enriched in Hydroxytyrosol and Tyrosol Modulates Host-Pathogen Interaction in IPEC-J2 Cells.

The dietary supplementation of olive oil by-products, including olive mill waste-water (OMWW) in animal diets, is a novel application that allows for their re-utilization and recycling and could potentially decrease the use of antibiotics, antimicrobial resistance risk in livestock species, and the occurrence of intestinal diseases. Salmonella serovar typhimurium is one of the most widespread intestinal pathogens in the world, causing enterocolitis in pigs. The aim of this study was to investigate the effect of an OMWW extract enriched in polyphenols (hydroxytyrosol and tyrosol) in the immune response of an intestinal porcine epithelial cell line (IPEC-J2) following S. typhimurium infection. Cells were pre-treated with OMWW-extract polyphenols (OMWW-EP, 0.35 and 1.4 µg) for 24 h and then infected with S. typhimurium for 1 h. We evaluated bacterial invasiveness and assayed IPEC-J2 gene expression with RT-qPCR and cytokine release with an ELISA test. The obtained results showed that OMWW-EP (1.4 µg) significantly reduced S. typhimurium invasiveness; 0.35 µg decreased the IPEC-J2 gene expression of IL1B, MYD88, DEFB1 and DEFB4A, while 1.4 µg down-regulated IL1B and DEFB4A and increased TGFB1. The cytokine content was unchanged in infected cells. This is the first study demonstrating the in vitro immunomodulatory and antimicrobial activity of OMWW extracts enriched in polyphenols, suggesting a protective role of OMWW polyphenols on the pig intestine and their potential application as feed supplements in farm animals such as pigs.

KLF4 interacts with TXNIP to modulate the pyroptosis in ulcerative colitis via regulating NLRP3 signaling.

Ulcerative colitis (UC) is one of the most common diseases in the gastrointestinal tract related to abnormal inflammation. Pyroptosis, which is characterized by the formation of inflammasome, activation of caspase-1, and separation of N- and C-terminus of gasdermin D (GSDMD), and may be involved in the pathogenesis of IBD. Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor expressed in differentiated epithelial cells. KLF4 mediates proinflammatory signaling in macrophages. Here, we tested whether KLF4 is functional in pyroptosis of UC. In human UC tissues and/or lipopolysaccharide (LPS)/adenosine 5-triphosphate (ATP) stimulation human colon epithelial cells, KLF4, TXNIP, Cleave-Caspase-1, and GSDMD expression were detected through quantitative reverse transcription polymerase chain reaction (PCR), immunohistochemical and western blot assay. Interleukin (IL)-1β and IL-18 levels were quantified by enzyme-linked immunosorbent assay. We successfully constructed a KLF4-silenced colon epithelial cell line using an adenovirus vector. We apply the UCSC and JASPAR to predict the KLF4 binding sites in the promoter region of TXNIP. In human UC tissues and/or LPS/ATP stimulation human colon epithelial cells, KLF4, TXNIP, Caspase-1, and GSDMD expression level were significantly elevated via quantitative reverse transcription PCR, immunohistochemical and western blot assay. Moreover, We identified that there is an interaction between KLF4 and TXNIP through Yeast double hybrid assay and CO-IP assay. We successfully constructed a KLF4-silenced human intestinal epithelial cell line. In LPS/ATP stimulation KLF4-silenced human intestinal epithelial cells, KLF4, TXNIP, Cleave Caspase-1, ASC, and GSDMD expression level were significantly decreased via quantitative reverse transcription PCR. Our results confirm that KLF4 can positively regulate the expression of TXNIP and regulate the pyroptosis process of UC through the TXNIP/NLRP3 pathway.

Explore Key Genes and Mechanisms Involved in Colon Cancer Progression Based on Bioinformatics Analysis.

To explore underlying mechanisms related to the progression of colon cancer and identify hub genes associated with the prognosis of patients with colon cancer. GSE10950 and GSE62932 were downloaded from the Gene Expression Omnibus (GEO) database. GEO2R was utilized to screen out the differentially expressed genes (DEGs). Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted on DEGs. Moreover, STRING and Cytoscape software were utilized for establishing the network of protein-protein interaction (PPI) and identifying hub genes. Afterward, data from The Cancer Genome Atlas (TCGA) was utilized for identifying prognosis-related hub genes by Kaplan-Meier survival analysis. Colon cancer cell line LOVO and human normal intestinal epithelial cell line NCM-460 were exploited to demonstrate the differential expression of selected hub genes through RT-qPCR and western blot. The LOVO cells were transfected to regulate expressions of prognosis-associated genes, followed by exploring the effects of those genes on prognosis by Cell Counting Kit-8 assay and colony-forming assay for cancer cell proliferation, cell scratch test and transwell migration assay for cancer cell migration and Annexin V-PE/7-AAD double staining as well as flow cytometry for cancer cell apoptosis. In this study, 266 common DEGs were obtained from the intersection of two datasets. The GO analysis suggested the common DEGs mainly participated in the one-carbon metabolic process, cell cycle G2/M phase transition, organelle fission, cell cycle phase transition regulation, and regulation of mitotic cell cycle phase transition. The KEGG analysis demonstrated the common DEGs were related to the p53 signaling pathway, nitrogen metabolism, mineral absorption, and cell cycle. 10 hub genes including CCNB1, KIF4A, TPX2, MT1F, PRC1, PLK4, CALD1, MMP9, CLCA1, and MMP1 were identified and CCNB1, CLCA1, and PLK4 were prognosis-related. Increased expression of CCNB1, CLCA1, and PLK4 restrained proliferation as well as migration of cancer cells and induced apoptosis of cancer cells. CCNB1, KIF4A, TPX2, MT1F, PRC1, PLK4, CALD1, MMP9, CLCA1, and MMP1 were identified as hub genes and CCNB1, CLCA1, and PLK4 could inhibit the progression of colon cancer through inhibiting proliferation as well as migration of the cancer cell and promoting apoptosis of cancer cell.

P054 R. intestinalis improves inflammatory bowel disease by preventing gut uric acid absorption through inhibiting METTL3 mediated m6A modification of SLC2A9

Abstract Background Inflammatory bowel disease (IBD) is a chronic inflammatory disease with persistent, recurrent, unsatisfactory response, and a poor prognosis. The incidence of Chinese IBD has increased rapidly, and Westernized diets such as purine-rich foods play an important role in its rapid growth. The alteration of purine-urate metabolism by microbiota may contribute to the development of IBD. Methods In this study, metagenomic and metabolomic analyses were performed on fecal samples from 30 untreated IBD patients and 30 healthy controls. R. intestinalis was used to observe the effect on serum uric acid levels in mice, respectively. Body weight, colon length, disease activity score, serum uric acid levels, and electron microscopy were all assessed. The RNAseq analysis of intestinal tissue from DSS-induced colitis mice intervened with R. intestinalis and co-cultured with R. intestinalis and the normal intestinal epithelial cell line NCM460, combined with m6A antibody dot blot assay, RM2Target database, and MeRIP sequencing data analysis, provided evidence that SLC2A9 was modified by METTL3, and SRAMP database predicted the potential for SLC2A9 mRNA to be modified by m6A after R. intestinalis intervention. Results Multi-omics analysis revealed that the abundance of R. intestinalis was significantly decreased in untreated IBD patients, and metabolomics revealed a correlation between high uric acid levels and disease severity, while the multi-omics integrated analysis suggested a significant negative correlation between R. intestinalis and uric acid. The research team confirmed that R. intestinalis can lower blood uric acid levels in mice and found that R. intestinalis intervention can alleviate the intestinal mucosal inflammation exacerbated by uric acid treatment in a DSS-induced mouse colitis model. Butyric acid has been reported to regulate m6A, and dot blot assays showed that R. intestinalis can reduce the m6A modification level of intestinal epithelial cells, METTL3, and SLC2A9. RM2Target database identified the protein-RNA interaction of the m6A regulator (WER) for SLC2A9 mRNA, and MeRIP sequencing data further indicated that METTL3 can bind to SLC2A9 mRNA through m6A modification. Meanwhile, based on sequence prediction, the research team identified 13 potential m6A modification sites on SLC2A9 mRNA, of which 4 sites had extremely high credibility . The highest credibility site had a score of 0.694, with a modification site sequence of GGACU located on an adenosine nucleotide. Conclusion R. intestinalis inhibit the METTL3 mediated m6A modification of intestinal epithelial SLC2A9, reducing the expression of SLC2A9, preventing the epithelial absorption of uric acid, and relieving IBD-related colitis